Differentiation of strains from the Bacillus cereus group by RFLP-PFGE genomic fingerprinting.

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Otlewska, Anna; Oltuszak-Walczak, Elzbieta; Walczak, Piotr

2013-11-01

Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus anthracis, Bacillus thuringiensis, and Bacillus cereus belong to the B. cereus group. The last three species are characterized by different phenotype features and pathogenicity spectrum, but it has been shown that these species are genetically closely related. The macrorestriction analysis of the genomic DNA with the NotI enzyme was used to generate polymorphism of restriction profiles for 39 food-borne isolates (B. cereus, B. mycoides) and seven reference strains (B. mycoides, B. thuringiensis, B. weihenstephanensis, and B. cereus). The PFGE method was applied to differentiate the examined strains of the B. cereus group. On the basis of the unweighted pair group method with the arithmetic mean method and Dice coefficient, the strains were divided into five clusters (types A-E), and the most numerous group was group A (25 strains). A total of 21 distinct pulsotypes were observed. The RFLP-PFGE analysis was successfully used for the differentiation and characterization of B. cereus and B. mycoides strains isolated from different food products. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prevalence, PFGE typing, and antibiotic resistance of Bacillus cereus group isolated from food in Morocco.

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Merzougui, Souad; Lkhider, Mustapha; Grosset, Noel; Gautier, Michel; Cohen, Nozha

2014-02-01

This article reports the prevalence and antibiotic resistance of the Bacillus cereus group isolated from different foods (milk and dairy products, spices, and rice salad) in Morocco. In total, 402 different food samples collected from 2008 to 2010 were analyzed by microbiological methods to isolate B. cereus. The strains were subjected to a polymerase chain reaction test in order to verify whether they belonged to the B. cereus group. Sixty-four of all isolates (15.9%) were found to be positive. Among the sources, B. cereus strains from milk and dairy products constituted the largest proportion of isolates (33/64; 51.6%) followed by spices (22/64; 34.4%) and salad with rice (9/64; 14.1%). The genetic diversity of the strains of B. cereus group was examined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with SmaI. The enzyme restriction profiles showed a high degree of polymorphism among the strains. The results showed that PFGE analysis could reveal the genetic differences among B. cereus strains. Investigation of antibiotic-resistance profiles showed that isolates were resistant to ampicillin (98.4%), tetracycline (90.6%), oxacillin (100%), cefepime (100%), and penicillin (100%), and were susceptible to chloramphenicol (67.2%), erythromycin (84.4%), and gentamicin (100%). The results of this study indicated that B. cereus could be a significant etiological agent of food poisoning in Morocco because of its high prevalence. Also, we demonstrated that the majority of strains came from milk and dairy products. However, additional research involving cytotoxicity tests is needed to more evaluate this sanitary risk.

Klebsiella Typing

DEFF Research Database (Denmark)

Hansen, D S; Skov, R; Benedí, J.V.

2002-01-01

OBJECTIVE: To compare pulsed-field gel electrophoresis (PFGE) typing and O:K-serotyping of Klebsiella in two different epidemiological settings. METHODS: One hundred and four bacteremia isolates without known epidemiological relation and 47 isolates from an outbreak in a neonatal intensive care...... unit (NICU) were K-typed by countercurrent immunoelectrophoresis (CCIE), O-typed by an inhibition enzyme-linked immunosorbent assay method, and typed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. RESULTS: Typing data for the 104 bacteremia isolates were compared...... with regard to typability, number of types, maximum number of isolates per type, and the Discriminative Index (DI). O-typing combined with K-typing (DI 0.98) as O:K-serotyping (DI 0.99) gave a very discriminative typing system, whereas O-typing alone was not very discriminative (DI 0.76). PFGE (DI 1...

Phage Types and Genotypes of Shiga Toxin-Producing Escherichia coli O157:H7 Isolates from Humans and Animals in Spain: Identification and Characterization of Two Predominating Phage Types (PT2 and PT8)

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Mora, Azucena; Blanco, Miguel; Blanco, Jesús E.; Alonso, M. Pilar; Dhabi, Ghizlane; Thomson-Carter, Fiona; Usera, Miguel A.; Bartolomé, Rosa; Prats, Guillermo; Blanco, Jorge

2004-01-01

Phage typing and DNA macrorestriction fragment analysis by pulsed-field electrophoresis (PFGE) were used for the epidemiological subtyping of a collection of Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains isolated in Spain between 1980 and 1999. Phage typing distinguished a total of 18 phage types among 171 strains isolated from different sources (67 humans, 82 bovines, 12 ovines, and 10 beef products). However, five phage types, phage type 2 (PT2; 42 strains), PT8 (33 strains), PT14 (14 strains), PT21/28 (11 strains), and PT54 (16 strains), accounted for 68% of the study isolates. PT2 and PT8 were the most frequently found among strains from both humans (51%) and bovines (46%). Interestingly, we detected a significant association between PT2 and PT14 and the presence of acute pathologies. A group of 108 of the 171 strains were analyzed by PFGE, and 53 distinct XbaI macrorestriction patterns were identified, with 38 strains exhibiting unique PFGE patterns. In contrast, phage typing identified 15 different phage types. A total of 66 phage type-PFGE subtype combinations were identified among the 108 strains. PFGE subtyping differentiated between unrelated strains that exhibited the same phage type. The most common phage type-PFGE pattern combinations were PT2-PFGE type 1 (1 human and 11 bovine strains), PT8-PFGE type 8 (2 human, 6 bovine, and 1 beef product strains), PT2-PFGE subtype 4A (1 human, 3 bovine, and 1 beef product strains). Nine (29%) of 31 human strains showed phage type-PFGE pattern combinations that were detected among the bovine strains included in this study, and 26 (38%) of 68 bovine strains produced phage type-PFGE pattern combinations observed among human strains included in this study, confirming that cattle are a major reservoir of strains pathogenic for humans. PT2 and PT8 strains formed two groups which differed from each other in their motilities, stx genotypes, PFGE patterns, and the severity of the illnesses that they caused

Multilocus Sequence Typing Scheme versus Pulsed-Field Gel Electrophoresis for Typing Mycobacterium abscessus Isolates

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Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate

2014-01-01

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. PMID:24899019

Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and NARMS

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Petermann Shana R

2011-06-01

Full Text Available Abstract Background Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA. PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. Results The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14 with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. Conclusion The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar.

Multilocus sequence typing scheme versus pulsed-field gel electrophoresis for typing Mycobacterium abscessus isolates.

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Machado, Gabriel Esquitini; Matsumoto, Cristianne Kayoko; Chimara, Erica; Duarte, Rafael da Silva; de Freitas, Denise; Palaci, Moises; Hadad, David Jamil; Lima, Karla Valéria Batista; Lopes, Maria Luiza; Ramos, Jesus Pais; Campos, Carlos Eduardo; Caldas, Paulo César; Heym, Beate; Leão, Sylvia Cardoso

2014-08-01

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparison of double-locus sequence typing (DLST) and multilocus sequence typing (MLST) for the investigation of Pseudomonas aeruginosa populations.

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Cholley, Pascal; Stojanov, Milos; Hocquet, Didier; Thouverez, Michelle; Bertrand, Xavier; Blanc, Dominique S

2015-08-01

Reliable molecular typing methods are necessary to investigate the epidemiology of bacterial pathogens. Reference methods such as multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are costly and time consuming. Here, we compared our newly developed double-locus sequence typing (DLST) method for Pseudomonas aeruginosa to MLST and PFGE on a collection of 281 isolates. DLST was as discriminatory as MLST and was able to recognize "high-risk" epidemic clones. Both methods were highly congruent. Not surprisingly, a higher discriminatory power was observed with PFGE. In conclusion, being a simple method (single-strand sequencing of only 2 loci), DLST is valuable as a first-line typing tool for epidemiological investigations of P. aeruginosa. Coupled to a more discriminant method like PFGE or whole genome sequencing, it might represent an efficient typing strategy to investigate or prevent outbreaks. Copyright © 2015 Elsevier Inc. All rights reserved.

SNP-based typing: a useful tool to study Bordetella pertussis populations.

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Marjolein van Gent

Full Text Available To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA. In this study, a single nucleotide polymorphism (SNP typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in The Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis.

SNP-Based Typing: A Useful Tool to Study Bordetella pertussis Populations

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van der Heide, Han G. J.; Heuvelman, Kees J.; Kallonen, Teemu; He, Qiushui; Mertsola, Jussi; Advani, Abdolreza; Hallander, Hans O.; Janssens, Koen; Hermans, Peter W.; Mooi, Frits R.

2011-01-01

To monitor changes in Bordetella pertussis populations, mainly two typing methods are used; Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). In this study, a single nucleotide polymorphism (SNP) typing method, based on 87 SNPs, was developed and compared with PFGE and MLVA. The discriminatory indices of SNP typing, PFGE and MLVA were found to be 0.85, 0.95 and 0.83, respectively. Phylogenetic analysis, using SNP typing as Gold Standard, revealed false homoplasies in the PFGE and MLVA trees. Further, in contrast to the SNP-based tree, the PFGE- and MLVA-based trees did not reveal a positive correlation between root-to-tip distance and the isolation year of strains. Thus PFGE and MLVA do not allow an estimation of the relative age of the selected strains. In conclusion, SNP typing was found to be phylogenetically more informative than PFGE and more discriminative than MLVA. Further, in contrast to PFGE, it is readily standardized allowing interlaboratory comparisons. We applied SNP typing to study strains with a novel allele for the pertussis toxin promoter, ptxP3, which have a worldwide distribution and which have replaced the resident ptxP1 strains in the last 20 years. Previously, we showed that ptxP3 strains showed increased pertussis toxin expression and that their emergence was associated with increased notification in the Netherlands. SNP typing showed that the ptxP3 strains isolated in the Americas, Asia, Australia and Europe formed a monophyletic branch which recently diverged from ptxP1 strains. Two predominant ptxP3 SNP types were identified which spread worldwide. The widespread use of SNP typing will enhance our understanding of the evolution and global epidemiology of B. pertussis. PMID:21647370

Correlation between PFGE Groups and mrp/epf/sly Genotypes of Human Streptococcus suis Serotype 2 in Northern Thailand

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Prasit Tharavichitkul

2014-01-01

Full Text Available Streptococcus suis infection is a severe zoonotic disease commonly found in Northern Thailand where people often consume raw pork and/or pig’s blood. The most frequent clinical presentations are meningitis, sepsis, and endocarditis with higher rate of mortality and hearing loss sequelae. To clarify the correlation between pulsed-field gel electrophoresis (PFGE groups and mrp/epf/sly genotypes of S. suis serotype 2, 62 patient and 4 healthy pig isolates from Northern Thailand were studied. By PFGE analysis, at 66% homology, most human isolates (69.4% and 1 pig isolate were in group A, whereas 14.5% of human isolates and 3 out of 4 pig isolates were in group D. According to mrp/epf/sly genotypes, 80.6% of human isolates were identified in mrp+epf−sly− and only 12.9% were in mrp−epf−sly+ genotypes; in contrast, 1 and 3 pig isolates were detected in these two genotypes, respectively. Interestingly, all isolates of S. suis serotype 2 classified in PFGE groups A, B, and E were set in mrp+epf−sly− genotypes. These data show a close correlation between PFGE groups and mrp/epf/sly genotypes of human S. suis serotype 2.

Correlation between PFGE Groups and mrp/epf/sly Genotypes of Human Streptococcus suis Serotype 2 in Northern Thailand.

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Tharavichitkul, Prasit; Wongsawan, Kanreuthai; Takenami, Naoki; Pruksakorn, Sumalee; Fongcom, Achara; Gottschalk, Marcelo; Khanthawa, Banyong; Supajatura, Volaluk; Takai, Shinji

2014-01-01

Streptococcus suis infection is a severe zoonotic disease commonly found in Northern Thailand where people often consume raw pork and/or pig's blood. The most frequent clinical presentations are meningitis, sepsis, and endocarditis with higher rate of mortality and hearing loss sequelae. To clarify the correlation between pulsed-field gel electrophoresis (PFGE) groups and mrp/epf/sly genotypes of S. suis serotype 2, 62 patient and 4 healthy pig isolates from Northern Thailand were studied. By PFGE analysis, at 66% homology, most human isolates (69.4%) and 1 pig isolate were in group A, whereas 14.5% of human isolates and 3 out of 4 pig isolates were in group D. According to mrp/epf/sly genotypes, 80.6% of human isolates were identified in mrp (+) epf (-) sly (-) and only 12.9% were in mrp (-) epf (-) sly (+) genotypes; in contrast, 1 and 3 pig isolates were detected in these two genotypes, respectively. Interestingly, all isolates of S. suis serotype 2 classified in PFGE groups A, B, and E were set in mrp (+) epf (-) sly (-) genotypes. These data show a close correlation between PFGE groups and mrp/epf/sly genotypes of human S. suis serotype 2.

Molecular Typing and Epidemiology of Human Listeriosis Cases, Denmark, 2002-2012

DEFF Research Database (Denmark)

Jensen, Anne Kvistholm; Björkman, Jonas T; Ethelberg, Steen

2016-01-01

Denmark has a high incidence of invasive listeriosis (0.9 cases/100,000 population in 2012). We analyzed patient data, clinical outcome, and trends in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of Listeria monocytogenes strains isolated in Denmark during 2002......-2012. We performed 2-enzyme PFGE and serotyping on 559 isolates and MLST on 92 isolates and identified some correlation between molecular type and clinical outcome and patient characteristics. We found 178 different PFGE types, but isolates from 122 cases belonged to just 2 closely related PFGE types......, clonal complex 8 and sequence type 8. These 2 types were the main cause of a peak in incidence of invasive listeriosis during 2005-2009, possibly representing an outbreak or the presence of a highly prevalent clone. However, current typing methods could not fully confirm these possibilities, highlighting...

Discriminatory usefulness of pulsed-field gel electrophoresis and sequence-based typing in Legionella outbreaks.

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Quero, Sara; García-Núñez, Marian; Párraga-Niño, Noemí; Barrabeig, Irene; Pedro-Botet, Maria L; de Simon, Mercè; Sopena, Nieves; Sabrià, Miquel

2016-06-01

To compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) in Legionella outbreaks for determining the infection source. Twenty-five investigations of Legionnaires' disease were analyzed by PFGE, SBT and Dresden monoclonal antibody. The results suggested that monoclonal antibody could reduce the number of Legionella isolates to be characterized by molecular methods. The epidemiological concordance PFGE-SBT was 100%, while the molecular concordance was 64%. Adjusted Wallace index (AW) showed that PFGE has better discriminatory power than SBT (AWSBT→PFGE = 0.767; AWPFGE→SBT = 1). The discrepancies appeared mostly in sequence type (ST) 1, a worldwide distributed ST for which PFGE discriminated different profiles. SBT discriminatory power was not sufficient verifying the infection source, especially in worldwide distributed STs, which were classified into different PFGE patterns.

Phene Plate (PhP) biochemical fingerprinting. A screening method for epidemiological typing of enterococcal isolates.

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Saeedi, B; Tärnberg, M; Gill, H; Hällgren, A; Jonasson, J; Nilsson, L E; Isaksson, B; Kühn, I; Hanberger, H

2005-09-01

Pulsed-field gel electrophoresis (PFGE) is currently considered the gold standard for genotyping of enterococci. However, PFGE is both expensive and time-consuming. The purpose of this study was to investigate whether the PhP system can be used as a reliable clinical screening method for detection of genetically related isolates of enterococci. If so, it should be possible to minimize the number of isolates subjected to PFGE typing, which would save time and money. Ninety-nine clinical enterococcal isolates were analysed by PhP (similarity levels 0.90-0.975) and PFGE (similarity levels PhP also belong to the same cluster according to PFGE, i.e. p(A(PFGE)=B(PFGE) * A(PhP)=B(PhP)), and the probability that a pair of isolates of different types according to PhP also belong to different clusters according to PFGE, i.e. p(A(PFGE) not equalB(PFGE) * A(PhP) not equalB(PhP)), was relatively high for E. faecalis (0.86 and 0.96, respectively), but was lower for E. faecium (0.51 and 0.77, respectively). The concordance which shows the probability that PhP and PFGE agree on match or mismatch was 86%-93% for E. faecalis and 54%-66% for E. faecium, which indicates that the PhP method may be useful for epidemiological typing of E. faecalis in the current settings but not for E. faecium.

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing.

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Magnússon, S H; Guðmundsdóttir, S; Reynisson, E; Rúnarsson, A R; Harðardóttir, H; Gunnarson, E; Georgsson, F; Reiersen, J; Marteinsson, V Th

2011-10-01

Campylobacter jejuni isolates from various sources in Iceland were genotyped with the aim of assessing the genetic diversity, population structure, source distribution and campylobacter transmission routes to humans. A collection of 584 Campylobacter isolates were collected from clinical cases, food, animals and environment in Iceland in 1999-2002, during a period of national Campylobacter epidemic in Iceland. All isolates were characterized by pulse field gel electrophoresis (PFGE), and selected subset of 52 isolates representing the diversity of the identified PFGE types was further genotyped using multilocus sequence typing (MLST) and fla-SVR sequencing to gain better insight into the population structure. The results show a substantial diversity within the Icelandic Campylobacter population. Majority of the human Campylobacter infections originated from domestic chicken and cattle isolates. MLST showed the isolates to be distributed among previously reported and common sequence type complexes in the MLST database. The genotyping of Campylobacter from various sources has not previously been reported from Iceland, and the results of the study gave a valuable insight into the population structure of Camp. jejuni in Iceland, source distribution and transmission routes to humans. The geographical isolation of Iceland in the north Atlantic provides new information on Campylobacter population dynamics on a global scale. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology No claim to Icelandic Government works.

Occurrence, species distribution, antimicrobial resistance and clonality of methicillin- and erythromycin-resistant staphylococci in the nasal cavity of domestic animals

DEFF Research Database (Denmark)

Bagcigil, Funda A.; Moodley, Arshnee; Baptiste, Keith E.

2007-01-01

beta-Lactams and macrolides are important antibiotics for treatment of staphylococcal infections in both humans and animals. The aim of the study was to investigate the occurrence, species distribution and clonality of methicillin and erythromycin-resistant staphylococci in the nasal cavity of dogs......, horses, pigs, and cattle in Denmark. Nasal swabs were collected from a total of 400 animals, including 100 individuals of each species. Methicillin and erythromycin-resistant staphylococci were isolated on selective media, identified by 16S rDNA sequencing, and typed by pulsed field gel electrophoresis...... (PFGE). Methicillin-resistant coagulase-negative staphylococci (MRCoNS) harbouring mecA were isolated from horses (50%) and dogs (13%), but not from food animals. The species identified were S. haemolyticus (n = 21), S. vitulinus (n = 19), S. sciuri (n = 13), S. epidermidis (n = 8), and S. warneri (n...

Molecular Typing of Salmonella Isolates in Poultry by Pulsed-Field Gel Electrophoresis in Iran

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Narges Golab

2014-11-01

Full Text Available Background: Salmonella is one of the most widespread zoonotic enter pathogenic microorganisms found in the global food chain. Poultryand Poultry products have been identified as one of the important foodborne sources of Salmonella. Pulsed-Field Gel Electrophoresis (PFGE is a gold standard typing method for identification of Salmonella isolates during outbreaks and epidemiological investigations. Objectives: The aim of this study was to carry out molecular typing of Salmonella enterica spp. by PFGE technique. Materials and Methods: All 47 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. Results: In the current work, PFGE and serotyping were used to subtype 47 Salmonella isolates belonging to 22 different serotypes and derived from poultry. Thirty-nine PFGE patterns out of 47 isolates were obtained. The Discrimination Index (DI by serotyping (0.93 was lower than PFGE (DI = 0.99. Conclusions: In conclusion, molecular methods such as PFGE can be used for epidemiological characterization of Salmonella serotypes.

Characterization of Listeria monocytogenes isolated from a fresh mixed sausage processing line in Pelotas-RS by PFGE

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von Laer, Ana Eucares; de Lima, Andréia Saldanha; Trindade, Paula dos Santos; Andriguetto, Cristiano; Destro, Maria Teresa; da Silva, Wladimir Padilha

2009-01-01

Listeria monocytogenes is a bacterium capable to adhere to the surfaces of equipment and utensils and subsequently form biofilms. It can to persist in the food processing environmental for extended periods of time being able to contaminate the final product. The aim of this study was to trace the contamination route of L. monocytogenes on a fresh mixed sausage processing line, from raw material to the final product. The isolates obtained were characterized by serotyping and molecular typing by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes ApaI and AscI. L. monocytogenes was detected in 25% of the samples. The samples of raw material were not contaminated, however, the microorganism was detected in 21% of the environmental samples (food contact and non-food contact), 20.8% of the equipments, 20% of the food worker’s hands, 40% of the mass ready to packaging and in all the final products samples, demonstrating that the contamination of final product occurred during the processing and the importance of cross contamination. PFGE yielded 22 pulsotypes wich formed 7 clusters, and serotyping yielded 3 serotypes and 1 serogroup, however, the presence of serotypes 4b and 1/2b in the final product is of great concern for public health. The tracing of contamination showed that some strains are adapted and persisted in the processing environment in this industry. PMID:24031402

Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana

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HADIWIYONO

2011-01-01

Full Text Available Hadiwiyono, Widada J, Subandiyah S, Fegan F (2011 Pulsed Field Gel Electrophoresis (PFGE: a DNA finger printing technique to study the genetic diversity of blood disease bacterium of banana. Biodiversitas 12: 12-16. Blood disease bacterium (BDB is the most important pathogen of bananas in Indonesia. In some field, the disease incidence reaches over 80%. Epidemiologically, the disease is similar to moko disease in South America and bugtok disease in the Philippines caused by Ralstonia solanacearum race 2. However, BDB is different in phenotype and genotype from the two diseases. Previously BDB was limited in South Sulawesi since 1920s – 1980s and recently was reported in 27 of 30 provinces in Indonesia. Pulsed-Field Gel Electrophoresis (PFGE is a genomic DNA fingerprinting method, which employs rare cutting restriction endonucleases to digest genome prior to electrophoresis using specialized condition to separate of large DNA fragments. The results showed that PFGE analysis was a discriminative tool to study the genetic diversity of BDB. Based on the PFGE analysis, BDB isolates obtained from different localities in Yogyakarta and Central Java were quit diverse.

Comparison of typing by whole genome sequencing (WGS) and pulsed field gel electrophoresis (PFGE) of isolates from a hospital outbreak with a CTX-M-15 producing Klebsiella pneumoniae

DEFF Research Database (Denmark)

Hansen, D. S.; Kaas, Rolf Sommer; Nielsen, E. M.

2013-01-01

strains, were investigated. The 44 isolates were phenotypic similar; ESBL-producers with reduced susceptibility to gentamicin and ciprofloxacin. PFGE was performed using XbaI; data handling in BioNumeric. For WGS a reference genome (outbreak isolate 2006-1-264) was assembled to a single scaffold using...

Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

Science.gov (United States)

Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

2015-01-01

Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

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Sulakvelidze, Alexander; Kekelidze, Merab; Gomelauri, Tsaro; Deng, Yingkang; Khetsuriani, Nino; Kobaidze, Ketino; De Zoysa, Aruni; Efstratiou, Androulla; Morris, J. Glenn; Imnadze, Paata

1999-01-01

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates. PMID:10488190

Enhanced discrimination of highly clonal ST22-methicillin-resistant Staphylococcus aureus IV isolates achieved by combining spa, dru, and pulsed-field gel electrophoresis typing data.

LENUS (Irish Health Repository)

Shore, Anna C

2010-05-01

ST22-methicillin-resistant Staphylococcus aureus type IV (ST22-MRSA-IV) is endemic in Irish hospitals and is designated antibiogram-resistogram type-pulsed-field group (AR-PFG) 06-01. Isolates of this highly clonal strain exhibit limited numbers of pulsed-field gel electrophoresis (PFGE) patterns and spa types. This study investigated whether combining PFGE and spa typing with DNA sequencing of the staphylococcal cassette chromosome mec element (SCCmec)-associated direct repeat unit (dru typing) would improve isolate discrimination. A total of 173 MRSA isolates recovered in one Irish hospital during periods in 2007 and 2008 were investigated using antibiogram-resistogram (AR), PFGE, spa, dru, and SCCmec typing. Isolates representative of each of the 17 pulsed-field group 01 (PFG-01) spa types identified underwent multilocus sequence typing, and all isolates were ST22. Ninety-seven percent of isolates (168 of 173) exhibited AR-PFG 06-01 or closely related AR patterns, and 163 of these isolates harbored SCCmec type IVh. The combination of PFGE, spa, and dru typing methods significantly improved discrimination of the 168 PFG-01 isolates, yielding 65 type combinations with a Simpson\\'s index of diversity (SID) of 96.53, compared to (i) pairwise combinations of spa and dru typing, spa and PFGE typing, and dru and PFGE typing, which yielded 37, 44, and 43 type combinations with SIDs of 90.84, 91.00, and 93.57, respectively, or (ii) individual spa, dru, and PFGE typing methods, which yielded 17, 17, and 21 types with SIDs of 66.9, 77.83, and 81.34, respectively. Analysis of epidemiological information for a subset of PFG-01 isolates validated the relationships inferred using combined PFGE, spa, and dru typing data. This approach significantly enhances discrimination of ST22-MRSA-IV isolates and could be applied to epidemiological investigations of other highly clonal MRSA strains.

Salmonella isolated from the feces of migrating cranes at the Izumi Plain (2002-2008): serotype, antibiotic sensitivity and PFGE type.

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Kitadai, Noriyuki; Ninomiya, Naoko; Murase, Toshiyuki; Obi, Takeshi; Takase, Kozo

2010-07-01

From November 2002 to February 2008, 2,251 crane feces were collected at the Izumi Plain in Kagoshima Prefecture. Salmonella enterica was isolated from 359 feces (15.9%), of which 332 (92.5%) were Salmonella Typhimurium (ST), 9 were S. Hvittingfoss/II, 4 were S. Abaetetuba, 3 were S. Enteritidis, 2 were S. Konstanz, 1 was S. Pakistan and 8 were untyped isolates, respectively. Against 12 antimicrobial agents, no resistant strains were found in 154 isolates examined, but one was found to be resistant to ampicillin. By pulsed-field gel electrophoresis (PFGE), all but one of the 68 ST isolates tested showed indistinguishable banding patterns; one had a different pattern. The results suggest that ST strains from the same origin would spread in crane flocks during their stay at Izumi Plain every winter.

In vitro and in vivo invasiveness of different pulsed-field get electrophoresis types of Listeria monocytogenes

DEFF Research Database (Denmark)

Larsen, Charlotte Nexmann; Nørrung, Birgit; Sommer, Helle Mølgaard

2002-01-01

The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences...

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand.

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Kazuhisa Okada

Full Text Available BACKGROUND: Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time. METHODS/FINDINGS: A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA, and PCR to detect Vibrio seventh pandemic island II (VSP-II related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009-2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1-2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area. CONCLUSIONS: MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.

Population Genetic Structure of Listeria monocytogenes Strains as Determined by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing

DEFF Research Database (Denmark)

Henri, Clémentine; Félix, Benjamin; Guillier, Laurent

2016-01-01

on the basis of different pulsed-field gel electrophoresis (PFGE) clusters, serotypes, and strain origins and typed by multilocus sequence typing (MLST), and the MLST results were supplemented with MLST data available from Institut Pasteur, representing human and additional food strains from France....... The distribution of sequence types (STs) was compared between food and clinical strains on a panel of 675 strains. High congruence between PFGE and MLST was found. Out of 73 PFGE clusters, the two most prevalent corresponded to ST9 and ST121. Using original statistical analysis, we demonstrated that (i...

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

DEFF Research Database (Denmark)

Garaizar, J.; Lopez-Molina, N.; Laconcha, I.

2000-01-01

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate...... showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.......0 software, Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance...

Discriminatory Indices of Typing Methods for Epidemiologic Analysis of Contemporary Staphylococcus aureus Strains.

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Rodriguez, Marcela; Hogan, Patrick G; Satola, Sarah W; Crispell, Emily; Wylie, Todd; Gao, Hongyu; Sodergren, Erica; Weinstock, George M; Burnham, Carey-Ann D; Fritz, Stephanie A

2015-09-01

Historically, a number of typing methods have been evaluated for Staphylococcus aureus strain characterization. The emergence of contemporary strains of community-associated S. aureus, and the ensuing epidemic with a predominant strain type (USA300), necessitates re-evaluation of the discriminatory power of these typing methods for discerning molecular epidemiology and transmission dynamics, essential to investigations of hospital and community outbreaks. We compared the discriminatory index of 5 typing methods for contemporary S. aureus strain characterization. Children presenting to St. Louis Children's Hospital and community pediatric practices in St. Louis, Missouri (MO), with community-associated S. aureus infections were enrolled. Repetitive sequence-based PCR (repPCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal protein A (spa), and staphylococcal cassette chromosome (SCC) mec typing were performed on 200 S. aureus isolates. The discriminatory index of each method was calculated using the standard formula for this metric, where a value of 1 is highly discriminatory and a value of 0 is not discriminatory. Overall, we identified 26 distinct strain types by repPCR, 17 strain types by PFGE, 30 strain types by MLST, 68 strain types by spa typing, and 5 strain types by SCCmec typing. RepPCR had the highest discriminatory index (D) of all methods (D = 0.88), followed by spa typing (D = 0.87), MLST (D = 0.84), PFGE (D = 0.76), and SCCmec typing (D = 0.60). The method with the highest D among MRSA isolates was repPCR (D = 0.64) followed by spa typing (D = 0.45) and MLST (D = 0.44). The method with the highest D among MSSA isolates was spa typing (D = 0.98), followed by MLST (D = 0.93), repPCR (D = 0.92), and PFGE (D = 0.89). Among isolates designated USA300 by PFGE, repPCR was most discriminatory, with 10 distinct strain types identified (D = 0.63). We identified 45

Genotypic Characterization of Coagulase-negative Staphylococci Isolated from Sheep Milk in Slovakia

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Ivana Pilipčincová

2010-01-01

Full Text Available Hitherto very few reports are available presenting identification and molecular characterization of the coagulase negative staphylococci (CNS from sheep milk in the subclinical stage of mastitis. Furthermore, very scanty data are available on the epidemiological status of CNS in different Slovak provinces. Milk samples from 54 sheep farms located in eastern Slovak region were screened. A total 240 CNS were identified with series of biochemical testes (STAPH-API and subjected further for genotyping with the help of pulse field gel electrophoresis (PFGE. The most frequently occurring CNS species according the biochemical characterization were: S. epidermidis (36.3 %, S. caprae (21.3 %, S. hominis (6.6 %, S. chromogenes (6.3 %, S. xylosus (5.8 %, S. warneri (5.0 % and S. capitis (4.6 %. Further PFGE-based characterization of these isolates revealed six pulsotypes of the S. epidermidis, two of S. caprae, three of S. chromogenes, nine of S. hominis, five of S. capitis and seven of S. xylosus. These results contribute to knowledge of the epidemiological situation of the CNS from the subclinical form of mastitis in Slovakia.

Clinical isolates of Acinetobacter baumannii from a Portuguese hospital: PFGE characterization, antibiotic susceptibility and biofilm-forming ability.

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Duarte, Andreia; Ferreira, Susana; Almeida, Sofia; Domingues, Fernanda C

2016-04-01

Acinetobacter baumannii is an emerging pathogen associated with nosocomial infections that in addition has shown an increasing resistance to antibiotics. In this work the genetic diversity of A. baumannii isolates from a Portuguese hospital, their antibiotic resistance profiles and ability to form biofilms was studied. Seventy-nine clinical A. baumannii isolates were characterized by pulsed-field gel electrophoresis (PFGE) with 9 different PFGE profiles being obtained. Concerning the antimicrobial susceptibility, all A. baumannii isolates were resistant to 12 of the 17 tested antibiotics and classified as multidrug-resistant (MDR). In addition, 74.7% of the isolates showed biofilm formation ability, however no statistical significance with antibiotic resistance was observed. In contrast, urine samples isolates were more likely to form biofilms than strains isolated from other sources. Our findings highlight the high number of MDR A. baumannii isolates and the importance of the formation of biofilms as a potential virulence factor. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gastropulmonary Route of Infection and the Prevalence of Microaspiration in the Elderly Patients with Ventilator-Associated Pneumonia Verified by Molecular Microbiology-GM-PFGE.

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Liu, Qing-hua; Zhang, Jing; Lin, Dian-jie; Mou, Xiao-yan; He, Li-xian; Qu, Jie-ming; Li, Hua-yin; Hu, Bi-jie; Zhu, Ying-min; Zhu, Du-ming; Gao, Xiao-dong

2015-04-01

Gastropulmonary route of infection was considered to be an important mechanism of ventilator-associated pneumonia (VAP). However there is little evidence to support this assumption. Moreover, the prevalence of microaspiration in elderly ventilated patients was not well understood. To confirm gastropulmonary infection route and investigate the prevalence of microaspiration in elderly ventilated patients using genome macrorestriction-pulsed field gel electrophoresis (GM-PFGE). Patients over 60 years old, expected to receive mechanical ventilation longer than 48 h, were prospectively enrolled from October 2009 to January 2012. Clinical data were collected and recorded until they died, developed pneumonia, or were extubated. Samples from gastric fluid, subglottic secretion and lower respiratory tract (LRT) were collected during the follow-up for microbiological examination. To evaluate the homogeneity, GM-PFGE was performed on strains responsible for VAP that had the same biochemical phenotype as those isolated from gastric juice and subglottic secretions sequentially. Among 44 VAP patients, 76 strains were isolated from LRT and considered responsible for VAP. Twenty-two isolates had the same biochemical phenotype with the corresponding gastric isolates. The homology was further confirmed using GM-PFGE in 12 episodes of VAP. Nearly 30% of VAPs were caused by microaspiration based on the analysis of bacterial phenotype or GM-PFGE. In addition, 58.3% patients with gastric colonization developed VAP, especially late-onset VAP (LOP). Gastropulmonary infection route exists in VAP especially LOP in elderly ventilated patients. It is one of the important mechanisms in the development of VAP.

Electroforesis en Gel de Campo Pulsado (PFGE para la diferenciación molecular de Listeria monocytogenes

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Angela Maria Cardoso Bernal

2013-08-01

Full Text Available The reporting of L. monocytogenes in food in Colombia is not a mandatory; however, foods considered high-risk are monitored, and the organism is only reported clinically as Gram-positive when it causes meningitis. L. monocytogenes is a foodborne, intracellular, pathogen which causes listeriosis, a disease lethal to humans and animals. Outbreaks of this disease worldwide can bring about human and economic losses. Only a few studies in Colombia have been able to identify and molecularly serotype isolates allowing only the theoretical distribution of serotypes by lineage. This review explains the characteristics of the pathogen, its importance in public health and in the food industry, and provides an overview of PFGE-CHEF; identifying the standard work protocol and the appropriate restriction enzymes to cut DNA. We found that the enzyme combination, XbaI-AscI, followed by ApaI offers the best results to differentiate isolates, by grouping them by lineages, and displaying intra-serotype variations. Additionally, we found that in several Latin American countries the results are analyzed using PulseNet; this ensures the comparison of PFGE patterns in equivalent conditions.

Staphylococcus aureus strains associated with food poisoning outbreaks in France: comparison of different molecular typing methods, including MLVA

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Roussel, Sophie; Felix, Benjamin; Vingadassalon, Noémie; Grout, Joël; Hennekinne, Jacques-Antoine; Guillier, Laurent; Brisabois, Anne; Auvray, Fréderic

2015-01-01

Staphylococcal food poisoning outbreaks (SFPOs) are frequently reported in France. However, most of them remain unconfirmed, highlighting a need for a better characterization of isolated strains. Here we analyzed the genetic diversity of 112 Staphylococcus aureus strains isolated from 76 distinct SFPOs that occurred in France over the last 30 years. We used a recently developed multiple-locus variable-number tandem-repeat analysis (MLVA) protocol and compared this method with pulsed field gel electrophoresis (PFGE), spa-typing and carriage of genes (se genes) coding for 11 staphylococcal enterotoxins (i.e., SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEP, SER). The strains known to have an epidemiological association with one another had identical MLVA types, PFGE profiles, spa-types or se gene carriage. MLVA, PFGE and spa-typing divided 103 epidemiologically unrelated strains into 84, 80, and 50 types respectively demonstrating the high genetic diversity of S. aureus strains involved in SFPOs. Each MLVA type shared by more than one strain corresponded to a single spa-type except for one MLVA type represented by four strains that showed two different-but closely related-spa-types. The 87 enterotoxigenic strains were distributed across 68 distinct MLVA types that correlated all with se gene carriage except for four MLVA types. The most frequent se gene detected was sea, followed by seg and sei and the most frequently associated se genes were sea-seh and sea-sed-sej-ser. The discriminatory ability of MLVA was similar to that of PFGE and higher than that of spa-typing. This MLVA protocol was found to be compatible with high throughput analysis, and was also faster and less labor-intensive than PFGE. MLVA holds promise as a suitable method for investigating SFPOs and tracking the source of contamination in food processing facilities in real time. PMID:26441849

[Molecular typing characterization of food-borne methicillin-resistant Staphylococcus aureus in China].

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Bai, Y; Wang, W; Yan, L; Yang, S R; Yan, S F; Dong, Y P; Zhao, B C; Zhao, Y Y; Xu, J; Hu, Y J; Li, F Q

2018-04-06

Objective: To analyses the antimicrobial resistance and molecular characterization of 21 MRSA isolates cultured from retail foods from different provinces in China, and evaluate the molecular typing methods. Methods: Twenty-one MRSA isolates were obtained from national foodborne pathogen surveillance network in 2012 (Chinese salad, n= 3; milk, n= 1; cake, n= 2; rice, n= 1; cold noodle, n= 1; spiced beef, n= 1; dumpling, n= 1; packed meal, n= 1; salad, n= 1; raw pork, n= 9). The antimicrobial resistance of 21 strains to 12 antimicrobial agents was tested by broth dilution method. Polymerase chain reaction (PCR) and DNA sequencing were performed to obtain the genetic types of MLST (ST) and spa typing. The clonal complex (CC) was assigned by eBURST soft and the MLVA type (MT) and MLVA complex (MC) were identified via the database of the MLVA website (http://www.mlva.net). Sma I pulsed-field gel electrophoresis ( Sma Ⅰ-PFGE) was also carried out to obtain the PFGE patterns of 21 strains. The genetic diversity and discriminatory power of typing were calculated by the Simpson's index of diversity (diversity index, DI) to find out the best genotyping method for MRSA. Results: All MRSA isolates showed multi-drug resistance(MDR), and were resistant to oxacillin, benzylpenicillin, clindamycin and erythromycin, and 71.4% (15/21), 47.6% (10/21), 42.9% (9/21) and 28.6% (6/21) of the MRSA isolates were resistant to tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole and gentamicin, respectively. Moreover, one strain was found to be resistant to all three antimicrobials of levofloxacin, moxifloxacin and rifampicin. Great diversity was found in these food-associated MRSA (6 STs, 7 spa types, and 9 MTs). PFGE patterns were more diverse than those of other three molecular typing methods (19 pulse types). The index of diversity (DI) of PFGE, MLVA, spa typing and MLST was 0.99, 0.80, 0.73, and 0.61, respectively. Among the MRSA isolates, CC9-ST9-t899-MT929-MC2236 (PFGE

Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses.

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Kern, Andrea; Perreten, Vincent

2013-06-01

To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment. Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively. MRCoNS were identified as Staphylococcus epidermidis (n=20), Staphylococcus haemolyticus (n=17), Staphylococcus hominis (n=3), Staphylococcus capitis (n=1), Staphylococcus cohnii (n=1) and Staphylococcus warneri (n=1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia, aph(3')-III] (n=34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n=31), tetracycline [tet(K)] (n=22), streptomycin [str, ant(6)-Ia] (n=20), trimethoprim [dfr(A), dfr(G)] (n=17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n=30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms. MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms.

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Steven Y C Tong

Full Text Available We have developed a single nucleotide polymorphism (SNP nucleated high-resolution melting (HRM technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE and an allele specific real-time PCR (AS kinetic PCR SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs and provides a Simpson's Index of Diversity (D of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

Carriage of Escherichia coli Producing CTX-M-Type Extended-Spectrum β-Lactamase in Healthy Vietnamese Individuals.

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Bui, Thi Mai Huong; Hirai, Itaru; Ueda, Shuhei; Bui, Thi Kim Ngan; Hamamoto, Kouta; Toyosato, Takehiko; Le, Danh Tuyen; Yamamoto, Yoshimasa

2015-10-01

Healthy carriage of CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Escherichia coli was examined by thrice collecting fecal samples from the same 199 healthy Vietnamese subjects every 6 months. Using pulsed-field gel electrophoresis (PFGE), identical PFGE patterns throughout the three samplings were not observed, although prevalence of E. coli in the subjects was around 50% in the three samplings. Our results suggested a short carriage period of the CTX-M-type ESBL-producing E. coli in healthy Vietnamese subjects. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

mec-associated dru typing in the epidemiological analysis of ST239 MRSA in Malaysia.

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Ghaznavi-Rad, E; Goering, R V; Nor Shamsudin, M; Weng, P L; Sekawi, Z; Tavakol, M; van Belkum, A; Neela, V

2011-11-01

The usefulness of mec-associated dru typing in the epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Malaysia was investigated and compared with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa and SCCmec typing. The isolates studied included all MRSA types in Malaysia. Multilocus sequence type ST188 and ST1 isolates were highly clonal by all typing methods. However, the dru typing of ST239 isolates produced the clearest discrimination between SCCmec IIIa and III isolates, yielding more subtypes than any other method. Evaluation of the discriminatory power for each method identified dru typing and PFGE as the most discriminatory, with Simpson's index of diversity (SID) values over 89%, including an isolate which was non-typeable by spa, but dru-typed as dt13j. The discriminatory ability of dru typing, especially with closely related MRSA ST239 strains (e.g., Brazilian and Hungarian), underscores its utility as a tool for the epidemiological investigation of MRSA.

Comparison of the DiversiLab Repetitive Element PCR System with spa Typing and Pulsed-Field Gel Electrophoresis for Clonal Characterization of Methicillin-Resistant Staphylococcus aureus▿

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Babouee, B.; Frei, R.; Schultheiss, E.; Widmer, A. F.; Goldenberger, D.

2011-01-01

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become an increasing problem worldwide in recent decades. Molecular typing methods have been developed to identify clonality of strains and monitor spread of MRSA. We compared a new commercially available DiversiLab (DL) repetitive element PCR system with spa typing, spa clonal cluster analysis, and pulsed-field gel electrophoresis (PFGE) in terms of discriminatory power and concordance. A collection of 106 well-defined MRSA strains from our hospital was analyzed, isolated between 1994 and 2006. In addition, we analyzed 6 USA300 strains collected in our institution. DL typing separated the 106 MRSA isolates in 10 distinct clusters and 8 singleton patterns. Clustering analysis into spa clonal complexes resulted in 3 clusters: spa-CC 067/548, spa-CC 008, and spa-CC 012. The discriminatory powers (Simpson's index of diversity) were 0.982, 0.950, 0.846, and 0.757 for PFGE, spa typing, DL typing, and spa clonal clustering, respectively. DL typing and spa clonal clustering showed the highest concordance, calculated by adjusted Rand's coefficients. The 6 USA300 isolates grouped homogeneously into distinct PFGE and DL clusters, and all belonged to spa type t008 and spa-CC 008. Among the three methods, DL proved to be rapid and easy to perform. DL typing qualifies for initial screening during outbreak investigation. However, compared to PFGE and spa typing, DL typing has limited discriminatory power and therefore should be complemented by more discriminative methods in isolates that share identical DL patterns. PMID:21307215

Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates

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Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

Efficacy of Pulsed-Field Gel Electrophoresis and Repetitive Element Sequence-Based PCR in Typing of Salmonella Isolates from Assam, India.

Science.gov (United States)

Gogoi, Purnima; Borah, Probodh; Hussain, Iftikar; Das, Leena; Hazarika, Girin; Tamuly, Shantanu; Barkalita, Luit Moni

2018-05-01

A total of 12 Salmonella isolates belonging to different serovars, viz , Salmonella enterica serovar Enteritidis ( n = 4), Salmonella enterica serovar Weltevreden ( n = 4), Salmonella enterica serovar Newport ( n = 1), Salmonella enterica serovar Litchifield ( n = 1), and untypeable strains ( n = 2) were isolated from 332 diarrheic fecal samples collected from animals, birds, and humans. Of the two molecular typing methods applied, viz , repetitive element sequence-based PCR (REP-PCR) and pulsed-field gel electrophoresis (PFGE), PFGE could clearly differentiate the strains belonging to different serovars as well as differentiate between strains of the same serovar with respect to their source of isolation, whereas REP-PCR could not differentiate between strains of the same serovar. Thus, it can be suggested that PFGE is more useful and appropriate for molecular typing of Salmonella isolates during epidemiological investigations than REP-PCR. Copyright © 2018 American Society for Microbiology.

Genetic relatedness of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated in south Asia.

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Talukder, Kaisar A; Khajanchi, Bijay K; Islam, M Aminul; Dutta, Dilip K; Islam, Zhahirul; Safa, Ashrafus; Khan, G Y; Alam, Khorshed; Hossain, M A; Malla, Sarala; Niyogi, S K; Rahman, Mustafizur; Watanabe, Haruo; Nair, G Balakrish; Sack, David A

2004-10-01

The aim of the present study was to determine the clonal relationships of ciprofloxacin-resistant Shigella dysenteriae type 1 strains isolated from south Asia, and S. dysenteriae 1 strains associated with epidemics in 1978, 1984 and 1994. The antimicrobial susceptibilities were examined by NCCLS methods. Molecular epidemiological characterization was performed by plasmid profiling, pulsed-field gel electrophoresis (PFGE) and mutation analysis of the quinolone resistance-determining region (QRDR) of gyrA by sequencing. Plasmid patterns of the current ciprofloxacin-resistant strains from India, Nepal and Bangladesh were very similar to those of the 1978, 1984 and 1994 epidemic isolates of S. dysenteriae 1, except for the presence of a new plasmid of approximately 2.6 MDa, which was found in one recent ciprofloxacin-resistant strain isolated in Bangladesh. PFGE analysis showed that the ciprofloxacin-resistant strains isolated in Bangladesh, India and Nepal belonged to a PFGE type (type A), which was possibly related to that of the 1984 and 1994 clone of S. dysenteriae 1, but different from 1978 epidemic strains. The current ciprofloxacin-resistant strains belong to five subtypes (A3-A7), all of which were found in India, but in Bangladesh and Nepal, only A3 existed. Mutation analysis of the QRDR of gyrA revealed that amino acid substitutions at positions 83 and 87 of ciprofloxacin-resistant strains isolated in Bangladesh were similar to those of the strains isolated in Nepal, but different (at position 87) from ciprofloxacin-resistant strains isolated in India. PFGE and mutation analysis of gyrA showed differences between the current ciprofloxacin-resistant S. dysenteriae 1 strains isolated in south Asia and those associated with epidemics in 1978, 1984 and 1994.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

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Glauber P Arêas

2014-11-01

Full Text Available Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10 and iMLSB (4. The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81 were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7. There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

Comparison of ompP5 sequence-based typing and pulsed-filed gel ...

African Journals Online (AJOL)

In this study, comparison of the outer membrane protein P5 gene (ompP5) sequence-based typing with pulsed-field gel electrophoresis (PFGE) for the genotyping of Haemophilus parasuis, the 15 serovar reference strains and 43 isolates were investigated. When comparing the two methods, 31 ompP5 sequence types ...

Listeria monocytogenes Circulating in Rabbit Meat Products and Slaughterhouses in Italy: Prevalence Data and Comparison Among Typing Results.

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De Cesare, Alessandra; Parisi, Antonio; Mioni, Renzo; Comin, Damiano; Lucchi, Alex; Manfreda, Gerardo

2017-03-01

Rabbit meat has outstanding dietetic and nutritional properties. However, few data on microbiological hazards associated with rabbit productions are available. In this study, the presence of Listeria monocytogenes was determined in 430 rabbit carcasses, 256 rabbit meat cuts and products, and 599 environmental sponges collected from four Italian rabbit slaughterhouses over a period of 1 year. Prevalence of L. monocytogenes among the 1285 rabbit meat and environmental samples was 11%, with statistically significant differences between slaughterhouses. The highest prevalence (33.6%) was observed in rabbit meat cuts and products; the majority of positive environmental samples were collected from conveyor belts. Overall, 27.9% and 14.3% of rabbit cuts and carcasses, respectively, had L. monocytogenes counts higher than 1 colony-forming unit (CFU)/10 g. A selection of 123 isolates from positive samples was genotyped and serotyped to determine genetic profiles and diversity among L. monocytogenes isolates contaminating different slaughterhouses and classes of products investigated. Discriminatory power and concordance among the results obtained using multilocus variable-number tandem-repeat analysis (MLVA), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), automated EcoRI ribotyping, and serotyping were assessed. The isolates selected for typing were classified into serotypes 1/2a (52.8%), 1/2c (32.5%), and 1/2b (14.6%). The majority of the isolates were classified as ST14 (34.1%), ST9 (35.5%), ST121 (17.9%), and ST224 (14.6%). The greatest discriminatory power was observed with the MLVA typing, followed by MLST, PFGE, and ribotyping. The best bidirectional concordance was achieved between PFGE and MLST. There was 100% correlation between both MLST and MLVA with serotype. Moreover, a high unidirectional correspondence was observed between MLVA and both MLST and PFGE, as well as between PFGE and both MLST and serotyping. The results of this

Cost-effectiveness and efficacy of spa, SCCmec, and PVL genotyping of methicillin-resistant Staphylococcus aureus as compared to pulsed-field gel Electrophoresis.

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Vincent Li

Full Text Available Pulsed-field gel electrophoresis (PFGE is a valuable molecular typing assay used for methicillin-resistant Staphylococcus aureus (MRSA surveillance and genotyping. However, there are several limitations associated with PFGE. In Alberta, Canada, the significant increase in the number of MRSA isolates submitted to the Provincial Laboratory for Public Health (ProvLab for PFGE typing led to the need for an alternative genotyping method. In this study, we describe the transition from PFGE to Staphylococcus protein A (spa, Staphylococcal cassette chromosome (SCCmec, and Panton-Valentine leukocidin (PVL typing. A total of 1915 clinical MRSA isolates collected from 2005 to 2009 were used to develop and validate an algorithm for assigning PFGE epidemic types using spa, SCCmec, and PVL typing and the resulting data was used to populate a new Alberta MRSA typing database. An additional 12620 clinical MRSA isolates collected from 2010 to 2012 as part of ongoing routine molecular testing at ProvLab were characterized using the new typing algorithm and the Alberta MRSA typing database. Switching to spa, SCCmec, and PVL from PFGE typing substantially reduced hands-on and turn-around times while maintaining historical PFGE epidemic type designations. This led to an approximate $77,000 reduction in costs from 2010 to 2012. PFGE typing is still required for a small subset of MRSA isolates that have spa types that are rare, novel, or associated with more than one PFGE epidemic type.

Pseudomonas aeruginosa intensive care unit outbreak: winnowing of transmissions with molecular and genomic typing.

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Parcell, B J; Oravcova, K; Pinheiro, M; Holden, M T G; Phillips, G; Turton, J F; Gillespie, S H

2018-03-01

Pseudomonas aeruginosa healthcare outbreaks can be time consuming and difficult to investigate. Guidance does not specify which typing technique is most practical for decision-making. To explore the usefulness of whole-genome sequencing (WGS) in the investigation of a P. aeruginosa outbreak, describing how it compares with pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) analysis. Six patient isolates and six environmental samples from an intensive care unit (ICU) positive for P. aeruginosa over two years underwent VNTR, PFGE and WGS. VNTR and PFGE were required to fully determine the potential source of infection and rule out others. WGS results unambiguously distinguished linked isolates, giving greater assurance of the transmission route between wash-hand basin water and two patients, supporting the control measures employed. WGS provided detailed information without the need for further typing. When allied to epidemiological information, WGS can be used to understand outbreak situations rapidly and with certainty. Implementation of WGS in real-time would be a major advance in day-to-day practice. It could become a standard of care as it becomes more widespread due to its reproducibility and lower costs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter pittii and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme.

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Hu, Yuan; Li, Bo Qing; Jin, Da Zhi; He, Li Hua; Tao, Xiao Xia; Zhang, Jian Zhong

2015-12-01

To develop a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Acinetobacter pittii typing. Polymorphic VNTRs were searched by Tandem Repeats Finder. The distribution and polymorphism of each VNTR locus were analyzed in all the A. pittii genomes deposited in the NCBI genome database by BLAST and were evaluated with a collection of 20 well-characterized clinical A. pittii strains and one reference strain. The MLVA assay was compared with pulsed-field gel electrophoresis (PFGE) for discriminating A. pittii isolates. Ten VNTR loci were identified upon bioinformatic screening of A. pittii genomes, but only five of them showed full amplifiability and good polymorphism. Therefore, an MLVA assay composed of five VNTR loci was developed. The typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. Compared with PFGE, the new optimized MLVA typing scheme provided the same and even greater discrimination. Compared with PFGE, MLVA typing is a faster and more standardized alternative for studying the genetic relatedness of A. pittii isolates in disease surveillance and outbreak investigation. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Molecular Typing of Acinetobacter Baumannii Clinical Strains in Tehran by Pulsed-Field Gel Electrophoresis

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Neda Farahani

2013-03-01

Full Text Available Background & Objective : Currently, Acinetobacter baumannii is an important nosocomial pathogen insofar as its hospital outbreaks have been described from various geographical areas. Since the discrimination of strains within a species is important for delineating nosocomial outbreaks, this study was conducted with the aim of genotyping the A. baumannii clinical strains in Tehran via the pulsed-field gel electrophoresis (PFGE method, which is the most accurate method used for the typing of bacterial species.   Materials & methods: This study was performed on 70 isolates of acinetobacter baumannii isolated from patients from Baqiyatallah, Rasoole Akram, and Milad hospitals in Tehran. Cultural and biochemical methods were used for the identification of the isolates in species level, and then susceptibility tests were carried out on 50 isolates of A. baumannii using the disk diffusion method. The PFGE method was performed on the isolates by Apa I restriction enzyme. Finally, the results of the PFGE were analyzed. Result: Acinetobacter baumannii strains isolated from hospitals in Tehran showed seven different genetic patterns, two of which were sporadic . Also, genotypic profiles were different in each hospital, and different patterns of genetic resistance to common antibiotics were observed. Conclusion: A lthough diversity was observed among the strains of A. baumannii by the PFGE method in Tehran, no epidemic strains were found among them.  

Molecular techniques for MRSA typing: current issues and perspectives

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P. A. Trindade

Full Text Available Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially as to therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE, techniques involving polymerase chain reaction and multilocus sequence typing (MLST. Repetitive DNA Sequence PCR (rep-PCR may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST, are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Molecular techniques for MRSA typing: current issues and perspectives

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Trindade P. A.

2003-01-01

Full Text Available Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially as to therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE, techniques involving polymerase chain reaction and multilocus sequence typing (MLST. Repetitive DNA Sequence PCR (rep-PCR may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST, are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Typing of O26 enterohaemorrhagic and enteropathogenic Escherichia coli isolated from humans and cattle with IS621 multiplex PCR-based fingerprinting.

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Mainil, J G; Bardiau, M; Ooka, T; Ogura, Y; Murase, K; Etoh, Y; Ichihara, S; Horikawa, K; Buvens, G; Piérard, D; Itoh, T; Hayashi, T

2011-09-01

This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. Two multiplex PCRs, targeting either the left (5') or right (3') IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had identical IS621 fingerprints and PFGE profiles. The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. The IS621 printing method represents a rapid (24 h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology. No claim to Belgian Government works.

The pathogenic potential of different pulsed-field gel electrophoresis types of Listeria monocytogenes strains isolated from food in Northeast Bosnia and Herzegovina.

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Hodžić, Snjezana; Hukić, Mirsada; Franciosa, Giovanna; Aureli, Paolo

2011-09-01

Listeria monocytogenes is often present in meat and meat products that are sold in the area of northeast Bosnia and Herzegovina. The major objective of this study was to examine the virulence of L. monocytogenes strains isolated from these types of food in that geographic area. Polymerase chain reaction was used to detect eight genes responsible for virulence of this pathogen, namely, prfA, inlA, inlB, hly, plcA, plcB, actA, and mpl. All examined isolates were confirmed to possess the eight virulence genes. Ten different pulsed-field gel electrophoresis (PFGE) macrorestriction profiles were recognized among 19 L. monocytogenes strains after restriction with two different endonucleases (ApaI and AscI). The pathogenicity of three different PFGE types of L. monocytogenes was confirmed through in vivo tests, which were performed on female white mice (Pasteur strain), and it ranged from 3.55 × 10(8) LD50 to 1.58 × 10(10) LD50. All of the three different PFGE types of L. monocytogenes were regarded as moderately virulent in relation to the reference strain L. monocytogenes Scott A. This result might be one of the reasons for the absence of reported listeriosis in northeast Bosnia and Herzegovina, despite the high degree of food contamination with this pathogen.

Sub-typing of extended-spectrum-β-lactamase-producing isolates from a nosocomial outbreak: application of a 10-loci generic Escherichia coli multi-locus variable number tandem repeat analysis.

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Nahid Karami

Full Text Available Extended-spectrum β-lactamase producing Escherichia coli (ESBL-E. coli were isolated from infants hospitalized in a neonatal, post-surgery ward during a four-month-long nosocomial outbreak and six-month follow-up period. A multi-locus variable number tandem repeat analysis (MLVA, using 10 loci (GECM-10, for 'generic' (i.e., non-STEC E. coli was applied for sub-species-level (i.e., sub-typing delineation and characterization of the bacterial isolates. Ten distinct GECM-10 types were detected among 50 isolates, correlating with the types defined by pulsed-field gel electrophoresis (PFGE, which is recognized to be the 'gold-standard' method for clinical epidemiological analyses. Multi-locus sequence typing (MLST, multiplex PCR genotyping of bla CTX-M, bla TEM, bla OXA and bla SHV genes and antibiotic resistance profiling, as well as a PCR assay specific for detecting isolates of the pandemic O25b-ST131 strain, further characterized the outbreak isolates. Two clusters of isolates with distinct GECM-10 types (G06-04 and G07-02, corresponding to two major PFGE types and the MLST-based sequence types (STs 131 and 1444, respectively, were confirmed to be responsible for the outbreak. The application of GECM-10 sub-typing provided reliable, rapid and cost-effective epidemiological characterizations of the ESBL-producing isolates from a nosocomial outbreak that correlated with and may be used to replace the laborious PFGE protocol for analyzing generic E. coli.

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

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Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Genotypic characterization of Salmonella by multilocus sequence typing, pulsed-field gel electrophoresis and amplified fragment length polymorphism

DEFF Research Database (Denmark)

Torpdahl, Mia; Skov, Marianne N.; Sandvang, Dorthe

2005-01-01

subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length...

New multilocus sequence typing of MRSA in São Paulo, Brazil

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M.S. Carmo

2011-10-01

Full Text Available An increased incidence of nosocomial and community-acquired infections caused by methicillin-resistant Staphylococcus aureus (MRSA has been observed worldwide. The molecular characterization of MRSA has played an important role in demonstrating the existence of internationally disseminated clones. The use of molecular biology methods in the surveillance programs has enabled the tracking of MRSA spread within and among hospitals. These data are useful to alert nosocomial infection control programs about the potential introduction of these epidemic clones in their areas. Four MRSA blood culture isolates from patients hospitalized at two hospitals in the city of São Paulo, Brazil, were analyzed; one of them was community acquired. The isolates were characterized as SCCmec, mecA and PVL by PCR, pulsed-field gel electrophoresis (PFGE profile and molecular sequence typing (MLST genotyping. The isolates presented type IV SCCmec, and none proved to be positive for PVL. The isolates showed a PFGE profile similar to the pediatric clone. MLST genotyping demonstrated that the isolates belonged to clonal complex 5 (CC5, showing a new yqiL allele gene, resulting in a new sequence typing (ST (1176. Our results showed that strains of MRSA carrying a new ST are emerging in community and nosocomial infections, including bacteremia, in São Paulo, Brazil.

Methicillin-resistant Staphylococcus aureus carrying SCCmec type II was more frequent than the Brazilian endemic clone as a cause of nosocomial bacteremia.

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Caiaffa-Filho, Helio Hehl; Trindade, Priscila A; Gabriela da Cunha, Paula; Alencar, Cecilia Salete; Prado, Gladys V B; Rossi, Flavia; Levin, Anna S

2013-08-01

Fifty consecutive MRSA blood isolates were evaluated: 30(60%) carried SCCmec type II (single PFGE clone; sequence type 5 or ST105); 12 (26%), IV; 5 (10%), III; 3 (6%), I. Brazilian endemic clone, carrying SCCmec type III, has been the main nosocomial clone in Brazil; however, this study showed that a clone carrying type II predominated. Copyright © 2013 Elsevier Inc. All rights reserved.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

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Alba, Patricia; Feltrin, Fabiola; Cordaro, Gessica

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex( CC) 1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected...... in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized...... by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (>= 90% to 100%) similarity with human...

Comparison and Evaluation of the Molecular Typing Methods for Toxigenic Vibrio cholerae in Southwest China.

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Liao, Feng; Mo, Zhishuo; Chen, Meiling; Pang, Bo; Fu, Xiaoqing; Xu, Wen; Jing, Huaiqi; Kan, Biao; Gu, Wenpeng

2018-01-01

Vibrio cholerae O1 strains taken from the repository of Yunnan province, southwest China, were abundant and special. We selected 70 typical toxigenic V. cholerae (69 O1 and one O139 serogroup strains) isolated from Yunnan province, performed the pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and MLST of virulence gene (V-MLST) methods, and evaluated the resolution abilities for typing methods. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. Seventy V. cholerae obtained 50 PFGE patterns, with a high resolution. The strains could be divided into three groups with predominance of strains isolated during 1980s, 1990s, and 2000s, respectively, showing a good consistency with the epidemiological investigation. We also evaluated two MLST method for V. cholerae , one was used seven housekeeping genes ( adk , gyrB , metE , pntA , mdh , purM , and pyrC ), and all the isolates belonged to ST69; another was used nine housekeeping genes ( cat , chi , dnaE , gyrB , lap , pgm , recA , rstA , and gmd ). A total of seven sequence types (STs) were found by using this method for all the strains; among them, rstA gene had five alleles, recA and gmd have two alleles, and others had only one allele. The virulence gene sequence typing method ( ctxAB , tcpA , and toxR ) showed that 70 strains were divided into nine STs; among them, tcpA gene had six alleles, toxR had five alleles, while ctxAB was identical for all the strains. The latter two sequences based typing methods also had consistency with epidemiology of the strains. PFGE had a higher resolution ability compared with the sequence based typing method, and MLST used seven housekeeping genes showed the lower resolution power than nine housekeeping genes and virulence genes methods. These two sequence typing methods

Comparison and Evaluation of the Molecular Typing Methods for Toxigenic Vibrio cholerae in Southwest China

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Feng Liao

2018-05-01

Full Text Available Vibrio cholerae O1 strains taken from the repository of Yunnan province, southwest China, were abundant and special. We selected 70 typical toxigenic V. cholerae (69 O1 and one O139 serogroup strains isolated from Yunnan province, performed the pulsed field gel electrophoresis (PFGE, multilocus sequence typing (MLST, and MLST of virulence gene (V-MLST methods, and evaluated the resolution abilities for typing methods. The ctxB subunit sequence analysis for all strains have shown that cholera between 1986 and 1995 was associated with mixed infections with El Tor and El Tor variants, while infections after 1996 were all caused by El Tor variant strains. Seventy V. cholerae obtained 50 PFGE patterns, with a high resolution. The strains could be divided into three groups with predominance of strains isolated during 1980s, 1990s, and 2000s, respectively, showing a good consistency with the epidemiological investigation. We also evaluated two MLST method for V. cholerae, one was used seven housekeeping genes (adk, gyrB, metE, pntA, mdh, purM, and pyrC, and all the isolates belonged to ST69; another was used nine housekeeping genes (cat, chi, dnaE, gyrB, lap, pgm, recA, rstA, and gmd. A total of seven sequence types (STs were found by using this method for all the strains; among them, rstA gene had five alleles, recA and gmd have two alleles, and others had only one allele. The virulence gene sequence typing method (ctxAB, tcpA, and toxR showed that 70 strains were divided into nine STs; among them, tcpA gene had six alleles, toxR had five alleles, while ctxAB was identical for all the strains. The latter two sequences based typing methods also had consistency with epidemiology of the strains. PFGE had a higher resolution ability compared with the sequence based typing method, and MLST used seven housekeeping genes showed the lower resolution power than nine housekeeping genes and virulence genes methods. These two sequence typing methods could

Strain typing of acetic acid bacteria responsible for vinegar production by the submerged elaboration method.

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Fernández-Pérez, Rocío; Torres, Carmen; Sanz, Susana; Ruiz-Larrea, Fernanda

2010-12-01

Strain typing of 103 acetic acid bacteria isolates from vinegars elaborated by the submerged method from ciders, wines and spirit ethanol, was carried on in this study. Two different molecular methods were utilised: pulsed field gel electrophoresis (PFGE) of total DNA digests with a number of restriction enzymes, and enterobacterial repetitive intergenic consensus (ERIC) - PCR analysis. The comparative study of both methods showed that restriction fragment PFGE of SpeI digests of total DNA was a suitable method for strain typing and for determining which strains were present in vinegar fermentations. Results showed that strains of the species Gluconacetobacter europaeus were the most frequent leader strains of fermentations by the submerged method in the studied vinegars, and among them strain R1 was the predominant one. Results showed as well that mixed populations (at least two different strains) occurred in vinegars from cider and wine, whereas unique strains were found in spirit vinegars, which offered the most stressing conditions for bacterial growth. Copyright © 2010 Elsevier Ltd. All rights reserved.

Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks

DEFF Research Database (Denmark)

Petersen, L.; Nielsen, E.M.; On, Stephen L.W.

2001-01-01

, 314 C jejuni and 32 C coli isolates from parent and broiler flocks and from the surroundings of broiler houses were typed by flagellin gene PCR/RFLP fla-typing), and selected isolates were also typed by serotyping and macrorestriction profiling using PFGE (MRP/PFGE). The combined typing results showed...... discriminated by fla-typing as well as by MRP/PFGE, except for a few cases where individual isolates belonging to two different clones were found to have altered fla-types. Similarly, one C coli clone showed pronounced fla-type variation. The present results lead to the conclusion that vertical transmission...

Optimization of a pulsed-field gel electrophoresis for molecular typing of Proteus mirabilis

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Alper Karagöz

2013-09-01

Full Text Available Objective: For the detection of outbreaks caused byProteus mirabilis, strains clonal relations are determinedmethods as “pulsed-field gel electrophoresis (PFGE”.The aim of this study was optimization of a pulsed-fieldgel electrophoresis for molecular typing of P. mirabilis.Methods: In this study, PFGE’ protocol is optimized foruse in molecular typing of P. mirabilis. Phylogenetic analyzesof strains were evaluated with Bionumerics softwaresystem (version 6.01; Applied Maths, Sint-Martens-Latem, Belgium.Results: This protocol compared with Gram-negativebacteria PFGE protocols, NotI enzyme is suitable for thisbacterium. Electrophoresis conditions should be revealedas; - block 1: initial pulse duration 1 sec, ending pulseduration 30 sec, striking angle 120°, the current 6 V/cm2,temperature 14°C, time 8 hours; - block 2: initial pulseduration 30 sec, ending pulse duration 70 sec, strikingangle 120°, the current 6 V/cm2, temperature 14°C, time16 hours; - TBE, pH=8.4.Conclusion: P. mirabilis strains were typed by PFGE andBionumerics analysis program were determined clonal relationships.The procedure was simple, reproducible andsuitable for these bacteria. Also it was evaluated, becauseof reducing time, the solution volumes and enzymes canbe economically. Outbreaks of nosocomial infections dueto bacteria studied assessment and the potential to provideuseful information about the degree of prevalence.This optimized protocol is allowed different centers’ PFGEresults to compare with other laboratories results. J ClinExp Invest 2013; 4 (3: 306-312Key words: Proteus mirabilis, molecular typing, pulsedfieldgel electrophoresis.

Comparison of Pulsed-Gel Electrophoresis and a Commercial Repetitive-Element PCR Method for Assessment of Methicillin-Resistant Staphylococcus aureus Clustering in Different Health Care Facilities

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Duster, Megan; Warrack, Simone; Maki, Dennis; Safdar, Nasia

2014-01-01

Pulsed-field gel electrophoresis (PFGE) is a common method used to type methicillin-resistant Staphylococcus aureus (MRSA) in nosocomial investigations and epidemiological studies but is time-consuming and methodologically challenging. We compared typing results obtained using a commercial repetitive-element PCR (rep-PCR) system with PFGE in a sample of 86 unique MRSA isolates recovered from subjects in an academic referral hospital and two nursing homes in the same geographic region. Both methods reliably assigned isolates to the same Centers for Disease Control and Prevention (CDC) pulsotype. PFGE was significantly more discriminatory (Simpson's index of diversity, 0.92 at the 95% strain similarity threshold) than the commercial rep-PCR system (Simpson's index of diversity, 0.58). The global (adjusted Rand coefficient, 0.10) and directional congruence (adjusted Wallace coefficientrepPCR→PFGE = 0.06; adjusted Wallace coefficientPFGE→repPCR = 0.52) between the two methods was low. MRSA strains recovered from study nursing homes that were clonal when typed by the commercial rep-PCR method were frequently noted to be genetically distinct when typed using PFGE. These data suggest that the commercial rep-PCR has less utility than PFGE in small-scale epidemiological assessments of MRSA in health care settings. PMID:24671801

Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat (MIRU-VNTR) Genotyping of Mycobacterium intracellulare for Strain Comparison with Establishment of a PCR-Based Database

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Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A.; Falkinham, Joseph O.; Williams, Myra D.; Vasireddy, Ravikiran; Wilson, Rebecca W.; Turenne, Christine

2013-01-01

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the “gold standard” of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible. PMID:23175249

Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping of mycobacterium intracellulare for strain comparison with establishment of a PCR-based database.

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Iakhiaeva, Elena; McNulty, Steven; Brown Elliott, Barbara A; Falkinham, Joseph O; Williams, Myra D; Vasireddy, Ravikiran; Wilson, Rebecca W; Turenne, Christine; Wallace, Richard J

2013-02-01

Strain comparison is important to population genetics and to evaluate relapses in patients with Mycobacterium avium complex (MAC) lung disease, but the "gold standard" of pulsed-field gel electrophoresis (PFGE) is time-consuming and complex. We used variable-number tandem repeats (VNTR) for fingerprinting of respiratory isolates of M. intracellulare from patients with underlying bronchiectasis, to establish a nonsequence-based database for population analysis. Different genotypes identified by PFGE underwent species identification using a 16S rRNA gene multiplex PCR. Genotypes of M. intracellulare were confirmed by internal transcribed spacer 1 (ITS1) sequencing and characterized using seven VNTR primers. The pattern of VNTR amplicon sizes and repeat number defined each specific VNTR type. Forty-two VNTR types were identified among 84 genotypes. PFGE revealed most isolates with the same VNTR type to be clonal or exhibit similar grouping of bands. Repetitive sequence-based PCR (rep-PCR) showed minimal pattern diversity between VNTR types compared to PFGE. Fingerprinting of relapse isolates from 31 treated patients using VNTR combined with 16S multiplex PCR unambiguously and reliably distinguished different genotypes from the same patient, with results comparable to those of PFGE. VNTR for strain comparison is easier and faster than PFGE, is as accurate as PFGE, and does not require sequencing. Starting with a collection of 167 M. intracellulare isolates, VNTR distinguished M. intracellulare into 42 clonal groups. Comparison of isolates from different geographic areas, habitats, and clinical settings is now possible.

Genes de enterotoxinas, multirresistência a antimicrobianos e caracterização molecular de espécies de Staphylococcus spp. isoladas de leite bovino orgânico

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Amanda Keller Siqueira

2017-05-01

Full Text Available A emergência de estafilococos multirresistentes e resistentes à meticilina, isolados de animais, alimentos e humanos é uma preocupação em saúde pública. Esses micro-organismos produzem diferentes toxinas relacionadas à intoxicação alimentar em humanos. Este estudo caracterizou Staphylococcus spp. isolados em duas fazendas orgânicas no Brasil. Foram coletadas 259 amostras de leite em duas propriedades leiteiras orgânicas, nas quais 58 (22,4% estirpes de Staphylococcus spp. foram isoladas. A maior sensibilidade dos isolados foi observada para ceftiofur e sulfametoxazol/trimetoprim em 96,6%. Em contraste, acima de 89% de resistência dos estafilicocos foi encontrada para penicilina G. O gene mecA foi identificado em 13,8% dos isolados. SEA e SEC foram as enterotoxinas mais comumente detectadas. PFGE revelou heterogeneidade genética entre S. intermedius e S. warneri, enquanto S. aureus demonstraram perfis semelhantes entre isolados dos dois rebanhos estudados. Relata-se pela primeira vez no Brasil a detecção de enterotoxinas, o gene mecA e diversidade genética em estafilococos isolados de vacas em produção orgânica.

SCCmec type IX element in Methicillin resistant Staphylococcus aureus spa-type t337 (CC9 isolated from pigs and pork in Thailand

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Martin eVestergaard

2012-03-01

Full Text Available Methicillin resistant Staphylococcus aureus (MRSA have emerged among livestock in several countries. In this study, we describe the results of a screening performed in pigs and raw pork samples in Thailand. Ten pork samples and 15 nasal swabs from pigs were collected from two markets and one pig farm in the Samuth Songkhram province in Thailand. MRSA were isolated using selective isolation procedures and confirmed by mecA PCR. The MRSA were characterized by antimicrobial susceptibility testing, PFGE, spa typing, SCCmec typing, and MLST. Resistance and virulence markers were screened using a microarray. Five of the pork samples and six pig nasal swabs were positive for MRSA. All 11 isolates belonged to spa type t337 and CC9 but showed diversity in antimicrobial resistance patterns and PFGE profiles. All isolates harbored SCCmec IX and were resistant to seven out of 14 tested antimicrobials; additional resistances were found in the pork isolates. Microarray analysis identified blaZ, aac-aphD, vga(A, tetM, and a tet efflux marker, in all strains and additionally ermB and aadD, cat and fex(A in the pork isolates. None of the isolates were found PVL-positive, but enterotoxins were identified in all isolates. This is, to our knowledge, the first description of MRSA in livestock and food products in Thailand and also the first observation of MRSA CC9 associated with SCCmec IX. This study indicates a likely widespread distribution of MRSA in pig and pork in Thailand and further investigation on the prevalence and importance of livestock associated MRSA in Thailand is needed.

Prospective Genotyping of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Isolates by Use of a Novel, Highly Discriminatory Binary Typing System

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Zhou, Fei; Sintchenko, Vitali; Gilbert, Gwendolyn L.

2012-01-01

In settings of high methicillin-resistant Staphylococcus aureus (MRSA) prevalence, detection of nosocomial transmission events can be difficult without strain typing. Prospective typing of all MRSA isolates could potentially identify transmission in a timely fashion, making infection control responses to outbreaks more effective. We describe the development and evaluation of a novel 19-target binary typing system for MRSA using the multiplex-PCR/reverse line blot hybridization platform. Pulse-field gel electrophoresis (PFGE), spa typing, and phage-derived open reading frame (PDORF) typing were performed for comparison. The system was utilized to identify transmission events in three general surgical wards over a 12-month period. Initial MRSA isolates from 273 patients were differentiated into 55 unique binary types. One or more potential contacts colonized with the same MRSA strain were identified in 69 of 87 cases (79%) in which definite or possible nosocomial MRSA acquisition had occurred. The discriminatory power of the typing system was similar to that of PFGE (Simpson's index of diversity [D] = 0.994, versus 0.987) and higher than that of spa typing (D = 0.926). Strain typing reduced the total number of potential MRSA-colonized source contacts from 859 to 212 and revealed temporal clustering of transmission events. Prospective MRSA typing using this novel binary typing method can rapidly identify nosocomial transmission events, even in high-prevalence settings, which allows timely infection control interventions. The system is rapid, inexpensive, discriminatory, and suitable for routine, high-throughput use in the hospital microbiology laboratory. PMID:22895043

Huellas digitales de cepas de Acinetobacter baumannii procedentes de pacientes hospitalizados en la Caja Petrolera de Salud de Obrajes, mediante el método de Pulsed Field Gel Electrophoresis (PFGE, La Paz, Bolivia. Marzo 2015

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García-Rada Giovanni

2017-08-01

Full Text Available Acinetobacter baumannii, worldwide is considered an opportunistic microorganism, present in several cases of hospital-acquired infections. In the Caja Petrolera de Salud of Obrajes Hospital was made the isolation of four Acinetobacter baumannii strains. Identification was confirmed by biochemical tests. Then, the PFGE molecular technique was applied for the identification of genomic fingerprints using Apa I restriction en-zyme.

MLVA Typing of Streptococcus pneumoniae Isolates with Emphasis on Serotypes 14, 9N and 9V: Comparison of Previously Described Panels and Proposal of a Novel 7 VNTR Loci-Based Simplified Scheme.

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Costa, Natália S; Pinto, Tatiana C A; Merquior, Vânia L C; Castro, Luciana F S; da Rocha, Filomena S P; Morais, Jaqueline M; Peralta, José M; Teixeira, Lúcia M

2016-01-01

Streptococcus pneumoniae remains as an important cause of community-acquired bacterial infections, and the nasopharynx of asymptomatic carriers is the major reservoir of this microorganism. Pneumococcal strains of serotype 14 and serogroup 9 are among the most frequently isolated from both asymptomatic carriers and patients with invasive disease living in Brazil. Internationally disseminated clones belonging to such serotypes have been associated with the emergence and spread of antimicrobial resistance in our setting, highlighting the need for epidemiological tracking of these isolates. In this scenario, Multiple Loci VNTR Analysis (MLVA) has emerged as an alternative tool for the molecular characterization of pneumococci, in addition to more traditional techniques such as Multi-Locus Sequence Typing (MLST) and Pulsed-Field Gel Electrophoresis (PFGE). In the present study, 18 VNTR loci, as well as other previously described reduced MLVA panels (7 VNTR loci), were evaluated as tools to characterize pneumococcal strains of serotypes 14, 9N and 9V belonging to international and regional clones isolated in Brazil. The 18 VNTR loci panel was highly congruent with MLST and PFGE, being also useful for indicating the genetic relationship with international clones and for discriminating among strains with indistinguishable STs and PFGE profiles. Analysis of the results also allowed deducing a novel shorter 7 VNTR loci panel, keeping a high discriminatory power for isolates of the serotypes investigated and a high congruence level with MLST and PFGE. The newly proposed simplified panel was then evaluated for typing pneumococcal strains of other commonly isolated serotypes. The results indicate that MLVA is a faster and easier to perform, reliable approach for the molecular characterization of S. pneumoniae isolates, with potential for cost-effective application, especially in resource-limited countries.

Combination of multilocus sequence typing and pulsed-field gel electrophoresis reveals an association of molecular clonality with the emergence of extensive-drug resistance (XDR) in Salmonella.

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Cao, Yongzhong; Shen, Yongxiu; Cheng, Lingling; Zhang, Xiaorong; Wang, Chao; Wang, Yan; Zhou, Xiaohui; Chao, Guoxiang; Wu, Yantao

2018-03-01

Salmonellae is one of the most important foodborne pathogens and becomes resistant to multiple antibiotics, which represents a significant challenge to food industry and public health. However, a molecular signature that can be used to distinguish antimicrobial resistance profile, particularly multi-drug resistance or extensive-drug resistance (XDR). In the current study, 168 isolates from the chicken and pork production chains and ill chickens were characterized by serotyping, antimicrobial susceptibility test, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The results showed that these isolates belonged to 13 serotypes, 14 multilocus sequence types (STs), 94 PFGE genotypes, and 70 antimicrobial resistant profiles. S. Enteritidis, S. Indiana, and S. Derby were the predominant serotypes, corresponding to the ST11, ST17, and ST40 clones, respectively and the PFGE Cluster A, Cluster E, and Cluster D, respectively. Among the ST11-S. Enteritidis (Cluster A) and the ST40-S. Derby (Cluster D) clones, the majority of isolates were resistant to 4-8 antimicrobial agents, whereas in the ST17S. Indiana (Cluster E) clone, isolates showed extensive-drug resistance (XDR) to 9-16 antimicrobial agents. The bla TEM-1-like gene was prevalent in the ST11 and ST17 clones corresponding to high ampicillin resistance. The bla TEM-1-like , bla CTX-M , bla OXA-1-like , sul1, aaC4, aac(6')-1b, dfrA17, and floR gene complex was highly prevalent among isolates of ST17, corresponding to an XDR phenotype. These results demonstrated the association of the resistant phenotypes and genotypes with ST clone and PFGE cluster. Our results also indicated that the newly identified gene complex comprising bla TEM-1-like , bla CTX-M , bla OXA-1-like , sul1, aaC4, aac(6')-1b, dfrA17, and floR, was responsible for the emergence of the ST17S. Indiana XDR clone. ST17 could be potentially used as a molecular signature to distinguish S. Indiana XDR clone. Copyright © 2017

MALDI-TOF MS typing of a nosocomial methicillin-resistant Staphylococcus aureus outbreak in a neonatal intensive care unit.

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Steensels, Deborah; Deplano, Ariane; Denis, Olivier; Simon, Anne; Verroken, Alexia

2017-08-01

The early detection of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak is decisive to control its spread and rapidly initiate adequate infection control measures. Therefore, prompt determination of epidemiologic relatedness of clinical MRSA isolates is essential. Genetic typing methods have a high discriminatory power but their availability remains restricted. In this study, we aimed to challenge matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a typing tool of a nosocomial MRSA outbreak in a neonatal intensive care unit. Over a 2-year period, 15 MRSA isolates were recovered from patients (n = 14) and health care workers (n = 1) at the neonatal intensive care unit. Five reference strains were included for comparison. Identification was performed by MALDI-TOF MS and susceptibility profiles determined by automated broth microdilution. Typing analysis by MALDI-TOF MS included mean spectrum profiles and subsequent dendrogram creation using BioNumerics software. Results were compared with spa typing and pulsed-field gel electrophoresis (PFGE). Our study showed good concordance (93%) between PFGE, spa typing, and MALDI-TOF MS for the outbreak-related MRSA strains. MALDI-TOF MS typing showed excellent typeability and discriminatory power but showed poor reproducibility. This study is one of the first to document the potential usefulness of MALDI-TOF MS with standardized data analysis as a typing tool for investigating a nosocomial MRSA outbreak. A concordance of 93% compared to reference typing techniques was observed. However, because of poor reproducibility, long-term follow-up of prospective isolated strains is not practical for routine use. Further studies are needed to confirm our observations.

Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing--a continuous attempt at species and serovar differentiation.

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Moreno, Luisa Z; Miraglia, Fabiana; Lilenbaum, Walter; Neto, José S F; Freitas, Julio C; Morais, Zenaide M; Hartskeerl, Rudy A; da Costa, Barbara L P; Vasconcellos, Silvio A; Moreno, Andrea M

2016-03-09

Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

OpenAIRE

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 33...

CC8 MRSA strains harboring SCCmec type IVc are predominant in Colombian hospitals.

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J Natalia Jiménez

Full Text Available BACKGROUND: Recent reports highlight the incursion of community-associated MRSA within healthcare settings. However, knowledge of this phenomenon remains limited in Latin America. The aim of this study was to evaluate the molecular epidemiology of MRSA in three tertiary-care hospitals in Medellín, Colombia. METHODS: An observational cross-sectional study was conducted from 2008-2010. MRSA infections were classified as either community-associated (CA-MRSA or healthcare-associated (HA-MRSA, with HA-MRSA further classified as hospital-onset (HAHO-MRSA or community-onset (HACO-MRSA according to standard epidemiological definitions established by the U.S. Centers for Disease Control and Prevention (CDC. Genotypic analysis included SCCmec typing, spa typing, PFGE and MLST. RESULTS: Out of 538 total MRSA isolates, 68 (12.6% were defined as CA-MRSA, 243 (45.2% as HACO-MRSA and 227 (42.2% as HAHO-MRSA. The majority harbored SCCmec type IVc (306, 58.7%, followed by SCCmec type I (174, 33.4%. The prevalence of type IVc among CA-, HACO- and HAHO-MRSA isolates was 92.4%, 65.1% and 43.6%, respectively. From 2008 to 2010, the prevalence of type IVc-bearing strains increased significantly, from 50.0% to 68.2% (p = 0.004. Strains harboring SCCmec IVc were mainly associated with spa types t1610, t008 and t024 (MLST clonal complex 8, while PFGE confirmed that the t008 and t1610 strains were closely related to the USA300-0114 CA-MRSA clone. Notably, strains belonging to these three spa types exhibited high levels of tetracycline resistance (45.9%. CONCLUSION: CC8 MRSA strains harboring SCCmec type IVc are becoming predominant in Medellín hospitals, displacing previously reported CC5 HA-MRSA clones. Based on shared characteristics including SCCmec IVc, absence of the ACME element and tetracycline resistance, the USA300-related isolates in this study are most likely related to USA300-LV, the recently-described 'Latin American variant' of USA300.

Distributions of Salmonella Subtypes Differ between Two U.S. Produce-Growing Regions

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Danyluk, Michelle D.; Worobo, Randy W.; Wiedmann, Martin

2014-01-01

Salmonella accounts for approximately 50% of produce-associated outbreaks in the United States, several of which have been traced back to contamination in the produce production environment. To quantify Salmonella diversity and aid in identification of Salmonella contamination sources, we characterized Salmonella isolates from two geographically diverse produce-growing regions in the United States. Initially, we characterized the Salmonella serotype and subtype diversity associated with 1,677 samples collected from 33 produce farms in New York State (NYS). Among these 1,677 samples, 74 were Salmonella positive, yielding 80 unique isolates (from 147 total isolates), which represented 14 serovars and 23 different pulsed-field gel electrophoresis (PFGE) types. To explore regional Salmonella diversity associated with production environments, we collected a smaller set of samples (n = 65) from South Florida (SFL) production environments and compared the Salmonella diversity associated with these samples with the diversity found among NYS production environments. Among these 65 samples, 23 were Salmonella positive, yielding 32 unique isolates (from 81 total isolates), which represented 11 serovars and 17 different PFGE types. The most common serovars isolated in NYS were Salmonella enterica serovars Newport, Cerro, and Thompson, while common serovars isolated in SFL were Salmonella serovars Saphra and Newport and S. enterica subsp. diarizonae serovar 50:r:z. High PFGE type diversity (Simpson's diversity index, 0.90 ± 0.02) was observed among Salmonella isolates across both regions; only three PFGE types were shared between the two regions. The probability of three or fewer shared PFGE types was Salmonella isolates were considerably different between the two sampled regions. These findings suggest the potential for PFGE-based source tracking of Salmonella in production environments. PMID:24747908

Genetic patterns of Streptococcus uberis isolated from bovine mastitis

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Elina B Reinoso

2015-06-01

Full Text Available The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE. Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection.

The limitations of pulsed-field gel electrophoresis for analysis of Yersinia enterocolitica isolates.

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Gilpin, B J; Robson, B; Lin, S; Hudson, J A; Weaver, L; Dufour, M; Strydom, H

2014-09-01

This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence. © 2013 Blackwell Verlag GmbH.

[Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

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Wang, Yue; Chen, Hong; Liu, Ying; Zhou, Jingzhu; Li, Shijun; Hang, Yan; Tang, Guangpeng; Wang, Dingming; Chen, Guichun

2015-09-01

To identify and characterize the Brucella strains from Guizhou province in 2010-2013. A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.

Application of Pulsed-Field Gel Electrophoresis and Binary Typing as Tools in Veterinary Clinical Microbiology and Molecular Epidemiologic Analysis of Bovine and Human Staphylococcus aureus Isolates

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Zadoks, Ruth; van Leeuwen, Willem; Barkema, Herman; Sampimon, Otlis; Verbrugh, Henri; Schukken, Ynte Hein; van Belkum, Alex

2000-01-01

Thirty-eight bovine mammary Staphylococcus aureus isolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) after SmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureus isolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison of S. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureus isolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus. PMID:10790124

Epidemiology of methicillin-resistant and -susceptible Staphylococcus aureus in Luanda, Angola: first description of the spread of the MRSA ST5-IVa clone in the African continent.

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Conceição, Teresa; Coelho, Céline; Santos-Silva, Isabel; de Lencastre, Hermínia; Aires-de-Sousa, Marta

2014-10-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen worldwide, and although surveillance studies are available in the most developed countries, data from Angola are inexistent. In June 2012, 295 inpatients and 199 healthcare workers from three hospitals in Luanda, Angola were nasal swabbed for S. aureus and MRSA carriage. A total of 117 individuals (23.7%) were S. aureus nasal carriers, out of which 68 (58.1%) were colonized with MRSA. The majority of the MRSA isolates (74%) belonged to a single clonal lineage, pulsed-field gel electrophoresis (PFGE) A-ST5-IVa associated with three spa types (spa types t105/t311/t11657), followed by PFGE C-ST88-IVa (spa types t186/t325/t786/t1951/t3869) (n=9; 12%); the other 11 MRSA isolates were representatives of 4 additional lineages. Almost half (49%) of the methicillin-susceptible S. aureus (MSSA) isolates belonged to three major clones: PFGE B-ST508 (spa types t050/t861/t1346/t1574/t2626/t12218), PFGE D-ST45 (spa types t939/t11656), and PFGE E-ST30 (spa types t1202/t9118). MSSA isolates presented a high variability of virulence factors, including Panton-Valentine leukocidine (7.9%). MRSA carriage in Luanda is considerably high, and the major clone corresponds to a worldwide epidemic lineage, so far scarcely reported in Africa. Additional infection control measures in this metropolis are mandatory for a global MRSA control.

Molecular characterization of Rhodococcus equi isolates from horses in Poland: pVapA characteristics and plasmid new variant, 85-kb type V.

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Witkowski, Lucjan; Rzewuska, Magdalena; Takai, Shinji; Chrobak-Chmiel, Dorota; Kizerwetter-Świda, Magdalena; Feret, Małgorzata; Gawryś, Marta; Witkowski, Maciej; Kita, Jerzy

2017-01-26

Rhodococcus equi is one of the most significant bacterial pathogens affecting foals up to 6 months of age worldwide. Rhodococcosis is present in Poland however information about molecular characterization of R. equi isolates is scarce. This study describes molecular characterization of Rhodococcus equi infection on 13 horse breeding farms in Poland between 2001 and 2012. Samples were collected by tracheobronchial aspiration from pneumonic foals or during necropsy. The R. equi isolates were genotyped by plasmid profiling and pulsed-field gel electrophoresis. Totally, 58 R. equi isolates were investigated. One isolate lost its plasmid. Among the 57 VapA-positive isolates, 48 contained 85-kb type I plasmid (82.8%), 8 contained 87-kb type I plasmid (13.8%). One isolate (1.7%) had a unique restriction cleavage pattern and the 2nd fragment of EcoRI digests of this plasmid DNA was about 2600 bases smaller than that of the 85 kb type I. This new plasmid variant was designated as the "85-kb type V". Among the 58 isolates typeable with VspI-PFGE, ten PFGE clusters were detected. The majority of foals were infected mostly with isolates of low genetic diversity. Most of clinical isolates of R. equi from foals in Poland contain pVapA 85-kb type I and 87-kb type I similarly to the other European countries and the United States. However, the new variant of pVapA 85-kb type V was identified. The chromosomal variability was detected among some of the investigated isolates and the presence of farm-specific isolates might be possible.

Genetic patterns of Streptococcus uberis isolated from bovine mastitis.

Science.gov (United States)

Reinoso, Elina B; Lasagno, Mirta C; Odierno, Liliana M

2015-01-01

The aim of this study was to evaluate the genotypic relationships among 40 Streptococcus uberis isolated from bovine mastitis by using pulsed-field gel electrophoresis (PFGE). Additionally, the association between PFGE patterns and virulence profiles was investigated. The isolates exhibited 17 PFGE patterns. Different strains were found within and among herds; however, a low number of isolates within the same herd shared an identical PFGE type. No association between PFGE patterns and virulence profiles was found. However, the detection of specific strains in some herds could indicate that some strains are more virulent than others. Further research needs to be undertaken to elucidate new virulence-associated genes that might contribute to the capability of these strains to produce infection. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Pulsed-field gel electrophoresis typing of Escherichia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women

DEFF Research Database (Denmark)

Ejrnaes, Karen; Sandvang, Dorthe; Lundgren, Bettina

2006-01-01

). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment......The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE......). In the pivmecillinam treatment group PFGE showed that among patients having a negative urine culture at the first follow-up 77% (46/60) had a relapse with the primary infecting E. coli strain and 23% (14/60) had reinfection with a new E. coli strain at the second follow-up. Among patients having E. coli at the first...

Pulsed-field gel electrophoresis typing of Eschericia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women

DEFF Research Database (Denmark)

Ejrnæs, K; Sandvang, D; Lundgren, Bettina

2006-01-01

). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment......The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE......). In the pivmecillinam treatment group PFGE showed that among patients having a negative urine culture at the first follow-up 77% (46/60) had a relapse with the primary infecting E. coli strain and 23% (14/60) had reinfection with a new E. coli strain at the second follow-up. Among patients having E. coli at the first...

Transmission of endemic ST22-MRSA-IV on four acute hospital wards investigated using a combination of spa, dru and pulsed-field gel electrophoresis typing.

LENUS (Irish Health Repository)

Creamer, E

2012-11-01

The transmission of meticillin-resistant Staphylococcus aureus (MRSA) between individual patients is difficult to track in institutions where MRSA is endemic. We investigated the transmission of MRSA where ST22-MRSA-IV is endemic on four wards using demographic data, patient and environmental screening, and molecular typing of isolates. A total of 939 patients were screened, 636 within 72 h of admission (on admission) and 303 >72 h after admission, and 1,252 environmental samples were obtained. Isolates were typed by spa, dru and pulsed-field gel electrophoresis (PFGE) typing. A composite dendrogram generated from the three sets of typing data was used to divide isolates into \\'dendrogram groups\\' (DGs). Ten percent of patients (92\\/939) were MRSA-positive; 7 % (44\\/636) on admission and 16 % (48\\/303) >72 h after admission (p = 0.0007). MRSA was recovered from 5 % of environmental specimens (65\\/1,252). Most isolates from patients (97 %, 85\\/88) and the environment (97 %, 63\\/65) exhibited the ST22-MRSA-IV genotype. Four DGs (DG1, DG4, DG16 and DG17) accounted for 58 % of ST22-MRSA-IV isolates from patients. Epidemiological evidence suggested cross-transmission among 44\\/92 patients (48 %) but molecular typing confirmed probable cross-transmission in only 11 instances (13 %, 11\\/88), with the majority of cross-transmission (64 %; 7\\/11) occurring on one ward. In the setting of highly clonal endemic MRSA, the combination of local epidemiology, PFGE, spa and dru typing provided valuable insights into MRSA transmission.

Pheno- and genotyping of Staphylococcus epidermidis isolated from bovine milk and human skin

DEFF Research Database (Denmark)

Thorberg, B. M.; Kuhn, I.; Aarestrup, Frank Møller

2006-01-01

showed one pattern, which was identical to the most common pattern found in the milk isolates. Isolates from herd 2 showed three to four patterns, two of these being identical to skin isolates from the milker. As dairy cows are not a natural host for S. epidermidis the results suggest a human source...... (PFGE) and 122 by ribotyping. PFGE showed single patterns in the human strains with one exception; one strain was categorised as the same clone as four of the milk strains. PFGE divided 73 of the milk strains into 62 different patterns. The PFGE method had high discriminatory power and shows that many...... different S. epidermidis types exist in milk samples. Antibiotic resistance patterns matched the SmaI profiles closely in the two herds, but poorly in the routinely collected milk samples. Isolates from herd I showed one to five patterns, depending on the typing method used. Isolates from the milker's skin...

Experiencias en la vigilancia epidemiológica de agentes patógenos transmitidos por alimentos a través de electroforesis en campo pulsado (PFGE en el Perú Experiences in the epidemiological surveillance of foodborne pathogens by pulsed field gel electrophoresis (PFGE in Peru

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María Luz Zamudio

2011-03-01

Full Text Available Las enfermedades transmitidas por alimentos (ETA y otras enfermedades entéricas infecciosas ocurren a menudo como brotes y son causa de morbilidad y mortalidad en todo el mundo. En el Perú, son un importante problema de salud pública y son causados por una gran variedad de agentes infecciosos. Para la investigación epidemiológica se utiliza una variedad de métodos de tipificación. Una de las herramientas más importantes en la subtipificación molecular de patógenos bacterianos es la técnica de la electroforesis en campo pulsado (PFGE, que es un método altamente resolutivo que permite la discriminación entre diferentes aislamientos bacterianos epidemiológicamente relacionados. El Instituto Nacional de Salud (INS del Perú integra las redes WHO Global Foodborne Infections Network y la Red PulseNet América Latina y Caribe, con quienes comparte los perfiles genéticos de las cepas patógenas aisladas, permitiendo comparar los genotipos de cepas semejantes halladas en diferentes países y reconocer la ocurrencia de brotes epidémicos en la región, fortaleciendo el sistema de vigilancia epidemiológica regional y generando una rápida respuesta conjunta entre países. Se presenta la experiencia de los dos últimos años sobre los avances en la utilización de estas herramientas estratégicas que nos ha permitido caracterizar patrones de genotipo de principales patógenos implicados en ETA a partir de aislamientos recuperados de la red de laboratorios del Perú.Foodborne diseases and other enteric infections often occur as outbreaks and cause morbidity and mortality all over the world. In Perú, they represent a serious public health problem, and are caused by a great variety of infectious agents. For epidemiological research, a wide array of typification methods are used. One of the most important tools for the molecular subtyping of bacterial pathogens is the Pulsed Field Gel Electrophoresis (PFGE, which is a highly precise method that

Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types.

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Oliveira, C J B; Tiao, N; de Sousa, F G C; de Moura, J F P; Santos Filho, L; Gebreyes, W A

2016-03-01

The aim of this study was to investigate the phenotypic and genotypic diversity and anti-microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north-eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti-microbial susceptibility by Kirby-Bauer disc diffusion method. Confirmation of methicillin-resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP-2a latex agglutination. Clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase-negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622-ST1625). The findings show that MRSA is prevalent in milk from semi-extensive dairy cows in north-eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm-to-table continuum is warranted. © 2015 Blackwell Verlag GmbH.

Methicillin-Resistant Staphylococcus aureus in the Community in Luanda, Angola: Blurred Boundaries with the Hospital Setting.

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Conceição, Teresa; Coelho, Céline; Santos Silva, Isabel; de Lencastre, Hermínia; Aires-de-Sousa, Marta

2016-01-01

Although the nosocomial prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Angola is over 60% and one of the highest in Africa, the extent of MRSA in the community is unknown. To fill this gap, we conducted a hospital-based study in which 158 children attending the emergency ward and ambulatory services of a pediatric hospital in Luanda, the capital of Angola, were screened for S. aureus nasal colonization. Overall, 70 (44.3%) individuals were colonized with S. aureus, of which 20 (28.6%) carried MRSA, resulting in a prevalence of 12.7% (20/158) of MRSA in the population screened. Molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and SCCmec typing distributed the isolates into two major MRSA clones and one dominant methicillin-susceptible S. aureus (MSSA) lineage, corresponding to the main clones circulating in hospitals in Luanda. The MRSA isolates mainly belonged to clones A (PFGE type A, spa type t105, ST5-IVa-65%) and B (PFGE B, t3869, ST88-IVa-30%), while MSSA isolates mainly belonged to clone L (PFGE type L, t861, ST508-42%). S. aureus isolates showed resistance to penicillin (96%), rifampin (87%), and trimethoprim-sulfamethoxazole (21%). In conclusion, the prevalence of MRSA among children in the community in Luanda is high and seems to originate from hospitals, warranting continuous monitoring and implementation of additional infection control measures.

Subtyping of Salmonella enterica isolated from humans and food animals using Pulsed-Field Gel Electrophoresis

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Golab, N.

2016-07-01

Full Text Available Salmonella infections are the second leading cause of zoonotic bacterial foodborne illness. Main source of infection in human is contaminated food products. The aim of this study was sub typing isolates of Salmonella enterica obtained during our previous study by Pulsed Field Gel Electrophoresis (PFGE technique. All 46 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. In this study, PFGE and serotyping were used to subtype 46 Salmonella isolates belonging to 27different serovars and derived from human and different food origins. Among these isolates, S. Typhimurium was found to be the most predominant serovar. 40 PFGE patterns out of 46 isolates were obtained. The Discrimination Index obtained by serotyping (DI = 0.93 was lower than PFGE (DI = 0.99. Subtyping of Salmonella enterica is very important and shows that animal origin can be one of a reservoir that potentially could be transferred to human through the food chain. In addition, results of this study also revealed that this procedure is a golden standard for genotyping of such salmonella serotypes.

Using BOX-PCR to exclude a clonal outbreak of melioidosis

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Ward Linda

2007-06-01

Full Text Available Abstract Background Although melioidosis in endemic regions is usually caused by a diverse range of Burkholderia pseudomallei strains, clonal outbreaks from contaminated potable water have been described. Furthermore B. pseudomallei is classified as a CDC Group B bioterrorism agent. Ribotyping, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST have been used to identify genetically related B. pseudomallei isolates, but they are time consuming and technically challenging for many laboratories. Methods We have adapted repetitive sequence typing using a BOX A1R primer for typing B. pseudomallei and compared BOX-PCR fingerprinting results on a wide range of well-characterized B. pseudomallei isolates with MLST and PFGE performed on the same isolates. Results BOX-PCR typing compared favourably with MLST and PFGE performed on the same isolates, both discriminating between the majority of multilocus sequence types and showing relatedness between epidemiologically linked isolates from various outbreak clusters. Conclusion Our results suggest that BOX-PCR can be used to exclude a clonal outbreak of melioidosis within 10 hours of receiving the bacterial strains.

Limited similarity between plasmids encoding CTX-M-1 β-lactamase in Escherichia coli from humans, pigs, cattle, organic poultry layers and horses in Denmark

DEFF Research Database (Denmark)

Jakobsen, Lotte; Bortolaia, Valeria; Bielak, Eliza Maria

2015-01-01

in Denmark between 2006 and 2010. In total, 65 CTX-M-1-producing isolates from patients (n=22), pigs (n=21), cattle (n=4), organic poultry layers (n=3) and horses (n=15) were typed by pulsed-field gel electrophoresis (PFGE). Plasmids harbouring blaCTX-M-1 were characterised by S1 PFGE, PCR-based replicon...

Distribution of Campylobacter jejuni isolates from turkey farms and different stages at slaughter using pulsed-field gel electrophoresis and flaA-short variable region sequencing.

Science.gov (United States)

Perko-Mäkelä, P; Alter, T; Isohanni, P; Zimmermann, S; Lyhs, U

2011-09-01

The aim of this study was to assess the diversity of thermotolerant Campylobacter spp. isolated from turkey flocks at six rearing farms 1-2 weeks prior to slaughter (360 faecal swab samples) and from 11 different stages at the slaughterhouse (636 caecal, environmental, neck skin and meat samples). A total of 121 Campylobacter isolates were identified to species level using a multiplex PCR assay and were typed by pulsed-field gel electrophoresis (PFGE) and flaA-short variable region (SVR) sequencing. All Campylobacter isolates were identified as Campylobacter jejuni. PFGE analysis with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA SVR typing yielded in nine flaA-SVR alleles. The Campylobacter-positive turkey flocks A, C and E were colonized by a limited number of Campylobacter clones at the farm and slaughter. The present study confirms the traceability of flock-specific strains (PFGE types I, V and IX; flaA types 21, 36 and 161) from the farm along the entire processing line to meat cuts. It seems that stress factors such as high temperature of the defeathering water (54-56 °C), drying of the carcass skin during air chilling (24 h at 2 °C), and oxygen in the air could not eliminate Campylobacter completely. Campylobacter-negative flocks became contaminated during processing by the same subtypes of Campylobacter introduced into the slaughter house by preceeding positive flocks even if they were slaughtered on subsequent days. Proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination, especially in countries with a low prevalence of Campylobacter spp. The majority of flaA SVR alleles displayed a distinct association with a specific PFGE type. However, a linear relationship for all strains among both typing methods could not be established. To specify genetic relatedness of strains, a combination of different genotyping methods, is needed. © 2011 Blackwell Verlag GmbH.

Pulsed-field gel electrophoresis and multi locus sequence typing for characterizing genotype variability of Yersinia ruckeri isolated from farmed fish in France.

Science.gov (United States)

Calvez, Ségolène; Fournel, Catherine; Douet, Diane-Gaëlle; Daniel, Patrick

2015-06-23

Yersinia ruckeri is a pathogen that has an impact on aquaculture worldwide. The disease caused by this bacterial species, yersiniosis or redmouth disease, generates substantial economic losses due to the associated mortality and veterinary costs. For predicting outbreaks and improving control strategies, it is important to characterize the population structure of the bacteria. The phenotypic and genetic homogeneities described previously indicate a clonal population structure as observed in other fish bacteria. In this study, the pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST) methods were used to describe a population of isolates from outbreaks on French fish farms. For the PFGE analysis, two enzymes (NotI and AscI) were used separately and together. Results from combining the enzymes showed the great homogeneity of the outbreak population with a similarity > 80.0% but a high variability within the cluster (cut-off value = 80.0%) with a total of 43 pulsotypes described and an index of diversity = 0.93. The dominant pulsotypes described with NotI (PtN4 and PtN7) have already been described in other European countries (Finland, Germany, Denmark, Spain and Italy). The MLST approach showed two dominant sequence types (ST31 and ST36), an epidemic structure of the French Y. ruckeri population and a preferentially clonal evolution for rainbow trout isolates. Our results point to multiple types of selection pressure on the Y. ruckeri population attributable to geographical origin, ecological niche specialization and movements of farmed fish.

A clinical and bacteriologic investigation of invasive streptococcal infections in Japan on the basis of serotypes, toxin production, and genomic DNA fingerprints.

Science.gov (United States)

Nakashima, K; Ichiyama, S; Iinuma, Y; Hasegawa, Y; Ohta, M; Ooe, K; Shimizu, Y; Igarashi, H; Murai, T; Shimokata, K

1997-08-01

In a survey of invasive streptococcal infections in Japan, we analyzed isolates of Streptococcus pyogenes collected between 1992 and 1994. Genomic DNA fingerprints produced by pulsed-field gel electrophoresis (PFGE) were compared by computer-assisted analysis. Conventional serologic M types were subdivided into PFGE types showing close genetic similarity. Among the 42 isolates from patients with invasive diseases, 16 PFGE types were identified and genetic diversity was clearly demonstrated. Identical fingerprints were observed in both invasive and noninvasive isolates. Only 43% of invasive isolates produced streptococcal pyrogenic exotoxin A (SPE A), and 31% did not contain the speA gene. These findings suggest that the dissemination of a specific clone is not sufficient to explain all cases of these diseases in Japan and pose a question as to the role of SPE A as a major virulent factor. Bacterial factors other than SPE A and host factors should be considered in evaluation of the pathogenesis of the diseases.

Genotypic Characterization of Streptococcus infantarius subsp. coli Isolates from Sea Otters with Infective Endocarditis and/or Septicemia and from Environmental Mussel Samples

Science.gov (United States)

Counihan-Edgar, Katrina L.; Gill, Verena A.; Doroff, Angela M.; Burek, Kathleen A.; Miller, Woutrina A.; Shewmaker, Patricia L.; Jang, Spencer; Goertz, Caroline E. C.; Tuomi, Pamela A.; Miller, Melissa A.; Jessup, David A.

2012-01-01

Pulsed-field gel electrophoresis (PFGE) was used to type 128 Streptococcus infantarius subsp. coli isolates from sea otters and mussels. Six SmaI PFGE groups were detected, with one predominant group representing 57% of the isolates collected over a wide geographic region. Several sea otter and mussel isolates were highly related, suggesting that an environmental infection source is possible. PMID:23052307

Effect of High Hydrostatic Pressure Combined with Moderate Heat to Inactivate Pressure-Resistant Bacteria in Water-Boiled Salted Duck.

Science.gov (United States)

Ye, Keping; Feng, Yulin; Wang, Kai; Bai, Yun; Xu, Xinglian; Zhou, Guanghong

2015-06-01

The objective of this work was to study the effect of high hydrostatic pressure combined with moderate heat to inactivate pressure-resistant bacteria in water-boiled salted duck meat (WBSDM), and to establish suitable procedures to improve the quality of WBSDM. The conditions (300 MPa/60 °C, 400 MPa/60 °C, and 500 MPa/50 °C) effectively inactivated the pressure-resistant bacteria (Bacillus cereus and Staphylococcus warneri) in WBSDM. Although more pressure-resistant than S. warneri, the above treatment conditions inactivated B. cereus more than 10(7) CFU/mL in buffer, and more than 10(6) CFU/g in WBSDM, and did not cause any changes in color, texture, or moisture content of products. The interaction between pressure and temperature is a more significant factor than only pressure in inactivating both B. cereus and S. warneri, the treatment of WBSDM at 400 MPa/ 60 °C/ 10 min is the most practical condition for postprocess of WBSDM after cooking. © 2015 Institute of Food Technologists®

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Acinetobacter baumannii and Interlaboratory Validation of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿†

Science.gov (United States)

Pourcel, Christine; Minandri, Fabrizia; Hauck, Yolande; D'Arezzo, Silvia; Imperi, Francesco; Vergnaud, Gilles; Visca, Paolo

2011-01-01

Acinetobacter baumannii is an important opportunistic pathogen responsible for nosocomial outbreaks, mostly occurring in intensive care units. Due to the multiplicity of infection sources, reliable molecular fingerprinting techniques are needed to establish epidemiological correlations among A. baumannii isolates. Multiple-locus variable-number tandem-repeat analysis (MLVA) has proven to be a fast, reliable, and cost-effective typing method for several bacterial species. In this study, an MLVA assay compatible with simple PCR- and agarose gel-based electrophoresis steps as well as with high-throughput automated methods was developed for A. baumannii typing. Preliminarily, 10 potential polymorphic variable-number tandem repeats (VNTRs) were identified upon bioinformatic screening of six annotated genome sequences of A. baumannii. A collection of 7 reference strains plus 18 well-characterized isolates, including unique types and representatives of the three international A. baumannii lineages, was then evaluated in a two-center study aimed at validating the MLVA assay and comparing it with other genotyping assays, namely, macrorestriction analysis with pulsed-field gel electrophoresis (PFGE) and PCR-based sequence group (SG) profiling. The results showed that MLVA can discriminate between isolates with identical PFGE types and SG profiles. A panel of eight VNTR markers was selected, all showing the ability to be amplified and good amounts of polymorphism in the majority of strains. Independently generated MLVA profiles, composed of an ordered string of allele numbers corresponding to the number of repeats at each VNTR locus, were concordant between centers. Typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. A database containing information and MLVA profiles for several A. baumannii strains is available from http://mlva.u-psud.fr/. PMID:21147956

Characteristics related to antimicrobial resistance and biofilm formation of widespread methicillin-resistant Staphylococcus epidermidis ST2 and ST23 lineages in Rio de Janeiro hospitals, Brazil.

Science.gov (United States)

Iorio, Natalia Lopes Pontes; Caboclo, Roberta Ferreira; Azevedo, Milena Borgo; Barcellos, Ariane Guimarães; Neves, Felipe Piedade Gonçalves; Domingues, Regina Maria Cavalcanti Pilotto; dos Santos, Kátia Regina Netto

2012-01-01

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in seriously ill or immunocompromised patients. Currently, the characteristics of methicillin-resistant S. epidermidis (MRSE) isolates from Rio de Janeiro hospitals are unknown. In this study, staphylococcal chromosomal cassette mec (SCCmec) types, antimicrobial susceptibility profiles, biofilm formation genes, and multilocus sequence types (MLST) were investigated in 35 MRSE clinical isolates. The collection of isolates was previously well characterized by pulsed-field gel electrophoresis (PFGE) into 2 main genotypes (A and B, 22 isolates) and 10 sporadic genotypes (13 isolates). MLST revealed a total of 8 different sequence types (STs), but ST2 and ST23, which were icaAB-positive, represented the majority (71.4%) of MRSE isolates tested. Almost all isolates (91.4%) belonged to clonal complex 2. SCCmec types III and IV were identified among 71.4% of the isolates, while the remaining was nontypeable. The predominant MRSE genotypes were defined as SCCmec type III/ST2 (PFGE type A) and SCCmec type IV/ST23 (PFGE type B) isolates, which were both associated with high antimicrobial resistance and presence of biofilm-related genes. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic diversity and microevolution of Burkholderia pseudomallei in the environment.

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Narisara Chantratita

2008-02-01

Full Text Available The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined.We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m(2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE and multilocus sequence typing (MLST. Twelve PFGE types and nine sequence types (STs were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively, only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93. Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone.We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

Detection of Listeria spp. in liquid egg products and in the egg breaking plants environment and tracking of Listeria monocytogenes by PFGE.

Science.gov (United States)

Rivoal, Katell; Fablet, Aurore; Courtillon, Céline; Bougeard, Stéphanie; Chemaly, Marianne; Protais, Jocelyne

2013-08-16

Human listeriosis, caused by Listeria monocytogenes, is a severe bacterial infection that can lead to meningitis, cerebromeningitis, bacteremia or septicemia, with acute lethality and potentially leading to death. A study has shown that 29.5% of the caged laying hens in France are contaminated by L. monocytogenes (Chemaly et al., 2008). However, very little information regarding egg and egg product contamination is currently available. The objective of this study is to determine the sanitary status of egg products and egg breaking plants in France regarding Listeria spp. and L. monocytogenes contaminations. The sampling scheme performed in five egg breaking plants in Western France during one year have revealed that 8.5% of raw egg products were contaminated by L. monocytogenes. No pasteurized egg products have been shown to be contaminated by L. monocytogenes. However, a high level of contamination by Listeria spp., and particularly by L. innocua, has been shown with 26.2% and 1.8% of raw and pasteurized egg products contaminated, respectively. This work has also revealed the presence of Listeria spp. and L. monocytogenes in the environment of egg breaking plants with 65.1% and 8.0% of contaminated samples, respectively. The typing of 253 isolates of L. monocytogenes by PFGE using ApaI and AscI enzymes has revealed a high diversity with 46 different pulsotypes and has shown that the raw material is a source of contamination of egg breaking plants. One L. monocytogenes cluster was dominant in the 5 egg-breaking plants during the four seasons studied. The issue of which strains are better adapted to egg products must be considered and studied in depth by comparing them to pulsotypes from strains of other chains. However, the traceability of L. monocytogenes in plants during the various seasons has also made it possible to highlight the presence of strains that are specific to egg breaking plants. The study of cleaning and disinfection methods in these plants as well

Genetic Variation among Staphylococcus aureus Strains from Norwegian Bulk Milk

Science.gov (United States)

Jørgensen, H. J.; Mørk, T.; Caugant, D. A.; Kearns, A.; Rørvik, L. M.

2005-01-01

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex. PMID:16332822

Prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan.

Science.gov (United States)

Hasegawa, Megumi; Iwabuchi, Eriko; Yamamoto, Shiori; Esaki, Hidetake; Kobayashi, Kazuhiko; Ito, Masahiko; Hirai, Katsuya

2013-02-01

This study was conducted to determine the prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan. We collected bovine colostrum samples from 210 dams from 21 dairy farms in Hokkaido prefecture (Japan) between March and June 2009. L. monocytogenes was detected in samples from 6 (28.6%) of the 21 farms. Of the 210 samples, 16 (7.6%) were positive for L. monocytogenes. We recovered 80 L. monocytogenes isolates; 44 (55%) isolates were classified as serotype 1/2b and 36 (45%) were classified as serotype 4b. The isolates were susceptible to penicillin, ampicillin, amoxicillin, gentamicin, kanamycin, streptomycin, erythromycin, vancomycin, tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole. Pulsed-field gel electrophoresis (PFGE) characterization of the 80 isolates revealed six PFGE types. Two PFGE types corresponded to human listeriosis cases. Most L. monocytogenes isolates possessed virulence-associated genes (actA, hly, iap, inlA, inlC, mpl, plcA, plcB, opuCA, prfA, and clpC). One PFGE type isolate possessed an epidemic clone II marker. Our findings suggest that isolates from bovine colostrum have the potential to cause human and animal listeriosis. This is the first study on the prevalence and characteristics of L. monocytogenes isolated from bovine colostrum obtained from dairy farms. Our results have important implications for improving public health and elucidating the epidemiology of L. monocytogenes in bovine colostrum.

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis.

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Cavanagh, Jorunn Pauline; Klingenberg, Claus; Hanssen, Anne-Merethe; Fredheim, Elizabeth Aarag; Francois, Patrice; Schrenzel, Jacques; Flægstad, Trond; Sollid, Johanna Ericson

2012-06-01

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Genetic diversity of Streptococcus uberis isolates from dairy cows with subclinical mastitis in Southern Xinjiang Province, China.

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Wang, Lijun; Chen, Wei; Zhang, Lili; Zhu, Yaxin

2013-01-01

Streptococcus uberis is a common cause of dairy cow mastitis throughout the world. The failure to control bovine mastitis caused by S. uberis is largely attributed to the little known about the epidemiology of this bacteria, especially strain differences in the same area. To define the local epidemiology of S. uberis in the south of Xinjiang, China, we explored the genetic diversity of 28 bovine subclinical mastitis field isolates of S. uberis, collected from 3 Chinese farms during 2009 and 2010, which was examined by using pulsed-field gel electrophoresis (PFGE) for clustering of the isolates and multilocus sequence typing (MLST) to assess the relationship between PFGE patterns and to identify genetic lineages. The 28 isolates were grouped into 13 pulsotypes (U1 to U13), and 1 PFGE type (U1) accounted for almost half of the isolates (13/28, 46.4%). This major type was herd specific, indicating either cow-to-cow transmission or infection with isolates from the same environmental reservoirs. The remaining 12 PFGE types of isolates were from different herds, strongly suggesting environmental sources of S. uberis infection. All 28 isolates were analyzed by MLST and clustered into 8 sequence types (STs), of which 7 STs were found to be novel, either with 5 new alleles of 6 housekeeping and virulence genes (ST158, ST159) or with different combinations of previously assigned alleles (ST153, ST154, ST155, ST156, ST157). To our knowledge, this is the first report that documents molecular typing studies of bovine isolates of S. uberis from southern Xinjiang Province, China, which were shown to represent novel genomic backgrounds of this pathogen.

Typing of Methicillin resistant Staphylococcus aureus: A technical review

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P L Mehndiratta

2012-01-01

Full Text Available Increasing prevalence of Methicillin-resistant Staphylococcus aureus (MRSA worldwide is a growing public health concern. MRSA typing is an essential component of an effective surveillance system to describe epidemiological trends and infection control strategies. Current challenges for MRSA typing are focused on selecting the most appropriate technique in terms of efficiency, reliability, ease of performance and cost involved. This review summarises the available information on application, potential and problems of various typing techniques in discriminating the strains and understanding the epidemiology of MRSA strains. The phenotypic methods in general are easier to perform, easier to interpret, cost effective and are widely available, however less discriminatory. The genotypic methods are expensive and technically demanding, however more discriminatory. Newer technologies involving sequencing of various genes are coming up as broadly applicable and high throughput typing systems. Still there is no consensus regarding the single best method for typing of MRSA strains. Phage typing is recommended as first line approach in epidemiological investigation of MRSA strains. PFGE remains the gold standard for characterisation of outbreak strains. DNA sequencing methods including MLST, spa typing, SCCmec typing and toxin gene profile typing are more practical methods for detecting evolutionary changes and transmission events. The choice of typing technique further depends on the purpose of the study, the facilities available and the utility of data generated to answer a desirable research question. A need for harmonisation of typing techniques by following standard protocols is emphasised to establish surveillance networks and facilitate global MRSA control.

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis.

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Campioni, Fábio; Falcão, Juliana P

2014-06-01

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Multilocus Variable-Number Tandem-Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Patterns in Discrimination of Sporadic and Outbreak-Related Strains of Yersinia enterocolitica

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Skurnik Mikael

2011-02-01

Full Text Available Abstract Background We assessed the potential of multilocus variable-number tandem-repeat analysis (MLVA, pulsed-field gel electrophoresis (PFGE, and antimicrobial susceptibility testing for discriminating 104 sporadic and outbreak-related Yersinia enterocolitica (YE bio/serotype 3-4/O:3 and 2/O:9 isolates. MLVA using six VNTR markers was performed in two separate multiplex PCRs, and the fluorescently labeled PCR products were accurately sized on an automated DNA sequencer. Results MLVA discriminated 82 sporadic YE 3-4/O:3 and 2/O:9 strains into 77 types, whereas PFGE with the restriction enzyme NotI discriminated the strains into 23 different PFGE pulsotypes. The discriminatory index for a sporadic strain was 0.862 for PFGE and 0.999 for MLVA. MLVA confirmed that a foodborne outbreak in the city of Kotka, Finland in 2003 had been caused by a multiresistant YE 4/O:3 strain that was distinctly different from those of epidemiologically unrelated strains with an identical PFGE pulsotype. The multiresistance of Y. enterocolitica strains (19% of the sporadic strains correlated significantly (p = 0.002 with travel abroad. All of the multiresistant Y. enterocolitica strains belonged to four PFGE pulsotypes that did not contain any susceptible strains. Resistance to nalidixic acid was related to changes in codons 83 or 87 that stemmed from mutations in the gyrA gene. The conjugation experiments demonstrated that resistance to CHL, STR, and SUL was carried by a conjugative plasmid. Conclusions MLVA using six loci had better discriminatory power than PFGE with the NotI enzyme. MLVA was also a less labor-intensive method than PFGE and the results were easier to analyze. The conjugation experiments demonstrated that a resistance plasmid can easily be transferred between Y. enterocolitica strains. Antimicrobial multiresistance of Y. enterocolitica strains was significantly associated with travel abroad.

Molecular Characterization of Streptococcus agalactiae Isolated from Bovine Mastitis in Eastern China

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Yang, Yongchun; Liu, Yinglong; Ding, Yunlei; Yi, Li; Ma, Zhe; Fan, Hongjie; Lu, Chengping

2013-01-01

One hundred and two Streptococcus agalactiae (group B streptococcus [GBS]) isolates were collected from dairy cattle with subclinical mastitis in Eastern China during 2011. Clonal groups were established by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), respectively. Capsular polysaccharides (CPS), pilus and alpha-like-protein (Alp) family genes were also characterized by molecular techniques. MLST analysis revealed that these isolates were limited to three clonal groups and were clustered in six different lineages, i.e. ST (sequence type) 103, ST568, ST67, ST301, ST313 and ST570, of which ST568 and ST570 were new genotypes. PFGE analysis revealed this isolates were clustered in 27 PFGE types, of which, types 7, 8, 14, 15, 16, 18, 23 and 25 were the eight major types, comprising close to 70% (71/102) of all the isolates. The most prevalent sequence types were ST103 (58% isolates) and ST568 (31% isolates), comprising capsular genotype Ia isolates without any of the detected Alp genes, suggesting the appearance of novel genomic backgrounds of prevalent strains of bovine S. agalactiae. All the strains possessed the pilus island 2b (PI-2b) gene and the prevalent capsular genotypes were types Ia (89% isolates) and II (11% isolates), the conserved pilus type providing suitable data for the development of vaccines against mastitis caused by S. agalactiae. PMID:23874442

Molecular typing and epidemiological investigation of clinical populations of Pseudomonas aeruginosa using an oligonucleotide-microarray

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Ballarini Annalisa

2012-07-01

Full Text Available Abstract Background Pseudomonas aeruginosa is an opportunistic pathogen which has the potential to become extremely harmful in the nosocomial environment, especially for cystic fibrosis (CF patients, who are easily affected by chronic lung infections. For epidemiological purposes, discriminating P.aeruginosa isolates is a critical step, to define distribution of clones among hospital departments, to predict occurring microevolution events and to correlate clones to their source. A collection of 182 P. aeruginosa clinical strains isolated within Italian hospitals from patients with chronic infections, i.e. cystic fibrosis (CF patients, and with acute infections were genotyped. Molecular typing was performed with the ArrayTube (AT multimarker microarray (Alere Technologies GmbH, Jena, Germany, a cost-effective, time-saving and standardized method, which addresses genes from both the core and accessory P.aeruginosa genome. Pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST were employed as reference genotyping techniques to estimate the ArrayTube resolution power. Results 41 AT-genotypes were identified within our collection, among which 14 were novel and 27 had been previously described in publicly available AT-databases. Almost 30% of the genotypes belonged to a main cluster of clones. 4B9A, EC2A, 3C2A were mostly associated to CF-patients whereas F469, 2C1A, 6C22 to non CF. An investigation on co-infections events revealed that almost 40% of CF patients were colonized by more than one genotype, whereas less than 4% were observed in non CF patients. The presence of the exoU gene correlated with non-CF patients within the intensive care unit (ICU whereas the pKLC102-like island appeared to be prevalent in the CF centre. The congruence between the ArrayTube typing and PFGE or MLST was 0.077 and 0.559 (Adjusted Rand coefficient, respectively. AT typing of this Italian collection could be easily integrated with the global P

The genotypic characterization of Cronobacter spp. isolated in China.

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Jinghua Cui

Full Text Available Cronobacter spp. (Enterobacter sakazakii is an important pathogen contaminating powdered infant formula (PIF. To describe the genotypic diversity of Cronobacter isolated in China, we identified the isolates using fusA allele sequencing, and subtyped all of the isolates using pulsed-field gel electrophoresis (PFGE, multi-locus sequence typing (MLST, and multiple-locus variable-number tandem-repeat analysis (MLVA. A total of 105 isolates were identified, which included C. sakazakii (58 isolates, C. malonaticus (30 isolates, C. dublinensis (11 isolates, C. turicensis (5 isolates, and C. muytjensii (1 isolate. These isolates were showed to have 85 PFGE-patterns, 71 sequence types (STs, and 55 MLVA-patterns. Comparisons among the three molecular subtyping methods revealed that the PFGE method was the most distinguishable tool in identifying clusters of Cronobacter spp. through DNA fingerprinting, and MLST method came second. However, ESTR-1, ESTR-2, ESTR-3, and ESTR-4 were not effective loci for subtyping Cronobacter spp. such that the MLVA method requires further improvement.

Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

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Jalal, S; Ciofu, O; Høiby, Niels

2000-01-01

Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the......Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates...

Evaluation of MLVA for epidemiological typing and outbreak detection of ESBL-producing Escherichia coli in Sweden.

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Helldal, Lisa; Karami, Nahid; Welinder-Olsson, Christina; Moore, Edward R B; Åhren, Christina

2017-01-06

To identify the spread of nosocomial infections and halt outbreak development caused by Escherichia coli that carry multiple antibiotic resistance factors, such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases, is becoming demanding challenges due to the rapid global increase and constant and increasing influx of these bacteria from the community to the hospital setting. Our aim was to assess a reliable and rapid typing protocol for ESBL-E. coli, with the primary focus to screen for possible clonal relatedness between isolates. All clinical ESBL-E. coli isolates, collected from hospitals (n = 63) and the community (n = 41), within a single geographical region over a 6 months period, were included, as well as clinical isolates from a polyclonal outbreak (ST131, n = 9, and ST1444, n = 3). The sporadic cases represented 36 STs, of which eight STs dominated i.e. ST131 (n = 33 isolates), ST648 (n = 10), ST38 (n = 9), ST12 and 69 (each n = 4), ST 167, 405 and 372 (each n = 3). The efficacy of multiple-locus variable number tandem repeat analysis (MLVA) was evaluated using three, seven or ten loci, in comparison with that of pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST). MLVA detected 39, 55 and 60 distinct types, respectively, using three (GECM-3), seven (GECM-7) or ten (GECM-10) loci. For GECM-7 and -10, 26 STs included one type and eleven STs each included several types, the corresponding numbers for GECM-3 were 29 and 8. The highest numbers were seen for ST131 (7,7 and 8 types, respectively), ST38 (5,5,8) and ST648 (4,5,5). Good concordance was observed with PFGE and GECM-7 and -10, despite fewer types being identified with MLVA; 78 as compared to 55 and 60 types. The lower discriminatory power of MLVA was primarily seen within the O25b-ST131 lineage (n = 34) and its H30-Rx subclone (n = 21). Epidemiologically unrelated O25b-ST131 isolates were clustered with O25b-ST131

Time-based distribution of Staphylococcus saprophyticus pulsed field gel-electrophoresis clusters in community-acquired urinary tract infections

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Viviane Santos de Sousa

2013-02-01

Full Text Available The epidemiology of urinary tract infections (UTI by Staphylococcus saprophyticus has not been fully characterised and strain typing methods have not been validated for this agent. To evaluate whether epidemiological relationships exist between clusters of pulsed field gel-electrophoresis (PFGE genotypes of S. saprophyticus from community-acquired UTI, a cross-sectional surveillance study was conducted in the city of Rio de Janeiro, Brazil. In total, 32 (16% female patients attending two walk-in clinics were culture-positive for S. saprophyticus. Five PFGE clusters were defined and evaluated against epidemiological data. The PFGE clusters were grouped in time, suggesting the existence of community point sources of S. saprophyticus. From these point sources, S. saprophyticus strains may spread among individuals.

Detection of enterotoxins and genotyping of Staphylococcus aureus strains isolated from Isfahan Educational Hospital, Iran

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Seyed Asghar Havaei

2017-10-01

Full Text Available Background and aims: Staphylococcus aureus is known as one of the most important nosocomial pathogens, which may lead to several infections. The aim of this study was determining the enterotoxins A, C, and TSST-1 and molecular characterization of S. aureus strains with PFGE and MLST typing methods. Materials and methods: In the present study during the sixmonths sampling, fifty S. aureus strains were isolated from patients admitted to Al-Zahra university hospital. Antimicrobial susceptibility testing, Multiplex PCR for detection of enterotoxin A, C and TSST-1, pulse field gel electrophoresis (PFGE and multilocus sequence typing (MLST were used for molecular typing. Results: In antibiogram the highest and lowest percentage of resistance was belonged to tetracycline and rifampin respectively. Multiplex PCR indicated that 30% of the strains harbored sea and 34% harbored sec genes. However, only 4% of our collected isolates had tsst gene. In PFGE method analysis on all S. aureus strains, a total of 19 different patterns were identified. Nine various sequence types in 27 selected S. aureus isolates were identified by MLST. Conclusions: Present study indicates a possible higher variability among our S. aureus strains by two different molecular typing methods; nevertheless four main common types (CT1, CT7, CT9, and CT11 with at least one toxin genes were determined.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans.

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Patricia Alba

Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA Sequence Type (ST1, Clonal Complex(CC1, SCCmec V is one of the major Livestock-Associated (LA- lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE, spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100% similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A mediated macrolide-lincosamide-streptograminB, and of vga(A-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans.

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Alba, Patricia; Feltrin, Fabiola; Cordaro, Gessica; Porrero, María Concepción; Kraushaar, Britta; Argudín, María Angeles; Nykäsenoja, Suvi; Monaco, Monica; Stegger, Marc; Aarestrup, Frank M; Butaye, Patrick; Franco, Alessia; Battisti, Antonio

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Dissemination of Multidrug-Resistant, Class I and II Integrons and Molecular Typing of CTX-M-producing Klebsiella pneumoniae.

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Akya, Alisha; Elahi, Azam; Chegenelorestani, Roya; Rezaee, Mahya

2018-01-01

Klebsiella pneumoniae ( K. pneumoniae ) is an important opportunistic pathogen causes serious community and hospital-acquired infections, which is highly resistant to antibiotics. We aimed to determine the frequency of multidrug resistant (MDR) and molecular typing of clinical isolates of K. pneumoniae . One hundred isolates of K. pneumoniae were collected from clinical samples in three general hospitals in Kermanshah. The antimicrobial susceptibility and extended-spectrum beta-lactamases (ESBL) production of isolates were determined using disk diffusion and combined disk methods, respectively. The bla CTX-M gene, class I and II integrons were detected using polymerase chain reaction. The bla CTX-M positive isolates were selected for genotyping using pulsed-field gel electrophoresis (PFGE). MDR phenotype was observed in 56% of isolates. The 40% of isolates were ESBL positive and 35 isolates contained bla CTX-M . Class I and II of integrons were detected in 50 (89.2%) and 39 (69.6%) of MDR isolates, respectively. PFGE patterns of K. pneumoniae bla CTX-M positive isolates indicated 19 clusters (X 1-19 ) with different genotype patterns. The study findings highlight the concern of circulating MDR strains of K. pneumoniae with bla CTX-M and class I and II integrons in Kermanshah hospitals. The presence of integrons among isolates may facilitate the spread of new resistance genes in this bacterium. Therefore, surveillance for the spread of MDR strains of this bacterium is recommended in hospitals.

High prevalence of EMRSA-15 in Portuguese public buses: a worrisome finding.

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Roméo Rocha Simões

Full Text Available BACKGROUND: The nosocomial prevalence of methicillin resistant Staphylococcus aureus (MRSA in Portugal remains one of the highest in Europe and is currently around 50%. Transmission of S. aureus, including MRSA, occurs principally by direct human-to-human skin contact. However, S. aureus can survive for long periods on inanimate objects, which may represent an important reservoir for dissemination as well. METHODOLOGY/PRINCIPAL FINDINGS: Between May 2009 and February 2010, handrails of 85 public urban buses circulating in Oporto, Portugal, were screened for the occurrence of MRSA. Twenty-two (26% buses showed MRSA contamination. The molecular characterization of a total of 55 MRSA, by pulsed-field gel electrophoresis (PFGE, staphylococcal cassette chromosome (SCC mec typing, spa typing, and multilocus sequence typing (MLST, clustered the isolates into three clonal types. However, the overwhelming majority (n = 50; 91% of the isolates belonged to a single clone (PFGE A, spa types t747, t032, t025 or t020, ST22, SCCmec type IVh that exhibits the characteristics of the pandemic EMRSA-15, currently the major lineage circulating in Portuguese hospitals, namely in the Oporto region. Two additional clones were found but in much lower numbers: (i PFGE B, ST5, spa type t002, SCCmec IVa (n = 3, and (ii PFGE C, spa type t008, ST8, SCCmec IVa (n = 2. None of the 55 isolates was PVL positive. CONCLUSIONS/SIGNIFICANCE: Public buses in Oporto seem to be an important reservoir of MRSA of nosocomial origin, providing evidence that the major hospital-associated MRSA clone in Portugal is escaping from the primary ecological niche of hospitals to the community environment. Infection control measures are urgently warranted to limit the spread of EMRSA-15 to the general population and future studies are required to assess the eventual increase of MRSA in the Portuguese community, which so far remains low.

Characteristics of invasive Haemophilus influenzae serotype a (Hia) from Nunavik, Canada and comparison with Hia strains in other North American Arctic regions.

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Tsang, Raymond S W; Proulx, Jean-Francois; Hayden, Kristy; Shuel, Michelle; Lefebvre, Brigitte; Boisvert, Andree-Anne; Moore, Dorothy

2017-04-01

This study examines the microbiological characteristics of invasive Haemophilus influenae serotype a (Hia) isolates from Nunavik (northern Quebec), Canada. The relationship between invasive Hia isolates from Nunavik, Nunavut, Canada, and Alaska, USA will be discussed. Twenty invasive Hia isolates were recovered from patients in Nunavik from 2010 to 2013 and characterized by biotype, multi-locus sequence typing, IS1016-bexA deletion, antibiotic susceptibility and pulsed field gel electrophoresis (PFGE). All 20 Hia isolates were biotype II, sequence type -23, did not have IS1016-bexA deletions and were susceptible to all antibiotics tested. PFGE showed only two patterns, with 19 isolates giving identical molecular fingerprints, and the remaining isolate gave a PFGE pattern >95% similar. One major clone of Hia appears to be causing invasive disease in Nunavik, Canada. Based on previous studies, Hia from Nunavut were also typed as ST-23, while invasive Hia isolates from Alaska belonged to either ST-23 or closely related STs. Thus invasive Hia in the North America Arctic belonged to the ST-23 clonal complex and lacked the IS1016-bexA partial deletion. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid

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Fu, Ying; Du, Xiaoxing; Shen, Yuqin; Yu, Yunsong

2015-01-01

The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of bla NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including bla KPC, bla IMP, bla VIM, bla OXA-48 and bla NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the bla KPC-2 genes, and five bla NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three bla NDM-1-positive K. pneumoniae isolates belonged to two different clones. S1-PFGE and southern blot suggested that the bla NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to Escherichia coli by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around bla NDM-1 (bla NDM-1-trpF- dsbC-cutA1-groEL-ΔInsE,) was detected. PCR mapping and sequencing demonstrated that four smaller bla NDM-1 plasmids contained a common gene environment around bla NDM-1 (IS5-bla NDM-1-trpF- dsbC-cutA1-groEL). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the bla NDM-1 gene among the CRE. PMID:26047502

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout, Onchorynchus mykiss (Walbaum), in Spain.

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Del Cerro, A; Márquez, I; Prieto, J M

2010-04-01

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed-field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 microg mL(-1), and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.

Study of methicillin resistant Staphylococcus aureus (MRSA) in Danish pigs at slaughter and in imported retail meat reveals a novel MRSA type in slaughter pigs

DEFF Research Database (Denmark)

Agersø, Yvonne; Hasman, Henrik; Cavaco, Lina M.

2012-01-01

Methicillin-resistant Staphylococcus aureus (MRSA), especially CC398, have emerged in livestock worldwide. We investigated the occurrence of MRSA in pigs at slaughter and in retail meat. During 2009, nasal swabs (n=789) were taken from pigs at slaughter. Moreover, 866 meat samples [Danish: pork...... (153), broiler meat (121), beef (142) and; imported: pork (173), broiler meat (193), and beef (84)] were randomly collected in retail stores and outlets. MRSA was isolated from nasal swabs or from meat samples after preenrichment (Mueller Hinton broth with 6.5% NaCl), selective enrichment (tryptone...... soya broth with 4mg/L cefoxitine and 75mg/L aztreonam) and selective plating on Brilliance Chromogenic MRSA agar. The presence of mecA was confirmed by PCR and the MRSA isolates were spa typed. Novel MRSA spa types were characterized by MLST, PFGE and SCCmec typing. Thirteen percent (101...

Changing epidemiology of methicillin-resistant Staphylococcus aureus in Iceland from 2000 to 2008: a challenge to current guidelines

DEFF Research Database (Denmark)

Holzknecht, B.J.; Hardardottir, H.; Haraldsson, Gustav Helgi

2010-01-01

, and screening for the Panton-Valentine leukocidin (PVL) gene. Two hundred twenty-six infected (60%) or colonized (40%) individuals were detected (annual incidence 2.5 to 16/100,000). From 2000 to 2003, two health care-associated outbreaks dominated (spa types t037 and t2802), which were successfully controlled...... with extensive infection control measures. After 2004, an increasing number of community-associated (CA) cases without relation to the health care system occurred. A great variety of clones (40 PFGE types and 49 spa types) were found, reflecting an influx of MRSA from abroad. The USA300 and Southwest Pacific...... and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the Staphylococcus protein A gene (spa typing), staphylococcal cassette chromosome mec (SCCmec) typing...

Pulsed-field gel electrophoresis typing of Escherichia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women

DEFF Research Database (Denmark)

Ejrnaes, Karen; Sandvang, Dorthe; Lundgren, Bettina

2006-01-01

The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE......). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment......, constituting a reservoir for recurrent UTI....

Pulsed-field gel electrophoresis typing of Eschericia coli strains from samples collected before and after pivmecillinam or placebo treatment of uncomplicated community-acquired urinary tract infection in women

DEFF Research Database (Denmark)

Ejrnæs, K; Sandvang, D; Lundgren, Bettina

2006-01-01

The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE......). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment......, constituting a reservoir for recurrent UTI....

Epidemiology of a Salmonella enterica subsp. Enterica serovar Typhimurium strain associated with a songbird outbreak.

Science.gov (United States)

Blehert, David S.; Hernandez, Sonia M.; Keel, Kevin; Sanchez, Susan; Trees, Eija; ,

2012-01-01

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.

Genotyping and study of the pauA and sua genes of Streptococcus uberis isolates from bovine mastitis.

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Perrig, Melina S; Ambroggio, María B; Buzzola, Fernanda R; Marcipar, Iván S; Calvinho, Luis F; Veaute, Carolina M; Barbagelata, María Sol

2015-01-01

This study aimed to determine the clonal relationship among 137 Streptococcus uberis isolates from bovine milk with subclinical or clinical mastitis in Argentina and to assess the prevalence and conservation of pauA and sua genes. This information is critical for the rational design of a vaccine for the prevention of bovine mastitis caused by S. uberis. The isolates were typed by random amplified polymorphic DNA (RAPD) analysis and by pulsed-field gel electrophoresis (PFGE). The 137 isolates exhibited 61 different PFGE types and 25 distinct RAPD profiles. Simpson's diversity index was calculated both for PFGE (0.983) and for RAPD (0.941), showing a high discriminatory power in both techniques. The analysis of the relationship between pairs of isolates showed 92.6% concordance between both techniques indicating that any given pair of isolates distinguished by one method tended to be distinguished by the other. The prevalence of the sua and pauA genes was 97.8% (134/137) and 94.9% (130/137), respectively. Nucleotide and amino acid sequences of the sua and pauA genes from 20 S. uberis selected isolates, based on their PFGE and RAPD types and geographical origin, showed an identity between 95% and 100% with respect to all reference sequences registered in GenBank. These results demonstrate that, in spite of S. uberis clonal diversity, the sua and pauA genes are prevalent and highly conserved, showing their importance to be included in future vaccine studies to prevent S. uberis bovine mastitis. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Biofilm synthesis and its relationship with genetic characteristics in clinical methicillin-resistant staphylococci

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Nikolaos Giormezis

2015-09-01

Full Text Available Staphylococcus aureus can cause a broad range of infections, including skin infections, pneumonia and bacteraemia. Coagulase-negative staphylococci (CNS, mainly S. epidermidis, have also emerged as important pathogens, especially in immunocompromised patients or those with prosthetic devices, such as intravascular catheters or biomaterials. Of great importance in the initiation of these infections is the ability of staphylococci to adhere to various surfaces, such as host tissues and prosthetic devices and to form biofilm. The staphylococcal adhesins are encoded by a number of genes such as fnbA (S. aureus fibronectin binding protein A, sasG (S. aureus surface protein G, aap (S. epidermidis accumulation associated protein, bhp (Bap homologue protein and fbe (fibrinogen binding protein epidermidis. In this study, 106 methicillin-resistant S. aureus (MRSA, 145 methicillin-resistant S. epidermidis (MRSE and 70 non-epidermidis methicillin-resistant CNS (MR-CNS; 58 S. haemolyticus, 10 S. hominis and two S. lugdunensis were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution and adhesin genes carriage. Isolates were classified into pulsotypes by PFGE and assigned to sequence types by MLST. In total, 121/321 isolates (37.7% produced biofilm and 219 (68.2% carried ica operon. The majority was multidrug resistant (94.7% and carried one or more adhesin genes. MRSE and all other MR-CNS prevailed in biofilm formation (P < 0.001 and antimicrobial resistance (P < 0.05 as compared to MRSA. MRSE also prevailed in ica carriage compared to the other methicillin-resistant staphylococci (P ≤ 0.007 Among MRSE, isolates from bacteraemias prevailed in biofilm formation (P = 0.031, whereas, strains from prosthetic device-associated infections carried more frequently aap (P = 0.003. Even though PFGE showed genetic diversity among MRSE, MLST revealed three major clones (ST2, ST5, ST16. MRSA isolates were less diverse, with five PFGE

Characterization of Enterococcus faecalis isolated from the cloaca of 'fancy breeds' and confined chickens

DEFF Research Database (Denmark)

Jørgensen, S L; Poulsen, L L; Thorndahl, Lotte

2017-01-01

%, while only 44 and 13% in two fancy breeder flocks. A total of 204 isolates were characterized by pulsed-field-gel-electrophoresis (PFGE) where 40 strains were selected for multi-locus sequence typing (MLST). In all, 19 PFGE patterns and 14 STs were obtained. Four STs were identified in each of the two...... conditions were unrelated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided new information on opportunistic E. faecalis in confined non-industrial chicken. Knowledge of population diversity and prevalence compared to conventional production is important to accesses, if certain clonal lineages...

Muusikauudiseid maailmast / Nele-Eva Steinfeld

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Steinfeld, Nele-Eva

2015-01-01

Lühisõnumeid maailmast: Chopini-nimelise konkursi Varssavis võitis Seong-Jin Cho. Martha Argerichile mitu auhinda. Tšaikovski konkursi laureaat Lucas Debargue Sony ja CAMi artist. Augustin Hadelich võitis Warneri preemia jne.

Time Warner veab aktsionäriga vägikaigast / Peeter Teder

Index Scriptorium Estoniae

Teder, Peeter

2005-01-01

Meediafirma Time Warner juhid tõrjuvad aktsionär Carl Icahni süüdistusi firma juhtkonna tegevusetuses ja halvas majandamises. Diagramm: Time Warneri aktsia. Vt. samas: Kes on miljardär Carl Icahn

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay

DEFF Research Database (Denmark)

Nielsen, J B; Skov, M N; Jørgensen, R L

2011-01-01

pneumoniae (K. pneumoniae) producing extended spectrum β-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla (TEM......), bla (SHV), and bla (CTX-M), and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla (CTX-M15), bla (SHV-28), and bla (TEM-1) positive by PCR. Ten out of 52 were ST16 and tested positive for bla (CTX-M15), bla (SHV-1), and bla (TEM-1...

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

Science.gov (United States)

Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

2013-04-01

Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

Characterization of the Brazilian endemic clone of methicillin-resistant Staphylococcus aureus (MRSA from hospitals throughout Brazil

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Geraldo A. Oliveira

Full Text Available The objective of this study was to characterize patterns of the Brazilian endemic clone of methicillin-resistant Staphylococcus aureus (MRSA from hospitals throughout Brazil. We studied 83 MRSA strains isolated from patients hospitalized in 27 public and private hospitals in 19 cities located in 14 Brazilian states from September, 1995, to June, 1997. The MRSA strains were typed using antibiograms, bacteriophage typing and pulsed field gel electrophoresis (PFGE. The analysis of genomic DNA by PFGE showed that 65 isolates presented the same PFGE pattern. This pattern was present in all of the hospitals studied indicating the presence of an endemic MRSA clone widely disseminated throughout Brazilian hospitals (BEC. All isolates belonging to the BEC proved to be resistant to ciprofloxacin, erythromycin, lincomycin, trimethoprim-sulphamethoxazole, and tetracycline. Variable susceptibility to these drugs was found only in isolates belonging to clones other than the BEC. The results show that, among MRSA, the BEC is common in Brazil. The best method for mapping changes in the frequency of this clone among MRSA is pulsed field gel electrophoresis. Use of molecular mapping is an important tool for monitoring the spread of potentially dangerous microbes.

Direct Repeat Unit (dru) Typing of Methicillin-Resistant Staphylococcus pseudintermedius from Dogs and Cats.

Science.gov (United States)

Kadlec, Kristina; Schwarz, Stefan; Goering, Richard V; Weese, J Scott

2015-12-01

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) has emerged in a remarkable manner as an important problem in dogs and cats. However, limited molecular epidemiological information is available. The aims of this study were to apply direct repeat unit (dru) typing in a large collection of well-characterized MRSP isolates and to use dru typing to analyze a collection of previously uncharacterized MRSP isolates. Two collections of MRSP isolates from dogs and cats were included in this study. The first collection comprised 115 well-characterized MRSP isolates from North America and Europe. The data for these isolates included multilocus sequence typing (MLST) and staphylococcal protein A gene (spa) typing results as well as SmaI macrorestriction patterns after pulsed-field gel electrophoresis (PFGE). The second collection was a convenience sample of 360 isolates from North America. The dru region was amplified by PCR, sequenced, and analyzed. For the first collection, the discriminatory indices of the typing methods were calculated. All isolates were successfully dru typed. The discriminatory power for dru typing (D = 0.423) was comparable to that of spa typing (D = 0.445) and of MLST (D = 0.417) in the first collection. Occasionally, dru typing was able to further discriminate between isolates that shared the same spa type. Among all 475 isolates, 26 different dru types were identified, with 2 predominant types (dt9a and dt11a) among 349 (73.4%) isolates. The results of this study underline that dru typing is a useful tool for MRSP typing, being an objective, standardized, sequence-based method that is relatively cost-efficient and easy to perform. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Methicillin-resistant Staphylococcus aureus DNA electrophoretic pattern: temporal changes in an endemic hospital environment Patrón electroforético del ADN de Staphylococcus aureus resistente a la meticilina: cambios temporales en un medio hospitalario endémico

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Gleice Cristina Leite

2011-12-01

Full Text Available OBJECTIVE: To describe the analysis of geographical and temporal distribution of DNA profiles determined by pulsedfield gel electrophoresis (PFGE of methicillinresistant Staphylococcus aureus (MRSA strains isolated from hospitalized patients in a tertiary care university hospital in Brazil. METHODS: Ninetynine samples of MRSA obtained from 89 patients in the period 1999- 2004 were studied. MRSA strains were isolated from central venous catheters (33 isolates and bloodstream infections (66 strains. PFGE with 20 units of SmaI restriction endonuclease was used for genomic typing. RESULTS: Analysis of DNA PFGE of 99 strains of MRSA revealed 26 profiles and their respective related profiles. The mean time interval for detecting MRSA infection was 26 days from hospital admission. Forty-nine patients (57.6% had a recent hospitalization. The DNA PFGE MRSA profiles were distributed in three clonal groups-I, II, and III-according to the period of time when the MRSA strains were isolated. DNA PFGE MRSA profiles were spread homogeneously through all hospital wards. CONCLUSIONS: Changes in the distribution of DNA PFGE MRSA profiles were largely temporal, with clonal groups being replaced over time, without predominance in any hospital ward or any specific area of the hospital.OBJETIVO: Analizar la distribución geográfica y temporal de los perfiles de ADN determinados mediante electroforesis en gel de campo pulsado (PFGE de cepas de Staphylococcus aureus resistente a la meticilina (SARM aisladas de pacientes internados en un hospital universitario de atención terciaria en el Brasil. MÉTODOS: Se estudiaron 99 muestras de SARM obtenidas 89 de pacientes en el período 1999-2004. Las cepas de SARM se aislaron de infecciones de catéteres venosos centrales (33 aislados y del torrente sanguíneo (66 cepas. Para la tipificación genómica se empleó PFGE con 20 unidades de endonucleasa de restricción SmaI. RESULTADOS: El análisis del ADN de 99 cepas de SARM

Genetic and serological diversity of Flavobacterium psychrophilum isolates from salmonids in United Kingdom.

Science.gov (United States)

Ngo, Thao P H; Bartie, Kerry L; Thompson, Kim D; Verner-Jeffreys, David W; Hoare, Rowena; Adams, Alexandra

2017-03-01

Flavobacterium psychrophilum is one of the most important bacterial pathogens affecting cultured rainbow trout (Oncorhynchus mykiss) and is increasingly causing problems in Atlantic salmon (Salmo salar L.) hatcheries. Little is known about the heterogeneity of F. psychrophilum isolates on UK salmonid farms. A total of 315 F. psychrophilum isolates, 293 of which were collected from 27 sites within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG) 5 -PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. Seven PFGE groups and 27 singletons were formed at a band similarity of 80%. PFGE group P (n=75) was found to be numerically predominant in eight sites within the UK. Two major PFGE clusters and 13 outliers were found at the band similarity of 40%. The predominant profileobserved within the F. psychrophilum isolates examined was PFGE cluster II - (GTG) 5 -PCR type r1-16S rRNA lineage II - serotype Th (70/156 isolates examined, 45%). Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, confounding the ability to control RTFS outbreaks. The occurrence over time (up to 11 years) of F. psychrophilum pulsotypes in three representative sites (Scot I, Scot III and Scot V) within Scotland was examined, potentially providing important epidemiological data for farm management and the development of site-specific vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

[Inter-laboratory reproducibility of pulsed-field electrophoresis for the study of 12 types of Pseudomonas aeruginosa].

Science.gov (United States)

Foissaud, V; Puyhardy, J M; Chapalain, J C; Salord, H; Depina, J J; Morillon, M; Nicolas, P; Perrier-Gros-Claude, J D

1999-12-01

The increasing hospital-to-hospital transmission of multiple drug-resistant bacteria is a major concern for bacteriology laboratories involved in nosocomial infection control. The interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) for Pseudomonas aeruginosa typing was evaluated by asking four hospital laboratories (two in Lyon, one in Brest, and one in Marseille) to study 11 P. aeruginosa isolates, some of which were epidemiologically related, and the reference strain ATCC 27853. Two laboratories used the Genepath system, one the Chef DR II, system, and one the Chef Mapper system, Bio-Rad, restriction/Spe I. Profiles were read visually and by computerized comparison of restriction band molecular weights (Taxotron, software, PAD Grimont, Pasteur Institute, Paris, France). These two methods led to similar epidemiological conclusions. However, centralization of the data showed poor center-to-center reproducibility due to inadequate standardization of the procedure.

Virulence-associated genes, antimicrobial resistance and molecular typing of Salmonella Typhimurium strains isolated from swine from 2000 to 2012 in Brazil.

Science.gov (United States)

Almeida, F; Medeiros, M I C; Kich, J D; Falcão, J P

2016-06-01

The aims of this study were to assess the pathogenic potential, antimicrobial resistance and genotypic diversity of Salmonella Typhimurium strains isolated in Brazil from swine (22) and the surrounding swine environment (5) from 2000 to 2012 and compare them to the profiles of 43 human strains isolated from 1983 to 2010, which had been previously studied. The presence of 12 SPI-1, SPI-2 and plasmid genes was assessed by PCR, the antimicrobial susceptibility to 13 antimicrobials was determined by the disc diffusion assay and genotyping was performed using pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number of tandem repeats analysis (MLVA) and ERIC-PCR. More than 77·8% of the swine strains carried 10 or more of the virulence markers. Ten (37%) strains isolated from swine were multi-drug resistant (MDR). All the molecular typing techniques grouped the strains in two main clusters. Some strains isolated from swine and humans were allocated together in the PFGE-B2, MLVA-A1, MLVA-B and ERIC-A1 clusters. The genotyping results suggest that some strains isolated from swine and humans may descend from a common subtype and may indicate a possible risk of MDR S. Typhimurium with high frequency of virulence genes isolated from swine to contaminate humans in Brazil. This study provided new information about the pathogenic potential, antimicrobial resistance and genotypic diversity of S. Typhimurium isolates from swine origin in Brazil, the fourth largest producer of pigs worldwide. © 2016 The Society for Applied Microbiology.

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis.

Science.gov (United States)

Lindahl, Susanne; Söderlund, Robert; Frosth, Sara; Pringle, John; Båverud, Viveca; Aspán, Anna

2011-11-21

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi. Copyright © 2011 Elsevier B.V. All rights reserved.

Staphylococci Related to Farm Ostriches and Their Sensitivity to Enterocins.

Science.gov (United States)

Kandričáková, Anna; Lauková, Andrea; Ščerbová, Jana

2016-03-01

In Slovakia, ostriches are reared mainly for their meat. There is still limited information related to microflora of ostriches, including staphylococci. Knowing the composition of microflora is very important for the recognition of potential pathogenic agents. Recently, a frequent problem in animals is the occurrence of bacteria resistant to antibiotics. The aim of this study was to detect staphylococcal species in feces of farm ostriches and to test their sensitivity to antibiotics and enterocins. Altogether 140 ostriches from three age groups were sampled (n = 18, faecal mixture samples from each group) on a farm in Slovakia or on Slovak farm. From 54 fecal samples, the staphylococcal count reached an average 4. 3 ± 0. 63 (log10) CFU/g. Twenty-four lactic acid producing strains were taxonomically classified to eight species of the genus Staphylococcus: Staphylococcus equorum, S. xylosus, S. epidermidis, S. haemolyticus, S. cohnii, S. succinus, S. warneri, and S. hominis. Strains were evaluated by secure probable species identification/probable species identification (score value up to 2.299) confirmed also by phenotypization. Most strains were sensitive to antibiotics. Four strains (S. haemolyticus SHae 111, S. haemolyticus SHAe 371, S. xylosus SX 2133, and S. warneri SW 292) were resistant to methicillin but sensitive to six or five of the seven enterocins tested (inhibitory activity 200-12,800 AU/mL). S. warneri SW 292 was sensitive to all enterocins (activity up to 12,800 AU/mL).

Characteristics and management of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemase in a tertiary hospital.

Science.gov (United States)

Pang, Feng; Jia, Xiu-Qin; Song, Zhen-Zhu; Li, Yan-Hua; Wang, Bin; Zhao, Qi-Gang; Wang, Chuan-Xin; Zhang, Yi; Wang, Le-Xin

2016-03-01

The emergence of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemases is rare. We report an occurrence of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemases in a Chinese tertiary care hospital from November 2010 to December 2012. The clinical characteristics of 30 patients were described. The genetic relationship of isolates was determined by pulsed-field gel electrophoresis (PFGE). Carbapenemases were detected by modified Hodge test (MHT) and polymerase chain reactions (PCRs). Amplicons were sequenced and blasted to determine the genotype. Most infected patients were from intensive care unit and had complex and serious underlying illnesses requiring mechanical ventilation. PFGE revealed that Klebsiella pneumoniae showed two major PFGE types. Two Klebsiella oxytoca had an indistinguishable PFGE pattern, while four Enterobacter cloacae were different strains. The sequencing studies showed Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemase in the 23 infected patients. The majority of patients had infections with the carbapenemase-producing Enterobacteriaceae (CPE) strain, most were successfully treated with a range of antibiotics and discharged. It is important to maintain a high index of suspicion to screen for carbapenemase-producing Enterobacteriaceae strains. Rapid identification of these strains and implementation of stringent procedures are the key to prevent major outbreaks in a hospital setting.

Human and Swine Hosts Share Vancomycin-Resistant Enterococcus faecium CC17 and CC5 and Enterococcus faecalis CC2 Clonal Clusters Harboring Tn1546 on Indistinguishable Plasmids

DEFF Research Database (Denmark)

Freitas, Ana R.; Coque, Teresa M.; Novais, Carla

2011-01-01

clonally related Enterococcus faecium clonal complex 5 (CC5) isolates (17 sequence type 6 [ST6], 6 ST5, 5 ST185, 1 ST147, and 1 ST493) were obtained from feces of swine and healthy humans. This collection included isolates widespread among pigs of European Union (EU) countries since the mid-1990s. Each ST...... comprised isolates showing similar pulsed-field gel electrophoresis (PFGE) patterns (≤6 bands difference; >82% similarity). Some CC5 PFGE subtype strains from swine were indistinguishable from hospital vancomycin-resistant enterococci (VRE) causing infections. A truncated variant of Tn1546 (encoding...... resistance to vancomycin) and tcrB (coding for resistance to copper) were consistently located on 150- to 190-kb plasmids (rep(pLG1)). E. faecium CC17 (ST132) isolates from pig manure and two clinical samples showed identical PFGE profiles and contained a 60-kb mosaic plasmid (rep(Inc18) plus rep...

Association of the shuffling of Streptococcus pyogenes clones and the fluctuation of scarlet fever cases between 2000 and 2006 in central Taiwan

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Wang Wan-Ling

2009-06-01

Full Text Available Abstract Background The number of scarlet fever occurrences reported between 2000 and 2006 fluctuated considerably in central Taiwan and throughout the nation. Isolates of Streptococcus pyogenes were collected from scarlet fever patients in central Taiwan and were characterized by emm sequencing and a standardized pulsed-field gel electrophoresis (PFGE method. National weekly report data were collected for investigating epidemiological trends. Results A total of 23 emm types were identified in 1,218 S. pyogenes isolates. The five most prevalent emm types were emm12 (50.4%, emm4 (23.2%, emm1 (16.4%, emm6 (3.8% and emm22 (3.0%. PFGE analysis with SmaI suggested that, with a few exceptions, strains with a common emm type belonged to the same clone. There were two large emm12 clones, one with DNA resistant to cleavage by SmaI. Each prevalent emm clone had major PFGE strain(s and many minor strains. Most of the minor strains emerged in the population and disappeared soon after. Even some major strains remained prevalent for only 2–3 years before declining. The large fluctuation of scarlet fever cases between 2000 and 2006 was associated with the shuffling of six prevalent emm clones. In 2003, the dramatic drop in scarlet fever cases in central Taiwan and throughout the whole country was associated with the occurrence of a severe acute respiratory syndrome (SARS outbreak that occurred between late-February and mid-June in Taiwan. Conclusion The occurrences of scarlet fever in central Taiwan in 2000–2006 were primarily caused by five emm types, which accounted for 96.8% of the isolates collected. Most of the S. pyogenes strains (as defined by PFGE genotypes emerged and lasted for only a few years. The fluctuation in the number of scarlet fever cases during the seven years can be primarily attributed to the shuffling of six prevalent emm clones and to the SARS outbreak in 2003.

Assessing the genome level diversity of Listeria monocytogenes from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States.

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Yi Chen

Full Text Available A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP-based whole genome sequencing (WGS analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE profiles and multilocus sequence typing (MLST sequence type (ST 5 of clonal complex 5 (CC5. WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5

Assessing the genome level diversity of Listeria monocytogenes from contaminated ice cream and environmental samples linked to a listeriosis outbreak in the United States.

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Chen, Yi; Luo, Yan; Curry, Phillip; Timme, Ruth; Melka, David; Doyle, Matthew; Parish, Mickey; Hammack, Thomas S; Allard, Marc W; Brown, Eric W; Strain, Errol A

2017-01-01

A listeriosis outbreak in the United States implicated contaminated ice cream produced by one company, which operated 3 facilities. We performed single nucleotide polymorphism (SNP)-based whole genome sequencing (WGS) analysis on Listeria monocytogenes from food, environmental and clinical sources, identifying two clusters and a single branch, belonging to PCR serogroup IIb and genetic lineage I. WGS Cluster I, representing one outbreak strain, contained 82 food and environmental isolates from Facility I and 4 clinical isolates. These isolates differed by up to 29 SNPs, exhibited 9 pulsed-field gel electrophoresis (PFGE) profiles and multilocus sequence typing (MLST) sequence type (ST) 5 of clonal complex 5 (CC5). WGS Cluster II contained 51 food and environmental isolates from Facility II, 4 food isolates from Facility I and 5 clinical isolates. Among them the isolates from Facility II and clinical isolates formed a clade and represented another outbreak strain. Isolates in this clade differed by up to 29 SNPs, exhibited 3 PFGE profiles and ST5. The only isolate collected from Facility III belonged to singleton ST489, which was in a single branch separate from Clusters I and II, and was not associated with the outbreak. WGS analyses clustered together outbreak-associated isolates exhibiting multiple PFGE profiles, while differentiating them from epidemiologically unrelated isolates that exhibited outbreak PFGE profiles. The complete genome of a Cluster I isolate allowed the identification and analyses of putative prophages, revealing that Cluster I isolates differed by the gain or loss of three putative prophages, causing the banding pattern differences among all 3 AscI-PFGE profiles observed in Cluster I isolates. WGS data suggested that certain ice cream varieties and/or production lines might have contamination sources unique to them. The SNP-based analysis was able to distinguish CC5 as a group from non-CC5 isolates and differentiate among CC5 isolates from

Occurrence and strain diversity of thermophilic campylobacters in cattle of different age groups in dairy herds

DEFF Research Database (Denmark)

Nielsen, Eva M.

2002-01-01

. Serotype 2 was especially prevalent among calves (68% of the positive calves). In eight of the 20 positive herds, all isolates had the same sero- and PFGE type while, in the other herds, two to five different types were isolated. Conclusions: Significant differences were found between age groups...

Concomitant genotyping revealed diverse spreading between methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus in central Taiwan.

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Ho, Cheng-Mao; Lin, Chien-Yu; Ho, Mao-Wang; Lin, Hsiao-Chuan; Peng, Ching-Tien; Lu, Jang-Jih

2016-06-01

Staphylococcus aureus is a versatile bacterium, which can lead to various infectious diseases. Various molecular typing methods are applied to the evolution and epidemiology surveys of S. aureus, mostly for methicillin-resistant S. aureus (MRSA). However, methicillin-susceptible S. aureus (MSSA) is still an important pathogen, but their molecular typing is evaluated infrequently. Pulsed-field gel electrophoresis (PFGE), spa typing, and detection of five virulent genes for 95 MRSA and 56 MSSA isolates (July-December 2008 and July 2008-December 2009, respectively) during an overlapping period were performed. More diversity was found in MSSA isolates (23 pulsotypes and 25 spa types, excluding 4 new-type and 1 nontypable isolates for spa typing) than in MRSA isolates (19 pulsotypes and 16 spa types, excluding 1 new-type and 1 nontypable isolates for spa typing). By spa typing, t002 (n = 30), t037 (n = 23), t437 (n = 21), t234 (n = 3), t1081 (n = 3), and t1094 (n = 3) were the six major MRSA clones. For MSSA isolates, t189 (n = 13), t437 (n = 4), t084 (n = 3), t213 (n = 3), t701 (n = 3), and t7200 (n = 3) were the six major types. Combining PFGE and spa typing, there were five combinations (pulsotype + spa type) that contained both MRSA and MSSA isolates (pulsotype 9-t437, pulsotype 15-t037, pulsotype 19-t002, pulsotype 21-t002, and pulsotype 28-t1081). For all 151 S. aureus or 95 MRSA isolates, the PFGE typing had more discrimination power, but spa typing had larger discrimination index for 56 MSSA isolates. In conclusion, there were different predominant MRSA and MSSA clones clinically. Continuing longitudinal tracking of molecular typing is necessary for elucidating the evolution of this important clinical pathogen. Copyright © 2014. Published by Elsevier B.V.

High prevalence of methicillin-resistant coagulase-negative staphylococci isolated from a university environment in Thailand.

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Seng, Rathanin; Leungtongkam, Udomluk; Thummeepak, Rapee; Chatdumrong, Wassana; Sitthisak, Sutthirat

2017-06-01

The present study was conducted to isolate and characterize the molecular epidemiology of the methicillin-resistant staphylococci in the general university environment, where all five locations; the library, restrooms, canteens, computer rooms and outdoor surfaces, are in common use by a large population of students. We used Mannitol Salt Agar (MSA) supplemented with 4 μg/ml of oxacillin to screen the methicillin-resistant staphylococci. The species level was identified by PCR of rdr (Staphylococcus epidermidis), groESL (Staphylococcus haemolyticus) and nuc (Staphylococcus aureus and Staphylococcus warneri) genes and DNA sequencing of tuf and dnaJ genes. The susceptibility patterns of the isolates were determined using the disk diffusion method. Antibiotic and disinfectant resistance genes, together with SCCmec types, were detected by the PCR method. The methicillin resistant-staphylococci were isolated from 41 of 200 samples (20.5%), and all of them were found to be methicillin-resistant coagulase negative staphylococci (MR-CoNS). The library had the highest percentage of contamination, with 43.3% of the samples found to be contaminated. All isolates belonged to 6 different species including S. haemolyticus, S. epidermidis, S. warneri, S. cohnii, S. saprophyticus and S. hominis. The antimicrobial resistance rates were highest against penicillin (100%), then cefoxitin (73.1%), erythromycin (73.1%) and oxacillin (68.3%). Altogether, the isolates were approximately 61.0% multidrug resistant (MDR), with the S. epidermidis isolates being the most multidrug resistant. The prevalence of the qacA/B gene was detected in 63.4% of the isolates, and SCCmec could be typed in 43.9% (18/41) of the isolates. The type range was: II (n = 1), IVd (n = 1), I (n = 2), V (n = 6), IVa (n = 8) and untypeable (n = 23). This result indicates that these university environments are contaminated with methicillin-resistant coagulase negative staphylococci that carry various SCCmec types and

Isolation and identification of a Staphylococcus warneri strain with ...

African Journals Online (AJOL)

Tuberculosis is the principal cause of death from infection in the world. The resurgence of tuberculosis and the increase in mycobacterial infections, as well as multidrug-resistance of mycobacteria to available antibiotics, has incentivized research on new antimycobacterial agents. Therefore, research based on water and ...

Isolation and identification of a Staphylococcus warneri strain with ...

African Journals Online (AJOL)

Ezedom Theresa

2013-10-16

Oct 16, 2013 ... due to an active substance secreted into the culture medium. Sequencing of the 16S rRNA .... were used, a negative control (T-) corresponds to a no template control, and a .... mastitis (Oliveira et al., 1998b). Hassi et al. (2007) ...

Pulsed-field gel electrophoresis of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections.

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Hanifah, Y A; Hiramatsu, K

1994-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) infection has been endemic in the University Hospital, Kuala Lumpur since the late 1970s. Fifty isolates of MRSA obtained from clinical specimens of patients with nosocomial infections associated with this organism have been studied by pulsed-field gel electrophoresis (PFGE) of its chromosomal DNA fragments to discrimate between strains and to identify the predominant strain. Twenty-one chromosomal patterns were observed which could be further grouped into nine types. The predominant strain was Type 9-b (40% of isolates) found mainly in the Orthopaedic and Surgical Units. Outbreak strains found in the Special Care Nursery were of Type 1, entirely different from those of the surgical ward S2, which were of Type 9-b. Type 8 strains were found mainly at one end of the hospital building where the maternity, paediatric and orthopaedic units were situated. Genomic DNA fingerprinting by PFGE is recommended as a useful and effective tool for the purpose of epidemiological studies of MSRA infections, particularly for nosocomial infections.

Reliability analysis of multi-trigger binary systems subject to competing failures

International Nuclear Information System (INIS)

Wang, Chaonan; Xing, Liudong; Levitin, Gregory

2013-01-01

This paper suggests two combinatorial algorithms for the reliability analysis of multi-trigger binary systems subject to competing failure propagation and failure isolation effects. Propagated failure with global effect (PFGE) is referred to as a failure that not only causes outage to the component from which the failure originates, but also propagates through all other system components causing the entire system failure. However, the propagation effect from the PFGE can be isolated in systems with functional dependence (FDEP) behavior. This paper studies two distinct consequences of PFGE resulting from a competition in the time domain between the failure isolation and failure propagation effects. As compared to existing works on competing failures that are limited to systems with a single FDEP group, this paper considers more complicated cases where the systems have multiple dependent FDEP groups. Analysis of such systems is more challenging because both the occurrence order between the trigger failure event and PFGE from the dependent components and the occurrence order among the multiple trigger failure events have to be considered. Two combinatorial and analytical algorithms are proposed. Both of them have no limitation on the type of time-to-failure distributions for the system components. Their correctness is verified using a Markov-based method. An example of memory systems is analyzed to demonstrate and compare the applications and advantages of the two proposed algorithms. - Highlights: ► Reliability of binary systems with multiple dependent functional dependence groups is analyzed. ► Competing failure propagation and failure isolation effect is considered. ► The proposed algorithms are combinatorial and applicable to any arbitrary type of time-to-failure distributions for system components.

[Genotypic variability and persistence of Legionella pulsed-field gel electrophoresis patterns in 16 cooling towers in Shanghai, China].

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Chen, Ming-liang; Wang, Gang-yi; Chen, Min; Zhou, Hai-jian; Shao, Zhu-jun; Zhang, Xi; Wu, Fan

2010-07-01

To investigate the genotypic characteristics and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns in 16 air-conditioner cooling towers in six different public sites of Shanghai. From May to October, continuous sampling was operated once per month in 2007. Legionella strains isolated from the 16 cooling towers were confirmed by serological and latex agglutination. PFGE was applied for the fingerprinting of the isolates, while the cluster results of PFGE were analyzed by BioNumerics software. 131 strains of Legionella were isolated, including L. pneumophila, L. bozemanae, L. micdadei and L. anisa. 52 distinguishable PFGE patterns were differentiated among the 16 cooling towers, with 37 patterns were owned by just one cooling tower, which was not shared with other cooling towers, while 15 patterns were shared by more than 2 cooling towers. All the cooling towers had ≥ 2 PFGE patterns, while in 13 cooling towers the same PFGE patterns were recovered during the six months. From June to October of 2007, 18 strains of Legionella belonging to the PFGE pattern of LPAs.SH0078 were isolated continuously from 6 cooling towers. This study demonstrated great genotypic diversity and complexity of Legionella in cooling towers. Persistence of the PFGE patterns was observed in 81.25% of the cooling towers. The PFGE pattern of LPAs. SH0078 was distributed widely, suggesting it might be the dominate strain in Shanghai.

IS21-558 insertion sequences are involved in the mobility of the multiresistance gene cfr

DEFF Research Database (Denmark)

Kehrenberg, Corinna; Aarestrup, Frank Møller; Schwarz, Stefan

2007-01-01

During a study of florfenicol-resistant porcine staphylococci from Denmark, the genes cfr and fexA were detected in the chromosomal DNA or on plasmids of Staphylococcus hyicus, Staphylococcus warneri, and Staphylococcus simulans. A novel variant of the phenicol resistance transposon Tn558...

Strain-specific transmission in an outbreak of ESBL-producing Enterobacteriaceae in the hemato-oncology care unit: a cohort study.

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Uemura, Makiko; Imataki, Osamu; Uchida, Shumpei; Nakayama-Imaohji, Haruyuki; Ohue, Yukiko; Matsuka, Harumi; Mori, Hatsune; Dobashi, Hiroaki; Kuwahara, Tomomi; Kadowaki, Norimitsu

2017-01-05

Extended-spectrum β-lactamase (ESBL)-producing bacteria are resistant to several types of antibiotics excluding carbapenems. A transmissibility of ESBL-producing Enterobacteriaceae would be depending on each bacterial property, however, that has not been elucidated in clinical setting. In this study, we attempted to identify the source of an outbreak of ESBL-producing bacteria in a medical oncology and immunology care unit. An ESBL-producing Enterobacteriaceae (ESBL-E) outbreak observed between July 2012 and August 2012 in Kagawa University Hospital was surveyed using various molecular microbiology techniques. We used Pulsed-field gel electrophoresis (PFGE), PCR-based ESBL gene typing, and direct sequence of ESBL gene as molecular microbiology typing method to distinguish each strain. The typical prevalence of ESBL-E isolation in the unit was 7.0 per month (1.7 per week). The prevalence of ESBL-E isolation during the target research period was 20.0 per month (5.0 per week). In total, 19 isolates (11 K. pneumoniae and 8 E. coli) were obtained from clinical samples, including four control strains (two each of both bacteria), that were physically different from those obtained from other inpatient units in our hospital. Pulsed-field gel electrophoresis (PFGE) for K. pneumoniae (digested by XbaI) produced similar patterns excluding one control strain. PCR classification of the ESBL gene for K. pneumoniae revealed that all strains other than the control strain carried SHV and CTX-M-9. This result was reconfirmed by direct DNA sequencing. Although the outbreak of K. pneumoniae was considered to be "clonal," PFGE and PCR classification of the ESBL genes for E. coli uncovered at least six different "non-clonal" strains possessing individual ESBL gene patterns. According to the result of an antibiogram, the pattern of antimicrobial susceptibility was more variable for K. pneumoniae than for E. coli. Typing by PFGE and ESBL gene PCR analysis is practical for discriminating

Molecular epidemiology of methicillin resistant staphylococcus aureus colonizing the anterior Nares of school children of Udupi Taluk

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S Govindan

2015-01-01

Full Text Available Context: Community associated methicillin resistant Staphylococcus aureus (CA-MRSA cause serious skin and soft tissue infections including necrotizing fasciitis and necrotizing pneumonia. Production of Panton Valentine Leucocidine (PVL toxin is implicated in its enhanced virulence. A variant of epidemic MRSA-15 (EMRSA-15 which produces PVL toxin has been isolated and characterized by pulsed-field gel electrophoresis (PFGE method from the Indian population both in hospital and community settings. Aims: Identify the epidemiological type of MRSA colonizing the anterior nares of school children in Udupi taluk. Settings and Design: The study population included children of the age group of 5-16 years belonging to the Udupi taluk of Karnataka, India. A total of 1503 children were screened for MRSA colonization during July 2009 to December 2010. Materials and Methods: PVL assay, Staphylococcal Cassette Chromosome (SCC mec typing and PFGE typing were carried out with all the MRSA isolates. Statistical Analysis Used: Frequency distribution of different variables was assessed by SPSS. Results: Among the 1.1% of MRSA, 58.8% (10/17 of isolates were positive for pvl and 41.7% (7/17 were identified as SCC mec type IV. PFGE patterns of all the strains were identical with Indian variant EMRSA-15; however they were different from classical EMRSA-15 in 3-4 bands. Conclusions: The Indian variant EMRSA-15 gains much epidemiological relevance owing to the acquisition of pvl gene. In spite of low prevalence of nasal colonization of MRSA, emergence of the virulent Indian variant EMRSA-15 in our community is a worrisome fact to be reckoned with.

Antimicrobial Agent of Susceptibilities and Antiseptic Resistance Gene Distribution among Methicillin-Resistant Staphylococcus aureus Isolates from Patients with Impetigo and Staphylococcal Scalded Skin Syndrome

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Noguchi, Norihisa; Nakaminami, Hidemasa; Nishijima, Setsuko; Kurokawa, Ichiro; So, Hiromu; Sasatsu, Masanori

2006-01-01

The susceptibilities to antimicrobial agents of and distributions of antiseptic resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated between 1999 and 2004 in Japan were examined. The data of MRSA strains that are causative agents of impetigo and staphylococcal scalded skin syndrome (SSSS) were compared with those of MRSA strains isolated from patients with other diseases. The susceptibilities to antiseptic agents in MRSA isolates from patients with impetigo and SSSS were higher than those in MRSA isolates from patients with other diseases. The distribution of the qacA/B genes in MRSA strains isolated from patients with impetigo and SSSS (1.3%, 1/76) was remarkably lower than that in MRSA strains isolated from patients with other diseases (45.9%, 95/207). Epidemiologic typings of staphylococcal cassette chromosome mec (SCCmec) and pulsed-field gel electrophoresis (PFGE) showed that MRSA strains isolated from patients with impetigo and SSSS had type IV SCCmec (75/76), except for one strain, and 64.5% (49/76) of the strains had different PFGE types. In addition, the patterns of restriction digestion of all tested qacA/B plasmid in MRSA isolates having different PFGE types were identical. The results showed that a specific MRSA clone carrying qacA/B was not prevalent, but qacA/B was spread among health care-associated MRSA strains. Therefore, it was concluded that the lower distribution rate of qacA/B resulted in higher susceptibilities to cationic antiseptic agents in MRSA isolated from patients with impetigo and SSSS. PMID:16757607

Carriage of Staphylococcus aureus among Portuguese nursing students: A longitudinal cohort study over four years of education.

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Teresa Conceição

Full Text Available Staphylococcus aureus is a major human pathogen that can colonize healthy people mainly in the anterior nares. The aim of the present study was to evaluate S. aureus nasal colonization over time among Portuguese nursing students, including methicillin-resistant S. aureus (MRSA.In this longitudinal cohort study, we collected 280 nasal swabs from nursing students at 14 time points over four years of schooling (2012-2016. The isolates were characterized by pulsed-field gel electrophoresis (PFGE, spa typing, multilocus sequence typing (MLST, and SCCmec typing for MRSA. Among 47 students, 20 (43% carried methicillin-susceptible S. aureus (MSSA at admission, but none was colonized with MRSA. A total of 19 students (40% became colonized after exposure during the nursing training, out of which five carried MRSA. Overall, 39 students (83% had S. aureus detected at least once during the study period. Among the 97 MSSA isolates, most (65% belonged to four clones: PFGE A-ST30 (21%, B-ST72 (20%, C-ST508 (13%, and D-ST398 (11%. Three of the five MRSA carriers were colonized with the predominant clone circulating in Portuguese hospitals (ST22-IVh and two with ST3162-II. Colonization of nursing students was highly dynamic with continuous appearance of strains with distinct PFGE types in the same individual.A considerable proportion of students became colonized by S. aureus, including MRSA, during the nursing education, evidencing this population represents an important reservoir of S. aureus. Therefore, education on infection control measures in nursing schools is of major importance.

Characterisation of recently emerged multiple antibiotic-resistant Salmonella enterica serovar typhimurium DT104 and other multiresistant phage types from Danish pig herds

DEFF Research Database (Denmark)

Baggesen, Dorte Lau; Aarestrup, Frank Møller

1998-01-01

electrophoresis (PFGE) using the restriction enzyme Xba I, Overall, 66 per cent of the 670 isolates were sensitive to all the antimicrobial agents tested. Eleven isolates of S typhimurium were resistant to ampicillin, streptomycin and tetracycline and also resistant to other antibiotics in different resistance...... patterns. Seven different multiresistant clones were identified, The most common clones were four isolates of DT104 and three isolates of DT193, TWO Of the three S typhimurium DT104 from 1994 and 1995 were sensitive to all the antimicrobials tested whereas the remaining isolate from 1994 was resistant......A total of 670 isolates of Salmonella enterica were isolated from Danish pig herds, phage typed and tested for susceptibility to amoxycillin + clavulanate, ampicillin, colistin, enrofloxacin, gentamicin, neomycin, spectinomycin, streptomycin, tetracyclines, and trimethoprim + sulphadiazine. S...

Longitudinal study of transmission of Escherichia coli from broiler breeders to broilers

DEFF Research Database (Denmark)

Poulsen, Louise Ladefoged; Thoefner, Ida; Bisgaard, Magne

2017-01-01

of E. coli, isolated from broiler breeders with salpingitis, to the progeny and the possibility of subsequent first week mortality. Four parent flocks were followed during the whole production period (20-60 weeks) by post mortem and bacteriological examination of randomly selected dead birds. Newly...... selected for Pulsed-Field-Gel-Electrophoresis (PFGE) and Multi-Locus-Sequence-Typing (MLST) to determine their clonal relationships. E. coli was the main cause of both salpingitis in parents and first week mortality in broilers, and E. coli dominated the bacterial flora of the cloaca of newly hatched...... chickens. PFGE of E. coli showed identical band patterns in isolates from the three different sources indicating a transmission of E. coli from parent birds to chickens. In conclusion, E. coli isolated from salpingitis in broiler parents were found to be transmitted to broilers in which some sequence types...

Characterization of Antimicrobial Resistance of Listeria monocytogenes Strains Isolated from a Pork Processing Plant and Its Respective Meat Markets in Southern China

DEFF Research Database (Denmark)

Li, Lili; Olsen, Rikke Heidemann; Ye, Lei

2016-01-01

A total of 78 Listeria monocytogenes isolates from a pork processing plant and the respective meat markets in southern China were examined. This number includes 60 isolates from pork at markets, 5 from cooked pork products at markets, 10 from pork at a processing plant, and 3 from food......, ampicillin/sulbactam, imipenem, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, and vancomycin. Two isolates were resistant to five antimicrobials. Twelve strains carried tet(M) and located on Tn916. PFGE analysis revealed genetic heterogeneity among individual serotypes. Two predominant PFGE...... types were found persistent from the processing plant to markets indicating that these two types of isolates were able to survive under environmental adverse conditions from the processing plant to markets, which need to be monitored. Compared to samples from the pork processing plant, the prevalence of...

An investigation of vancomycin minimum inhibitory concentration creep among methicillin-resistant Staphylococcus aureus strains isolated from pediatric patients and healthy children in Northern Taiwan

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Chia-Ning Chang

2017-06-01

Conclusion: Vancomycin MIC creeps existed in both clinical MRSA isolates and colonized MRSA strains. Great diversity of PFGE typing was in both strains collected. There was no association between the clinical and colonized MRSA isolates with vancomycin MIC creep.

Antibiotic resistance in bacteria Staphylococcus spp. isolated from samples of raw sheep's milk

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Milan Vasiľ

2016-12-01

Full Text Available From samples of raw sheep's milk were determined results of bacteriological examination from two herds in region of Eastern Slovakia in three years lasting study. The occurrence of Staphylococcus spp. 41.6% (124 was determined from 298 samples. The seven species of staphylococci were on a regular basis isolated: S. epidermidis (34, S. chromogenes (26, S. aureus (16. Alternately have been recorded S. warneri (16, S. schleiferi (15, S. haemolyticus (9 and S. xylosus (8. All isolated pathogens were tested by in vitro test on Mueller-Hinton agar by disc methods on resistance to 10 types of antibiotics.  Highest value of resistance was determined to Penicilin 21.0%, Neomycin 10.5% and Novobiocin 9.7%. Lower resistance was in to Oxacilin 7.2% and Amoxicilin 6.5%. Minimal resistance was founded to Cefoxitin 0.8%, Linkomycin 2.4%, Erytromycin, and Streptomycin 3.2%. Was founded total resistance (21.0% to all antibiotics in S. epidermidis (34 during the three years, S. chromogenes (26 showed resistance to 8 types of antibiotics (12.9%, S. aureus (16 to 6 antibiotics (10.5% and S. warneri (16 to 4 antibiotics (5.6%. It was confirmed that sheep's milk remains a major source of staphylococci. Bacteria in comparison with isolates from cows' raw milk, showed lower values of resistance, but were resistant to more than two antibiotics. Recorded occurrence of resistance in staphylococci may be connected with a minimum use of antibiotics in the treatment of mastitis and other diseases in sheep herds. Reported resistance to the tested antibiotics became the basis for the recommendation to use preparations to treat mastitis in sheep principally by the detection of resistance to antibiotics contained.

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

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Jeannot, Katy; Diancourt, Laure; Vaux, Sophie; Thouverez, Michelle; Ribeiro, Amandina; Coignard, Bruno; Courvalin, Patrice; Brisse, Sylvain

2014-01-01

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla OXA-23 was the most frequently detected (82%), followed by bla OXA-24 (11%) and bla OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones. PMID:25517732

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms.

Science.gov (United States)

Riboli, Danilo Flávio Moraes; Lyra, João César; Silva, Eliane Pessoa; Valadão, Luisa Leite; Bentlin, Maria Regina; Corrente, José Eduardo; Rugolo, Ligia Maria Suppo de Souza; da Cunha, Maria de Lourdes Ribeiro de Souza

2014-05-22

Catheter-related bloodstream infections (CR-BSIs) have become the most common cause of healthcare-associated bloodstream infections in neonatal intensive care units (ICUs). Microbiological evidence implicating catheters as the source of bloodstream infection is necessary to establish the diagnosis of CR-BSIs. Semi-quantitative culture is used to determine the presence of microorganisms on the external catheter surface, whereas quantitative culture also isolates microorganisms present inside the catheter. The main objective of this study was to determine the sensitivity and specificity of these two techniques for the diagnosis of CR-BSIs in newborns from a neonatal ICU. In addition, PFGE was used for similarity analysis of the microorganisms isolated from catheters and blood cultures. Semi-quantitative and quantitative methods were used for the culture of catheter tips obtained from newborns. Strains isolated from catheter tips and blood cultures which exhibited the same antimicrobial susceptibility profile were included in the study as positive cases of CR-BSI. PFGE of the microorganisms isolated from catheters and blood cultures was performed for similarity analysis and detection of clones in the ICU. A total of 584 catheter tips from 399 patients seen between November 2005 and June 2012 were analyzed. Twenty-nine cases of CR-BSI were confirmed. Coagulase-negative staphylococci (CoNS) were the most frequently isolated microorganisms, including S. epidermidis as the most prevalent species (65.5%), followed by S. haemolyticus (10.3%), yeasts (10.3%), K. pneumoniae (6.9%), S. aureus (3.4%), and E. coli (3.4%). The sensitivity of the semi-quantitative and quantitative techniques was 72.7% and 59.3%, respectively, and specificity was 95.7% and 94.4%. The diagnosis of CR-BSIs based on PFGE analysis of similarity between strains isolated from catheter tips and blood cultures showed 82.6% sensitivity and 100% specificity. The semi-quantitative culture method showed higher

Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda.

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Asiimwe, Benon B; Baldan, Rossella; Trovato, Alberto; Cirillo, Daniela M

2017-06-13

Staphylococcus aureus strains are now regarded as zoonotic agents. In pastoral settings where human-animal interaction is intimate, multi-drug resistant microorganisms have become an emerging zoonotic issue of public health concern. The study of S. aureus prevalence, antimicrobial resistance and clonal lineages in humans, animals and food in African settings has great relevance, taking into consideration the high diversity of ethnicities, cultures and food habits that determine the lifestyle of the people. Little is known about milk carriage of methicillin resistant S. aureus strains (MRSA) and their virulence factors in Uganda. Here, we present the prevalence of MRSA in bulk can milk and raw milk products in pastoral communities of south-west Uganda. We also present PFGE profiles, spa-types, as well as frequency of enterotoxins genes. S. aureus was identified by the coagulase test, susceptibility testing by the Kirby-Bauer disc diffusion and E-test methods and MRSA by detection of the mecA gene and SCCmec types. The presence of Panton - Valentine Leucocidin (PVL) genes and staphylococcal enterotoxins was determined by PCR, while genotyping was by PFGE and spa typing. S. aureus were isolated from 30/148 (20.3%) milk and 11/91(12%) sour milk samples. mecA gene carriage, hence MRSA, was detected in 23/41 (56.1%) of the isolates, with 21 of the 23 (91.3%) being SCCmec type V; while up to 30/41 (73.2%) of the isolates were resistant to tetracycline. Only five isolates carried the PVL virulence gene, while PFGE typing revealed ten clusters (ranging from two seven isolates each) that comprised 83% of the sample, and only eight isolates with unique pulsotypes. The largest PFGE profile (E) consisted of seven isolates while t7753, t1398, and t2112 were the most common spa-types. Thirty seven of the 41 strains (90.2%) showed at least one of the eight enterotoxin genes tested, with sem 29 (70.7%), sei 25 (61%) and seg 21 (51.2%) being the most frequently observed genes. This

Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period.

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Couto, Natacha; Monchique, Cláudia; Belas, Adriana; Marques, Cátia; Gama, Luís T; Pomba, Constança

2016-06-01

The objective of this study was to investigate the evolution of resistance to antimicrobials, corresponding mechanisms and molecular characteristics of Staphylococcus spp., between 1999 and 2014. Susceptibility to 38 antimicrobials was determined for 632 clinical staphylococcal isolates obtained from companion animals (dogs, cats, horses and other animals). Twenty antimicrobial resistance genes, including mecA and mecC, were screened by PCR. Methicillin-resistant staphylococci were characterized by spa (Staphylococcus aureus), SCCmec, MLST and PFGE typing. Statistical analyses were performed using SAS v9.3 and differences were considered relevant if P ≤ 0.05. The mecA gene was identified in 74 staphylococcal isolates (11.6%): 11 MRSA (40.7%), 40 methicillin-resistant Staphylococcus pseudintermedius (MRSP; 8.7%) and 23 methicillin-resistant CoNS (26.7%). Resistance to the majority of antimicrobials and the number of mecA-positive isolates increased significantly over time. Eighteen spa types were identified, including two new ones. MRSA isolates were divided into three PFGE clusters that included ST22-IV, ST105-II, ST398-V and ST5-VI. Most methicillin-resistant Staphylococcus epidermidis isolates were of clonal complex (CC) 5, including a new ST, and clustered in eight PFGE clusters. MRSP were grouped into five PFGE clusters and included ST45-NT, ST71-II-III, ST195-III, ST196-V, ST339-NT, ST342-IV and the new ST400-III. Methicillin-resistant Staphylococcus haemolyticus clustered in two PFGE clusters. The significant increase in antimicrobial-resistant and mecA-positive isolates in recent years is worrying. Furthermore, several isolates are MDR, which complicates antimicrobial treatment and increases the risk of transfer to humans or human isolates. Several clonal lineages of MRSA and methicillin-resistant S. epidermidis circulating in human hospitals and the community were found, suggesting that companion animals can become infected with and contribute to the

Control of Listeria species food safety at a poultry food production facility.

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Fox, Edward M; Wall, Patrick G; Fanning, Séamus

2015-10-01

Surveillance and control of food-borne human pathogens, such as Listeria monocytogenes, is a critical aspect of modern food safety programs at food production facilities. This study evaluated contamination patterns of Listeria species at a poultry food production facility, and evaluated the efficacy of procedures to control the contamination and transfer of the bacteria throughout the plant. The presence of Listeria species was studied along the production chain, including raw ingredients, food-contact, non-food-contact surfaces, and finished product. All isolates were sub-typed by pulsed-field gel electrophoresis (PFGE) to identify possible entry points for Listeria species into the production chain, as well as identifying possible transfer routes through the facility. The efficacy of selected in-house sanitizers against a sub-set of the isolates was evaluated. Of the 77 different PFGE-types identified, 10 were found among two or more of the five categories/areas (ingredients, food preparation, cooking and packing, bulk packing, and product), indicating potential transfer routes at the facility. One of the six sanitizers used was identified as unsuitable for control of Listeria species. Combining PFGE data, together with information on isolate location and timeframe, facilitated identification of a persistent Listeria species contamination that had colonized the facility, along with others that were transient. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inapparent Streptococcus agalactiae infection in adult/commercial tilapia.

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Sun, Jiufeng; Fang, Wei; Ke, Bixia; He, Dongmei; Liang, Yuheng; Ning, Dan; Tan, Hailing; Peng, Hualin; Wang, Yunxin; Ma, Yazhou; Ke, Changwen; Deng, Xiaoling

2016-05-24

We report on inapparent infections in adult/commercial tilapia in major tilapia fish farms in Guangdong. A total of 146 suspected isolates were confirmed to be S. agalactiae using an API 20 Strep system and specific PCR amplification. All isolates were identified as serotype Ia using multiplex serotyping PCR. An MLST assay showed single alleles of adhP (10), atr (2), glcK (2), glnA (1), pheS (1), sdhA (3) and tkt (2), and this profile was designated 'unique ST 7'. The analysis of virulence genes resulted in 10 clusters, of which dltr-bca-sodA-spb1-cfb-bac (62, 42.47%) was the predominant virulence gene profile. The PFGE analysis of S. agalactiae yielded 6 distinct PFGE types (A, B, C, D, F and G), of which Pattern C (103) was the predominant type, accounting for approximately 70.55% (103/146) of the total S. agalactiae strains. Therefore, unlike what has been found in juvenile tilapia, in which PFGE pattern D/F is the major prevalent pattern, we found that pattern C was the major prevalent pattern in inapparent infected adult/commercial tilapia in Guangdong, China. In conclusion, we close a gap in the current understanding of S. agalactiae epidemiology and propose that researchers should be alert for inapparent S. agalactiae infections in adult/commercial tilapia to prevent a potential threat to food safety.

Outbreak of carbapenem-resistant Klebsiella pneumoniae: two-year epidemiologic follow-up in a tertiary hospital

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Graziella Hanna Pereira

2013-02-01

Full Text Available This study describes a carbapenem-resistant Klebsiella pneumoniae (CRKP outbreak that occurred from October 2008-December 2010. Polymerase chain reaction assays were performed to detect the blaKPC gene and molecular typing was performed using pulsed-field gel electrophoresis (PFGE. There were 33 CRKP infections; PFGE revealed five genotypes: genotype A in five (15%, B in 18 (55%, C in eight (24% and two unique profiles. Genotype B was disseminated in all hospital units and belonged to the same clone identified in 11 different hospitals in the state of São Paulo. Sixteen (48% patients died. Seven isolates (21% were resistant to polymyxin B and 45% were resistant to tigecycline and amikacin.

Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

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Blomfeldt, A; Larssen, K W; Moghen, A; Haugum, K; Steen, T W; Jørgensen, S B; Aamot, H V

2017-03-01

The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Staphylococcus pseudintermedius colonization patterns and strain diversity in healthy dogs

DEFF Research Database (Denmark)

Paul, Narayan Chandra; Bärgman, Sofia Cathrine; Moodley, Arshnee

2012-01-01

This is the first large-scale study of Staphylococcus pseudintermedius colonization and diversity in healthy dogs where samples were collected over a long time and strains were identified by PCR according to the current taxonomy of the S. intermedius group and typed by a highly discriminatory...... method such as pulsed field gel electrophoresis (PFGE). A cross-sectional study of nasal, oral, perineal and inguinal carriage in 119 healthy dogs was followed by a longitudinal study where oral and perineal carriage was examined in 16 dogs for 10 times over a period of 1 year. Altogether we collected...... 762 samples and 285 S. pseudintermedius isolates, 182 of which were typed by PFGE to determine spatial and temporal strain diversity within individual carriers. In the cross-sectional study, S. pseudintermedius was isolated from at least one body site in 82 (69%) of the 119 dogs. The most frequent...

Antimicrobial effect of proteflazid extract on microflora of peri-implant areas in infectious and inflammatory complications after dental implantation

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M. M. Ananieva

2017-12-01

Full Text Available The purpose of this study was to investigate the proteflazid extraction antimicrobial action onto causative agents of infectious and inflammatory diseases occurring during dental implant placement. Materials and Methods. We studied clinical strains of Streptococcus sanguinis, Staphylococcus warneri and Kocuria kristinae, isolated from oral mucosa of patients who were diagnosed with peri-implant mucositis and peri-implantitis. The cultivation of strains was carried out by standard method. Final identification was carried out with an automatic bacteriological analyzer Viteck 2 compact bioMérieux (France according to manufacturer's instructions. The sensitivity of derived strains to proteflazid and to 96 % ethyl alcohol assessment was done by double serial dilutions according to the standard procedure. Results. The clinical strains of Streptococcus sanguinis, Staphylococcus warneri and Kocuria kristinae were found to be sensitive to effects of proteflazid extract. K. kristinae was proven to be the least susceptible strain. But the clinical strain of Streptococcus sanguinis was found to be the most sensitive to proteflazid extract. 96 % ethyl alcohol, which is a component of the extract, showed a bactericidal effect on the clinical strains in the study, but its minimal bactericidal concentration was significantly lower than minimal bactericidal concentration of the proteflazid extract. Conclusions. The proteflazid extract can produce antimicrobial effect on the clinical strains of Streptococcus sanguinis, Staphylococcus warneri and Kocuria kristinae, which colonize the oral mucosa of patients with infectious and inflammatory complications following dental implant placement. Antimicrobial action of the proteflazid extract is much higher than action of 96% ethyl alcohol that confirms the direct antimicrobial action of flavonoids, which are components of the studied extract.

An investigation of vancomycin minimum inhibitory concentration creep among methicillin-resistant Staphylococcus aureus strains isolated from pediatric patients and healthy children in Northern Taiwan.

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Chang, Chia-Ning; Lo, Wen-Tsung; Chan, Ming-Chin; Yu, Ching-Mei; Wang, Chih-Chien

2017-06-01

The phenomenon of vancomycin minimum inhibitory concentration (MIC) creep is an increasingly serious problem in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. In this study, we investigated the vancomycin and daptomycin MIC values of MRSA strains isolated from pediatric patients and MRSA colonized healthy children. Then, we assessed whether there was evidence of clonal dissemination for strains with an MIC to vancomycin of ≥ 1.5 μg/mL. We collected clinical MRSA isolates from pediatric patients and from healthy children colonized with MRSA during 2008-2012 at a tertiary medical center in northern Taiwan and obtained vancomycin and daptomycin MIC values using the Etest method. Pulse-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome (SCCmec) typing were used to assess clonal dissemination for strains with an MIC to vancomycin of ≥ 1.5 μg/mL. A total 195 MRSA strains were included in this study; 87 were isolated patients with a clinical MRSA infection, and the other 108 strains from nasally colonized healthy children. Vancomycin MIC≥1.5 μg/mL was seen in more clinical isolates (60/87, 69%) than colonized isolates (32/108, 29.6%), p < 0.001. The PFGE typing of both strains revealed multiple pulsotypes. Vancomycin MIC creeps existed in both clinical MRSA isolates and colonized MRSA strains. Great diversity of PFGE typing was in both strains collected. There was no association between the clinical and colonized MRSA isolates with vancomycin MIC creep. Copyright © 2016. Published by Elsevier B.V.

Review and International Recommendation of Methods for Typing Neisseria gonorrhoeae Isolates and Their Implications for Improved Knowledge of Gonococcal Epidemiology, Treatment, and Biology

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Unemo, Magnus; Dillon, Jo-Anne R.

2011-01-01

Summary: Gonorrhea, which may become untreatable due to multiple resistance to available antibiotics, remains a public health problem worldwide. Precise methods for typing Neisseria gonorrhoeae, together with epidemiological information, are crucial for an enhanced understanding regarding issues involving epidemiology, test of cure and contact tracing, identifying core groups and risk behaviors, and recommending effective antimicrobial treatment, control, and preventive measures. This review evaluates methods for typing N. gonorrhoeae isolates and recommends various methods for different situations. Phenotypic typing methods, as well as some now-outdated DNA-based methods, have limited usefulness in differentiating between strains of N. gonorrhoeae. Genotypic methods based on DNA sequencing are preferred, and the selection of the appropriate genotypic method should be guided by its performance characteristics and whether short-term epidemiology (microepidemiology) or long-term and/or global epidemiology (macroepidemiology) matters are being investigated. Currently, for microepidemiological questions, the best methods for fast, objective, portable, highly discriminatory, reproducible, typeable, and high-throughput characterization are N. gonorrhoeae multiantigen sequence typing (NG-MAST) or full- or extended-length porB gene sequencing. However, pulsed-field gel electrophoresis (PFGE) and Opa typing can be valuable in specific situations, i.e., extreme microepidemiology, despite their limitations. For macroepidemiological studies and phylogenetic studies, DNA sequencing of chromosomal housekeeping genes, such as multilocus sequence typing (MLST), provides a more nuanced understanding. PMID:21734242

Prevalence of quinolone resistance determinant qnrA6 among broad- and extended-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates with sul1-type class 1 integron association in a Tunisian Hospital.

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Mahrouki, Sihem; Perilli, Mariagrazia; Bourouis, Amel; Chihi, Hela; Ferjani, Mustapha; Ben Moussa, Mohamed; Amicosante, Gianfranco; Belhadj, Omrane

2013-08-01

The aim of this study was to investigate the prevalence and the emergence of plasmid-mediated quinolone resistance among broad-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates recovered in the Military Hospital in Tunisia. Of 200 strains examined, 50 exhibited resistance to quinolones. Quinolone resistance determinants (qnr and aac(6')-Ib-cr) were characterized by multiplex PCR and sequencing. Chromosomal quinolone resistance mutations in the quinolone resistance-determining region (QRDR) and class 1 integron characterization were analysed by PCR and sequencing. The clonal relationship between the isolates was studied by pulsed-field gel electrophoresis (PFGE). Fourteen isolates harboured qnrA6 and among them 8 (57%) were extended-spectrum beta-lactamase (ESBL) producers, whilst 12 (85%) isolates harboured blaDHA-1. Mutations in the QRDR were detected in gyrA (Ser83Ile, Glu87Lys), gyrB (Ser464Phe), and parC (Ser80Ile). qnrA6 and blaDHA-1 genes were found embedded in complex sul1-type class 1 integrons. A gene cassette carrying aac(6')-Ib-cr was found located in the class 1 integron upstream of the qacEΔ1 gene. According to the PFGE analysis, the isolates were clonally unrelated. This is the first description in North Africa of class 1 integrons carrying blaDHA-1, qnrA6 gene, and aac(6')-Ib-cr determinants in clinical strains of Proteus mirabilis and Morganella morganii.

Human Staphylococcus aureus lineages among Zoological Park residents in Greece

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E. Drougka

2015-10-01

Full Text Available Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL and tst (toxic shock syndrome toxin-1 were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST, spa type and Pulsed-Field Gel Electrophoresis (PFGE. Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred.

Molecular and clinical characteristics of clonal complex 59 methicillin-resistant Staphylococcus aureus infections in Mainland China.

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Juan Li

Full Text Available Detailed molecular analyses of Clonal Complex 59 (CC59 methicillin-resistant Staphylococcus aureus (MRSA isolates from children in seven major cities across Mainland China were examined. A total of 110 CC59 isolates from invasive and non-invasive diseases were analyzed by multilocus sequence typing (MLST, Staphylococcus cassette chromosome mec (SCCmec typing, staphylococcal protein A (spa typing and pulsed-field gel electrophoresis (PFGE. Antibiotics susceptibilities, carriage of plasmids and 42 virulence genes and the expression of virulence factors were examined. ST59 (101/110, 91.8% was the predominant sequence type (ST, while single locus variants (SLVs belonging to ST338 (8/110, 7.3% and ST375 (1/110, 0.9% were obtained. Three SCCmec types were found, namely type III (2.7%, type IV (74.5% and type V (22.7%. Seven spa types including t437, which accounted for 87.3%, were determined. Thirteen PFGE types were obtained. PFGE types A and B were the major types totally accounting for 81.8%. The dominant clone was ST59-t437-IVa (65.5%, followed by ST59-t437-V (14.5%. The positive rate of luks-PV and lukF-PV PVL encoding (pvl gene was 55.5%. Plasmids were detected in 83.6% (92/110 of the strains. The plasmid size ranging from 23.4 kb to 50 kb was most prevalent which accounted for 83.7% (77/92. A significantly lower expression of hla was found in ST59-t437-IVa compared with ST59-t437-V. Among the 110 cases, 61.8% of the patients were less than 1 year old. A total of 90 cases (81.8% were community-associated (CA infections whereas 20 cases (18.2% were hospital-associated (HA infections. Out of the 110 patients, 36.4% (40/110 were diagnosed with invasive infectious diseases in which ST59-t437-IVa accounted for 67.5% (27/40. In brief, ST59-t437-IVa was proved as the dominant clone in CC59 MRSA strains. The carriage rate of pvl gene was high. CC59 MRSA could result in CA and HA infections. The majortiy of MRSA infection children were in young age.

Molecular typing of Clostridium perfringens isolated from swine in slaughterhouses from São Paulo State, Brazil Tipagem molecular de Clostridium perfringens isolados de suínos em abatedouros do estado de São Paulo, Brasil

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Thais Sebastiana Porfida Ferreira

2012-08-01

Full Text Available Clostridium perfringens is an anaerobic Gram-positive bacterium known as common pathogen for humans, for domestic and wildlife animals. Although infections caused by C. perfringens type C and A in swine are well studied, just a few reports describe the genetic relationship among strains in the epidemiological chain of swine clostridioses, as well as the presence of the microorganism in the slaughterhouses. The aim of the present study was to isolate C. perfringens from feces and carcasses from swine slaughterhouses, characterize the strains in relation to the presence of enterotoxin, alpha, beta, epsilon, iota and beta-2 toxins genes, using polymerase chain reaction (PCR and comparing strains by means of Pulsed field gel electrophoresis (PFGE. Clostridium perfringens isolation frequencies in carcasses and finishing pig intestines were of 58.8% in both types of samples. According to the polymerase chain reaction assay, only alfa toxin was detected, being all isolates also negative to enterotoxin and beta2 toxin. Through PFGE technique, the strains were characterized in 35 pulsotypes. In only one pulsotype, the isolate from carcass sample was grouped with fecal isolate of the same animal, suggesting that the risk of cross-contamination was low. Despite the high prevalence of C. perfringens in swine carcasses from the slaughterhouses assessed, the risk of food poisoning to Brazilian pork consumers is low, since all strains were negative to cpe-gene, codifying enterotoxin.Clostridium perfringens é uma bactéria Gram positiva anaeróbica, conhecida por infectar os seres humanos, animais domésticos e de vida selvagem. Apesar de as infecções causadas por C. perfringens tipo C e A em suínos serem bastante estudadas, poucos relatos descrevem a relação genética entre as linhagens envolvidas na cadeia epidemiológica da clostridiose suína, bem como a presença do microorganismo em abatedouros. O objetivo do presente estudo foi isolar C

PCR melting profile (PCR MP - a new tool for differentiation of Candida albicans strains

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Nowak Magdalena

2009-11-01

Full Text Available Abstract Background We have previously reported the use of PCR Melting Profile (PCR MP technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains. Methods In total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE and RAPD techniques. Results The genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD. Conclusion Data presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

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Luiz, L M P; Chuat, V; Madec, M N; Araújo, E A; de Carvalho, A F; Valence, F

2016-10-01

The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.

Isolation and characterization of new Leptospira genotypes from patients in Mayotte (Indian Ocean.

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Pascale Bourhy

Full Text Available BACKGROUND: Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean. To date, Leptospira isolates have never been isolated in this endemic region. METHODS AND FINDINGS: Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA, and pulsed field gel electrophoresis (PFGE. Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars. CONCLUSIONS: Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.

Stenotrophomonas maltophilia isolated from patients exposed to invasive devices in a university hospital in Argentina: molecular typing, susceptibility and detection of potential virulence factors.

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Alcaraz, Eliana; Garcia, Carlos; Papalia, Mariana; Vay, Carlos; Friedman, Laura; Passerini de Rossi, Beatriz

2018-05-25

The aim of this work was to investigate the presence of selected potential virulence factors, susceptibility and clonal relatedness among 63 Stenotrophomonas maltophilia isolates recovered from patients exposed to invasive devices in a university hospital in Argentina between January 2004 and August 2012. Genetic relatedness was assessed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and pulsed-field gel electrophoresis (PFGE). Isolates were characterized by antimicrobial resistance, the presence and/or expression of potential virulence determinants, and virulence in the Galleria mellonella model.Results/Key findings. ERIC-PCR generated 52 fingerprints, and PFGE added another pattern. Resistance to trimethoprim-sulfamethoxazole (6.35 %), levofloxacin (9.52 %) and ciprofloxacin (23.80 %) was detected. All isolates were susceptible to minocycline. All isolates were lipase, protease and siderophore producers, while all but Sm61 formed biofilms. However, 11/63 isolates did not amplify the major extracellular protease-coding gene (stmPr1). Sm61 is an stmPr1-negative isolate, and showed (as did Sm13 and the reference strain K279a) strong proteolysis and siderophore production, and high resistance to hydrogen peroxide. The three isolates were virulent in the G. mellonella model, while Sm10, a low-resistance hydrogen peroxide stmPr1-negative isolate, and weak proteolysis and siderophore producer, was not virulent. This is the first epidemiological study of the clonal relatedness of S. maltophilia clinical isolates in Argentina. Great genomic diversity was observed, and only two small clusters of related S. maltophilia types were found. Minocycline and trimethoprim-sulfamethoxazole were the most active agents. S. maltophilia virulence in the G. mellonella model is multifactorial, and further studies are needed to elucidate the role of each potential virulence factor.

Molecular Characterization of Mycobacterium massiliense and Mycobacterium bolletii in Isolates Collected from Outbreaks of Infections after Laparoscopic Surgeries and Cosmetic Procedures▿

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Viana-Niero, Cristina; Lima, Karla Valéria Batista; Lopes, Maria Luiza; da Silva Rabello, Michelle Christiane; Marsola, Lourival Rodrigues; Brilhante, Vânia Cristina Ribeiro; Durham, Alan Mitchel; Leão, Sylvia Cardoso

2008-01-01

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belém, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak, all of which were originally attributed to a single strain of M. abscessus. PMID:18174307

Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm

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N. Indrawattana

2013-01-01

Full Text Available Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd, PFGE types, accessory gene regulator (agr groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates were methicillin resistant (MRSA and 8–10 (36 isolates were methicillin sensitive (MSSA. One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq, and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70% but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.

Mida mõtlevad tippjuhid? / interv. John A. Byrne

Index Scriptorium Estoniae

2008-01-01

USA majandusliku olukorra ja tulevikuprognooside üle arutlevad FedExi juht Fred Smith, Chrysleri tegevjuht Robert Nardell, endine General Electric'u aseesimees Dennis Dammerman, Caritas Christi Health Care tegevjuht Ralph de la Torre, Abbott Labs tegevjuht Miles White, FPL Group tegevjuht Lewis Hay, BorgWarneri tegevjuht Timothy Manganello ning Schering-Plough tegevjuht Fred Hassan

Identification of Burkholderia cepacia in patients with cystic fibrosis by pulsed-field gel electrophoresis

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Mohammad Mehdi Soltan Dallal

2014-05-01

Conclusion: Application of PFGE and identification of pulse-type is a potential tool to enhance the investigation of apparent nosocomial outbreaks of B.cepacia. Similar type of pulse patterns was observed in this study means that all of infection has been from one source; therefore the hypothesis of transferring person to person will be rejected. Base on these results environmental sources sampling should be considered in future investigation.

Changing epidemiology of methicillin-resistant Staphylococcus aureus in Iceland from 2000 to 2008: a challenge to current guidelines

DEFF Research Database (Denmark)

Holzknecht, B.J.; Hardardottir, H.; Haraldsson, Gustav Helgi

2010-01-01

and microbiological data of all MRSA patients from the years 2000 to 2008 were collected prospectively. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), sequencing of the repeat region of the Staphylococcus protein A gene (spa typing), staphylococcal cassette chromosome mec (SCCmec) typing...... setting. However, MRSA in Iceland is now shifting into the community, challenging the current Icelandic guidelines, which are tailored to the health care system....

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

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Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Evaluación de dos técnicas de subtipificación molecular para el estudio de Pasteurella multocida Evaluation of two techniques of molecular subtyping to study Pasteurella multocida

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G. A. Leotta

2006-12-01

Full Text Available Se determinó la tipibilidad, la reproducibilidad y el poder discriminatorio de ERIC-PCR y ApaI-PFGE para establecer la relación genética de cepas de Pasteurella multocida. Se estudiaron 49 cepas de diferente origen, subespecie, biotipo, grupo capsular, serotipo somático y perfil de resistencia antimicrobiana. Por ERIC-PCR se establecieron 31 patrones, los que presentaron entre 10 y 14 bandas en un rango comprendido entre 0,2 y 1,2 kb. Por ApaI-PFGE se detectaron 37 patrones de restricción, los cuales presentaron entre 7 y 15 bandas bien definidas de 34 a 450 kb. La tipibilidad de ERIC-PCR fue del 100% (T=1 y la de ApaI-PFGE del 94% (T=0,94. La reproducibilidad de ambas técnicas fue del 100% (R=1; sin embargo, el poder discriminatorio de ERIC-PCR fue 93% (D=0,93 y el de ApaI-PFGE 98% (D=0,98. Mediante ambas técnicas fue posible agrupar las cepas con relación epidemiológica y diferenciar claramente las cepas no relacionadas. Se demostró el valor de ERIC-PCR y ApaI-PFGE para complementar estudios epidemiológicos, principalmente si las cepas en estudio son analizadas por ambas técnicas.Typeability, reproducibility, and discriminatory power of ERIC-PCR and ApaI-PFGE to establish the genetic relation of P. multocida strains were determined. Forty-nine strains of different source, biotype, capsular group, somatic serotype, and resistance to antimicrobials were studied. By ERIC-PCR, 31 patterns were defined with 10 to 14 bands in a rank of 0.2 and 1.2 kb. By ApaI-PFGE, 37 restriction patterns were established with 7 to 15 bands of 34 to 450 kb. Typeability was 100% (T=1 for ERIC-PCR, and 94% (T=0.94 for ApaI-PFGE. Reproducibility of both techniques was 100% (R=1. Discriminatory power was 93% (D=0.93 for ERIC-PCR, and 98% (D=0.98 for ApaI-PFGE. By using both techniques, epidemiologically related strains were grouped, and unrelated strains were clearly differentiated. The value of ERIC-PCR and ApaI-PFGE as complements to epidemiologic studies

Occurrence and genetic diversity of Arcobacter butzleri in an artisanal dairy plant in Italy.

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Giacometti, Federica; Lucchi, Alex; Manfreda, Gerardo; Florio, Daniela; Zanoni, Renato Giulio; Serraino, Andrea

2013-11-01

The present study aimed to investigate the presence, distribution, and persistence of Arcobacter spp. in an artisanal dairy plant and to test the isolates to determine their different genotypes in the processing plant and in foods. Samples were collected in an artisanal cheese factory on four occasions between October and December 2012. Food samples (raw milk, ricotta cheese, mozzarella cheese, and conditioning liquid), water samples, and environmental samples were analyzed by the culture method; isolates were identified by multiplex PCR and genotyped by pulsed-field gel electrophoresis (PFGE) analysis. Arcobacter butzleri was isolated from 29 out of 59 samples (46.6%), 22 of which were from environmental samples and 7 of which were from food samples. Cluster analysis divided the strains into 47 PFGE patterns: 14 PFGE clusters and 33 unique types. Our findings indicate that the plant harbored numerous A. butzleri pulsotypes and that the manual cleaning and sanitation in the studied dairy plant do not effectively remove Arcobacter. The recurrent isolation of A. butzleri suggests that the environmental conditions in the dairy plant constitute a good ecological niche for the colonization of this microorganism. In some cases, the presence of indistinguishable strains isolated from the same facilities on different sampling days showed that these strains were persistent in the processing environment.

Occurrence and characterization of Shiga toxin-producing Escherichia coli in raw meat, raw milk, and street vended juices in Bangladesh.

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Islam, Mohammad A; Mondol, Abdus S; Azmi, Ishrat J; de Boer, Enne; Beumer, Rijkelt R; Zwietering, Marcel H; Heuvelink, Annet E; Talukder, Kaisar A

2010-11-01

The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of 213 food samples, including 90 raw meat samples collected from retail butcher shops, 20 raw milk samples from domestic cattle, and 103 fresh juice samples from street vendors in Dhaka city. We found that more than 68% (n = 62) of the raw meat samples were positive for the stx gene(s); 34% (n = 21) of buffalo meats and 66% (n = 41) of beef. Approximately 10% (n = 2) of the raw milk and 8% (n = 8) of the fresh juice samples were positive for stx. We isolated STEC O157 from seven meat samples (7.8%), of which two were from buffalo meats and five from beef; and no other STEC serotypes could be isolated. We could not isolate STEC from any of the stx-positive raw milk and juice samples. The STEC O157 isolates from raw meats were positive for the stx(2), eae, katP, etpD, and enterohemorrhagic E. coli hly virulence genes, and they belonged to three different phage types: 8 (14.3%), 31 (42.8%), and 32 (42.8%). Pulsed-field gel electrophoresis (PFGE) typing revealed six distinct patterns among seven isolates of STEC O157, suggesting a heterogeneous clonal diversity. Of the six PFGE patterns, one was identical and the other two were ≥90% related to PFGE patterns of STEC O157 strains previously isolated from animal feces, indicating that raw meats are readily contaminated with fecal materials. This study represents the first survey of STEC in the food chain in Bangladesh.

Escherichia coli Contamination of Lettuce Grown in Soils Amended with Animal Slurry

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Jensen, Annette Nygaard; Storm, Christina; Forslund, Anita

2013-01-01

A pilot study was conducted to assess the transfer of Escherichia coli from animal slurry fertilizer to lettuce, with E. coli serving as an indicator of fecal contamination and as an indicator for potential bacterial enteric pathogens. Animal slurry was applied as fertilizer to three Danish agric...... types between slurry, soil, and lettuce. The frequent finding of fecal-contaminated lettuce indicates that human pathogens such as Salmonella and Campylobacter can be present and represent food safety hazards.......A pilot study was conducted to assess the transfer of Escherichia coli from animal slurry fertilizer to lettuce, with E. coli serving as an indicator of fecal contamination and as an indicator for potential bacterial enteric pathogens. Animal slurry was applied as fertilizer to three Danish....... coli. A relatively higher frequency of E. coli in lettuce compared with the soil samples at harvest suggests environmental sources of fecal contamination, e.g., wildlife. The higher frequency was supported by the finding of 21 different PFGE types among the E. coli isolates, with only a few common PFGE...

Characterization of resistance mechanisms and genetic relatedness of carbapenem-resistant Acinetobacter baumannii isolated from blood, Italy.

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Migliavacca, Roberta; Espinal, Paula; Principe, Luigi; Drago, Monica; Fugazza, Giulia; Roca, Ignasi; Nucleo, Elisabetta; Bracco, Silvia; Vila, Jordi; Pagani, Laura; Luzzaro, Francesco

2013-02-01

The aim of this study was to characterize the resistance mechanisms and genetic relatedness of 21 carbapenem-resistant Acinetobacter baumannii blood isolates collected in Italy during a 1-year multicenter prospective surveillance study. Genes coding for carbapenemase production were identified by polymerase chain reaction (PCR) and sequencing. Pulsed-field gel electrophoresis (PFGE), multiplex PCRs for group identification, and multilocus sequence typing (MLST) were used to determine genetic relationships. Carbapenem resistance was consistently related to the production of oxacillinases, mostly the plasmid-mediated OXA-58 enzyme. Strains producing the OXA-23 enzyme (chromosomally mediated) were also detected. Seven PFGE clones were identified, some of which being related to international (ICL- I and ICL-II) or national clonal lineages. Multiplex PCRs identified 4 different groups (group 2 being dominant), further distinguishable in 6 sequence types by MLST. The heterogeneity of profiles highlights the diffusion of international and national clonal lineages in Italy. Continuous surveillance is needed for monitoring the spread of these worrisome strains equipped with multiple drug resistance mechanisms. Copyright © 2013 Elsevier Inc. All rights reserved.

Potensi Ekstrak Tumbuhan Sebagai Pengawet Produk Pangan

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I Nengah Kencana Putra

2015-04-01

Full Text Available Nowadays, the use of natural antimicrobials such as plants extracts to preserve food get much attention of the researchers. This is related to the increasing of public concern over synthetic chemical food preservatives. Various types of plants had been declared can produce extracts that effectively inhibited the growth of food-contaminating microbes, such as methanol and ethanol extracts of bark Saccoglottis gabonensis, effectively inhibited the growth of Leuconostoc mesenteroides and Lactobacillus plantarum; extracts of onion and red pepper inhibited Candida crucei and Candida utilis; ethanol and water extracts of Eugenia Jambos had antimicrobial properties against bacteria such as Staphylococcus aureus, Yersenia enterocolitica, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus warneri; Picung seed extract (Pagium edule inhibited the growth of Staphylococcus aureus; and ethanol extract of Salvia pratensis leaves inhibited Escherichia coli, Bacillus cereus and Saccharomyces cerevisiae. Extraction of antimicrobial compounds from plant material could be done by using different types of solvents. Among the various types of solvents, ethanol and methanol solvents are most commonly used.Keyword: antimicrobial, plant extract, food preservative

Identification and characterization of a bioactive lantibiotic produced by Staphylococcus warneri

DEFF Research Database (Denmark)

Petersen, Jørgen; Boysen, Anders; Fogh, Lotte

2009-01-01

Lantibiotics are a group of potent antibacterial agents that contain unusual amino acids, such as the thioether amino acids lanthionine and methyllanthionine, and the didehydroamino acids didehydroalanine and didehydro-aminobutyric acid. Here, we report on an antibacterial lantibiotic peptide nam...

International spread of multidrug-resistant Salmonella Schwarzengrund in food products

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Aarestrup, Frank Møller; Hendriksen, Rene S.; Lockett, Jana

2007-01-01

We compared 581 Salmonella enterica serotype Schwarzengrund isolates from persons, food, and food animals in Denmark, Thailand, and the United States by antimicrobial drug susceptibility and pulsed-field gel electrophoresis (PFGE) typing. Resistance, including resistance to nalidixic acid......, was frequent among isolates from persons and chickens in Thailand, persons in the United States, and food imported from Thailand to Denmark and the United States. A total of 183 PFGE patterns were observed, and 136 (23.4%) isolates had the 3 most common patterns. Seven of 14 isolates from persons in Denmark...... had patterns found in persons and chicken meat in Thailand; 22 of 390 human isolates from the United States had patterns found in Denmark and Thailand. This study suggests spread of multidrug-resistant S. Schwarzengrund from chickens to persons in Thailand, and from imported Thai food products...

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods Análisis de la relación clonal entre aislamientos clínicos de Salmonella enterica serovar Infantis mediante diferentes métodos de tipificación

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Luis A. Merino

2003-06-01

Full Text Available Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP PCR, enterobacterial repetitive intergenic consensus (ERIC PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE. Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.Salmonella Infantis ha sido el segundo serovar más común en la Argentina en los últimos dos años, siendo aislada principalmente, a partir de pacientes pediátricos hospitalizados. La relación clonal entre 15 aislamientos de Salmonella Infantis obtenidos de heces de pacientes pediátricos en Argentina se estudió mediante la susceptibilidad antimicrobiana, el perfil plasmídico, amplificación por reacción en cadena de la polimerasa (PCR de las secuencias repetitivas REP y ERIC, y electroforesis de ADN total en campo pulsátil (PFGE. Cuatro cepas españolas fueron incluidas como control de diversidad clonal. El antibiotipo y el perfil plasmídico no fueron herramientas útiles en la tipificación. PFGE y REP-PCR fueron capaces de discriminar entre las cepas argentinas y españolas de Salmonella Infantis, permitiendo detectar cepas gen

Diversity of Listeria monocytogenes isolates from cold-smoked salmon produced in different smokehouses as assessed by Random Amplified Polymorphic DNA analyses

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Vogel, Birte Fonnesbech; Jørgensen, Lasse Vigel; Ojeniyi, B.

2001-01-01

a standardised RAPD analysis by four primers separately. The grouping of the 148 strains was exactly the same for the four primers used. For a sub-set of 20 strains typed by Pulsed Field Gel Electrophoresis (PFGE), only one strain was allocated into a different group as compared to the grouping by RAPD typing....... Different RAPD types dominated in products from different smokehouses. Some identical RAPD types were isolated in several smokehouses. In each of four smokehouses, one particular RAPD type could he repeatedly isolated from products. Each smokehouse/product carried its own specific RAPD type and this may...

Molecular epidemiology and emergence of worldwide epidemic clones of Neisseria meningitidis in Taiwan

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Chang Hsiu-Li

2006-02-01

Full Text Available Abstract Background Meningococcal disease is infrequently found in Taiwan, a country with 23 million people. Between 1996 and 2002, 17 to 81 clinical cases of the disease were reported annually. Reported cases dramatically increased in 2001–2002. Our record shows that only serogroup B and W135 meningococci have been isolated from patients with meningococcal disease until 2000. However, serogroup A, C and Y meningococci were detected for the first time in 2001 and continued to cause disease through 2002. Most of serogroup Y meningococcus infections localized in Central Taiwan in 2001, indicating that a small-scale outbreak of meningococcal disease had occurred. The occurrence of a meningococcal disease outbreak and the emergence of new meningococcal strains are of public health concern. Methods Neisseria meningitidis isolates from patients with meningococcal disease from 1996 to 2002 were collected and characterized by serogrouping, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST. The genetic relatedness and clonal relationship between the isolates were analyzed by using the PFGE patterns and the allelic profiles of the sequence types (STs. Results Serogroups A, B, C, W135, Y, and non-serogroupable Neisseria meningitidis were, respectively, responsible for 2%, 50%, 2%, 35%, 9%, and 2% of 158 culture-confirmed cases of meningococcal disease in 1996–2002. Among 100 N. meningitidis isolates available for PFGE and MLST analyses, 51 different PFGE patterns and 30 STs were identified with discriminatory indices of 0.95 and 0.87, respectively. Of the 30 STs, 21 were newly identified and of which 19 were found in serogroup B isolates. A total of 40 PFGE patterns were identified in 52 serogroup B isolates with the patterns distributed over several distinct clusters. In contrast, the isolates within each of the serogroups A, C, W135, and Y shared high levels of PFGE pattern similarity. Analysis of the allelic profile of the

A reliable combination method to identification and typing of epidemic and endemic clones among clinical isolates of Acinetobacter baumannii.

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Piran, Arezoo; Shahcheraghi, Fereshteh; Solgi, Hamid; Rohani, Mahdi; Badmasti, Farzad

2017-10-01

The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of bla OXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of bla OXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and bla OXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and bla OXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded bla OXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE. Copyright © 2017 Elsevier B.V. All rights reserved.

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

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Denayer, Sarah; Nia, Yacine; Botteldoorn, Nadine

2017-01-01

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented. PMID:29261162

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection.

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Denayer, Sarah; Delbrassinne, Laurence; Nia, Yacine; Botteldoorn, Nadine

2017-12-20

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented.

Characterization of Clostridium difficile Strains in British Columbia, Canada: A Shift from NAP1 Majority (2008 to Novel Strain Types (2013 in One Region

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Agatha N. Jassem

2016-01-01

Full Text Available Background. Clostridium difficile is a major cause of gastrointestinal illness. Epidemic NAP1 strains contain toxins A and B, a deletion in repressor tcdC, and a binary toxin. Objectives. To determine the molecular epidemiology of C. difficile in British Columbia and compare between two time points in one region. Methods. C. difficile isolates from hospital and community laboratories (2008 and one Island Health hospital laboratory (2013 were characterized by pulsed-field gel electrophoresis, PCR-ribotyping, toxin possession, tcdC genotype, and antimicrobial susceptibility. Results. In 2008, 42.7% of isolates had NAP1 designation. Hospital-collected isolates were associated with older patients and more NAP1 types. Unlike other isolates, most NAP1 isolates possessed binary toxin and a 19 bp loss in tcdC. All isolates were susceptible to metronidazole and vancomycin. A 2013 follow-up revealed a 28.9% decrease in NAP1 isolates and 20.0% increase in isolates without NAP designation in one region. Then, community-associated cases were seen in younger patients, while NAP types were evenly distributed. Isolates without NAP designation did not cluster with a PFGE pattern or ribotype. Conclusions. Evaluation of C. difficile infections within British Columbia revealed demographic associations, epidemiological shifts, and characteristics of strain types. Continuous surveillance of C. difficile will enable detection of emerging strains.

Epidemiologic analysis of sporadic Salmonella typhi isolates and those from outbreaks by pulsed-field gel electrophoresis.

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Thong, K L; Cheong, Y M; Puthucheary, S; Koh, C L; Pang, T

1994-05-01

Pulsed-field gel electrophoresis (PFGE) was used to compare and analyze 158 isolates of Salmonella typhi from five well-defined outbreaks of typhoid fever in Malaysia and also isolates involved in sporadic cases of typhoid fever occurring during the same period. Digestion of chromosomal DNAs from these S. typhi isolates with the restriction endonucleases XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3') and then PFGE produced restriction endonuclease analysis (REA) patterns consisting of 11 to 24 DNA fragments ranging in size from 20 to 630 kbp. Analysis of the REA patterns generated by PFGE after digestion with XbaI and SpeI indicated that the S. typhi isolates obtained from sporadic cases of infection were much more heterogeneous (at least 13 different REA patterns were detected; Dice coefficient, between 0.73 and 1.0) than those obtained during outbreaks of typhoid fever. The clonal nature and the close genetic identities of isolates from outbreaks in Alor Setar, Penang, Kota Kinabalu, Johor Bahru, and Kota Bahru were suggested by the fact that only a limited number of REA patterns, which mostly differed by only a single band, were detected (one to four patterns; Dice coefficient, between 0.82 and 1.0), although a different pattern was associated with each of these outbreaks. Comparison of REA patterns with ribotyping for 18 S. typhi isolates involved in sporadic cases of infection showed a good correlation, in that 72% of the isolates were in the same group. There was no clear correlation of phage types with a specific REA pattern. We conclude that PFGE of s. typhi chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for comparing and differentiating S. typhi isolates for epidemiological purposes.

Response to Bile Salts in Clinical Strains of Acinetobacter baumannii Lacking the AdeABC Efflux Pump: Virulence Associated with Quorum Sensing

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Maria López

2017-05-01

Full Text Available Introduction:Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce.Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR in the response to bile salts were carried out in isogenic (A. baumanni ΔadeB ATCC 17978 and A. baumannii ΔadeL ATCC 17978 and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump.Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii ΔadeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1.Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1 lacking the main RND efflux pump (AdeABC. Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1 displayed a new clinical profile (increased invasiveness possibly associated with the response to stress conditions (such as the presence of bile salts.

Response to Bile Salts in Clinical Strains of Acinetobacter baumannii Lacking the AdeABC Efflux Pump: Virulence Associated with Quorum Sensing.

Science.gov (United States)

López, Maria; Blasco, Lucia; Gato, Eva; Perez, Astrid; Fernández-Garcia, Laura; Martínez-Martinez, Luis; Fernández-Cuenca, Felipe; Rodríguez-Baño, Jesús; Pascual, Alvaro; Bou, German; Tomás, Maria

2017-01-01

Introduction: Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection) host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce. Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR) in the response to bile salts were carried out in isogenic ( A. baumanni Δ adeB ATCC 17978 and A. baumannii Δ adeL ATCC 17978) and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump. Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii Δ adeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System) which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1. Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1) lacking the main RND efflux pump (AdeABC). Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1) displayed a new clinical profile (increased invasiveness) possibly associated with the response to stress conditions (such as the presence of bile salts).

Prevalence of Campylobacter spp. in skinless, boneless retail broiler meat from 2005 through 2011 in Alabama, USA

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Williams Aretha

2012-08-01

Full Text Available Abstract Background The prevalence of Campylobacter spp. in 755 skinless, boneless retail broiler meat samples (breast, tenderloins and thighs collected from food stores in Alabama, USA, from 2005 through 2011 was examined. Campylobacter spp. were isolated using enrichment and plate media. Isolates were identified with multiplex PCR assays and typed with pulsed field gel electrophoresis (PFGE. Data were analyzed by nominal variables (brand, plant, product, season, state and store that may affect the prevalence of these bacteria. Results The average prevalence of Campylobacter spp. in retail broiler meat for these years was 41%, with no statistical differences in the prevalence by year (P > 0.05. Seasons did not affect the prevalence of C. jejuni but statistically affected the prevalence of C. coli (P P P C. coli and C. jejuni had an average prevalence of 28% and 66%, respectively. The prevalence of C. coli varied by brand, plant, season, state, store and year, while the prevalence of C. jejuni varied by brand, product, state and store. Tenderloins had a lower prevalence of Campylobacter spp. than breasts and thighs (P P > 0.05 were observed in the prevalence of C. jejuni by season, the lowest prevalence of C. coli was recorded from October through March. A large diversity of PFGE profiles was found for C. jejuni, with some profiles from the same processing plants reappearing throughout the years. Conclusions The prevalence of Campylobacter spp. did not change during the seven years of the study; however, it did change when analyzed by brand, product and state. Seasons did not affect the prevalence of C. jejuni, but they did affect the prevalence of C. coli. Larger PFGE databases are needed to assess the temporal reoccurrence of PFGE profiles to help predict the risk associated with each profile.

Listeria monocytogenes and Listeria spp. contamination patterns in retail delicatessen establishments in three U.S. states.

Science.gov (United States)

Simmons, Courtenay; Stasiewicz, Matthew J; Wright, Emily; Warchocki, Steven; Roof, Sherry; Kause, Janell R; Bauer, Nathan; Ibrahim, Salam; Wiedmann, Martin; Oliver, Haley F

2014-11-01

Postprocessing contamination in processing plants has historically been a significant source of Listeria monocytogenes in ready-to-eat delicatessen meats, and therefore a major cause of human listeriosis cases and outbreaks. Recent risk assessments suggest that a majority of human listeriosis cases linked to consumption of contaminated deli meats may be due to L. monocytogenes contamination that occurs at the retail level. To better understand the ecology and transmission of Listeria spp. in retail delicatessens, food and nonfood contact surfaces were tested for L. monocytogenes and other Listeria spp. in a longitudinal study conducted in 30 retail delis in three U.S. states. In phase I of the study, seven sponge samples were collected monthly for 3 months in 15 delis (5 delis per state) prior to start of daily operation; in phase II, 28 food contact and nonfood contact sites were sampled in each of 30 delis during daily operation for 6 months. Among the 314 samples collected during phase I, 6.8% were positive for L. monocytogenes. Among 4,503 samples collected during phase II, 9.5% were positive for L. monocytogenes; 9 of 30 delis showed low L. monocytogenes prevalence (Listeria spp. isolates, including 184 Listeria innocua, 48 Listeria seeligeri, and 13 Listeria welshimeri were characterized. Pulsed-field gel electrophoresis (PFGE) was used to characterize 446 L. monocytogenes isolates. PFGE showed that for 12 of 30 delis, one or more PFGE types were isolated on at least three separate occasions, providing evidence for persistence of a given L. monocytogenes subtype in the delis. For some delis, PFGE patterns for isolates from nonfood contact surfaces were distinct from patterns for occasional food contact surface isolates, suggesting limited cross-contamination between these sites in some delis. This study provides longitudinal data on L. monocytogenes contamination patterns in retail delis, which should facilitate further development of control strategies in

Pulsed-Field Gel Electrophoresis Analysis of Bordetella pertussis Isolates Circulating in Europe from 1998 to 2009

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Advani, Abdolreza; Hallander, Hans O.; Dalby, Tine; Krogfelt, Karen Angeliki; Guiso, Nicole; Njamkepo, Elisabeth; von Könnig, Carl Heinz Wirsing; Riffelmann, Marion; Mooi, Frits R.; Sandven, Per; Lutyńska, Anna; Fry, Norman K.; Mertsola, Jussi

2013-01-01

Between 1998 and 2009, Bordetella pertussis clinical isolates were collected during three periods, i.e., 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), and 2007 to 2009 (n = 140), from nine countries with distinct vaccination programs, i.e., Denmark, Finland, France, Germany, The Netherlands, Norway, Poland, Sweden, and the United Kingdom. Pulsed-field gel electrophoresis (PFGE) analysis was performed according to standardized recommendations for epidemiological typing of B. pertussis. There were 81 different PFGE profiles, five of which (BpSR3, BpSR5, BpSR10, BpSR11, and BpSR12) were observed in 61% of the 396 isolates and shown to be predominant in almost all countries. The major profile, BpSR11, showed a decreasing trend from 25% to 30% in 1998 to 2005 to 13% in 2007 to 2009, and there were increases in BpSR3 and BpSR10 from 0% and 8% to 21% and 22%, respectively. One difference between these profiles is that BpSR11 contains isolates harboring the fim3-2 allele and BpSR3 and BpSR10 contain isolates harboring the fim3-1 allele. The total proportion of the five predominant profiles increased from 44% in 1998 to 2001 to 63% in 2004 to 2005 to 70% in 2007 to 2009. In conclusion, common PFGE profiles were identified in B. pertussis populations circulating in European countries with different vaccination programs and different vaccine coverages. These prevalent isolates contain the novel pertussis toxin promoter ptxP3 allele. However, there is evidence for diversifying selection between ptxP3 strains characterized by distinct PFGE profiles. This work shows that, even within a relatively short time span of 10 years, successful isolates which spread through Europe and cause large shifts in B. pertussis populations may emerge. PMID:23175253

Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids.

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Park, Miseon; Deck, Joanna; Foley, Steven L; Nayak, Rajesh; Songer, J Glenn; Seibel, Janice R; Khan, Saeed A; Rooney, Alejandro P; Hecht, David W; Rafii, Fatemeh

2016-04-01

Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes. Published by Elsevier Ltd.

Epidemiological investigation of Salmonella enterica serovar Kedougou in Thailand.

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Pornruangwong, Srirat; Hendriksen, Rene S; Pulsrikarn, Chaiwat; Bangstrakulnonth, Aroon; Mikoleit, Matthew; Davies, Rob H; Aarestrup, Frank M; Garcia-Migura, Lourdes

2011-02-01

Salmonella enterica serovar Kedougou is among the top 10 serovars reported in northern Thailand. The objective of this study was to identify risk factors associated with Salmonella Kedougou infection in Thailand and to compare the molecular types and antimicrobial resistance with Salmonella Kedougou isolates of human origin from United States and of animal origin from the United Kingdom. Data from 13,976 Salmonella infections of which 253 were Salmonella Kedougou collected in Thailand between 2002 and 2008 were analyzed by logistic regression. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed on selected Salmonella Kedougou strains causing infections in Thailand (n = 66), and compared to isolates from the United States (n = 5) and the United Kingdom (n = 20). Logistic analysis revealed season (hot/dry; p = 0.023), region (northern Thailand; p Thailand were resistant to third-generation cephalosporins: two harbored bla(CTX-M-63) and one bla(CMY-2). PFGE revealed 45 unique clusters. Isolates obtained from humans in Thailand and the United States presented identical PFGE profiles suggesting a travel association, whereas the majority of the animal isolates from United Kingdom clustered separately. This study reveals Salmonella Kedougou as a major cause of human infections in northern Thailand especially during the hot period and suggests a global spread probably due to travel. The clonal types causing infections in humans differed from those observed in animals in United Kingdom, which suggests the absence of an epidemiological link and could suggest differences in virulence. The high frequency of antimicrobial resistance, including emergence of resistance to fluoroquinolones and third-generation cephalosporins, might pose problems for treatment of infections.

Molecular and epidemiological characterisation of clinical isolates of carbapenem-resistant Acinetobacter baumannii from public and private sector intensive care units in Karachi, Pakistan.

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Irfan, S; Turton, J F; Mehraj, J; Siddiqui, S Z; Haider, S; Zafar, A; Memon, B; Afzal, O; Hasan, R

2011-06-01

The purpose of this study was to identify molecular and epidemiological characteristics of hospital-acquired carbapenem-resistant Acinetobacter baumannii (CRAB) from two different intensive care unit (ICU) settings in Karachi, Pakistan. A cross-sectional study was performed in the adult ICUs of a private sector tertiary care hospital (PS-ICU) and of a government sector hospital (GS-ICU) between November 2007 and August 2008. Deduplicated CRAB isolates from clinical specimens were examined for carbapenemase and class 1 integrase genes. Isolates were typed using sequence-based multiplex polymerase chain reaction, pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR). A total of 50 patients (33 from PS-ICU and 17 from GS-ICU) were recruited. There were statistically significant differences between patients in the two ICUs in terms of mean age, comorbidities, the presence of central venous pressure lines, urinary catheters, and average length of stay. bla(OxA-23-like) acquired-oxacillinase genes were found in 47/50 isolates. Class 1 integrase genes were found in 50% (25/50) of the organisms. The majority of isolates belonged to strains of European clones I and II. PFGE typing grouped the isolates into eight distinct clusters, three of which were found in both hospitals. Most of the isolates within each PFGE cluster shared identical or highly similar VNTR profiles, suggesting close epidemiological association. Irrespective of differences in risk factors and infection control policies and practices, the extent of clonality among CRAB isolates was very similar in both ICU settings. Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Molecular characterization of Streptococcus agalactiae and Streptococcus uberis isolates from bovine milk.

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Shome, Bibek Ranjan; Bhuvana, Mani; Mitra, Susweta Das; Krithiga, Natesan; Shome, Rajeswari; Velu, Dhanikachalam; Banerjee, Apala; Barbuddhe, Sukhadeo B; Prabhudas, Krishnamshetty; Rahman, Habibar

2012-12-01

Streptococci are one among the major mastitis pathogens which have a considerable impact on cow health, milk quality, and productivity. The aim of the present study was to investigate the occurrence and virulence characteristics of streptococci from bovine milk and to assess the molecular epidemiology and population structure of the Indian isolates using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Out of a total of 209 bovine composite milk samples screened from four herds (A-D), 30 Streptococcus spp. were isolated from 29 milk samples. Among the 30 isolates, species-specific PCR and partial 16S rRNA gene sequence analysis identified 17 Streptococcus agalactiae arising from herd A and 13 Streptococcus uberis comprising of 5, 7, and 1 isolates from herds B, C, and D respectively. PCR based screening for virulence genes revealed the presence of the cfb and the pavA genes in 17 and 1 S. agalactiae isolates, respectively. Similarly, in S. uberis isolates, cfu gene was present in six isolates from herd C, the pau A/skc gene in all the isolates from herds B, C, and D, whereas the sua gene was present in four isolates from herd B and the only isolate from herd D. On MLST analysis, all the S. agalactiae isolates were found to be of a novel sequence type (ST), ST-483, reported for the first time and is a single locus variant of the predicted subgroup founder ST-310, while the S. uberis isolates were found to be of three novel sequence types, namely ST-439, ST-474, and ST-475, all reported for the first time. ST-474 was a double locus variant of three different STs of global clonal complex ST-143 considered to be associated with clinical and subclinical mastitis, but ST-439 and ST-475 were singletons. Unique sequence types identified for both S. agalactiae and S. uberis were found to be herd specific. On PFGE analysis, identical or closely related restriction patterns for S. agalactiae ST-483 and S. uberis ST-439 in herds A and B

First report of blaNDM-1-producing Acinetobacter baumannii isolated in Lebanon from civilians wounded during the Syrian war.

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Rafei, Rayane; Dabboussi, Fouad; Hamze, Monzer; Eveillard, Matthieu; Lemarié, Carole; Mallat, Hassan; Rolain, Jean-Marc; Joly-Guillou, Marie-Laure; Kempf, Marie

2014-04-01

The emergence of carbapenem-resistant Acinetobacter baumannii has been observed worldwide. We describe the first detection of A. baumannii carrying the blaNDM-1 gene in Lebanon, isolated from Syrian patients wounded during the civil war. Four carbapenem-resistant A. baumannii strains isolated in 2012 in the Tripoli Government Hospital, Lebanon, from civilians wounded during the Syrian war, were analysed. Susceptibility was determined by disk diffusion testing, and resistance to carbapenems was confirmed by Etest. The presence of blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, and blaNDM was investigated by PCR. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and blaOXA-51 sequence-based typing. All isolates harboured the blaNDM-1 gene and were negative for other tested carbapenemases. They all belonged to the sequence type 85 and formed a single cluster by PFGE. Finally, blaOXA-51-like gene sequencing revealed the presence of the blaOXA-94 variant in all four isolates. These findings show that Syria constitutes a reservoir for NDM-1-producing bacteria. These results also highlight the need for effective measures to stop the threatening spread of such strains. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

The isolated Leptospira Spp. Identification by molecular biological techniques

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Duangjai Suwancharoen

2017-01-01

Full Text Available Leptospirosis is a zoonotic disease caused by the bacteria of Leptospira spp. Identification of this bacterium relies on serotyping and genotyping. Data base for animal causative serovars in Thailand is limited. As the unknown serovars are found in the laboratory, they need to be sent overseas for referent identification. To reduce the cost, this research intended to develop a leptospiral identification method which is user–friendly and able to classify efficiently. Ten Leptospira isolations were cultured from urine samples. They were identified by three molecular biological techniques, including Pulsed-Field Gel Electrophoresis (PFGE, Variable Number Tandem Repeat (VNTR and Multilocus Sequence Typing (MLST. These methods were developed and compared to find the most suitable one for leptospiral identification. VNTR was found to be inappropriate since it could not identify the agents and it did not show the PCR product. PFGE and MLST gave the same results of the unknown 1 and 2 which were L.weilii sv Samin st Samin. Unknown 4 showed different results by each technique. Unknown 5 to 10 were likely to be L.meyeri sv Ranarum st ICF and Leptonema illini sv Illini st 3055 by PFGE but MLST could not identify the serovar. However, molecular biological technique for Leptospira identification should be done by several methods in order to confirm the result of each other.

Genetic Relatedness of Salmonella Serovars Isolated from Catfish (Clarias gariepinus) and Tilapia (Tilapia mossambica) Obtained from Wet Markets and Ponds in Penang, Malaysia.

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Budiati, Titik; Rusul, Gulam; Wan-Abdullah, Wan Nadiah; Chuah, Li-Oon; Ahmad, Rosma; Thong, Kwai Lin

2016-04-01

A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovismorbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.

Genotypic and serotypic stability of Campylobacter jejuni strains during in vitro and in vivo passage

DEFF Research Database (Denmark)

Nielsen, Eva M.; Engberg, J.; Fussing, V.

2001-01-01

The stability of four typing methods and the sero- and genotypic stability of three Campylobacter jejuni strains were evaluated after subculturing 50 times in triplicate and after colonising mice for up to 26 days. The employed methods were Penner heat-stable serotyping; automated ribotyping (Ribo......Printing) using HaeIII restriction enzyme; pulsed-field gel electrophoresis (PFGE) using SmaI, SalI and KpnI; and random amplified polymorphic DNA analysis (RAPD) using primers 1254, 1281 and HLWL85. No changes in any of the DNA profiles or in the reactions to heat-stable antigens were identified among...... these strains after the in vitro and in vivo passages. However, one isolate became untypeable with RAPD after passage in one of the mice. In addition, eleven other C. jejuni strains of four different serotypes were subcultured ten times to screen for instability. Neither of these showed instability using PFGE...

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

Science.gov (United States)

Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

2016-01-01

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Typing of Panton-Valentine Leukocidin-encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China

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Huanqiang Zhao

2016-08-01

Full Text Available Panton-Valentine leucocidin (PVL, encoded by lukSF-PV genes, a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus (S. aureus have been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec typing, staphylococcal protein A (spa gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE typing, accessory gene regulator (agr locus typing and multilocus sequence typing (MLST. Seventy eight (78/1175, 6.6% isolates possessed the lukSF-PV genes and 59.0% (46/78 of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n=13 and ΦPVL (n=12 were the most prevalent among them. While 25 (25/78, 32.1% isolates, belonging to ST30 and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Molecular epidemiology of extended-spectrum β-lactamase-producing Escherichia coli in the community and hospital in Korea: emergence of ST131 producing CTX-M-15

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Park Sun

2012-06-01

Full Text Available Abstract Background The prevalence of extended-spectrum β-lactamase (ESBL-producing Escherichia coli has been increased not only in the hospital but also in the community worldwide. This study was aimed to characterize ESBL- producing E. coli isolates and to investigate the molecular epidemiology of community isolates in comparison with hospital isolates at a single center in Korea. Methods A total of 142 ESBL-producing E. coli isolates were collected at Daejeon St Mary’s Hospital in Korea from January 2008 to September 2009. The ESBLs were characterized by PCR sequencing using specific primers. The genetic relatedness was determined by pulsed field gel electrophoresis (PFGE and multilocus sequence typing (MLST. Results Of 142 isolates, 139 were positive for CTX-M type ESBLs; CTX-M-14 (n = 69, 49.6 %, CTX-M-15 (n = 53, 38.1 % and both CTX-M-14 and -15 (n = 17, 12.2 %. CTX-M-14 and CTX-M-15 were detected in both community and hospital isolates whereas isolates producing both CTX-M14 and-15 were mainly identified in the hospital. CTX-M producing E. coli isolates were genetically heterogeneous, revealing 75 distinct PFGE types. By MLST, 21 distinctive STs including 5 major STs (ST131, ST405, ST38, ST10, and ST648 were identified. Major STs were distributed in both community and hospital isolates, and ST131 was the predominant clone regardless of the locations of acquisition. No specific major STs were confined to a single type of ESBLs. However, ST131 clones were significantly associated with CTX-M-15 and the majority of them were multidrug-resistant. Distinctively, we identified a hospital epidemic caused by the dissemination of an epidemic strain, ST131-PFGE type 10, characterized by multidrug resistance and co-producing both CTX-Ms with OXA-1 or TEM-1b. Conclusions The epidemiology of ESBL-producing E. coli is a complex and evolving phenomenon attributed to the horizontal transfer of genetic elements and clonal spread of

Typing of Staphylococcus aureus in order to determine the spread of drug resistant strains inside and outside hospital environment.

Science.gov (United States)

Pobiega, Monika; Wójkowska-Mach, Jadwiga; Heczko, Piotr B

2013-01-01

Staphylococcus aureus is one of the most important etiological factors of both nosocomial and community-acquired infections. Multidrug-resistant S. aureus is frequently isolated nowadays. Antibiotics used on the hospital ward exert a selective pressure on the strains and favor resistant strains. Multidrug-resistant and highly virulent strains can spread not only within the hospital but also between hospitals. Numerous studies show a predominance of one clone on a specific territory. The spread of such dangerous clones to neighboring countries and the entire continent is possible. Typing methods are very useful in infection control and prevention. Modern methods which are based on sequencing are necessary in rationalizing of infection control programs. Typing of Staphylococcus aureus includes methods that allow to determine the spread of drug-resistant pathogens. 'Gold standard' is pulsed-field gel electrophoresis (PFGE), which relies on separating the DNA fragments after restriction cutting. MLST (Multi Locus Sequence Typing) is based on a comparison of"housekeeping" gene sequences controlling the basic cell functions. With the MLST method, it is possible to demonstrate a broad, international spread of the specific clones. However, for epidemiological investigations, MLST seems to be too time-consuming and expensive to be used as a basic typing tool. The complementary method is spa typing, based on the sequencing of short repetitive sequences of the polymorphic X region from the gene encoding protein A. This method can be used for studying molecular evolution of S. aureus, as well as for testing for hospital outbreaks. It is faster and cheaper than MLST. It is also necessary to subtype the elements responsible for methycillin resistance (SCCmec), which allows to distinguish MRSA (Methicillin-resistant Staphylococcus aureus) clones with a common ancestor, but different epidemiological origin. All of those methods have their specific advantages and disadvantages and

Insights to genetic characterization tools for epidemiological tracking of Francisella tularensis in Sweden.

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Tara Wahab

Full Text Available Tularaemia, caused by the bacterium Francisella tularensis, is endemic in Sweden and is poorly understood. The aim of this study was to evaluate the effectiveness of three different genetic typing systems to link a genetic type to the source and place of tularemia infection in Sweden. Canonical single nucleotide polymorphisms (canSNPs, MLVA including five variable number of tandem repeat loci and PmeI-PFGE were tested on 127 F. tularensis positive specimens collected from Swedish case-patients. All three typing methods identified two major genetic groups with near-perfect agreement. Higher genetic resolution was obtained with canSNP and MLVA compared to PFGE; F. tularensis samples were first assigned into ten phylogroups based on canSNPs followed by 33 unique MLVA types. Phylogroups were geographically analysed to reveal complex phylogeographic patterns in Sweden. The extensive phylogenetic diversity found within individual counties posed a challenge to linking specific genetic types with specific geographic locations. Despite this, a single phylogroup (B.22, defined by a SNP marker specific to a lone Swedish sequenced strain, did link genetic type with a likely geographic place. This result suggests that SNP markers, highly specific to a particular reference genome, may be found most frequently among samples recovered from the same location where the reference genome originated. This insight compels us to consider whole-genome sequencing (WGS as the appropriate tool for effectively linking specific genetic type to geography. Comparing the WGS of an unknown sample to WGS databases of archived Swedish strains maximizes the likelihood of revealing those rare geographically informative SNPs.

Development of simple and rapid PCR-fingerprinting methods for Vibrio cholerae on the basis of genetic diversity of the superintegron.

Science.gov (United States)

Chowdhury, N; Asakura, M; Neogi, S B; Hinenoya, A; Haldar, S; Ramamurthy, T; Sarkar, B L; Faruque, S M; Yamasaki, S

2010-07-01

To develop simple and rapid PCR-fingerprinting methods for Vibrio cholerae O1 (El Tor and classical biotypes) and O139 serogroup strains which cause major cholera epidemics, on the basis of the diversity of superintegron (SI) carried by these strains. PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed targeting region between integrase gene in the SI and its nearby ORF, followed by BglI digestion. Besides, a V. cholerae repeat-amplified fragment length polymorphism (VCR-AFLP) assay was also developed. In the PCR-RFLP, 94 El Tor, 29 classical and 54 O139 strains produced nine, three and six different DNA fingerprints, respectively. On the other hand, VCR-AFLP distinguished these El Tor, classical and O139 strains into five, nine and two DNA fingerprints, respectively. Combining both assays the El Tor, classical and O139 strains could be differentiated into 11, 10 and seven different types, respectively. In a comparative study, pulsed-field gel electrophoresis (PFGE) showed similar differentiation for El Tor (11 types), but lower discrimination for O139 (two types) and classical strains (five types). The PCR assays based on SI diversity can be used as a useful typing tool for epidemiological studies of V. cholerae. This newly developed method is more discriminatory, simple, rapid and cost-effective in comparison with PFGE, and thus can be widely applicable. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1, VIM-1, and OXA-23/72 in a Bulgarian Hospital.

Science.gov (United States)

Pfeifer, Yvonne; Trifonova, Angelina; Pietsch, Michael; Brunner, Magdalena; Todorova, Iva; Gergova, Ivanka; Wilharm, Gottfried; Werner, Guido; Savov, Encho

2017-04-01

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different β-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes bla OXA-23 or bla OXA-72 ; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Prevalence and relatedness of Escherichia coli O157:H7 strains in the feces and on the hides and carcasses of U.S. meat goats at slaughter.

Science.gov (United States)

Jacob, M E; Foster, D M; Rogers, A T; Balcomb, C C; Sanderson, M W

2013-07-01

We determined the prevalences of Escherichia coli O157:H7 in feces, hide, and carcasses of meat goats at a U.S. processing plant. Prevalences were 11.1%, 2.7%, and 2.7%, respectively. Sixteen pulsed-field gel electrophoresis (PFGE) subtypes were identified among 49 E. coli O157:H7 isolates, some of which were present on multiple sample types or collection days.

High Resolution Typing by Whole Genome Mapping Enables Discrimination of LA-MRSA (CC398) Strains and Identification of Transmission Events

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Bosch, Thijs; Verkade, Erwin; van Luit, Martijn; Pot, Bruno; Vauterin, Paul; Burggrave, Ronald; Savelkoul, Paul; Kluytmans, Jan; Schouls, Leo

2013-01-01

After its emergence in 2003, a livestock-associated (LA-)MRSA clade (CC398) has caused an impressive increase in the number of isolates submitted for the Dutch national MRSA surveillance and now comprises 40% of all isolates. The currently used molecular typing techniques have limited discriminatory power for this MRSA clade, which hampers studies on the origin and transmission routes. Recently, a new molecular analysis technique named whole genome mapping was introduced. This method creates high-resolution, ordered whole genome restriction maps that may have potential for strain typing. In this study, we assessed and validated the capability of whole genome mapping to differentiate LA-MRSA isolates. Multiple validation experiments showed that whole genome mapping produced highly reproducible results. Assessment of the technique on two well-documented MRSA outbreaks showed that whole genome mapping was able to confirm one outbreak, but revealed major differences between the maps of a second, indicating that not all isolates belonged to this outbreak. Whole genome mapping of LA-MRSA isolates that were epidemiologically unlinked provided a much higher discriminatory power than spa-typing or MLVA. In contrast, maps created from LA-MRSA isolates obtained during a proven LA-MRSA outbreak were nearly indistinguishable showing that transmission of LA-MRSA can be detected by whole genome mapping. Finally, whole genome maps of LA-MRSA isolates originating from two unrelated veterinarians and their household members showed that veterinarians may carry and transmit different LA-MRSA strains at the same time. No such conclusions could be drawn based spa-typing and MLVA. Although PFGE seems to be suitable for molecular typing of LA-MRSA, WGM provides a much higher discriminatory power. Furthermore, whole genome mapping can provide a comparison with other maps within 2 days after the bacterial culture is received, making it suitable to investigate transmission events and

The dnd operon for DNA phosphorothioation modification system in Escherichia coli is located in diverse genomic islands.

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Ho, Wing Sze; Ou, Hong-Yu; Yeo, Chew Chieng; Thong, Kwai Lin

2015-03-17

Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands. The dndBCDE genes (dnd operon) were detected in all Dnd(+) E. coli strains by PCR. The addition of thiourea improved the typeability of Dnd(+) E. coli strains to 100% using PFGE and the Dnd(+) phenotype can be observed in both clonal and genetically diverse E. coli strains. Genomic analysis of 101 dnd operons from genome sequences of Enterobacteriaceae revealed that the dnd operons of the same bacterial species were generally clustered together in the phylogenetic tree. Further analysis of dnd operons of 52 E. coli genomes together with their respective immediate genetic environments revealed a total of 7 types of genetic organizations, all of which were found to be associated with genomic islands designated dnd-encoding GIs. The dnd-encoding GIs displayed mosaic structure and the genomic context of the 7 islands (with 1 representative genome from each type of genetic organization) were also highly variable, suggesting multiple recombination events. This is also the first report where two dnd operons were found within a strain although the biological implication is unknown. Surprisingly, dnd operons were frequently found in pathogenic E. coli although their link with virulence has not been explored. Genomic islands likely play an important role in facilitating the horizontal

Clonal dissemination, emergence of mutator lineages and antibiotic resistance evolution in Pseudomonas aeruginosa cystic fibrosis chronic lung infection.

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Carla López-Causapé

Full Text Available Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF. We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146 detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE revealed that a single genotype (clone FQSE-A produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089 was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination

Molecular epidemiology of Klebsiella pneumoniae K1 and K2 isolates in Japan.

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Harada, Sohei; Ishii, Yoshikazu; Saga, Tomoo; Aoki, Kotaro; Tateda, Kazuhiro

2018-03-20

Although severe infections caused by hypervirulent Klebsiella pneumoniae isolates, such as K1 isolates belonging to sequence type (ST) 23, have been a significant problem in Asian countries, epidemiology of these isolates in Japan remains unclear. We performed a nationwide molecular epidemiological study of K. pneumoniae K1 and K2 isolates in Japan. Of the 259K. pneumoniae isolates collected, 14 and 16 isolates were identified as capsular genotypes K1 and K2, respectively. All K1 isolates were ST23 or its closely related clones and showed high genetic similarity by pulsed-field gel electrophoresis (PFGE) and the DiversiLab system (DL). K2 isolates, belonging to ST14, ST25, ST65, ST86, and ST110, were more genetically diverse than K1 isolates. Isolates belonging to a specific ST showed identical virulence gene profiles with a few exceptions. PFGE and DL results using K1 and K2 isolates were generally in agreement. Copyright © 2018. Published by Elsevier Inc.

A ten-year surveillance study of carbapenemase-producing Klebsiella pneumoniae in a tertiary care Greek university hospital: predominance of KPC- over VIM- or NDM-producing isolates.

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Spyropoulou, Aikaterini; Papadimitriou-Olivgeris, Matthaios; Bartzavali, Christina; Vamvakopoulou, Sophia; Marangos, Markos; Spiliopoulou, Iris; Anastassiou, Evangelos D; Christofidou, Myrto

2016-03-01

Resistance patterns and carbapenemase gene presence among Klebsiella pneumoniae isolates from the University General Hospital of Patras, Greece during a ten-year period were analysed under a surveillance programme for multi-drug-resistant bacteria. From 2005 to 2014, K. pneumoniae isolates from clinically significant specimens were identified by the Vitek 2 Advanced Expert System. Antibiotic susceptibility testing was performed by the agar disc diffusion method and Etest. The strains were tested for the presence of blaVIM, blaIMP, blaKPC, blaNDM and blaOXA-48 genes by PCR. PFGE of chromosomal Xbal DNA digests was performed. A total of 3449 K. pneumoniae isolates were recovered during the last decade. Among them, 1668 (48 %) were carbapenemase-producing: 1333 (80%) K. pneumoniae carbapenemase (KPC)-, 286 (17%) Verona imipenemase (VIM), 45 (3%) KPC- and VIM-, and four New Delhi metallo-beta-lactamase (NDM)-producing. Their resistance rates to gentamicin, colistin and tigecycline were 41%, 23% and 16%, respectively. VIM-producing K. pneumoniae were isolated in 2005 and since 2008 have been endemic. KPC-producing K. pneumoniae (KPC-Kp) isolates were introduced in 2008 and until now represent the predominant carbapenemase-producing K. pneumoniae in our institution. PFGE of 97 KPC-Kp strains identified three types: A, 84 (87%); B, 11 (11%); and E, two (2%). Eleven co-producing KPC and VIM K. pneumoniae isolates belonged to PFGE B. The four NDM-positives were classified to type F. The number of K. pneumoniae bacteraemias increased during the study period, which may be solely attributed to the increase of carbapenemase-producing isolates. The threat of carbapenemase-producing K. pneumoniae emphasizes the urgent need for implementation of infection control measures and budgetary allocations to infection control.

[Report of Relapse Typhoid Fever Cases from Kolkata, India: Recrudescence or Reinfection?

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Samajpati, Sriparna; Das, Surojit; Ray, Ujjwayini; Dutta, Shanta

2018-05-24

Three relapse cases were reported out of 107 hospital-attending typhoid cases within a period of 2 years (2014-2016) from Apollo Gleneagles Hospital, Kolkata, India. During the first episode of typhoid fever, 2 of the 3 cases were treated with ceftriaxone (CRO) for 7 days, and 1 was treated for 14 days. Six Salmonella Typhi (S. Typhi) isolates, obtained from the 3 patients during both typhoid episodes, were subjected to antimicrobial susceptibility testing, detection of quinolone resistance-determining region (QRDR) mutation and molecular subtyping by pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), and H58 haplotyping. Pairs of the S. Typhi strains isolated from two of the patients during the 1st and 2nd episodes were similar with respect to the antimicrobial resistance (AMR) profiles, QRDR mutations, and molecular subtypes; whereas, the S. Typhi strain pair isolated from the 3rd patient were different in their AMR profiles, QRDR mutations, and MLVA profiles. From these observations, it may be concluded that in spite of treating typhoid cases with CRO for 7-14 days, relapse of typhoid fever might occur. The article also showed the advantage of MLVA typing over PFGE, MLST, and CRISPR typing for the discrimination of strains isolated from the same patient in case of relapse of typhoid fever.

Dissemination of a Multidrug-Resistant VIM-1- and CMY-99-Producing Proteus mirabilis Clone in Bulgaria.

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Markovska, Rumyana; Schneider, Ines; Keuleyan, Emma; Ivanova, Dobrinka; Lesseva, Magdalena; Stoeva, Temenuga; Sredkova, Mariya; Bauernfeind, Adolf; Mitov, Ivan

2017-04-01

The aim of this study was to analyze the beta-lactamases and the molecular epidemiology of 19 clinically significant isolates of Proteus mirabilis with decreased susceptibility to imipenem, which have been collected from seven hospitals, located in different Bulgarian towns (Sofia, Varna, and Pleven). The isolates were obtained from blood, urine, tracheal and wound specimens. One additional isolate from hospital environment was included. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and PFGE. Integron mapping was performed by PCR and sequencing. All isolates showed a multidrug-resistance profile, but remained susceptible to piperacillin/tazobactam, cefepime, meropenem, and fosfomycin. They produced identical beta-lactamases, namely: TEM-1, VIM-1, and CMY-99. PCR mapping revealed that the bla VIM-1 gene was part of a class 1 integron that additionally included the aac(6')-I, dhfrA1, and ant(3″)-Ia genes. In addition, 17 of the isolates carried the armA gene. Conjugation experiments and plasmid replicon typing were unsuccessful. The isolates were clonally related according to RAPD and PFGE typing. This study reveals the nationwide distribution of a multidrug-resistant P. mirabilis clone producing VIM-1 and CMY-99 along with the presence of different aminoglycoside resistance mechanisms.

Collection of Listeria monocytogenes Isolates from Milk, Dairy Products and Food Processing Environments in Slovakia for the Purposes of European Molecular Database

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Kubicová Z.

2017-03-01

Full Text Available The molecular typing of Listeria monocytogenes isolates is an important tool for monitoring the spread of the strains in food chains, providing evidence for epidemiological investigations and for the detection of out-breaks. The demand of European typing data centralization, collection and sharing stimulated the generation of “EURL L. monocytogenes Database (EURL Lm DB” in 2012 led by the European Union Reference Laboratory (EURL for L. monocytogenes (ANSES Maisons-Alfort Laboratory for Food Safety, France in close collaboration with Applied Maths. This database includes the typing results and epidemiological information on strains isolated from food, environmental or animal samples and it is in connection with human strains database TESSy (The European Surveillance System led by the ECDC (European Centre for Disease Prevention and Control. In total 147 L. monocytogenes isolates were examined by PFGE (pulsed field gel electrophoresis in 2014—2015 in VFI Dolny Kubin from different sources. Nearly half (68 of the 147 isolates in the national Slovak database came from milk or dairy products samples and the related manufacturing environment. In this work, 68 isolates associated with milk were selected and divided into 27 clusters (95 % similarity level after combined comparison analysis (AscI and ApaI by BioNumerics 6.6 software. Eight clusters included three or more similar PFGE profiles.

Characterization of Salmonella spp. isolated from an integrated broiler chicken operation in Korea.

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Lee, Young Ju; Kim, Hyun Jung; Park, Cheong Kyu; Kim, Ki Seuk; Bae, Dong Hwa; Kang, Min Su; Cho, Jae Keun; Kim, Ae Ran; Kim, Jong Wan; Kim, Byoung Han

2007-04-01

The purpose of this study was to investigate the biological and genetic characterization of persistent Salmonella isolates in an integrated broiler chicken operation, in an attempt to elucidate the source of contamination. From the breeder farm, the hatchery, the broiler farm and the chicken slaughter house of an integrated broiler chicken operation, a total of 6 serotypes were observed. Although S. Heidelberg was not detected in the broiler farm, it was consistently found in the breeder farm, the hatchery and the chicken slaughter house. Also, S. Enteritidis and S. Senftenberg were found in the hatchery and the chicken slaughter house, and the hatchery and the broiler farm, respectively. S. Gallinarum and S. Blockley were found only in the broiler farm, and S. Virchow was only recovered in the chicken slaughter house. Isolated S. Heidelberg, S. Enteritidis and S. Senftenberg strains were divided into 3, 5 and 7 types, respectively, on the basis of all properties. Especially, S. Senftenberg isolates, divided into four types by their antimicrobial resistance patterns, were all obviously the XbaI PFGE pattern. Also, four S. Enteritidis isolates resistant to nalidixic acid showed a difference in phage type and PFGE pattern. Such a different pattern was shown despite Salmonella isolates originating from an integrated broiler operation, suggesting that further epidemiological studies on many integrated chicken companies in Korea are needed.

Virulence properties of methicillin-susceptible Staphylococcus aureus food isolates encoding Panton-Valentine Leukocidin gene.

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Sudagidan, Mert; Aydin, Ali

2010-04-15

In this study, three Panton-Valentine Leukocidin gene carrying methicillin-susceptible Staphylococcus aureus (MSSA) strains (M1-AAG42B, PY30C-b and YF1B-b) were isolated from different food samples in Kesan-Edirne, Turkey. These strains were characterized on the basis of MLST type, spa type, virulence factor gene contents, antibiotic susceptibilities against 21 antibiotics and biofilm formation. The genetic relatedness of the strains was determined by PFGE. In addition, the complete gene sequences of lukS-PV and lukF-PV were also investigated. All strains were found to be susceptible to tested antibiotics and they were mecA negative. Three strains showed the same PFGE band pattern, ST152 clonal type and t355 spa type. In the detection of virulence factor genes, sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, seu, eta, etb, set1, geh and tst genes were not detected. All strains showed the positive results for alpha- and beta-haemolysin genes (hla and hlb), protease encoding genes (sspA, sspB and aur), lukE and lukD leukocidin genes (lukED). The strains were found to be non-biofilm formers. By this study, the virulence properties of the strains were described and this is one of the first reports regarding PVL-positive MSSA strains from food. (c) 2010 Elsevier B.V. All rights reserved.

The First Outbreak Caused by Acinetobacter baumannii ST208 and ST195 in China

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Junyan Qu

2016-01-01

Full Text Available This study aimed to analyze the clinical characteristics of patients and molecular mechanisms of the first outbreak mainly caused by sequence types (STs 208 multidrug resistant (MDR Acinetobacter baumannii in China. A total of 10 clinical samples were collected from 5 patients who were involved in the outbreak. Bacterial identification and antibiotic sensitivity tests were performed by the VITEK-2 COMPACT automated system. MICs of tigecycline for clinical isolates were determined using broth microdilution. The clonal relatedness of A. baumannii clinical isolates in our local settings was determinated by pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST. A total of 7 A. baumannii strains were isolated and all were MDR strains; two of them were carbapenem-nonsusceptible strains. blaOXA-23 was the only acquired carbapenemase gene in the isolates. The isolates belonged to a single clonal pulsotype determined by PFGE and two sequences types (STs determined by MLST. The isolates belonged to the globally disseminated clonal complex 92, among which ST195 and ST208 were the most common sequence types (71.43% and 28.57%. The outbreak was successfully controlled by stringent infection control measures, especially improving the hand hygiene compliance and enhancing antimicrobial stewardship. In conclusion, this is the first description of an outbreak caused mainly by A. baumannii of ST208 in China. Infection control measures should be strengthened when infection outbreaks in hospital.

Urease-positive thermophilic strains of Campylobacter isolated from seagulls (Larus spp.).

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Kaneko, A; Matsuda, M; Miyajima, M; Moore, J E; Murphy, P G

1999-07-01

Three strains of urease-positive thermophilic Campylobacter (UPTC), designated A1, A2 and A3, were identified by biochemical characterization after isolation from faeces of seagulls in Northern Ireland in 1996. The biochemical characteristics of the strains were identical to those of strains described previously. Analysis by pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI and SmaI demonstrated that the respective PFGE profiles were indistinguishable. The PFGE analysis also suggested that the genomes were approximately 1810 kb in length. This is the first example of the isolation of UPTC from flying homoiothermal animals, i.e. from seagulls (Larus spp.).

Serratia marcescens harbouring SME-type class A carbapenemases in Canada and the presence of blaSME on a novel genomic island, SmarGI1-1.

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Mataseje, L F; Boyd, D A; Delport, J; Hoang, L; Imperial, M; Lefebvre, B; Kuhn, M; Van Caeseele, P; Willey, B M; Mulvey, M R

2014-07-01

An increasing prevalence since 2010 of Serratia marcescens harbouring the Ambler class A carbapenemase SME prompted us to further characterize these isolates. Isolates harbouring bla(SME) were identified by PCR and sequencing. Phenotypic analysis for carbapenemase activity was carried out by a modified Hodge test and a modified Carba NP test. Antimicrobial susceptibilities were determined by Etest and Vitek 2. Typing was by PFGE of macrorestriction digests. Whole-genome sequencing of three isolates was carried out to characterize the genomic region harbouring the bla(SME)-type genes. All S. marcescens harbouring SME-type enzymes could be detected using a modified Carba NP test. Isolates harbouring bla(SME) were resistant to penicillins and carbapenems, but remained susceptible to third-generation cephalosporins, as well as fluoroquinolones and trimethoprim/sulfamethoxazole. Isolates exhibited diverse genetic backgrounds, though 57% of isolates were found in three clusters. Analysis of whole-genome sequence data from three isolates revealed that the bla(SME) gene occurred in a novel cryptic prophage genomic island, SmarGI1-1. There has been an increasing occurrence of S. marcescens harbouring bla(SME) in Canada since 2010. The bla(SME) gene was found on a genomic island, SmarGI1-1, that can be excised and circularized, which probably contributes to its dissemination amongst S. marcescens. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Sequence variation in the alpha-toxin encoding plc gene of Clostridium perfringens strains isolated from diseased and healthy chickens

DEFF Research Database (Denmark)

Abildgaard, L; Engberg, RM; Pedersen, Karl

2009-01-01

The aim of the present study was to analyse the genetic diversity of the alpha-toxin encoding plc gene and the variation in a-toxin production of Clostridium perfringens type A strains isolated from presumably healthy chickens and chickens suffering from either necrotic enteritis (NE) or cholangio......-hepatitis. The a-toxin encoding plc genes from 60 different pulsed-field gel electrophoresis (PFGE) types (strains) of C perfringens were sequenced and translated in silico to amino acid sequences and the a-toxin production was investigated in batch cultures of 45 of the strains using an enzyme...

ASTRO Research Fellow Presentation - A comparison of the comet assay and pulsed-field gel electrophoresis as a predictive assay for radiosensitivity in human fibroblasts

International Nuclear Information System (INIS)

Sarkaria, Jann N.; Eady, John J.; Peacock, John H.; Steel, G. Gordon

1996-01-01

Purpose/Objective: To determine whether neutral lysis single-cell gel electrophoresis (comet assay) or pulsed-field gel electrophoresis (PFGE) can be used as a predictive assay for tissue response to radiotherapy as an alternative to clonogenic survival measurements. Materials and Methods: The comet assay has been widely used to measure DNA double strand breaks (dsb) in individual cells, and it has been suggested that it could be used as an alternative to clonogenic assays to measure radiosensitivity. Previous studies in this lab have demonstrated the ability of pulsed-field gel electrophoresis, which also measures DNA dsb, to accurately predict the radiosensitivity of a panel of fibroblasts based on determination of residual DNA dsb. As part of an ongoing study examining the relationship between fibroblast radiosensitivity and normal-tissue radiation reactions, we have compared the sensitivity and accuracy of the comet assay and PFGE on a different panel of non-transformed fibroblasts derived from breast cancer patients who developed severe radiation late effects and from case-matched controls. For the measurement of initial damage, cells were suspended in PBS and irradiated on ice for the comet assay and irradiated in agarose plugs on ice for pFGE. Residual damage was measured following irradiation of confluent cultures at 37 degree sign C and subsequent incubation for four hours prior to preparation of agarose slides and plugs. All irradiations were performed with a 59 TBq 60 Co source at a dose rate of 1.7 Gy/min. Electrophoresis was performed following neutral pH cell lysis. Comet images were captured and analyzed using Optimas software with DNA damage quantitated by the comet moment. PFGE gels were analyzed using a phosphor-image analysis system and damage was quantitated based on the percent of activity released from the well. Results: The comet assay was able to detect initial DNA damage at a threshold of 5 Gy and exhibited a linear dose

Phenotypic and genotypic characterization of Streptococcus uberis isolated from bovine subclinical mastitis in Argentinean dairy farms

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Mirta C Lasagno

2011-09-01

Full Text Available The aim of this study was to investigate the phenotypic and genotypic characteristics of Streptococcus uberis isolated from subclinical mastitis (SCM cases, and to examine the possible association between both characteristics. A total of 32 S. uberis were isolated from 772 quarter milk samples (SCM > 250,000 cells/ml collected from 195 cows selected randomly from 18 dairy farms located in Argentina. The S. uberis strains were characterized phenotypically by the presence of virulence factors as plasminogen activator factor (PAF, hyaluronidase (HYA, capsule (CAP and CAMP factor, and were further characterized genotypically by pulsed-field gel electrophoresis (PFGE. S. uberis strains expressed plasminogen activator factor, hyaluronidase or capsule (65.5 %, 56.3 %, 59.4 %, respectively, but only 25 % of isolates were CAMP factor positive. Thirteen different virulence profiles were identified on the basis of the combination of virulence factors. Eighteen PFGE patterns with 90% of similarity were identified among 32 S. uberis. A great diversity of virulence profiles and PFGE patterns were present among dairy farms. S. uberis strains with the same PFGE pattern showed different virulence profiles. Bovine S. uberis strains causing SCM included in the present study showed heterogeneity in regard to their phenotypic and genotypic characteristics, and the PFGE patterns are not associated with the virulence profiles.Caracterización fenotípica y genotípica de Streptococcus uberis aislados de mastitis bovina subclínica en tambos de Argentina. El objetivo de este estudio fue investigar las características fenotípicas y genotípicas de Streptococcus uberis aislados de casos de mastitis subclínica (MSC y examinar la posible asociación entre ambas características. Un total de 32 cepas de S. uberis fueron aisladas de 772 muestras de leche de cuartos mamarios (MSC > 25 0000 células/ml colectadas de 195 vacas seleccionadas al azar pertenecientes a 18 tambos

Outbreak of Salmonella Oslo Infections Linked to Persian Cucumbers - United States, 2016.

Science.gov (United States)

Bottichio, Lyndsay; Medus, Carlota; Sorenson, Alida; Donovan, Danielle; Sharma, Reeti; Dowell, Natasha; Williams, Ian; Wellman, Allison; Jackson, Alikeh; Tolar, Beth; Griswold, Taylor; Basler, Colin

2016-12-30

In April 2016, PulseNet, the national molecular subtyping network for foodborne disease surveillance, detected a multistate cluster of Salmonella enterica serotype Oslo infections with an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern (XbaI PFGE pattern OSLX01.0090).* This PFGE pattern was new in the database; no previous infections or outbreaks have been identified. CDC, state and local health and agriculture departments and laboratories, and the Food and Drug Administration (FDA) conducted epidemiologic, traceback, and laboratory investigations to identify the source of this outbreak. A total of 14 patients in eight states were identified, with illness onsets occurring during March 21-April 9, 2016. Whole genome sequencing, a highly discriminating subtyping method, was used to further characterize PFGE pattern OSLX01.0090 isolates. Epidemiologic evidence indicates Persian cucumbers as the source of Salmonella Oslo infections in this outbreak. This is the fourth identified multistate outbreak of salmonellosis associated with cucumbers since 2013. Further research is needed to understand the mechanism and factors that contribute to contamination of cucumbers during growth, harvesting, and processing to prevent future outbreaks.

Prevalence and Characteristics of Salmonella and Campylobacter in Retail Poultry Meat in Japan.

Science.gov (United States)

Furukawa, Ichiro; Ishihara, Tomoe; Teranishi, Hiroshi; Saito, Shioko; Yatsuyanagi, Jun; Wada, Eriko; Kumagai, Yuko; Takahashi, Shiho; Konno, Takayuki; Kashio, Hiroko; Kobayashi, Akihiko; Kato, Naoki; Hayashi, Ken-Ichi; Fukushima, Keisuke; Ishikawa, Kazuhiko; Horikawa, Kazumi; Oishi, Akira; Izumiya, Hidemasa; Ohnishi, Takahiro; Konishi, Yoshiko; Kuroki, Toshiro

2017-05-24

This study was performed to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Salmonella enterica subsp. enterica and Campylobacter spp. in poultry meat, and to analyze the association of genetic types of these bacteria with their geographical distribution and antimicrobial resistance profiles. Salmonella and Campylobacter isolates have been detected, respectively, in 54 and 71 samples out of 100 samples tested. Nine Salmonella serotypes were found, including S. enterica subsp. enterica serovar Infantis (33%), Schwarzengrund (12%), Manhattan (9%), and others. Campylobacter jejuni and C. coli were detected in 64 (64%) and 14 (14%) samples, respectively. S. enterica subsp. enterica isolates were very frequently resistant to tetracycline (78.3%) and streptomycin (68.3%). Many C. jejuni and C. coli isolates were resistant to sulfamethoxazole/trimethoprim (90.5%), nalidixic acid (47.3%), ampicillin (45.9%), and ciprofloxacin (40.5%). Cluster analysis was performed for the Salmonella isolates using pulsed-field gel electrophoresis (PFGE) data. For Campylobacter isolates, the cluster analysis was based on both PFGE and comparative genomic fingerprinting. The molecular typing results were compared with the information about antimicrobial resistance and geographical locations in which the poultry meat was produced. This analysis revealed that C. jejuni strains with a particular genotype and antimicrobial resistance profile are spreading in specific areas of Japan.

Importance of Multifaceted Approaches in Infection Control: A Practical Experience from an Outbreak Investigation.

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Nina Katharina Stock

Full Text Available This study presents the results of a multidisciplinary, nosocomial MRSA outbreak investigation in an 8-bed medical intensive care unit (ICU. The identification of seven MRSA positive patients in the beginning of 2014 led to the closure of the ward for several weeks. A multidisciplinary, retrospective investigation was initiated in order to identify the reason and the source for the outbreak, describe MRSA transmission in the department and identify limitations in infection control.The investigation comprised an epidemiological description of MRSA cases from 2012 to 2014 and a characterization of MRSA isolates, including phage-, spa- and PFGE-typing. Additionally, MRSA screening was performed from the hospital staff and the environment. To identify the reason for the outbreak, work-related, psychological and behavioral factors were investigated by impartial audits and staff interviews.Thirty-one MRSA cases were registered during the study period, and 36 isolates were investigated. Molecular typing determined the outbreak strain (phage type 54/812, PFGE type A4, spa type t003 and identified the probable index case. Nasal carriage in one employee and a high environmental contamination with the outbreak strain was documented. Important gaps in nursing procedures and general management were identified. Elevated stress levels and communication problems preceded the outbreak. Compliance with hand hygiene and isolation procedures was evaluated as appropriate.This study demonstrates the complexity of controlling hospital-associated infections. The combined use of different typing methods is beneficial for outbreak investigations. Psychological, behavioral and other work-related factors have an important impact on the spread of nosocomial pathogens. These factors should be addressed and integrated in routine infection control practice.

Horizontal gene transfer of a ColV plasmid has resulted in a dominant avian clonal type of Salmonella enterica serovar Kentucky.

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Timothy J Johnson

Full Text Available Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%, Typhimurium (15.0% and Heidelberg (1.7%. We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

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Balart, Josep; Pueyo, Gemma; Llobet, Lara I de; Baro, Marta; Sole, Xavi; Marin, Susanna; Casanovas, Oriol; Mesia, Ricard; Capella, Gabriel

2011-01-01

Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay

Four-year prospective study of STAPH-IDENT system and conventional method for reference identification of Staphylococcus, Stomatococcus, and Micrococcus spp..

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Rhoden, D L; Miller, J M

1995-01-01

A 4-year prospective study compared the accuracy of the STAPH-IDENT system (bioMérieux Vitek, Inc., Hazelwood, Mo.) with that of the reference procedure of the Centers for Disease Control and Prevention for the identification of Staphylococcus species, Stomatococcus mucilaginosus, and Micrococcus species. The study compared the results from 1,106 cultures (500 eye cultures, 217 strains submitted for reference identification, and 389 known stock strains) representing 21 species of the family Micrococcaceae. The overall agreement of genus and species identifications was 81.1%. The percent agreement for the five most common clinical isolates was as follows: Staphylococcus epidermidis, 97.1% (517 isolates); Staphylococcus hominis, 82.5% (57 isolates); Staphylococcus aureus, 77.2% (162 isolates); Staphylococcus haemolyticus, 75.8% (61 isolates); and Staphylococcus warneri, 64.1% (39 isolates). The lowest percent agreement was with Staphylococcus cohnii (11.1%; (9 isolates). Of the 217 isolates sent to the Centers for Disease Control and Prevention for identification, 60.4% (131) were correctly identified by the STAPH-IDENT system. Of these, S. epidermidis accounted for 23.9%, S. aureus accounted for 15.6%, S. warneri accounted for 6.9%, Staphylococcus lugdunensis accounted for 6.5%, S. haemolyticus accounted for 5.5%, and S. hominis accounted for 4.1%. The STAPH-IDENT system did not perform adequately when dealing with commonly encountered organisms and is unsuitable for identifying uncommon isolates. PMID:7699074

Distribution of pathogenic microorganisms isolated from dental hospital workers in Korea

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Su Jung Kim

2013-04-01

Full Text Available With the significant rise in hospital infection management in dental hospitals as well as in hospitals, and in order to identify the distribution of pathogenic bacteria on hands and nasal cavity of workers in a dental hospital, bacteria from the hands and nasal cavities of six dentists and 44 dental hygienists from four dental hospitals were investigated. The results showed Staphylococcus aureus (13, Staphylococcus capitis (1, Staphylococcus epidermidis (12, Staphylococcus hominis (4, Staphylococcus warneri (3, Staphylococcus xylosus (14, Staphylococcus. Lugdunensis (1, and Neisseria spp. (2 were isolated from the nasal cavity and Staphylococcus aureus (9, Staphylococcus capitis (4, Staphylococcus epidermidis (16, Staphylococcus hominis (8, Staphylococcus warneri (5, Staphylococcus xylosus (22, Staphylococcus leutus (3, Micrococcus spp. (4, Staphylococcus cohnii (1, Serratia marcescens (2, Pseudomonas aeruginosa (3, Klebsiella pneumonia (2 and Pseudomonas pneumotropica (1 from the hands. An antimicrobial disk diffusion test was conducted on Staphylococcus aureus isolated from the hands and nasal cavity to detect MRSA by means of oxacillin. Two strains were detected. When the genes of penicillin binding protein 2 (mecA were detected from the 2 strains, MRSA was found from both strains. The results of this investigation on the distribution of various pathogenic bacteria and MRSA on hands and nasal cavity of workers of a dental hospital, will contribute to the basic data for the future infection management in a dental hospital.

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts.

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Shimamura, Yuko; Kidokoro, Shiho; Murata, Masatsune

2006-07-01

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.

Occurrence of Listeria spp. in retail meat and dairy products in the area of Addis Ababa, Ethiopia.

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Derra, Firehiwot Abera; Karlsmose, Susanne; Monga, Dharam P; Mache, Abebe; Svendsen, Christina Aaby; Félix, Benjamin; Granier, Sophie A; Geyid, Abera; Taye, Girum; Hendriksen, Rene S

2013-06-01

Listeriosis, a bacterial disease in humans and animals, is mostly caused by ingestion of Listeria monocytogenes via contaminated food and/or water, or by a zoonotic infection. Globally, listeriosis has in general a low incidence but a high case fatality rate. The objective of this study was to investigate the occurrence, antimicrobial profiles, and genetic relatedness of L. monocytogenes from raw meat and dairy products (raw milk, cottage cheese, cream cake), collected from the capital and five neighboring towns in Ethiopia. Two hundred forty food samples were purchased from July to December 2006 from food vendors, shops, and supermarkets, using a cross-sectional study design. L. monocytogenes were isolated and subjected to molecular serotyping. The genetic relatedness and antimicrobial susceptibility patterns were investigated using pulsed-field gel electrophoresis (PFGE) and minimum inhibitory concentration determinations. Of 240 food samples tested, 66 (27.5%) were positive for Listeria species. Of 59 viable isolates, 10 (4.1%) were L. monocytogenes. Nine were serotype 4b and one was 2b. Minimum inhibitory concentration determination and PFGE of the 10 L. monocytogenes isolates showed low occurrence of antimicrobial resistance among eight different PFGE types. The findings in this study correspond to similar research undertaken in Ethiopia by detecting L. monocytogenes with similar prevalence rates. Public education is crucial as regards the nature of this organism and relevant prevention measures. Moreover, further research in clinical samples should be carried out to estimate the prevalence and carrier rate in humans, and future investigations on foodborne outbreaks must include L. monocytogenes.

Characterisation of drug resistance of nosocomial ESBL-producing E. coli isolates obtained from a Turkish university hospital between 2009 and 2012 by pulsed field gel electrophoresis and antibiotic resistance tests.

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Karagöz, Alper; Sunnetcioglu, Mahmut; Ceylan, Mehmet Resat; Bayram, Yasemin; Yalcin, Gozde; Kocak, Nadir; Suvak, Burak; Andac, Cenk A

2016-01-01

In this study, drug resistance of 28 ESBL-producing Escherichia coli isolates obtained from 144 patients hospitalized at the Yüzüncüyil University Hospital at Van (YUH), Turkey, between 2009 and 2012 were characterized by pulsed field gel electrophoresis and antibiotic susceptibility tests. Antibiotic resistance profile was determined by Phoenix automated system (BD, USA). The ratio of ESBL-producing E. coli strains was determined to be 19.4% (28 out of 144 E. coli isolates). It was determined that the anaesthesiology, paediatrics and thoracic medicine intensive care units in YUH were cross-contaminated between 2009 and 2012 by ESBL-producing E. coli strains, which is a sign of nosocomial infection in YUH. Analysis of PFGE results gave rise to two main PFGE profiles, profile-A with four subprofiles and profile-B with three subprofiles, where profile-A predominates over profile-B (14%). Comparison of the antibiotic resistance profile with the PFGE profile yielded similarities while some differences also exist due to either identical restriction enzyme cutting sites with slightly different genetic sequences in between the cutting sites or newly formed restriction enzyme cutting sites that do not affect antibiotic resistance genes. Enterobacteriaceae, particularly E. coli, have developed resistance in YUH by producing ESBLs against oxyimino and non-oxyimino cephalosporins, and penicillin-type antibiotics. Therefore, more effective antibiotics such as cefoxitin or cefoperazone-sulbactam should be used for the treatment of future nosocomial infections in YUH while hospital staff should take care with hygiene, such as hand washing.

Rhesus macaques (Macaca mulatta are natural hosts of specific Staphylococcus aureus lineages.

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Sanne van den Berg

Full Text Available Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE, spa repeat sequencing and multi-locus sequence typing (MLST, and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs. Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention.

Diversity of staphylococcal cassette chromosome mec structures in coagulase-negative staphylococci and relationship to drug resistance.

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Garza-González, Elvira; López, Daniel; Pezina, Cesar; Muruet, Walter; Bocanegra-García, Virgilio; Muñoz, Ivan; Ramírez, Camilo; Llaca-Díaz, Jorge M

2010-03-01

The objective of this study was to determine the distribution of staphylococcal cassette chromosome mec (SCCmec) elements in meticillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from a tertiary-care hospital in Mexico and to examine the relationship to drug resistance. Fifty selected MR-CoNS isolates collected from catheters (n=15), blood (n=15), bone (n=9), bronchial lavage (n=2) and urine (n=2) and one isolate each from an abscess, cerebrospinal fluid, eye, pleural effusion, synovial fluid, tracheal aspirate and wound secretion were examined. Susceptibility testing was performed by the broth microdilution method. SCCmec types were determined by multiplex PCR and PFGE was carried out as described previously for Staphylococcus aureus. Among the MR-CoNS strains studied, the most frequently isolated species were Staphylococcus epidermidis (n=26) and Staphylococcus haemolyticus (n=13). Staphylococcus cohnii (n=5), Staphylococcus hominis (n=3), Staphylococcus sciuri (n=1), Staphylococcus pasteuri (n=1) and the recently described species Staphylococcus pettenkoferi (n=1) were also identified. The most frequent MR-CoNS genotype identified was SCCmec type IVa in S. epidermidis isolates, which also showed a high diversity in their PFGE patterns. A clone was found that amplified both SCCmec III and V elements in five isolates examined. The single MR S. pettenkoferi isolate harboured SCCmec type IVd and the single MR S. pasteuri isolate harboured SCCmec type I. The carriage of SCCmec type III was associated with resistance or intermediate resistance to meropenem (P <0.05). These results confirm the high prevalence of S. epidermidis SCCmec IVa and the high genetic diversity among MR-CoNS strains. As far as is known, this is the first report describing the newly identified S. pettenkoferi possessing SCCmec IVd and S. pasteuri harbouring SCCmec type I. MR-CoNS harbouring SCCmec type III were found to be more resistant to meropenem.

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei.

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Chen, Pei-Shih; Chen, Yao-Shen; Lin, Hsi-Hsun; Liu, Pei-Ju; Ni, Wei-Fan; Hsueh, Pei-Tan; Liang, Shih-Hsiung; Chen, Chialin; Chen, Ya-Lei

2015-06-01

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km2 area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.

Comparison of Staphylococcus aureus recovered from personnel in a poultry hatchery and in broiler parent farms with those isolated from skeletal disease in broilers.

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Rodgers, J D; McCullagh, J J; McNamee, P T; Smyth, J A; Ball, H J

1999-09-15

Personnel from one broiler hatchery, and workers on 18 separate broiler parent farms which supply the hatchery, were tested for hand and nasal carriage of Staphylococcus aureus. In both locations, nasal carriage of S. aureus was more common than hand carriage. A total of 63 S. aureus strains were characterised by biotyping, protein A analysis and pulsed field gel electrophoresis (PFGE) typing. Of these, 36 were recovered from broiler hatchery personnel, 14 from broiler parent farm personnel and 13 from cases of skeletal disease in commercial broilers. Biotyping and protein A analysis indicated that none of the strains recovered from hatchery personnel were of the poultry biotype, but that two strains recovered from the hands of two broiler parent farm personnel could be grouped together with 12/13 of strains recovered from skeletal disease in broilers, as poultry biotypes. PFGE-typing could not distinguish 9/13 strains recovered from skeletal disease in broilers and one of the strains from the broiler parent farm personnel from isolate 24 (I. 24), which is the predominant S. aureus strain type associated with clinical disease in N. Ireland broiler flocks. The present study found no evidence of nasal carriage of S. aureus strains of poultry biotype by humans. The finding of hand carriage by broiler parent farm personnel, suggests that handling by personnel may contribute to the dissemination of I. 24 or other S. aureus strains associated with skeletal disease in broilers.

Antimicrobial resistance and population structure of Staphylococcus epidermidis recovered from animals and humans.

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Argudín, M Angeles; Vanderhaeghen, Wannes; Vandendriessche, Stien; Vandecandelaere, Ilse; André, François-Xavier; Denis, Olivier; Coenye, Tom; Butaye, Patrick

2015-07-09

While Staphylococcus epidermidis, as part of the commensal flora, is a well-known human opportunistic pathogen, only little is known about the genetic relatedness of S. epidermidis carriage isolates from animal and human origin. This study aimed to compare S. epidermidis recovered from livestock, livestock-farmers and humans associated with the hospital environment. A total of 193 S. epidermidis isolates from three populations [animals (n=33), farmers (n=86) and hospital-associated (n=74)] were characterized by broth microdilution antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The overall S. epidermidis nasal colonization rate was low in animals (1-9%) but high among farmers (75%). High levels of multi-resistance were found in all populations. Tetracycline resistance was high in animal and farmer isolates; resistance to erythromycin, clindamycin and trimethoprim was high in animal and hospital-associated isolates. Methicillin-resistant S. epidermidis - MRSE isolates were found in all collections, with 22 (67%) MRSE in animals, 44 (51%) MRSE in farmers and 42 (57%) MRSE associated with the hospital-setting. Known SCCmec types and variants were detected in 79% of MRSE; the rest were non-typeable cassettes. In total 79 PFGE-types were found, of which 22 were shared between livestock, farmers and the hospital settings. Clonal complex 2 was predominant in all three populations and most STs corresponded to types previously observed in community and nosocomial S. epidermidis populations. S. epidermidis isolates from livestock, farmers and hospital-setting showed a high level of diversity, but some clones can be found in humans as well as in animals. Copyright © 2015 Elsevier B.V. All rights reserved.

Similarities between Salmonella Enteritidis isolated from humans and captive wild animals in South Africa.

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Smith, Anthony M; Ismail, Husna; Henton, Maryke M; Keddy, Karen H

2014-12-15

Salmonella is well recognized as an aetiological agent of gastrointestinal and diarrhoeal disease. Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) is one of the commonest serotypes associated with foodborne illness. In South Africa, we compared Salmonella Enteritidis strains isolated from humans with gastroenteritis and strains isolated from captive wild animals, between June 2011 and July 2012. Bacteria were phenotypically characterized using standard microbiological techniques. Genotypic relatedness of isolates was investigated by pulsed-field gel electrophoresis (PFGE) analysis. a diversity of 27 PFGE patterns amongst 196 human non-invasive isolates was shown; two PFGE patterns predominated and accounted for 74% of all human isolates. Human isolates showed a 12% prevalence rate for nalidixic acid resistance. Animal isolates from 5 different sources were investigated. With the exception of an isolate from a ground hornbill, all animal isolates (jaguar, crocodile, lion and poultry) showed PFGE pattern matches to a human isolate. Animal isolates showed susceptibility to all antimicrobial agents tested, with the exception of nalidixic acid resistance in isolates from the lion and poultry source. Our data showed similarities between Salmonella Enteritidis strains isolated from humans and captive wild animals, suggesting a probable common source for strains from humans and animals.

Meta-analysis of pulsed-field gel electrophoresis fingerprints based on a constructed Salmonella database.

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Wen Zou

Full Text Available A database was constructed consisting of 45,923 Salmonella pulsed-field gel electrophoresis (PFGE patterns. The patterns, randomly selected from all submissions to CDC PulseNet during 2005 to 2010, included the 20 most frequent serotypes and 12 less frequent serotypes. Meta-analysis was applied to all of the PFGE patterns in the database. In the range of 20 to 1100 kb, serotype Enteritidis averaged the fewest bands at 12 bands and Paratyphi A the most with 19, with most serotypes in the 13-15 range among the 32 serptypes. The 10 most frequent bands for each of the 32 serotypes were sorted and distinguished, and the results were in concordance with those from distance matrix and two-way hierarchical cluster analyses of the patterns in the database. The hierarchical cluster analysis divided the 32 serotypes into three major groups according to dissimilarity measures, and revealed for the first time the similarities among the PFGE patterns of serotype Saintpaul to serotypes Typhimurium, Typhimurium var. 5-, and I 4,[5],12:i:-; of serotype Hadar to serotype Infantis; and of serotype Muenchen to serotype Newport. The results of the meta-analysis indicated that the pattern similarities/dissimilarities determined the serotype discrimination of PFGE method, and that the possible PFGE markers may have utility for serotype identification. The presence of distinct, serotype specific patterns may provide useful information to aid in the distribution of serotypes in the population and potentially reduce the need for laborious analyses, such as traditional serotyping.

Genetic diversity of clinical and environmental strains of Salmonella enterica serotype Weltevreden isolated in Malaysia.

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Thong, K L; Goh, Y L; Radu, S; Noorzaleha, S; Yasin, R; Koh, Y T; Lim, V K E; Rusul, G; Puthucheary, S D

2002-07-01

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia.

Genetic Diversity of Clinical and Environmental Strains of Salmonella enterica Serotype Weltevreden Isolated in Malaysia

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Thong, K. L.; Goh, Y. L.; Radu, S.; Noorzaleha, S.; Yasin, R.; Koh, Y. T.; Lim, V. K. E.; Rusul, G.; Puthucheary, S. D.

2002-01-01

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of Salmonella serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with Salmonella serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of Salmonella serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of Salmonella serotype Weltevreden in Malaysia. PMID:12089269

Emergence of carbapenem resistant Escherichia coli isolates producing blaNDM and blaOXA-48-like carried on IncA/C and IncL/M plasmids at two Iranian university hospitals.

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Solgi, Hamid; Giske, Christian G; Badmasti, Farzad; Aghamohammad, Shadi; Havaei, Seyed Asghar; Sabeti, Shahram; Mostafavizadeh, Kamyar; Shahcheraghi, Fereshteh

2017-11-01

The emergence of carbapenem resistance among Escherichia coli is a serious threat to public health. The objective of this study was to investigate resistance genes and clonality of carbapenem resistant E. coli in Iran. Between February 2015 and July 2016, a total of 32 non-duplicate E. coli isolates that were ertapenem resistant or intermediate (R/I-ETP) were collected from patient clinical or surveillance cultures (rectal swabs) at two university hospitals. Resistance genes were identified by PCR and sequencing. Conjugation experiments, PCR-based replicon typing, PFGE and multilocus sequence typing (MLST) were performed. PCR assays showed, among the 32 isolates, twenty-nine strains produced carbapenemase genes. The predominant carbapenemase was bla OXA-48 (82.8%), followed by bla NDM-1 (31%), bla NDM-7 (6.9%) and bla OXA-181 (3.4%). Seven of the bla NDM positive isolates co-harbored bla OXA-48 carbapenemases. The bla NDM and bla OXA-48 were found in IncA/C and IncL/M conjugative plasmids, respectively. The bla CTX-M-15 , qnrA and intI1 genes were also present in most isolates. The PFGE revealed genetic diversity among the 28 E. coli isolates, which belonged to six minor PFGE clusters and 14 isolates were singletons. The 26 isolates were distributed into 18 STs, of which two were dominant (ST648 and ST167). We identified one bla NDM-1 -positive ST131 E. coli isolates that harbor the bla CTX-M-15 and bla TEM genes. Horizontal transfer of IncA/C and IncL/M plasmids has likely facilitated the spread of the bla OXA-48 and bla NDM genes among E. coli. Their clonal diversity and the presence of faecal carriers in isolates suggest an endemic spread of OXA-48 and NDM. Therefore, it emphasizes the critical importance of monitoring and controlling the spread of carbapenem resistant E. coli. Copyright © 2017. Published by Elsevier B.V.

Distribution and Diversity of Salmonella Strains in Shipments of Hatchling Poultry, United States, 2013.

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Habing, G G; Kessler, S E; Mollenkopf, D F; Wittum, T E; Anderson, T C; Barton Behravesh, C; Joseph, L A; Erdman, M M

2015-08-01

Multistate outbreaks of salmonellosis associated with live poultry contact have been occurring with increasing frequency. In 2013, multistate outbreaks of salmonellosis were traced back to exposure to live poultry, some of which were purchased at a national chain of farm stores (Farm store chain Y). This study was conducted at 36 stores of Farm store chain Y and was concurrent with the timing of exposure for the human outbreaks of salmonellosis in 2013. We used environmental swabs of arriving shipment boxes of hatchling poultry and shipment tracking information to examine the distribution, diversity and anti-microbial resistance of non-typhoidal Salmonella (NTS) across farm stores and hatcheries. Isolates recovered from shipment boxes underwent serotyping, anti-microbial resistance (AMR) testing and pulsed-field gel electrophoresis (PFGE). Postal service tracking codes from the shipment boxes were used to determine the hatchery of origin. The PFGE patterns were compared with the PFGE patterns of NTS causing outbreaks of salmonellosis in 2013. A total of 219 hatchling boxes from 36 stores in 13 states were swabbed between 15 March 2013 and 18 April 2013. NTS were recovered from 59 (27%) of 219 hatchling boxes. Recovery was not significantly associated with species of hatchlings, number of birds in the shipment box, or the presence of dead, injured or sick birds. Four of the 23 PFGE patterns and 23 of 50 isolates were indistinguishable from strains causing human outbreaks in 2013. For serotypes associated with human illnesses, PFGE patterns most frequently recovered from shipment boxes were also more frequent causes of human illness. Boxes positive for the same PFGE pattern most frequently originated from the same mail-order hatchery. Only one of 59 isolates was resistant to anti-microbials used to treat Salmonella infections in people. This study provides critical information to address recurrent human outbreaks of salmonellosis associated with mail-order hatchling

Molecular epidemiology of Escherichia coli producing extended-spectrum {beta}-lactamases in Lugo (Spain): dissemination of clone O25b:H4-ST131 producing CTX-M-15.

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Blanco, Miguel; Alonso, Maria Pilar; Nicolas-Chanoine, Marie-Hélène; Dahbi, Ghizlane; Mora, Azucena; Blanco, Jesús E; López, Cecilia; Cortés, Pilar; Llagostera, Montserrat; Leflon-Guibout, Véronique; Puentes, Beatriz; Mamani, Rosalía; Herrera, Alexandra; Coira, María Amparo; García-Garrote, Fernando; Pita, Julia María; Blanco, Jorge

2009-06-01

Having shown that the Xeral-Calde Hospital in Lugo (Spain) has been concerned by Escherichia coli clone O25:H4-ST131 producing CTX-M-15 (Nicolas-Chanoine et al. J Antimicrob Chemother 2008; 61: 273-81), the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates and also to molecularly characterize the E. coli isolates producing ESBL other than CTX-M-15. In the first part of this study, 105 ESBL-producing E. coli isolates (February 2006 to March 2007) were characterized with regard to ESBL enzymes, serotypes, virulence genes, phylogenetic groups, multilocus sequence typing (MLST) and PFGE. In the second part of this study, 249 ESBL-producing E. coli isolates (April 2007 to May 2008) were investigated only for the detection of clone O25b:H4-ST131 producing CTX-M-15 using a triplex PCR developed in this study and based on the detection of the new operon afa FM955459 and the targets rfbO25b and 3' end of the bla(CTX-M-15) gene. Of the 105 ESBL-producing E. coli isolates, 60 (57.1%) were positive for CTX-M-14, 23 (21.9%) for CTX-M-15, 10 (9.5%) for SHV-12 and 7 (6.7%) for CTX-M-32. Serotypes, virulence genes, phylogenetic groups and molecular typing by PFGE demonstrated high homogeneity within those producing CTX-M-15 and high diversity within E. coli producing CTX-M-14 and other ESBLs. By PFGE, CTX-M-15-producing E. coli isolates O25b:H4 belonging to the phylogenetic group B2 and MLST profile ST131 were grouped in the same cluster. The epidemic strain of clone O25b:H4-ST131 represented 23.1%, 22.5% and 20.0% of all ESBL-producing E. coli isolated in 2006, 2007 and 2008, respectively. CTX-M-type ESBLs, primarily CTX-M-14 and CTX-M-15, have emerged as the predominant types of ESBL produced by E. coli isolates in Lugo. In view of the reported findings, long-term care facilities for elderly people may represent a significant reservoir for E. coli clone O25b:H4-ST131 producing CTX

Diversity of genotypes in CTX-M-producing Klebsiella pneumoniae isolated in different hospitals in Brazil

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Thiago Pavoni Gomes Chagas

Full Text Available OBJECTIVE: The present study was undertaken to characterize CTX-M ESBL-producing Klebsiella pneumoniae collected from hospitals in different cities of Brazil. MATERIAL AND METHODS: Eighty-five K. pneumoniae strains isolated from hospitalized patients in six different hospitals of three cities of Brazil were analyzed. ESBL production was confirmed by the standard double-disk synergy test and the Etest®. The MIC50 and MIC90 for ESBL-producing isolates were determined by the Etest® method. The antimicrobial susceptibilities of bacterial isolates were determined using the agar diffusion method according to the CLSI. Screening for blaTEM, blaSHV, blaCTX-M genes and class 1 integron was performed by PCR amplification. To determine the genomic diversity of CTX-M-producers, isolates were analyzed by macrorestriction profile analysis following PFGE. RESULTS AND DISCUSSION: Seventy-one K. pneumoniae isolates were ESBL-producing. PCR and sequencing experiments detected 38 CTX-M-producing K. pneumoniae belonged to groups CTX-M 1, CTX-M 2, CTX-M 8 and CTX-M 9. The association of different types ESBL (CTX-M, SHV and TEM was frequent. All K. pneumoniae isolates carried class 1 integron. PFGE analysis revealed thirty-one clonal types among CTX-M-producing isolates. The data presented herein illustrate the diversity of genotypes of CTX-M producing K. pneumoniae among Brazilians hospitals.

Epidemiological investigation of nosocomial outbreak of staphylococcal skin diseases in neonatal ward.

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Kurlenda, J; Grinholc, M; Krzysztoń-Russjan, J; Wiśniewska, K

2009-05-01

During a 1-month period, eight neonates developed staphylococcal skin disease diagnosed as a bullous impetigo in the maternity unit of the Provincial Hospital in Gdansk. An epidemiological investigation based on phenotyping and genotyping methods was performed. All neonates involved in the outbreak, their mothers and 15 staff members were screened for carriage of Staphylococcus aureus by nasal swabs. Isolated strains were compared with strains cultured from affected skin and purulent conjunctiva of infected newborns. Isolates were analyzed for the presence of the etA and etB genes using polymerase chain reaction and genotyped by pulsed-field gel electrophoresis (PFGE) and coa gene polymorphism. The analyzed S. aureus strains were methicillin-sensitive and could be divided into two groups according to antibiotyping, phage typing, coa polymorphism and PFGE pattern. The first group consisted of etA and etB negative strains, and the second one involved only the etB positive ones. Our results have shown that there were two different clusters of infection caused by two populations of S. aureus strains. Among the 15 medical staff members screened we have found seven carriers. However, phage typing revealed that distinct strains unrelated to the outbreak isolates were carried. Although we have not been able to establish the source of bacteria involved in the outbreak, our results suggest that for both groups, mothers could be the source of the infecting strains.

Genetic and phenotypic evidence of the Salmonella enterica serotype Enteritidis human-animal interface in Chile

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Patricio eRetamal

2015-05-01

Full Text Available Salmonella enterica serotype Enteritidis is a worldwide zoonotic agent that has been recognized as a very important food-borne bacterial pathogen, mainly associated with consumption of poultry products. The aim of this work was to determine genotypic and phenotypic evidence of S. Enteritidis transmission among seabirds, poultry and humans in Chile. Genotyping was performed using PCR-based virulotyping, pulse-field gel electrophoresis (PFGE and multi-locus sequence typing (MLST. Pathogenicity-associated phenotypes were determined with survival to free radicals, acidic pH, starvation, antimicrobial resistance, and survival within human dendritic cells. As result of PCR and PFGE assays, some isolates from the three hosts showed identical genotypic patterns, and through MLST it was determined that all of them belong to sequence type 11. Results of phenotypic assays showed diversity of survival capabilities among isolates. When results were analyzed according to bacterial host, statistical differences were identified in starvation and dendritic cells survival assays. In addition, isolates from seabirds showed the highest rates of resistance to gentamycin, tetracycline and ampicillin. Overall, the very close genetic and phenotypic traits shown by isolates from humans, poultry and seabirds suggest the inter-species transmission of S. Enteritidis bacteria between hosts, likely through anthropogenic environmental contamination that determines infection of seabirds with bacteria that are potentially pathogenic for other susceptible organism, including humans.

Vancomycin resistant enterococci (VRE in Swedish sewage sludge

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Aspan Anna

2009-05-01

Full Text Available Abstract Background Antimicrobial resistance is a serious threat in veterinary medicine and human healthcare. Resistance genes can spread from animals, through the food-chain, and back to humans. Sewage sludge may act as the link back from humans to animals. The main aims of this study were to investigate the occurrence of vancomycin resistant enterococci (VRE in treated sewage sludge, in a Swedish waste water treatment plant (WWTP, and to compare VRE isolates from sewage sludge with isolates from humans and chickens. Methods During a four month long study, sewage sludge was collected weekly and cultured for VRE. The VRE isolates from sewage sludge were analysed and compared to each other and to human and chicken VRE isolates by biochemical typing (PhenePlate, PFGE and antibiograms. Results Biochemical typing (PhenePlate-FS and pulsed field gel electrophoresis (PFGE revealed prevalence of specific VRE strains in sewage sludge for up to 16 weeks. No connection was found between the VRE strains isolated from sludge, chickens and humans, indicating that human VRE did not originate from Swedish chicken. Conclusion This study demonstrated widespread occurrence of VRE in sewage sludge in the studied WWTP. This implies a risk of antimicrobial resistance being spread to new farms and to the society via the environment if the sewage sludge is used on arable land.

Epidemiology of Methicillin-Susceptible Staphylococcus aureus in a Neonatology Ward.

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Achermann, Yvonne; Seidl, Kati; Kuster, Stefan P; Leimer, Nadja; Durisch, Nina; Ajdler-Schäffler, Evelyne; Karrer, Stephan; Senn, Gabriela; Holzmann-Bürgel, Anne; Wolfensberger, Aline; Leone, Antonio; Arlettaz, Romaine; Zinkernagel, Annelies S; Sax, Hugo

2015-11-01

In-hospital transmission of methicillin-susceptible Staphylococcus aureus (MSSA) among neonates remains enigmatic. We describe the epidemiology of MSSA colonization and infection in a 30-bed neonatal ward. Multimodal outbreak investigation A public 800-bed tertiary care university hospital in Switzerland Investigations in 2012-2013, triggered by a MSSA infection cluster, included prospective MSSA infection surveillance, microbiologic screening of neonates and environment, onsite observations, and a prospective cohort study. MSSA isolates were characterized by pulsed-field gel electrophoresis (PFGE) and selected isolates were examined for multilocus sequence type (MLST) and virulence factors. Among 726 in 2012, 30 (4.1%) patients suffered from MSSA infections including 8 (1.1%) with bacteremia. Among 655 admissions in 2013, 13 (2.0%) suffered from MSSA infections including 2 (0.3%) with bacteremia. Among 177 neonates screened for S. aureus carriage, overall 77 (44%) tested positive. A predominant PFGE-1-ST30 strain was identified in 6 of 30 infected neonates (20%) and 30 of 77 colonized neonates (39%). This persistent clone was pvl-negative, tst-positive and belonged to agr group III. We found no environmental point source. MSSA carriage was associated with central vascular catheter use but not with a particular midwife, nurse, physician, or isolette. Observed healthcare worker behavior may have propagated transmission via hands and fomites. Despite multimodal interventions, clonal transmission and colonization continued and another clone, PFGE-6-ST5, became predominant. Hospital-acquired MSSA clones represent a high proportion of MSSA colonization but not MSSA infections in neonate inpatients. In contrast to persisting MSSA, transmission infection rates decreased concurrently with interventions. It remains to be established whether eradication of hospital-acquired MSSA strains would reduce infection rates further.

Intensified colonisation screening according to the recommendations of the German Commission for Hospital Hygiene and Infectious Diseases Prevention (KRINKO): identification and containment of a Serratia marcescens outbreak in the neonatal intensive care unit, Jena, Germany, 2013-2014.

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Dawczynski, Kristin; Proquitté, Hans; Roedel, Jürgen; Edel, Brigit; Pfeifer, Yvonne; Hoyer, Heike; Dobermann, Helke; Hagel, Stefan; Pletz, Mathias W

2016-12-01

In 2013, the German Commission for Hospital Hygiene and Infectious Disease Prevention (KRINKO) stated that extending weekly colonisation screening from very low birth weight (VLBW) infants (Serratia marcescens. Strains were typed by Pulsed Field Gel Electrophoresis (PFGE). Over 6 months, 19 out of 159 infants acquired S. marcescens. Twelve of the nineteen patients with S. marcescens were non-VLBW infants, and they were colonised significantly earlier than were VLBW infants (median 17 vs. 28 days; p marcescens.

Research note: Molecular subtyping of Salmonella enterica serovar Tshiongwe recently isolated in Malaysia during 2001-2002.

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Thong, Kwai Lin; Bakeri, Shamsilawani Ahmad; Lai, Kin Seng; Koh, Yin Tee; Taib, Mohd Zainuldin; Lim, V K E; Yasin, Rohani Md

2004-03-01

Pulsed field gel electrophoresis (PFGE) and antimicrobial susceptibility analysis were undertaken on twenty-three strains of Salmonella enterica serovar Tshiongwe, an unusual serovar, which recently emerged in Malaysia. Antimicrobial susceptibility analysis showed that all the strains were sensitive to ampicilin, chloramphenicol, cotrimoxazole, and kanamycin. Twenty (87%) and 8 (3.5%) strains had resistance to tetracycline and streptomycin respectively. PFGE analysis subtyped 23 strains into 10 profiles (Dice coefficient of similarity, F = 0.7-1.0). The predominant profile, X1 was found in both clinical and environmental isolates and was widely distributed in different parts of Malaysia during the study period. In addition, isolates recovered from food, a hand-towel, apron and the surface of a table-top in one particular location had unique, indistinguishable profiles (X4/4a) and identical antibiograms. Similarly, isolates from cooked meat and a chopping board had PFGE profiles similar to some human isolates. These probably indicated cross-contamination and poor hygiene in food practices, hence contributing to Salmonellosis. Factors causing the emergence of this rare Salmonella serovar being responsible for food poisoning episodes during the study period remained unclear. The study reiterated the usefulness and versatility of PFGE in the molecular subtyping of this rare Salmonella serovar in Malaysia.

Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis

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Khan, Sharik R.; Kuzminov, Andrei

2013-01-01

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235

Genetic lineages of Salmonella enterica serovar Kentucky spreading in pet reptiles.

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Zając, Magdalena; Wasyl, Dariusz; Hoszowski, Andrzej; Le Hello, Simon; Szulowski, Krzysztof

2013-10-25

The purpose of the study was to define genetic diversity of reptilian Salmonella enterica serovar (S.) Kentucky isolates and their epidemiological relations to the ones from poultry, food, and environmental origin in Poland. Between 2010 and 2012 twenty-four S. Kentucky isolates derived from snakes (N=8), geckos (N=7), chameleons (N=4), agamas (N=1), lizard (N=1), and environmental swabs taken from reptile exhibition (N=3) were identified. They were characterized with antimicrobial minimal inhibitory concentration testing, XbaI-PFGE and MLST typing. The profiles compared to S. Kentucky available in BioNumerics local laboratory database (N=40) showed 67.3% of relatedness among reptile isolates. Three genetic lineages were defined. The first lineage gathered 20 reptile isolates with 83.4% of similarity and wild-type MICs for all antimicrobials tested but streptomycin in single case. The remaining three reptilian and one post-exhibition environment S. Kentucky isolates were clustered (87.2%) with isolates originating from poultry, mainly turkey, food, and environment and presented variable non-wild type MICs to numerous antimicrobials. The third S. Kentucky lineage was composed of two isolates from feed (96.3%). The results suggest diverse sources and independent routes of infection. Most of the isolates belonged to reptile-associated clones spread both horizontally and vertically. Simultaneously, PFGE profiles and MLST type indistinguishable from the ones observed in poultry point out carnivore reptiles as possible vector of infection with multidrug and high-level ciprofloxacin resistant (MIC≥8 mg/L) S. Kentucky. Public awareness and education are required to prevent potential reptile-associated S. Kentucky infections in humans. Copyright © 2013 Elsevier B.V. All rights reserved.

Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

International Nuclear Information System (INIS)

Kinashi, Yuko; Nagasawa, Hatsumi; Little, J.B.; Okayasu, Ryuichi; Iliakis, G.E.

1995-01-01

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing. 32 refs., 6 figs., 3 tabs

AVRUPA KAHVERENGİ TAVŞANI (Lepus europaeus Pallas,1778)’NIN DIŞKISINDA BULUNAN BAKTERİ TÜRLERİ

OpenAIRE

Özen, Ahmet Selçuk; Ercan Barış, Zeynep Maide; Yılmaz, Ayhan

2018-01-01

Bu çalışma, Avrupa Kahverengi tavşanının (Lepuseuropaeus) dışkısında bulunan bakteri türlerinin tespitine dayanmaktadır.Dışkıların laboratuvar analizi iki farklı periyotta gerçekleştirilmiştir.Sonuçta, Gram positifler (+) olarak Kocuira rosea, Aerococcus viridans,Staphylococcus warneri, Staphylococcus saprophyticus ile Bacillus cereus veGram negatif (-) olarak Rhizobium radiobacter, Klebsiella pneumoniae,Klebsiella oxytoca ve Echerichia coli olarak toplam dokuz bakteri türü tespit edilmiştir....

Antimicrobial susceptibility of Staphylococcus saprophyticus and urethral staphylococci.

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Marrie, T J; Kwan, C

1982-01-01

The activity of eight antimicrobial agents was determined against 115 isolates of Staphylococcus saprophyticus. All were susceptible to ampicillin, cephalexin, and trimethoprim-sulfamethoxazole and resistant to nalidixic acid and novobiocin. A bimodal pattern of susceptibility to erythromycin was observed: 80% were inhibited by 0.25 microgram/ml, whereas 13% required greater than or equal to 128 micrograms/ml. The following urethral staphylococci were susceptible to ampicillin, cephalexin, and nitrofurantoin but resistant to nalidixic acid: S. epidermidis, S. hominis, S. haemolyticus, S. warneri, S. simulans, and S. cohnii. PMID:6982679

Genomics of Staphylococcus

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Lindsay, Jodi A.

The staphylococci are Gram-positive cocci that divide to form clusters that look like grapes. By 16S ribosomal sequencing, they are most closely related to the Gram-positive, low G+C content Bacillus-Lactobacillus-Staphylococcus genera (Woese, 1987). There are over 30 species of staphylococci identified, and they are typically found on the skin and mucous membranes of mammals. About a dozen species are frequently carried on humans, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus cohnii, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus.

Isolation of coagulase-positive staphylococci from bitches' colostrum and milk and genetic typing of methicillin-resistant Staphylococcus pseudintermedius strains.

Science.gov (United States)

Rota, Ada; Corrò, Michela; Drigo, Ilenia; Bortolami, Alessio; Börjesson, Stefan

2015-07-23

Among the coagulase-positive, potentially pathogenic staphylococci, Staphylococcus pseudintermedius has been frequently isolated from bitches' milk. This organism colonizes the mammary gland or causes infection, while S. aureus has been only occasionally reported. The objective of this study was to investigate the occurrence and persistence of coagulase-positive staphylococci in the colostrum and milk of postpartum bitches, either treated or untreated with antimicrobials, and to assess the incidence, antibiotic resistance profile and genetic type of the methicillin-resistant strains. On postpartum D1, D7 and D15, drops of secretion were collected from the mammary glands of 27 postpartum bitches, nine of which were treated with antimicrobials. Coagulase-positive staphylococci were identified, antimicrobial susceptibility and the presence of mecA were tested and the genetic profile of methicillin-resistant strains was assessed. Staphylococcus pseudintermedius was the only coagulase-positive staphylococcus isolated, and its presence was detected in 21 out of 27 bitches and in 66 out of 145 swabs. In a single bitch, it caused puerperal mastitis. In untreated bitches, the frequency of isolation was lower in colostrum than in milk. All of the isolates except one were resistant to at least three antimicrobial classes, while 14 out of 66 S. pseudintermedius strains were methicillin-resistant mecA positive (MRSP) and were isolated from eight bitches housed in the same breeding kennel. A significant association was found between antimicrobial treatment and the presence of MRSP. Six of the 12 typed isolates belonged to spa-type t02 carrying SCCmec II/III, and another six were non-typeable with spa carrying SCCmec IV. The t02-SCCmec II/III isolates were sequence type (ST) 71; four NT-SCCmec IV isolates were ST258 and two were ST369. PFGE showed that isolates from the same dog had identical band patterns, while isolates from different dogs had unique band patterns. MRSP strains

Nasal carriage of Methicillin-resistant Staphylococcus aureus among healthy population of Kashmir, India

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B A Fomda

2014-01-01

Full Text Available Background: Nasal colonisation with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA is being increasingly reported, especially in places where people are in close contact and where hygiene is compromised. The aim of this study was to find out prevalence of methicillin resistant S.aureus (MRSA colonising anterior nares of healthy subjects. Materials and Methods: Nasal swabs of healthy subjects were collected aseptically and cultured using standard microbiological protocols. Antibiotic susceptibility was done by Kirby-Bauer disc diffusion method according to CLSI guidelines. Methicillin resistance was detected by cefoxitin disc diffusion method and confirmed by minimum inhibitory concentration (MIC and amplification of mecA gene by PCR. Strain typing of MRSA strains was done by PFGE. Results: Out of 820 samples, S.aureus was isolated from 229 (27.92% subjects. Of the 229 isolates, 15 were methicillin resistant. All S. aureus isolates were susceptible to vancomycin. Nasal carriage of MRSA was found to be 1.83% among healthy population. The isolates were found to be polyclonal by PFGE analysis. Conclusion: High prevalence of MRSA is a cause of concern and strategies to interrupt transmission should be implemented.

Molecular Analysis of Mycobacterium avium Isolates by Using Pulsed-Field Gel Electrophoresis and PCR

Science.gov (United States)

Pestel-Caron, Martine; Graff, Gabriel; Berthelot, Gilles; Pons, Jean-Louis; Lemeland, Jean-François

1999-01-01

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships. PMID:10405383

Genotyping of Methicillin Resistant Staphylococcus aureus Strains Isolated from Hospitalized Children

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Mouna Ben Nejma

2014-01-01

Full Text Available Community associated methicillin resistant Staphylococcus aureus (CA-MRSA is an emerging pathogen increasingly reported to cause skin and soft tissue infections for children. The emergence of highly virulencet CA-MRSA strains in the immunodeficiency of young children seemed to be the basic explanation of the increased incidence of CA-MRSA infections among this population. The subjects of this study were 8 patients hospitalized in the Pediatric Department at the University Hospital of Monastir. The patients were young children (aged from 12 days to 18 months who were suffering from MRSA skin infections; two of them had the infections within 72 h of their admission. The isolates were classified as community isolates as they all carried the staphylococcal cassette chromosome mec (SCCmec IV and pvl genes. Epidemiological techniques, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST, were applied to investigate CA-MRSA strains. Analysis of molecular data revealed that MRSA strains were related according to PFGE patterns and they belonged to a single clone ST80. Antimicrobial susceptibility tests showed that all strains were resistant to kanamycin and 2 strains were resistant to erythromycin.

Transposons and integrons in colistin-resistant clones of Klebsiella pneumoniae and Acinetobacter baumannii with epidemic or sporadic behaviour.

Science.gov (United States)

Arduino, Sonia M; Quiroga, María Paula; Ramírez, María Soledad; Merkier, Andrea Karina; Errecalde, Laura; Di Martino, Ana; Smayevsky, Jorgelina; Kaufman, Sara; Centrón, Daniela

2012-10-01

Multiple transposons, integrons and carbapenemases were found in Klebsiella pneumoniae colistin-resistant isolates as well as a genomic resistance island of the AbaR type in Acinetobacter baumannii colistin-resistant isolates from different hospitals from Buenos Aires City. PFGE analysis showed a polyclonal dissemination of antimicrobial resistance mechanisms among K. pneumoniae isolates, while in A. baumannii isolates the epidemic clone 1 from South America was found. Resistance determinants associated with horizontal gene transfer are contributing to the evolution to pandrug resistance in both epidemic and sporadic clones.

Characterization of Extended spectrum beta-lactamases (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of 3rd or 4th generation cephalosporins

DEFF Research Database (Denmark)

Hammerum, Anette M.; Larsen, Jesper; Dalhoff Andersen, Vibe

2014-01-01

-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-b-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST......Objectives: To compare and characterize extended-spectrum b-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. Methods: Twenty farms with no third- or fourth...

Genetic Diversity of Clinical and Environmental Strains of Salmonella enterica Serotype Weltevreden Isolated in Malaysia

OpenAIRE

Thong, K. L.; Goh, Y. L.; Radu, S.; Noorzaleha, S.; Yasin, R.; Koh, Y. T.; Lim, V. K. E.; Rusul, G.; Puthucheary, S. D.

2002-01-01

The incidence of food-borne salmonellosis due to Salmonella enterica serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of Salmonella serotype Weltevreden strains from humans and the environment. PFGE of XbaI-digested chromosomal DNA from 95 strains of Salmonella serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.2...

Clonal spread of MRSA CC398 sublineages within and between Danish pig farms

DEFF Research Database (Denmark)

Espinosa-Gongora, Carmen; Larsen, Jesper; Moodley, Arshnee

Background: Methicillin-resistant Staphylococcus aureus (MRSA) CC398 is non-typeable by standard pulsed-field gel electrophoresis (PFGE) due to methylation of the SmaI site. This makes it difficult to study the epidemiology of this livestock-associated MRSA clone. In this study, we employed...... a recently developed PFGE protocol using Cfr9I, a neoschizomer of SmaI, to investigate the diversity of MRSA CC398 in Danish pig farms. The PFGE profiles displayed by isolates from pigs, environmental samples and farm workers were compared in order to understand whether farms are contaminated with multiple...... MRSA CC398 sublineages and whether specific sublineages may occur on different farms. Methods: A cross sectional study was performed in five Danish pig farms where farm workers had been shown to carry MRSA CC398 in the previous year. A total of 75 environmental and 308 animal samples were collected...

Characterization of Clostridium perfringens isolates from healthy turkeys and from turkeys with necrotic enteritis

DEFF Research Database (Denmark)

Lyhs, Ulrike; Perko-Mäkelä, P.; Kallio, H.

2013-01-01

from 1998 to 2012. Furthermore, C. perfringens isolates from healthy and diseased turkeys were characterized and their genetic diversity was investigated using pulsed-field gel electrophoresis (PFGE). Isolates (n = 212) from birds with necrotic gut lesions and from healthy flocks of 30 commercial...... turkey farms were characterized for the presence of cpa, cpb, iA, etx, cpb2, and cpe and netB genes. A total of 93 C. perfringens isolates, including 55 from birds with necrotic gut lesions and 38 from healthy birds from 13 different farms, were analyzed with PFGE. All contract turkey farmers (n = 48......) of a turkey company that produces 99% of domestic turkey meat in Finland were interviewed about background information, management at the farm, and stress factors related to NE outbreaks. Pulsed-field gel electrophoresis analysis with SmaI restriction enzyme resulted in 30 PFGE patterns among the 92 C...

Pulsed-field gel electrophoresis of Staphylococcus hyicus and Staphylococcus chromogenes genomic DNA and its taxonomic, epidemiologic and ecologic applications in veterinary medicine.

Science.gov (United States)

Shimizu, A; Kloos, W E; Berkhoff, H A; George, C G; Ballard, D N

1997-06-01

One hundred and thirty-eight strains of Staphylococcus hyicus and 21 strains of S. chromogenes isolated from animals were analyzed by pulsed-field gel electrophoresis (PFGE) after restriction endonuclease Smal digestion of chromosomal DNA. Eighty-eight strains of S. hyicus from pigs with or without exudative epidermitis (EE) generated 16 to 26 fragments in the size range of chromogenes from pigs and cows generated 17 to 24 fragments ranging from chromogenes strains were more highly conserved than those of S. hyicus. S. chromogenes strains could be distinguished from S. hyicus strains by fragments within the range of 305 to 545 kb. The results indicate that PFGE analysis could be used to distinguish between S. hyicus and S. chromogenes. We conclude that PFGE analysis is a useful tool not only for species or strain identification but also for epidemiologic or ecologic studies of S. hyicus and S. chromogenes.

[Optimization of cluster analysis based on drug resistance profiles of MRSA isolates].

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Tani, Hiroya; Kishi, Takahiko; Gotoh, Minehiro; Yamagishi, Yuka; Mikamo, Hiroshige

2015-12-01

We examined 402 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from clinical specimens in our hospital between November 19, 2010 and December 27, 2011 to evaluate the similarity between cluster analysis of drug susceptibility tests and pulsed-field gel electrophoresis (PFGE). The results showed that the 402 strains tested were classified into 27 PFGE patterns (151 subtypes of patterns). Cluster analyses of drug susceptibility tests with the cut-off distance yielding a similar classification capability showed favorable results--when the MIC method was used, and minimum inhibitory concentration (MIC) values were used directly in the method, the level of agreement with PFGE was 74.2% when 15 drugs were tested. The Unweighted Pair Group Method with Arithmetic mean (UPGMA) method was effective when the cut-off distance was 16. Using the SIR method in which susceptible (S), intermediate (I), and resistant (R) were coded as 0, 2, and 3, respectively, according to the Clinical and Laboratory Standards Institute (CLSI) criteria, the level of agreement with PFGE was 75.9% when the number of drugs tested was 17, the method used for clustering was the UPGMA, and the cut-off distance was 3.6. In addition, to assess the reproducibility of the results, 10 strains were randomly sampled from the overall test and subjected to cluster analysis. This was repeated 100 times under the same conditions. The results indicated good reproducibility of the results, with the level of agreement with PFGE showing a mean of 82.0%, standard deviation of 12.1%, and mode of 90.0% for the MIC method and a mean of 80.0%, standard deviation of 13.4%, and mode of 90.0% for the SIR method. In summary, cluster analysis for drug susceptibility tests is useful for the epidemiological analysis of MRSA.

Molecular epidemiology and spatiotemporal analysis of hospital-acquired Acinetobacter baumannii infection in a tertiary care hospital in southern Thailand.

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Chusri, S; Chongsuvivatwong, V; Rivera, J I; Silpapojakul, K; Singkhamanan, K; McNeil, E; Doi, Y

2017-01-01

Acinetobacter baumannii is a major hospital-acquired pathogen in Thailand that has a negative effect on patient survival. The nature of its transmission is poorly understood. To investigate the genotypic and spatiotemporal pattern of A. baumannii infection at a hospital in Thailand. The medical records of patients infected with A. baumannii at an 800-bed tertiary care hospital in southern Thailand between January 2010 and December 2011 were reviewed retrospectively. A. baumannii was identified at the genomospecies level. Carbapenemase genes were identified among carbapenem-resistant isolates associated with A. baumannii infection. A spatiotemporal analysis was performed by admission ward, time of infection and pulsed-field gel electrophoresis (PFGE) groups of A. baumannii. Nine PFGE groups were identified among the 197 A. baumannii infections. All A. baumannii isolates were assigned to International Clonal Lineage II. bla OXA-23 was the most prevalent carbapenemase gene. Outbreaks were observed mainly in respiratory and intensive care units. The association between PFGE group and hospital unit was significant. Spatiotemporal analysis identified 20 clusters of single PFGE group infections. Approximately half of the clusters involved multiple hospital units simultaneously. A. baumannii transmitted both within and between hospital wards. Better understanding and control of the transmission of A. baumannii are needed. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Ready-to-eat street-vended food as a potential vehicle of bacterial pathogens and antimicrobial resistance: An exploratory study in Porto region, Portugal.

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Campos, Joana; Gil, Joana; Mourão, Joana; Peixe, Luísa; Antunes, Patrícia

2015-08-03

The ready-to-eat street vending commerce, as street mobile food vendors, has grown exponentially worldwide, representing in some countries a significant proportion of food consumed by the urban population. However, the microbiological food safety hazards of mobile vending units in industrialized countries are scarcely evaluated. To assess the microbiological quality and safety of this type of food and try to achieve the connection of its contamination with hygienic conditions of food-handlers, we analyzed hotdogs (n = 10), hamburgers (n = 10) and hands (n = 9) from ten street-vending trailers in the Porto region. Food and food-handler samples were tested for Enterobacteriaceae and coliform counts, Escherichia coli and coagulase-positive staphylococci counts/detection and presence of Salmonella. Aerobic mesophilic counts and detection of Listeria monocytogenes (Pulsed Field Gel Electrophoresis-PFGE and serotyping) were also tested in food samples. E. coli isolates were confirmed by MALDI-TOF and characterized for clonality (phylogenetic groups-PhG, PFGE and Multilocus Sequence Typing), antibiotic resistance (disk diffusion, PCR/sequencing) and intestinal pathogenic virulence factors (PCR/sequencing). All food samples presented poor microbiological quality (100% Enterobacteriaceae and coliforms; 20% E. coli (4 hamburgers, 4 trailers) and 20% (2 hamburgers/2 hotdogs, 3 trailers) were positive for L. monocytogenes (2 PFGE-types belonging to serotype 1/2a and 4b). Salmonella and coagulase-positive staphylococci were not detected. Food-handlers carried Enterobacteriaceae and coliforms (100%), E. coli (11%) and/or coagulase-positive staphylococci (44%). E. coli was detected in 12 samples (n = 30-food/food-handlers; phylogenetic groups A0/A1/B1) with 33% resistant to one or more antibiotics. Two multidrug resistant atypical E. coli pathotype strains (astA-ST165(CC165)/food-handler, eaeA-ST327/food) were detected. Three out of eight E. coli clonal lineages [ST409/ST976(CC10

Diversity of Campylobacter in retail meat and liver of lambs and goat kids.

Science.gov (United States)

Lazou, Thomai; Dovas, Chrysostomos; Houf, Kurt; Soultos, Nikolaos; Iossifidou, Eleni

2014-04-01

The presence, genetic diversity, and antimicrobial susceptibility profile of Campylobacter spp. in retail lamb and goat kid carcasses were assessed. A total of 200 samples consisting of 100 meat and 100 liver surface swabs were collected from 47 lamb and 53 goat kid carcasses at 23 retail markets in Northern Greece, and 125 Campylobacter isolates were recovered from 32 meat surfaces (32%) and 44 liver surfaces (44%). Multiplex polymerase chain reaction and restriction fragment length polymorphism analysis specified Campylobacter coli as the most frequently detected species (59.2%) followed by C. jejuni (40.8%). Pulsed-field gel electrophoresis (PFGE) was applied in order to typify a subset of randomly selected isolates (n=80). SmaI-PFGE successfully clustered the 80 isolates in 38 SmaI-PFGE types, indicating high heterogeneity among the analyzed Campylobacter isolates, and provided data regarding the dissemination of Camplobacter among carcasses stored in the same retail market. Antimicrobial susceptibility profiles of Campylobacter isolates, assessed by the disk-diffusion method, indicated that 31 isolates (24.8%) were multidrug resistant, and the most common profile was the concurrent resistance to tetracycline and streptomycin. Overall, 56.8% of isolates (n=71, multidrug-resistant isolates included) exhibited resistance to at least one antimicrobial (tetracycline 34.4%, quinolones 27.2%, and streptomycin 20.8%). However, all isolates were susceptible to erythromycin and gentamicin. The findings of this study verify the contamination of retail lamb and goat kid carcasses with a heterogeneous population of thermotolerant campylobacters. These data underscore the fact that retail meat and liver of small ruminants could serve as vehicles for consumer contamination with Campylobacter and that further investigation is necessary in order to evaluate the risk imposed by such products within the epidemiology of human campylobacteriosis cases.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle.

Science.gov (United States)

Pringle, Märit; Bergsten, Christer; Fernström, Lise-Lotte; Höök, Helena; Johansson, Karl-Erik

2008-10-20

Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

Directory of Open Access Journals (Sweden)

Höök Helena

2008-10-01

Full Text Available Abstract Background Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. Methods Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE, random amplified polymorphic DNA (RAPD and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. Results Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. Conclusion This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.

Stacking gels: A method for maximising output for pulsed-field gel electrophoresis

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Heng See

2009-01-01

Full Text Available Pulsed field gel electrophoresis (PFGE, the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.

Genotyping of methicillin-resistant Staphylococcus aureus in the Sultan Qaboos University Hospital, Oman reveals the dominance of Panton–Valentine leucocidin-negative ST6-IV/t304 clone

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E.E. Udo

2014-07-01

Full Text Available The objective of this study was to determine the prevalence and distribution of methicillin-resistant Staphylococcus aureus (MRSA genotypes circulating at a tertiary hospital in the Sultanate of Oman. A total of 79 MRSA isolates were obtained from different clinical samples and investigated using antibiogram, pulsed-field gel electrophoresis (PFGE, staphylococcal chromosome cassette mec (SCCmec, Spa typing and multilocus sequence typing (MLST. The isolates were susceptible to linezolid, vancomycin, teicoplanin, tigecycline and mupirocin but were resistant to tetracycline (30.4%, erythromycin (26.6%, clindamycin (24.1%, trimethoprim (19.0%, ciprofloxacin (17.7%, fusidic acid (15.2% and gentamicin (12.7%. Molecular typing revealed 19 PFGE patterns, 26 Spa types and 21 sequence types. SCCmec-IV (86.0% was the dominant SCCmec type, followed by SCCmec-V (10.1%. SCCmec-III (2.5% and SCCmec-II (1.3% were less common. ST6-IV/t304 (n = 30 and ST1295-IV/t690 (n = 12 were the dominant genotypes followed by ST772-V/t657 (n = 5, ST30-IV/t019/t021 (n = 5, ST22-IV/t852 (n = 4, ST80-IV/t044 (n = 3 and 18 single genotypes that were isolated sporadically. On the basis of SCCmec typing and MLST, 91.2% of the isolates were classified as community-associated MRSA and 8.8% of the isolates (consisting of four ST22-IV/t852, one ST239-III/t632, one ST5-III/t311 and one ST5-II/t003 were classified as healthcare-associated MRSA. The study has revealed the dominance of a Panton–Valentine leucocidin-negative ST6-IV/t304 clone and provided insights into the distribution of antibiotic resistance in MRSA at the tertiary hospital in Oman. It also highlights the importance of surveillance in detecting the emergence of new MRSA clones in a healthcare facility.

Demonstration of persistent contamination of a cooked egg product production facility with Salmonella enterica serovar Tennessee and characterization of the persistent strain.

Science.gov (United States)

Jakočiūnė, D; Bisgaard, M; Pedersen, K; Olsen, J E

2014-08-01

The aim of this study was to investigate whether continuous contamination of light pasteurized egg products with Salmonella enterica serovar Tennessee (S. Tennessee) at a large European producer of industrial egg products was caused by persistent contamination of the production facility and to characterize the persistent strains. Seventy-three S. Tennessee isolates collected from products over a 3-year period with intermittent contamination, and 15 control strains were compared by pulsed field gel electrophoresis (PFGE) using two enzymes. Forty-five case isolates distributed throughout the full period were shown to belong to one profile type. Isolates representing different PFGE profiles were all assigned to ST 319 by multilocus sequence typing (MLST). The case isolates did not show a higher ability to form biofilm on a plastic surface than noncase isolates. Characteristically, members of the persistent clone were weak producers of H2 S in laboratory medium. S. Tennessee isolated from the case was able to grow better in pasteurized egg product compared with other serovars investigated. It was concluded that the contamination was caused by a persistent strain in the production facility and that this strain apparently had adapted to grow in the relevant egg product. S. Tennessee has previously been associated with persistence in hatching facilities. This is the first report of persistent contamination of an egg production facility with this serovar. © 2014 The Society for Applied Microbiology.

Microbiological and molecular characterization of human clinical isolates of Staphylococcus cohnii, Staphylococcus hominis, and Staphylococcus sciuri.

Science.gov (United States)

Garza-González, Elvira; Morfin-Otero, Rayo; Martínez-Vázquez, Manuel A; Gonzalez-Diaz, Esteban; González-Santiago, Omar; Rodríguez-Noriega, Eduardo

2011-12-01

The incidence of coagulase-negative staphylococci reported as causative agents of nosocomial infections has risen in the last decade. The aim of this study was to characterize biofilm formation, antibiotic resistance, SCCmec type, and genetic relatedness in clinical isolates of Staphylococcus cohnii, Staphylococcus hominis, and Staphylococcus sciuri recovered from humans. Clinically relevant isolates of S. cohnii (n = 15), S. hominis (n = 9), and S. sciuri (n = 6), were collected from patients. Biofilm formation was evaluated using crystal violet staining, drug susceptibility was assessed using the broth microdilution method, and methicillin resistance was measured using the cefoxitin disk test. SCCmec was typed using 2 different methodologies, and genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Sixty percent (9/15) of S. cohnii, 33% (3/9) of S. hominis, and 50% (3/6) of S. sciuri isolates were categorized as weak producers of biofilm. None of the isolates were resistant to vancomycin or linezolid. All 3 species showed a high resistance (> 66%) to ampicillin, levofloxacin, erythromycin, and ceftriaxone, and the majority of the isolates were methicillin-resistant. PFGE revealed that the S. cohnii isolates comprised 1 dominant clone. The S. cohnii, S. hominis, and S. sciuri isolates analyzed in this study showed a high methicillin resistance and resistance to other antimicrobials. The results of this study strongly suggest that coagulase-negative staphylococci harbour new SCCmec elements. We report the first case of a clone of S. cohnii associated with human disease.

Epidemiology and resistance features of Acinetobacter baumannii isolates from the ward environment and patients in the burn ICU of a Chinese hospital.

Science.gov (United States)

Gong, Yali; Shen, Xiaodong; Huang, Guangtao; Zhang, Cheng; Luo, Xiaoqiang; Yin, Supeng; Wang, Jing; Hu, Fuquan; Peng, Yizhi; Li, Ming

2016-08-01

Acinetobacter baumannii is an important opportunistic pathogen that causes severe nosocomial infections, especially in intensive care units (ICUs). Over the past decades, an everincreasing number of hospital outbreaks caused by A. baumannii have been reported worldwide. However, little attention has been directed toward the relationship between A. baumannii isolates from the ward environment and patients in the burn ICU. In this study, 88 A. baumannii isolates (26 from the ward environment and 62 from patients) were collected from the burn ICU of the Southwest Hospital in Chongqing, China, from July through December 2013. Antimicrobial susceptibility testing results showed that drug resistance was more severe in isolates from patients than from the ward environment, with all of the patient isolates being fully resistant to 10 out of 19 antimicrobials tested. Isolations from both the ward environment and patients possessed the β-lactamase genes bla OXA-51, bla OXA-23, bla AmpC, bla VIM, and bla PER. Using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), these isolates could be clustered into 4 major PFGE types and 4 main sequence types (ST368, ST369, ST195, and ST191) among which, ST368 was the dominant genotype. Epidemiologic and molecular typing data also revealed that a small-scale outbreak of A. baumannii infection was underway in the burn ICU of our hospital during the sampling period. These results suggest that dissemination of β-lactamase genes in the burn ICU might be closely associated with the high-level resistance of A. baumannii, and the ICU environment places these patients at a high risk for nosocomial infection. Cross-contamination should be an important concern in clinical activities to reduce hospitalacquired infections caused by A. baumannii.

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain.

Science.gov (United States)

Solà-Ginés, Marc; Cameron-Veas, Karla; Badiola, Ignacio; Dolz, Roser; Majó, Natalia; Dahbi, Ghizlane; Viso, Susana; Mora, Azucena; Blanco, Jorge; Piedra-Carrasco, Nuria; González-López, Juan José; Migura-Garcia, Lourdes

2015-01-01

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-2. Two strains harboured qnrA, and two qnrA together with aac(6')-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured blaCMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain.

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Marc Solà-Ginés

Full Text Available Avian pathogenic Escherichia coli (APEC are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE and multi-locus sequence typing (MLST. The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-2. Two strains harboured qnrA, and two qnrA together with aac(6'-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured blaCMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.

Foods from black market at EU border as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

Science.gov (United States)

Oniciuc, Elena-Alexandra; Ariza-Miguel, Jaime; Bolocan, Andrei-Sorin; Diez-Valcarce, Marta; Rovira, Jordi; Hernández, Marta; Fernández-Natal, Isabel; Nicolau, Anca Ioana; Rodríguez-Lázaro, David

2015-09-16

The illegal entrance of foods to EU through black markets at the EU borders can constitute a neglected route of dissemination of foodborne pathogens, and in particular of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we have assessed the presence of MRSA in foods sold in a black market at an EU border (the southeast part of Romania, on the border with Republic of Moldavia). We performed a search for MRSA among 200 food samples collected from 2012 to 2013. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST) and SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 32 S. aureus isolates were recovered from 16 food samples (8%). One isolate detected in a pork lard sample was MRSA (0.5%). PFGE with the restriction enzyme SmaI revealed 12 genotypes among the 32 S. aureus isolates. The MRSA isolate belonged to sequence type 398, harbored SCCmec type V, tested negative for the presence of the PVL genes and was resistant to ciprofloxacin, tetracycline and cefazolin, besides all β-lactams. Among 31 methicillin-sensitive S. aureus (MSSA), 29% were resistant to penicillin, 9.7% to tetracycline and 3.2% to ciprofloxacin. In conclusion, in this study we report the presence of livestock-associated MRSA in foods sold in a black market at an EU border: ST398-MRSA-V. These results confirm the potential role of food in the dissemination of MRSA lineages among population, and the potential role of illegally introduced food to EU in the prevalence and evolution of MRSA clones in the community. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells

International Nuclear Information System (INIS)

Wlodek, D.; Banath, J.; Olive, P.L.

1991-01-01

Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5Gy of radiation. Low voltage (ca.0.6V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage. (author)

Contamination of lettuce with antibiotic resistant E. coli after slurry application

DEFF Research Database (Denmark)

Jensen, Annette Nygaard; Storm, Christina; Baggesen, Dorte Lau

2011-01-01

Due to disease outbreaks associated with contaminated vegetables it has been speculated to what extent this may be linked with application of animal manure as fertilizer, which is particularly practiced in organic vegetable production where conventional fertilizers are prohibited. A field survey......, where 20% of faecally contaminated samples contained >100 E. coli/g. This indicates that fecal contamination of crops originated from alternative sources such as contaminated water or wildlife. This was supported by genotyping of E. coli, where half of the 21 PFGE types were found on single occasions...

Pathogenicity Assay of Vibrio harveyi in Tiger Shrimp Larvae Employing Rifampicin-Resistant as A Molecular Marker

OpenAIRE

. Widanarni; D. Meha; Sri Nuryati; . Sukenda; A. Suwanto

2007-01-01

Rifampicin-resistant marker was employed as a reporter to assay pathogenicity of Vibrio harveyi  in shrimp larvae.  V. harveyi M. G3 and G7 that difference not schizotyping as shown by Pulsed-Filed Gel Electrophoresis (PFGE) used in this study. Spontaneous mutation was conducted to generate V. harveyi resistant to rifampicin. Two groups of shrimp post-larvae (PL5) were immersed for 30 min in 106 CFU/ml of mutants and wild type of V. harveyi, respectively; and then placed in a 2 liter shrimp r...

Methods to classify bacterial pathogens in cystic fibrosis

DEFF Research Database (Denmark)

Bjarnsholt, Thomas; Nielsen, Xiaohui Chen; Johansen, Ulla

2011-01-01

for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aeruginosa. A commercially available ELISA method is described for measuring IgG antibodies against P. aeruginosa in CF......Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch's criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients...

Clonal outbreaks of [ Pasteurella] pneumotropica biovar Heyl in two mouse colonies

DEFF Research Database (Denmark)

Adhikary, Sadhana; Bisgaard, Magne; Dagnæs-Hansen, Frederik

2017-01-01

The aim of this study was to document the pathogenic role of biovar Heyl of [Pasteurella] pneumotropica in mouse colonies. Fifty-three isolates associated with mastitis and orbital, cutaneous and vaginal abscesses as well as isolates from the nose and vagina of healthy mice were investigated...... strains with the same rpoB sequence type, as shown by the PFGE profiles. The investigation documented that members of biovar Heyl of [P.] pneumotropica caused disease outbreaks in mouse colonies since the clonality indicated a primary role of [P.] pneumotropica biovar Heyl in the infections observed....

Prevalence and genotypic relatedness of methicillin resistant Staphylococcus aureus in a tertiary care hospital

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B A Fomda

2014-01-01

Full Text Available Background: Methicillin-resistant Staphylococcus aureus (MRSA is the most common multidrug-resistant pathogen causing nosocomial infections across the world. MRSA is not only associated with significant mortality and morbidity but also places a large economic strain on our health care system. MRSA isolates are also typically resistant to multiple, non-β-lactam antibiotics. We conducted a prospective study in a tertiary care hospital, to determine the prevalence of MRSA and to establish the clonal distribution of MRSA isolates recovered from various clinical specimens. Materials and Methods: Clinical samples were cultured and S. aureus was identified as per standard microbiological procedures. Susceptibility testing was done by agar disk diffusion and minimum inhibitory concentration (MIC method as recommended by CLSI. Methicillin resistance was detected by phenotypic methods namely, oxacillin disc diffusion (ODD, minimum inhibitory concentration (MIC of oxacillin, cefoxitin disk diffusion (CDD, and MIC of cefoxitin. Amplification of mecA gene by PCR was used as gold standard for detection of methicillin resistance. Pulsed field gel electrophoresis (PFGE typing was performed for MRSA isolates. Results: Out of 390 S. aureus isolates, 154 (39.48% isolates were MRSA and 236 (60.51% isolates were MSSA. Penicillin was the least effective antibacterial drug against the hospital associated S. aureus isolates with 85.64% resistance rate. All the isolates were susceptible to vancomycin. The MRSA showed a high level of resistance to all antimicrobials in general in comparison to the MSSA and the difference was statistically significant (P < 0.05. Multiplex PCR performed for all strains showed amplification of both the mecA and nucA genes in MRSA strains whereas MSSA strains showed amplification of only nucA gene. PFGE of these isolates showed 10 different patterns. Conclusion: Prevalence of MRSA in our hospital was 39.48%. Most of these isolates were

A study of coagulase-negative staphylococci (CoNS isolated from bovine mastitis for the presence of penicillin and methicillin resistance-encoding genes in the north west of Iran

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Dastmalchi Saei, H.

2014-11-01

Full Text Available Coagulase-negative staphylococci (CoNS are often associated with bovine mastitis and may be resistant to antimicrobial therapy. The aim of the current study was to investigate the presence of blaZ (responsible for penicillin resistance and mecA (responsible for methicillin resistance genes among 108 CoNS belonging to 9 different species isolated from bovine mastitis in seven dairy herds (H1-H7. Of 108 CoNS isolates, 44 were Staphylococcus haemolyticus, 17 S. chromogenes, 11 S. epidermidis, 11 S. warneri, 11 S. cohnii, 6 S. simulans, 4 S. hominis, 3 S. capitis, and 1 S. xylosus. The blaZ was detected in 65.7% (n=71 of all Staphylococcus spp. isolates. Five isolates were positive for the presence of mecA gene (4.6%, including 2 S. hominis, 1 S. haemolyticus, 1 S. epidermidis, and 1 S. warneri. All mecA-carrying CoNS were also positive for the blaZ gene and were recovered from two studied herds (H3 and H6. Some variations were also observed in distribution of both blaZ and mecA genes between CoNS species. This study demonstrates that CoNS from bovine mastitis can be reservoirs of blaZ gene. This study also provides evidence of the presence of methicillin resistant CoNS (MR-CoNS and emphasizes the need for their epidemiological monitoring, in order to prevent the risk of spread to human through direct contact and/or consumption of contaminated food.

Antibiotic Susceptibility of Coagulase-Negative Staphylococci Isolated from Bovine Subclinical Mastitis in Turkey

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Beytullah Kenar*, Yahya Kuyucuoğlu and Esra Şeker

2012-06-01

Full Text Available A total of 572 California Mastitis Test (CMT positive milk samples were collected from 423 lactating cows on 18 private farms in the Middle Western Anatolia. Coagulase–negative staphylococci colonies and CNS species identification was performed based on conventional biochemical techniques and using the API Staph test. Slime production was detected by Congo Red Agar (CRA method. The antibiotic susceptibility was determined according to the National Committee for Clinical Laboratory Standards guidelines (NCCLS. A total of 67 (11.7% coagulase-negative staphylococci (CNS were isolated from CMT positive milk samples. In total, 11 CNS species: S. epidermidis (n=18, S. simulans (n=14, S. warneri (n=10, S. hominis (n=5, S. chromogenes (n=4, S. caprae (n=4, S. xylosus (n=3, S. haemolyticus (n=3, S. hyicus (n=3, S. cohnii (n=2, and S. capitis (n=1 were identified. The most commonly identified CNS species were Staphylococcus epidermidis (26.8% and Staphylococcus simulans (20.8% followed by Staphylococcus warneri (14.9%. Out of 67 CNS isolates, slime production was found in 37 (55.2% CNS strains. CNS isolates were the most resistance to trimethoprim+sulphamethoxazole (76.2%, erythromycin (73.2%, oxacillin and ampicillin (70.2% followed by penicillin (58.3%, gentamicin (53.8%, tetracycline (52.3%, vancomycin (51.8%, ciprofloxacin (26.9%, cefoxitim (23.9%, and cephalothin (13.5%. These results indicate that CNS species are resistant at high rates to the beta-lactam antibiotics which are intensively used in the prevention and treatment of mastitis without any antibiotic susceptibility test in the Middle Western of Turkey.

Phenotypical and Genotypical Antimicrobial Resistance of Coagulase-negative staphylococci Isolated from Cow Mastitis.

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Klimiene, I; Virgailis, M; Pavilonis, A; Siugzdiniene, R; Mockeliunas, R; Ruzauskas, M

2016-09-01

The objectives of this study were to determine the prevalence and antimicrobial resistance of coagulase-negative staphylococci (CNS) isolated from dairy cows with subclinical mastitis. Antimicrobial resistance in staphylococci were evaluated by breakpoint values specific to the species (EU-CAST). The presence of resistance-encoding genes was detected by multiplex PCR. A total of 191 CNS isolates were obtained. The CNS isolates were typically resistant to penicillin (67.4%), tetracyc-line (18.9%), and erythromycin (13.7%). CNS isolates (78.0%) were resistant to at least one antimicrobial compound, and 22.0% were multiresistant. The multiresistant isolates were predominantly Staphylococcus chromogenes (28.6%), Staphylococcus warneri (19%) and Staphylococcus haemolyticus (14.3%). According to MIC pattern data, multiresistant isolates showed the highest resistance (p<0.05) rates to penicillin (85.7%), tetracycline (66.7%), and erythromycin (48.2%), but all of them were sensitive to daptomycin, oxacillin, qiunupristin/dalfopristin, and vancomycin. S. chromogenes (9.5%), S. haemolyticus (4.8%), and S. capitis ss capitis (2.4%) strains were resistant to methicillin; their resistance to oxacillin and penicillin was more than 8 mg/l. A high rate of resistance to penicillin was linked to a blaZ gene found in 66.6% of the isolated multiresistant CNS strains. Resistance to tetracycline via the tetK (38.1%) gene and penicillin via the mecA (23.8%) gene were detected less frequently. Gene msrAB was responsible for macrolides and lincosamides resistance and detected in 28.6% of the CNS isolates. Antimicrobial resistance genes were identified more frequently in S. epidermidis, S. chromogenes, and S. warneri.

VNTR analysis reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of contagious agalactia

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Ayling Roger D

2008-11-01

Full Text Available Abstract Background Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. Previous studies of M. agalactiae have shown it to be unusually homogeneous and there are currently no available epidemiological techniques which enable a high degree of strain differentiation. Results We have developed variable number tandem repeat (VNTR analysis using the sequenced genome of the M. agalactiae type strain PG2. The PG2 genome was found to be replete with tandem repeat sequences and 4 were chosen for further analysis. VNTR 5 was located within the hypothetical protein MAG6170 a predicted lipoprotein. VNTR 14 was intergenic between the hypothetical protein MAG3350 and the hypothetical protein MAG3340. VNTR 17 was intergenic between the hypothetical protein MAG4060 and the hypothetical protein MAG4070 and VNTR 19 spanned the 5' end of the pseudogene for a lipoprotein MAG4310 and the 3' end of the hypothetical lipoprotein MAG4320. We have investigated the genetic diversity of 88 M. agalactiae isolates of wide geographic origin using VNTR analysis and compared it with pulsed field gel electrophoresis (PFGE and random amplified polymorphic DNA (RAPD analysis. Simpson's index of diversity was calculated to be 0.324 for PFGE and 0.574 for VNTR analysis. VNTR analysis revealed unexpected diversity within M. agalactiae with 9 different VNTR types discovered. Some correlation was found between geographical origin and the VNTR type of the isolates. Conclusion VNTR analysis represents a useful, rapid first-line test for use in molecular epidemiological analysis of M. agalactiae for outbreak tracing and control.

Two Distinct Yersinia pestis Populations Causing Plague among Humans in the West Nile Region of Uganda.

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Respicio-Kingry, Laurel B; Yockey, Brook M; Acayo, Sarah; Kaggwa, John; Apangu, Titus; Kugeler, Kiersten J; Eisen, Rebecca J; Griffith, Kevin S; Mead, Paul S; Schriefer, Martin E; Petersen, Jeannine M

2016-02-01

Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Since the 1990s, Africa has accounted for the majority of reported human cases. In Uganda, plague cases occur in the West Nile region, near the border with Democratic Republic of Congo. Despite the ongoing risk of contracting plague in this region, little is known about Y. pestis genotypes causing human disease. During January 2004-December 2012, 1,092 suspect human plague cases were recorded in the West Nile region of Uganda. Sixty-one cases were culture-confirmed. Recovered Y. pestis isolates were analyzed using three typing methods, single nucleotide polymorphisms (SNPs), pulsed field gel electrophoresis (PFGE), and multiple variable number of tandem repeat analysis (MLVA) and subpopulations analyzed in the context of associated geographic, temporal, and clinical data for source patients. All three methods separated the 61 isolates into two distinct 1.ANT lineages, which persisted throughout the 9 year period and were associated with differences in elevation and geographic distribution. We demonstrate that human cases of plague in the West Nile region of Uganda are caused by two distinct 1.ANT genetic subpopulations. Notably, all three typing methods used, SNPs, PFGE, and MLVA, identified the two genetic subpopulations, despite recognizing different mutation types in the Y. pestis genome. The geographic and elevation differences between the two subpopulations is suggestive of their maintenance in highly localized enzootic cycles, potentially with differing vector-host community composition. This improved understanding of Y. pestis subpopulations in the West Nile region will be useful for identifying ecologic and environmental factors associated with elevated plague risk.

Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016.

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Usein, Codruţa-Romaniţa; Ciontea, Adriana Simona; Militaru, Cornelia Mãdãlina; Condei, Maria; Dinu, Sorin; Oprea, Mihaela; Cristea, Daniela; Michelacci, Valeria; Scavia, Gaia; Zota, Lavinia Cipriana; Zaharia, Alina; Morabito, Stefano

2017-11-01

IntroductionAt the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae , ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae , with or without stx1a , while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered.

Improved protocol for isolation of Campylobacter spp. from retail broiler meat and use of pulsed field gel electrophoresis for the typing of isolates.

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Oyarzabal, Omar A; Williams, Aretha; Zhou, Ping; Samadpour, Mansour

2013-10-01

To improve the detection of Campylobacter spp. in retail broiler meat, a reference method (R subsamples) based on the enrichment of 25 g of meat in Bolton broth at 42°C under microaerobiosis was compared with an alternative method (A subsamples) consisting in the rinsing of meat samples for 30s in buffered peptone water with antimicrobials with incubation at 42°C under aerobiosis. One piece of meat (breasts, tenderloins and thighs) was rinse in experiment 1 (A1) and two pieces in experiment 2 (A2). Campylobacter spp. were isolated on agar plates and identified by PCR. Retail samples in Alabama had less prevalence (P ≤ 0.05) than samples in the state of Washington. The percentage of positive was higher (P ≤ 0.05) in A than in R subsamples and rinsing two pieces of meat yielded the highest percentage of positive subsamples. R subsamples showed variations in the prevalence by product. However, A subsamples had similar prevalence of positives among products compare to the result from reference method. More Campylobacter coli isolates were collected in A2 subsamples. Pulse field gel electrophoresis (PFGE) was used as subtyping method to study the genome similarity among the isolates from all methods. A larger diversity of isolates were detected by PFGE in A2 subsamples. Denaturing gradient gel electrophoresis analysis suggested that the initial bacterial populations of the meat samples impact the final bacterial profile after enrichment. Rinsing broiler meats was less time consuming, required less sample preparation and was more sensitive than the reference method for the isolation of naturally occurring Campylobacter spp. This new method could help with epidemiological and intervention studies to control Campylobacter spp. Copyright © 2013 Elsevier B.V. All rights reserved.

Typing of Ochrobactrum anthropi clinical isolates using automated repetitive extragenic palindromic-polymerase chain reaction DNA fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

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Quirino, Angela; Pulcrano, Giovanna; Rametti, Linda; Puccio, Rossana; Marascio, Nadia; Catania, Maria Rosaria; Matera, Giovanni; Liberto, Maria Carla; Focà, Alfredo

2014-03-22

Ochrobactrum anthropi (O. anthropi), is a non-fermenting gram-negative bacillus usually found in the environment. Nevertheless, during the past decade it has been identified as pathogenic to immunocompromised patients. In this study, we assessed the usefulness of the automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR-based DiversiLab™ system, bioMèrieux, France) and of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF MS) for typing of twentythree O. anthropi clinical isolates that we found over a four-months period (from April 2011 to August 2011) in bacteriemic patients admitted in the same operative unit of our hospital. Pulsed-field gel electrophoresis (PFGE), commonly accepted as the gold standard technique for typing, was also used. Analysis was carried out using the Pearson correlation coefficient to determine the distance matrice and the unweighted pair group method with arithmetic mean (UPGMA) to generate dendogram. Rep-PCR analysis identified four different patterns: three that clustered together with 97% or more pattern similarity, and one whose members showed < 95% pattern similarity. Interestingly, strains isolated later (from 11/06/2011 to 24/08/2011) displayed a pattern with 99% similarity. MALDI-TOF MS evaluation clustered the twentythree strains of O. anthropi into a single group containing four distinct subgroups, each comprising the majority of strains clustering below 5 distance levels, indicating a high similarity between the isolates. Our results indicate that these isolates are clonally-related and the methods used afforded a valuable contribution to the epidemiology, prevention and control of the infections caused by this pathogen.

High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.

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Woksepp, Hanna; Jernberg, Cecilia; Tärnberg, Maria; Ryberg, Anna; Brolund, Alma; Nordvall, Michaela; Olsson-Liljequist, Barbro; Wisell, Karin Tegmark; Monstein, Hans-Jürg; Nilsson, Lennart E; Schön, Thomas

2011-12-01

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.

High-Resolution Melting-Curve Analysis of Ligation-Mediated Real-Time PCR for Rapid Evaluation of an Epidemiological Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli ▿

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Woksepp, Hanna; Jernberg, Cecilia; Tärnberg, Maria; Ryberg, Anna; Brolund, Alma; Nordvall, Michaela; Olsson-Liljequist, Barbro; Wisell, Karin Tegmark; Monstein, Hans-Jürg; Nilsson, Lennart E.; Schön, Thomas

2011-01-01

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE. PMID:21956981

Irrigation water quality and the benefits of implementing good agricultural practices during tomato (Lycopersicum esculentum) production.

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Estrada-Acosta, M; Jiménez, M; Chaidez, C; León-Félix, J; Castro-Del Campo, N

2014-07-01

The implementation of good agricultural practices (GAP) from irrigation water to the tomato packaging process enhances the safety of fresh produce and its value throughout the food chain. The aim of the present study was to show that fresh produce farms that apply and enforce GAP could reduce the presence of Salmonella in finished produce. Samples were collected biweekly from six packing houses from the central region of Sinaloa, México, for the isolation of Salmonella spp by the ISO 6579:2002 method, and the isolated strains were serotyped and genotyped by the Kauffmman-White scheme and pulsed field gel electrophoresis (PFGE), respectively. Salmonella strains were detected in 13 (36.1 %) irrigation water samples, while only two tomato samples were positive (5.5 %). Eight different serotypes were identified in irrigation water, and Salmonella Oranienburg (34 %) was the most prevalent; however, only Salmonella Agona and Salmonella Weltevreden were present on tomatoes. Salmonella Oranienburg was the most widely dispersed and variable serotype, with 10 different PFGE profiles. Salmonella Weltevreden was isolated from both types of samples, albeit with distinct genetic profiles, implying that the sources of contamination differ. These results confirm the utility of implementing good agricultural practices to reduce Salmonella contamination in irrigation water and the packaging process.

A Comparison of Non-Typhoidal Salmonella from Humans and Food Animals Using Pulsed-Field Gel Electrophoresis and Antimicrobial Susceptibility Patterns

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Sandt, Carol H.; Fedorka-Cray, Paula J.; Tewari, Deepanker; Ostroff, Stephen; Joyce, Kevin; M’ikanatha, Nkuchia M.

2013-01-01

Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoidal Salmonella isolated from ill humans in Pennsylvania and from food animals before retail. Human clinical isolates were received from 2005 through 2011 during routine public health operations in Pennsylvania. Isolates from cattle, chickens, swine and turkeys were recovered during the same period from federally inspected slaughter and processing facilities in the northeastern United States. We found that subtyping Salmonella isolates by PFGE revealed differences in antimicrobial susceptibility patterns and, for human Salmonella, differences in sources and invasiveness that were not evident from serotyping alone. Sixteen of the 20 most common human Salmonella PFGE patterns were identified in Salmonella recovered from food animals. The most common human Salmonella PFGE pattern, Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS), was associated with more cases of invasive salmonellosis than all other patterns. In food animals, this pattern was almost exclusively (99%) found in Salmonella recovered from chickens and was present in poultry meat in every year of the study. Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS) was associated with susceptibility to all antimicrobial agents tested in 94.7% of human and 97.2% of food animal Salmonella isolates. In contrast, multidrug resistance (resistance to three or more classes of antimicrobial agents) was observed in five PFGE patterns. Typhimurium patterns JPXX01.0003 (JPXX01.0003 ARS) and JPXX01.0018 (JPXX01.0002 ARS), considered together, were associated with resistance to five or more classes of antimicrobial agents: ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT), in 92% of human and 80% of food

Antibiotic resistance determinants and genetic analysis of Salmonella enterica isolated from food in Morocco.

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Murgia, Manuela; Bouchrif, Brahim; Timinouni, Mohammed; Al-Qahtani, Ahmed; Al-Ahdal, Mohammed N; Cappuccinelli, Pietro; Rubino, Salvatore; Paglietti, Bianca

2015-12-23

Antimicrobial-resistant non-typhoidal Salmonella (NTS) are an important cause of infection in Africa, but there is a lack of information on their molecular mechanisms of resistance and epidemiology. This study contributes to fill this gap through the characterization by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), plasmid profiling and analysis of antibiotic-resistance determinants of 94 Salmonella enterica strains isolated from food in Morocco. PFGE revealed considerable heterogeneity among the strains, showing 32 pulsotypes. MLST of strains representative of the different serovars evidenced 13 sequence types (STs), three of which were newly identified (ST1694, ST1768 and ST1818) and nine not previously reported in Morocco. Thirty-four strains harbored from one to four plasmids, of IncI1 group in S. Mbandaka, IncFIIA in S. Typhimurium, IncL/M in S. Hadar and S. Blockley. For the first time in Morocco an intact Salmonella Genomic Island 1 (SGI1) carrying the resistance genes aadA2, floR, tetG, blaPSE-1 and sul1 was detected in S. Typhimurium DT104. In serovar Hadar resistance to ampicillin, tetracycline and streptomycin was associated to blaTEM-1, tetA and strA genes respectively, whereas one mutation in gyrA (Asp87Asn) and one in parC (Thr54Ser) genes conferred resistance to nalidixic acid. These findings improve the information on foodborne Salmonella in Morocco, evidencing the presence of MDR strains potentially dangerous to humans, and provide useful data for future studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of Plasmid-Mediated Quinolone Resistance Determinants in High-Level Quinolone-Resistant Enterobacteriaceae Isolates from the Community: First Report of qnrD Gene in Algeria.

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Yanat, Betitera; Machuca, Jesús; Díaz-De-Alba, Paula; Mezhoud, Halima; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

2017-01-01

The objective was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR)-producing isolates in a collection of quinolone-resistant Enterobacteriaceae of community origin isolated in Bejaia, Algeria. A total of 141 nalidixic acid-resistant Enterobacteriaceae community isolates were collected in Bejaia (Northern Algeria) and screened for PMQR genes using polymerase chain reaction (PCR). For PMQR-positive strains, antimicrobial susceptibility testing was performed by broth microdilution and disk diffusion. Mutations in the quinolone resistance-determining regions of the target genes, gyrA and parC, were detected with a PCR-based method and sequencing. Southern blotting, conjugation and transformation assays and molecular typing by pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing were also performed. The prevalence of PMQR-producing Enterobacteriaceae isolates was 13.5% (19/141); 11 of these isolates produced Aac(6')-Ib-cr and 8 were qnr-positive (4 qnrB1-like, 2 qnrS1-like, and 2 qnrD1-like), including the association with aac(6')-Ib-cr gene in three cases. PMQR gene transfer by conjugation was successful in 6 of 19 isolates tested. PFGE revealed that most of the PMQR-positive Escherichia coli isolates were unrelated, except for two groups comprising two and four isolates, respectively, including the virulent multidrug-resistant clone E. coli ST131 that were clonally related. Our findings indicate that PMQR determinants are prevalent in Enterobacteriaceae isolates from the community studied. We describe the first report of the qnrD gene in Algeria.

Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center.

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Bozdogan, Bülent; Ednie, Lois; Credito, Kim; Kosowska, Klaudia; Appelbaum, Peter C

2004-12-01

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.

Molecular epidemiology of bla OXA-23 -producing carbapenem-resistant Acinetobacter baumannii in a single institution over a 65-month period in north China.

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Ning, Nian-Zhi; Liu, Xiong; Bao, Chun-Mei; Chen, Su-Ming; Cui, En-Bo; Zhang, Ju-Ling; Huang, Jie; Chen, Fang-Hong; Li, Tao; Qu, Fen; Wang, Hui

2017-01-05

Carbapenem-resistant Acinetobacter baumannii poses a significant threat to hospitalized patients, as few therapeutic options remain. Thus, we investigated the molecular epidemiology and mechanism of resistance of carbapenem-resistant A.baumannii isolates in Beijing, China. Carbapenem-resistant A.baumannii isolates (n = 101) obtained between June 2009 and November 2014 were used. Multilocus sequence typing (MLST) and PCR assays for class C and D β-lactamase were performed on all isolates. S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to identify the resistance gene location. All 101 A.baumannii isolates were highly resistant to frequently used antimicrobials, and were considered multidrug resistant. A total of 12 sequence types (STs) were identified, including 10 reported STs and 2 novel STs. Eighty-seven isolates were classified to clonal complex 92 (CC92), among which ST191 and ST195 were the most common STs. The bla OXA-23 gene was positive in most (n = 95) of the A.baumannii isolates. Using S1-nuclease digestion PFGE and Southern blot hybridization, 3 patterns of plasmids carrying bla OXA-23 were confirmed. ST191 and ST195 (both harboring bla OXA-23 ) caused outbreaks during the study period, and this is the first report of outbreaks caused by ST191 and ST195 in north China. bla OXA-23 -producing A.baumannii ST191 and ST 195 isolates can disseminate in a hospital and are potential nosocomial outbreak strains. Surveillance of imipenem-resistant A.baumannii and antimicrobial stewardship should be strengthened.

Characterisation of successive Acinetobacter baumannii isolates from a deceased haemophagocytic lymphohistiocytosis patient.

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Choi, Hyeon Jin; Kil, Min Cheol; Choi, Ji-Young; Kim, Sun Ju; Park, Ki-Sup; Kim, Yae-Jean; Ko, Kwan Soo

2017-01-01

In this study, 38 Acinetobacter baumannii isolates successively isolated from blood, skin swabs and tracheal aspirates from a single patient who died from haemophagocytic lymphohistiocytosis were investigated. The isolates were collected between March 2012 and August 2012. A. baumannii genotypes were determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In vitro antimicrobial susceptibility testing was performed and colistin heteroresistance and persistence were evaluated. The structure of AbaR resistance islands was explored, and serum sensitivity was determined. Based on MLST analysis, all 38 A. baumannii isolates showed the same sequence type (ST138). However, PFGE analysis showed that isolates from blood samples belonged to different genotypes depending on the isolation time: whilst blood isolates obtained at the early stages showed restriction patterns similar to those of isolates from other sources, isolates obtained at later stages exhibited a distinct pattern. All isolates were resistant to imipenem, cefepime, ciprofloxacin and piperacillin/tazobactam. Five isolates from tracheal aspirates and one from a skin swab were resistant to polymyxins, and two isolates from skin swabs and one from another source were non-susceptible to tigecycline. All colistin-susceptible isolates showed heteroresistance to colistin, and four were persisters. Isolates from blood showed higher survival rates against human serum than those from other sources. This study shows that the patient was infected with more than one A. baumannii strain. Heteroresistance, persistence or evasion of the innate immune response may explain the failure of antimicrobial treatments in this patient. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Influence of Therapeutic Ceftiofur Treatments of Feedlot Cattle on Fecal and Hide Prevalences of Commensal Escherichia coli Resistant to Expanded-Spectrum Cephalosporins, and Molecular Characterization of Resistant Isolates

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Griffin, Dee; Kuehn, Larry A.; Brichta-Harhay, Dayna M.

2013-01-01

In the United States, the blaCMY-2 gene contained within incompatibility type A/C (IncA/C) plasmids is frequently identified in extended-spectrum-cephalosporin-resistant (ESCr) Escherichia coli strains from both human and cattle sources. Concerns have been raised that therapeutic use of ceftiofur in cattle may increase the prevalence of ESCr E. coli. We report that herd ESCr E. coli fecal and hide prevalences throughout the residency of cattle at a feedlot, including during the period of greatest ceftiofur use at the feedlot, were either not significantly different (P ≥ 0.05) or significantly less (P E. coli shedding that follows ceftiofur injection abated, ceftiofur-injected cattle were no more likely than untreated members of the same herd to shed ESCr E. coli. Pulsed-field gel electrophoresis (PFGE) genotyping, antibiotic resistance phenotyping, screening for presence of the blaCMY-2 gene, and plasmid replicon typing were performed on 312 ESCr E. coli isolates obtained during six sampling periods spanning the 10-month residence of cattle at the feedlot. The identification of only 26 unique PFGE genotypes, 12 of which were isolated during multiple sampling periods, suggests that clonal expansion of feedlot-adapted blaCMY-2 E. coli strains contributed more to the persistence of blaCMY-2 than horizontal transfer of IncA/C plasmids between E. coli strains at this feedlot. We conclude that therapeutic use of ceftiofur at this cattle feedlot did not significantly increase the herd prevalence of ESCr E. coli. PMID:23354706

The population genetics of Pseudomonas aeruginosa isolates from different patient populations exhibits high-level host specificity.

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Rosa van Mansfeld

Full Text Available OBJECTIVE: To determine whether highly prevalent P. aeruginosa sequence types (ST in Dutch cystic fibrosis (CF patients are specifically linked to CF patients we investigated the population structure of P. aeruginosa from different clinical backgrounds. We first selected the optimal genotyping method by comparing pulsed-field gel electrophoresis (PFGE, multilocus sequence typing (MLST and multilocus variable number tandem-repeat analysis (MLVA. METHODS: Selected P. aeruginosa isolates (n = 60 were genotyped with PFGE, MLST and MLVA to determine the diversity index (DI and congruence (adjusted Rand and Wallace coefficients. Subsequently, isolates from patients admitted to two different ICUs (n = 205, from CF patients (n = 100 and from non-ICU, non-CF patients (n = 58, of which 19 were community acquired were genotyped with MLVA to determine distribution of genotypes and genetic diversity. RESULTS: Congruence between the typing methods was >79% and DIs were similar and all >0.963. Based on costs, ease, speed and possibilities to compare results between labs an adapted MLVA scheme called MLVA9-Utrecht was selected as the preferred typing method. In 363 clinical isolates 252 different MLVA types (MTs were identified, indicating a highly diverse population (DI  = 0.995; CI  = 0.993-0.997. DI levels were similarly high in the diverse clinical sources (all >0.981 and only eight genotypes were shared. MTs were highly specific (>80% for the different patient populations, even for similar patient groups (ICU patients in two distinct geographic regions, with only three of 142 ICU genotypes detected in both ICUs. The two major CF clones were unique to CF patients. CONCLUSION: The population structure of P. aeruginosa isolates is highly diverse and population specific without evidence for a core lineage in which major CF, hospital or community clones co-cluster. The two genotypes highly prevalent among Dutch CF patients appeared unique to CF patients

Correlation between γ-ray-induced DNA double-strand breakage and cell killing after biologically relevant doses: analysis by pulsed-field gel electrophoresis

International Nuclear Information System (INIS)

Murray, D.

1994-01-01

We examined the degree of correlation between γ-ray-induced lethality and DNA double-strand breaks (dsbs) after biologically relevant doses of radiation. Radiation lethality was modified by treating 14 C-labelled Chinese hamster ovary cells with either of two aminothiols (WR-1065 or WR-255591) and the associated effect on dsb induction was determined by pulsed-field gel electrophoresis (PFGE). The use of phosphorimaging to analyse the distribution of 14 C-activity in the gel greatly improved the low-dose resolution of the PFGE assay. Both WR-1065 and WR-255591 protected against dsb induction and lethality to a similar extent after low doses of radiation. although this correlation broke down when supralethal doses were used to induce dsbs. Thus, the level of dsbs induced in these cells as measured by PFGE after survival-curve doses of γ-radiation is consistently predictive of the degree of lethality obtained, implying a cause-effect relationship between these two parameters and confirming previous results obtained using the neutral filter elution assay for dsbs. (author)

Characterization of Antimicrobial Resistance Patterns and Detection of Virulence Genes in Campylobacter Isolates in Italy

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Di Giannatale, Elisabetta; Di Serafino, Gabriella; Zilli, Katiuscia; Alessiani, Alessandra; Sacchini, Lorena; Garofolo, Giuliano; Aprea, Giuseppe; Marotta, Francesca

2014-01-01

Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones and fluoroquinolones, becoming a significant public health hazard. A total of 145 strains derived from raw milk, chicken faeces, chicken carcasses, cattle faeces and human faeces collected from various Italian regions, were screened for antimicrobial susceptibility, molecular characterization (SmaI pulsed-field gel electrophoresis) and detection of virulence genes (sequencing and DNA microarray analysis). The prevalence of C. jejuni and C. coli was 62.75% and 37.24% respectively. Antimicrobial susceptibility revealed a high level of resistance for ciprofloxacin (62.76%), tetracycline (55.86%) and nalidixic acid (55.17%). Genotyping of Campylobacter isolates using PFGE revealed a total of 86 unique SmaI patterns. Virulence gene profiles were determined using a new microbial diagnostic microarray composed of 70-mer oligonucleotide probes targeting genes implicated in Campylobacter pathogenicity. Correspondence between PFGE and microarray clusters was observed. Comparisons of PFGE and virulence profiles reflected the high genetic diversity of the strains examined, leading us to speculate different degrees of pathogenicity inside Campylobacter populations. PMID:24556669

SHV-type extended-spectrum beta-lactamase (ESBL are encoded in related plasmids from enterobacteria clinical isolates from Mexico beta-Lactamasas de espectro extendido (BLEE tipo SHV están codificadas en plásmidos relacionados en aislamientos clínicos de enterobacterias en México

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Ulises Garza-Ramos

2007-12-01

Full Text Available OBJECTIVE: In this work we report the molecular characterization of beta-lactam antibiotics resistance conferred by genes contained in plasmids from enterobacteria producing extended-spectrum beta-lactamases (ESBL. MATERIAL AND METHODS: Fourteen enterobacterial clinical isolates selected from a group of strains obtained from seven different hospitals in Mexico during 1990-1992 and 1996-1998 were analyzed at the Bacterial Resistance Laboratory (National Institute Public Health, Cuernavaca. Molecular characterization included PFGE, IEF of beta-lactamases, bacterial conjugation, PCR amplification and DNA sequencing, plasmid extraction and restriction. RESULTS: Isolates were genetically unrelated. ESBL identified were SHV-2 (5/14 and SHV-5 (9/14 type. Cephalosporin-resistance was transferable in 9 of 14 (64% clinical isolates with only one conjugative plasmid, DNA finger printing showed a similar band pattern in plasmids. CONCLUSIONS: The dissemination of cephalosporin resistance was due to related plasmids carrying the ESBL genes.OBJETIVO: En este trabajo se reporta la caracterización molecular de la resistencia a antibiótico beta-lactámicos conferida por genes contenidos en plásmidos de enterobacterias productoras de beta-lactamasas de espectro extendido (BLEEs. MATERIAL Y MÉTODOS: Catorce aislamientos clínicos de enterobacterias fueron seleccionados por conveniencia de un banco de cepas obtenidas de siete diferentes hospitales de México durante los periodos 1990-1992 y 1996-1998 y fueron procesados en el Laboratorio de Resistencia Bacteriana (Instituto Nacional de Salud Pública, Cuernavaca. En la caracterización se empleó PFGE, IEF para beta-lactamasas, conjugación bacteriana, amplificación por PCR y secuenciación de DNA, extracción y restricción de plásmidos. RESULTADOS: Las 14 cepas fueron no relacionadas genéticamente. Se identificaron BLEEs tipo SHV-2 (5/14 y SHV-5 (9/14. La resistencia a cefalosporinas fue transferida por

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Bermuda.

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Patrick Eberechi Akpaka

Full Text Available Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST. Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17 that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.

Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients.

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Khosravi, Azar Dokht; Vatani, Shideh; Feizabadi, Mohammad Mehdi; Abasi Montazeri, Effat; Jolodar, Abbas

2014-05-01

Mycobacterium tuberculosis genotyping can effectively improve tuberculosis (TB) control programs by controlling disease transmission. Pulsed field gel electrophoresis (PFGE) is a particularly powerful tool for determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. The aim of present study was to investigate the genetic diversity of M. tuberculosis strains in Khuzestan province by the PFGE technique. In total, 80 M. tuberculosis positive cultures were obtained from tuberculosis patients. PFGE was performed on 60 PCR-confirmed isolates by using DraI and XbaI restriction enzymes according to standard protocols. Plugs containing digested DNA were then loaded on agarose gels and run using contour-clamped homogenous electric fields. Fifty distinct DNA banding patterns were obtained by digestion of DNA with DraI and 38 DNA banding patterns by digestion with XbaI restriction enzymes. The patterns comprised of 17 different clusters in which cluster I was the major one, containing six strains. Three clusters contained three strains each and the 13 remaining clusters comprised of two strains each. Digestion with DraI yielded 15-20 DNA fragments with 50-485 kb size, while digestion by XbaI produced DNA fragments with a size smaller than 50-242 kb. Despite the ability of PFGE for study of genetic diversity of many mycobacterial species and it being considered as a robust and useful tool, in this study we only found a 15% epidemiological relationship amongst the isolates. Thus, for higher discrimination of genotypic clusters among M. tuberculosis clinical isolates, the application of more sophisticated complementary techniques is required.

Molecular analysis of Salmonella enteritidis isolates from the Caribbean by pulsed-field gel electrophoresis

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Abiodun Adesiyun

2000-11-01

Full Text Available Using pulsed-field gel electrophoresis (PFGE, between 1987 and 1996 we analyzed Salmonella enteritidis isolates from gastroenteritis cases in four Caribbean countries: Barbados, Saint Kitts and Nevis, Saint Lucia, and Trinidad and Tobago. We also determined the resistance of the isolates to 12 antimicrobial agents. Of the 129 isolates of S. enteritidis available for testing, DNA digested by XbaI revealed 13 distinctive PFGE patterns. The most prevalent XbaI PFGE patterns were group 1 (88 of 129 isolates, 68.2% and group 2 (26 of 129, 20.2%. The patterns found among S. enteritidis isolates correlated with the geographical origin of the isolates. Of the 28 isolates from Barbados, 20 of them (71.4% belonged to XbaI PFGE group 2, and of the 93 isolates from Trinidad and Tobago, 78 of them (83.9% belonged to group 1. SpeI digestion of S. enteritidis genome was not as discriminatory as XbaI. Overall, of the 129 isolates, 67 of them (51.9% exhibited resistance to one or more of the 12 antimicrobial agents that we tested. The prevalence of resistance was 53.8% for the S. enteritidis isolates tested from Trinidad and Tobago, 50.0% for those from Barbados, 28.6% for those from Saint Lucia, and 100.0% for one isolate from the island of Saint Kitts. Resistance was highest to triple sulfur (59 of 129 isolates, 45.7%, followed by furadantoin (10 of 129, 7.8%, ampicillin (7 of 129, 5.4%, and carbamycin (5 of 129, 3.9%.

Molecular analysis of Salmonella enteritidis isolates from the Caribbean by pulsed-field gel electrophoresis.

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Adesiyun, A; Carson, A; McAdoo, K; Bailey, C

2000-11-01

Using pulsed-field gel electrophoresis (PFGE), between 1987 and 1996 we analyzed Salmonella enteritidis isolates from gastroenteritis cases in four Caribbean countries: Barbados, Saint Kitts and Nevis, Saint Lucia, and Trinidad and Tobago. We also determined the resistance of the isolates to 12 antimicrobial agents. Of the 129 isolates of S. enteritidis available for testing, DNA digested by XbaI revealed 13 distinctive PFGE patterns. The most prevalent XbaI PFGE patterns were group 1 (88 of 129 isolates, 68.2%) and group 2 (26 of 129, 20.2%). The patterns found among S. enteritidis isolates correlated with the geographical origin of the isolates. Of the 28 isolates from Barbados, 20 of them (71.4%) belonged to XbaI PFGE group 2, and of the 93 isolates from Trinidad and Tobago, 78 of them (83.9%) belonged to group 1. SpeI digestion of S. enteritidis genome was not as discriminatory as XbaI. Overall, of the 129 isolates, 67 of them (51.9%) exhibited resistance to one or more of the 12 antimicrobial agents that we tested. The prevalence of resistance was 53.8% for the S. enteritidis isolates tested from Trinidad and Tobago, 50.0% for those from Barbados, 28.6% for those from Saint Lucia, and 100.0% for one isolate from the island of Saint Kitts. Resistance was highest to triple sulfur (59 of 129 isolates, 45.7%), followed by furadantoin (10 of 129, 7.8%), ampicillin (7 of 129, 5.4%), and carbamycin (5 of 129, 3.9%).

Molecular analysis of Salmonella enteritidis isolates from the Caribbean by pulsed-field gel electrophoresis

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Adesiyun Abiodun

2000-01-01

Full Text Available Using pulsed-field gel electrophoresis (PFGE, between 1987 and 1996 we analyzed Salmonella enteritidis isolates from gastroenteritis cases in four Caribbean countries: Barbados, Saint Kitts and Nevis, Saint Lucia, and Trinidad and Tobago. We also determined the resistance of the isolates to 12 antimicrobial agents. Of the 129 isolates of S. enteritidis available for testing, DNA digested by XbaI revealed 13 distinctive PFGE patterns. The most prevalent XbaI PFGE patterns were group 1 (88 of 129 isolates, 68.2% and group 2 (26 of 129, 20.2%. The patterns found among S. enteritidis isolates correlated with the geographical origin of the isolates. Of the 28 isolates from Barbados, 20 of them (71.4% belonged to XbaI PFGE group 2, and of the 93 isolates from Trinidad and Tobago, 78 of them (83.9% belonged to group 1. SpeI digestion of S. enteritidis genome was not as discriminatory as XbaI. Overall, of the 129 isolates, 67 of them (51.9% exhibited resistance to one or more of the 12 antimicrobial agents that we tested. The prevalence of resistance was 53.8% for the S. enteritidis isolates tested from Trinidad and Tobago, 50.0% for those from Barbados, 28.6% for those from Saint Lucia, and 100.0% for one isolate from the island of Saint Kitts. Resistance was highest to triple sulfur (59 of 129 isolates, 45.7%, followed by furadantoin (10 of 129, 7.8%, ampicillin (7 of 129, 5.4%, and carbamycin (5 of 129, 3.9%.

Multistate outbreaks of Salmonella infections associated with live poultry--United States, 2007.

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2009-01-23

During June 2007, the Minnesota Department of Health (MDH) Public Health Laboratory examined specimens from two ill persons and identified Salmonella Montevideo isolates with the same pulsed-field gel electrophoresis pattern (PFGE pattern 1). MDH officials interviewed the patients and determined that both had been exposed to chickens originating from the same Iowa hatchery (hatchery A). In September 2007, a cluster of seven additional infections with PFGE pattern 1 was identified in North Dakota; all seven patients had been exposed to baby chicks obtained from hatchery A. A subsequent nationwide investigation identified a total of 65 S. Montevideo isolates with PFGE pattern 1 during 2007 and a likely association with exposure to live poultry purchased at feed stores or by mail order from hatchery A or seven other hatcheries in four states. Meanwhile, throughout 2007, a separate outbreak was occurring that involved infections with a different S. Montevideo strain (PFGE pattern 2). A total of 64 of those isolates were identified in 23 states during 2007. Exposure to live poultry from a hatchery in New Mexico (hatchery B) and a hatchery in Ohio (hatchery C) was associated with those infections. This report describes two distinct and unrelated outbreaks, which demonstrate the ongoing risk for Salmonella infection from live poultry purchased from agricultural feed stores or directly from mail order hatcheries. The mail order hatchery industry is a source of bird-associated human pathogens, such as Salmonella, and comprehensive infection-control strategies are needed to prevent additional illnesses resulting from live poultry contact.

Clonal emergence of Klebsiella pneumoniae ST14 co-producing OXA-48-type and NDM carbapenemases with high rate of colistin resistance in Dubai, United Arab Emirates.

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Moubareck, Carole Ayoub; Mouftah, Shaimaa F; Pál, Tibor; Ghazawi, Akela; Halat, Dalal H; Nabi, Anju; AlSharhan, Mouza A; AlDeesi, Zulfa O; Peters, Christabel C; Celiloglu, Handan; Sannegowda, Manjunath; Sarkis, Dolla K; Sonnevend, Ágnes

2018-03-09

Few studies have addressed the molecular epidemiology of carbapenem resistant Enterobacteriaceae (CRE) isolates in the Arabian Peninsula, and such investigations have been missing from Dubai, a major economical, tourism and medical centre of the region. The antibiotic susceptibility, the carbapenemase type produced, and the clonality of 89 CRE strains isolated in five major Dubai hospitals in June 2015 - June 2016 were determined. Thirty three percent of the collection of 70 K. pneumoniae, 13 E. coli and 6 other Enterobacteriaceae were extremely drug resistant, 27% were resistant to colistin, and 4.5% (four K. pneumoniae isolates) were resistant to all antibiotics tested. The colistin resistance rate in K. pneumoniae was 31.4%. None of the isolates carried mobile colistin resistance genes. Seventy-seven isolates produced carbapenemase: 53.3% OXA-48-like, 24.7% NDM, and 22.1% both OXA-48-like and NDM, respectively. PFGE clustered 50% of K. pneumoniae into a 35-membered group, which showed significant association with double carbapenemase production, with extreme drug resistance, and with being isolated from Emirati patients. Members of the cluster belonged to sequence type ST14. The rate of colistin resistance in K. pneumoniae ST14 was 37.1% vs. 27.1% of K. pneumoniae isolates outside of the cluster. Two of the panresistant K. pneumoniae isolates also belonged to ST14, whereas the other two were ST15 and ST231, respectively. In conclusion, beyond the overall high colistin resistance rate in CRE, the emergence of a highly resistant clone of K. pneumoniae ST14 in all Dubai hospitals investigated is a serious problem requiring immediate attention. Copyright © 2018. Published by Elsevier B.V.

Staphylococcal species associated with bovine mastitis in the North West of Iran: Emerging of coagulase-negative staphylococci

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Somayyeh Hosseinzadeh

2014-06-01

Full Text Available Staphylococcal mastitis is a major and costly problem of dairy cattle all over the world. The objective of this study was to isolate and identify the main staphylococcal species causing bovine mastitis in 7 dairy herds located in 4 different areas of East and West Azerbaijan provinces, Iran. Of the 158 mastitic milk samples collected, 113 staphylococcal isolates were identified (71.5% on the basis of cultural and biochemical features as well as by genus specific PCR. Then, species level identification of staphylococcal isolates was carried out using restriction fragment length polymorphism (RFLP analysis of the gap gene (933 bp. On the basis of polymerase chain reaction-RFLP, 10 different patterns were identified. Of 113 isolates, 5 (4.4% were Staphylococcus aureus and 108 (95.6% were coagulase-negative staphylococci (CoNS. Overall, nine different species of CoNS were identified as: 44 Staphylococcus haemolyticus (40.7%, 17 Staphylococcus chromogenes (15.7%, 11 Staphylococcus epidermidis, Staphylococcus warneri and Staphylococcus cohnii each (10.2%, 6 Staphylococcus simulans (5.5%, 4 Staphylococcus hominis (3.7%, 3 Staphylococcus capitis (2.7% and 1 Staphylococcus xylosus (0.9%. S. haemolyticus, S. chromogenes and S. warneri were the only species identified from clinical mastitis. No significant difference in staphylococcal IMI was found among the studied herds and regions. This study demonstrated that CoNS, especially S. haemolyticus and S. chromogenes, were predominant and thus be considered as emerging pathogens causing mastitis in the North West of Iran. Our results also revealed that the gap PCR-RFLP was useful for identifying staphylococcal isolates derived from bovine mastitis at species level.

Characterization of pig-associated methicillin-resistant Staphylococcus aureus.

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Li, Jun; Jiang, Nansong; Ke, Yuebin; Feßler, Andrea T; Wang, Yang; Schwarz, Stefan; Wu, Congming

2017-03-01

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have been reported in various countries worldwide. However, although China is one of the biggest pig and pork producers, large-scale studies on pig-associated LA-MRSA from China are scarce. The aims of this study were to analyze 2420 non-duplicate samples collected from pigs at swine farms and slaughterhouses in different regions in China during 2014 for the prevalence of pig-associated MRSA and to determine the antimicrobial resistance pheno- and genotypes of the respective isolates. MRSA isolates were identified in 270 (11.2%) samples. The isolates were characterized by antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, pulsed-field gel electrophoresis (PFGE) and screening for resistance genes. All MRSA isolates belonged to the clonal complex 9 and spa type t899, but showed variable PFGE patterns. All isolates were non-susceptible to oxacillin, cefoxitin, clindamycin, chloramphenicol, florfenicol, ciprofloxacin, and valnemulin. High rates of resistance were also observed for tetracycline (99.6%), erythromycin (97.0%), quinupristin-dalfopristin (97.0%), and gentamicin (80.4%). Three linezolid-non-susceptible isolates containing the multi-resistance gene cfr and nine rifampicin-non-susceptible isolates with mutations in rpoB were detected. Resistance to β-lactams was exclusively associated with mecA, while phenicol resistance was mainly attributable to fexA, except in the three cfr-positive isolates. The pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) was identified in all MRSA isolates, and no other pleuromutilin resistance genes, except cfr in three isolates, were detected. Pigs are the most important hosts of LA-MRSA in China. Screening for pig-associated MRSA is necessary to monitor changes in epidemiology and characteristics of these important pathogens. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of new multilocus variable number of tandem repeat analysis (MLVA) for Listeria innocua and its application in a food processing plant.

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Takahashi, Hajime; Ohshima, Chihiro; Nakagawa, Miku; Thanatsang, Krittaporn; Phraephaisarn, Chirapiphat; Chaturongkasumrit, Yuphakhun; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

2014-01-01

Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE). The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Development of new multilocus variable number of tandem repeat analysis (MLVA for Listeria innocua and its application in a food processing plant.

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Hajime Takahashi

Full Text Available Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE. The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany

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Hain Torsten

2009-06-01

Full Text Available Abstract Background Multi-drug-resistant, extended-spectrum β-lactamase (ESBL-producing Enterobacteriaceae, constitute an emerging public-health concern. Little data on the molecular epidemiology of ESBL producing Escherichia coli is available in Germany. Here we describe the prevalence and molecular epidemiology of ESBL producing-Escherichia coli isolates at a German University hospital. Methods We analysed 63 non-duplicate clinical ESBL isolates obtained over an 8-month period using PCR and sequence-based ESBL allele typing, plasmid replicon typing, phylogenetic group typing. Pulsed-field gel electrophoresis (PFGE based genotyping and plasmid profiling was performed, as well as confirmatory DNA-based hybridization assays. Results Examination of the 63 Escherichia coli isolates revealed an almost equal distribution among the E. coli phylogenetic groups A, B1, B2 and D. High prevalence (36/63 of the CTX-M-15 gene was observed and an analysis of PFGE-based patterns revealed the presence of this CTX-M allele in multiple clones. Resistance to cefotaxime was a transferable trait and a commonly occurring 145.5 kb conjugative IncFI plasmid was detected in 65% of E. coli carrying the CTX-M-15 allele. The rate of transferable antibiotic resistances for GM, SXT, TET, GM-SXT-TET, SXT-TET and GM-TET was 33%, 61%, 61%, 27%, 44% and 11%, respectively. The remaining strains did not have a common IncFI plasmid but harboured transferable IncFI plasmids with sizes that ranged from 97 to 242.5 kb. Conclusion Our data demonstrate the presence of IncFI plasmids within the prevailing E. coli population in a hospital setting and suggest that the dissemination of CTX-M-15 allele is associated to lateral transfer of these well-adapted, conjugative IncFI plasmids among various E. coli genotypes.

Presence, distribution, and molecular epidemiology of methicillin-resistant Staphylococcus aureus in a small animal teaching hospital: a year-long active surveillance targeting dogs and their environment.

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van Balen, Joany; Kelley, Christina; Nava-Hoet, Rocio C; Bateman, Shane; Hillier, Andrew; Dyce, Jonathan; Wittum, Thomas E; Hoet, Armando E

2013-05-01

Methicillin-resistant Staphylococcus aureus (MRSA) is known to be present in small animal veterinary clinical environments. However, a better understanding of the ecology and dynamics of MRSA in these environments is necessary for the development of effective infectious disease prevention and control programs. To achieve this goal, a yearlong active MRSA surveillance program was established at The Ohio State University (OSU) Veterinary Medical Center to describe the spatial and molecular epidemiology of this bacterium in the small animal hospital. Antimicrobial susceptibility testing, staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE) typing, and dendrogram analysis were used to characterize and analyze the 81 environmental and 37 canine-origin MRSA isolates obtained during monthly sampling events. Overall, 13.5% of surfaces were contaminated with MRSA at 1 or more sampling times throughout the year. The majority of the environmental and canine isolates were SCCmec type II (93.8% and 86.5%, respectively) and USA100 (90.1% and 86.5%, respectively). By PFGE analysis, these isolates were found to be closely related, which reflects a low diversity of MRSA strains circulating in the hospital. For 5 consecutive months, 1 unique pulsotype was the most prevalent across the medical services and was recovered from a variety of surfaces and hospital locations. Carts/gurneys, doors, and examination tables/floors were the most frequently contaminated surfaces. Some surfaces maintained the same pulsotypes for 3 consecutive months. Molecular analysis found that incoming MRSA-positive dogs were capable of introducing a new pulsotype into the hospital environment during the surveillance period. Our results suggest that once a MRSA strain is introduced into the hospital environment, it can be maintained and spread for extended periods of time. These findings can aid in the development of biosecurity and biocontainment protocols aimed at

First Detection of GES-5 Carbapenemase-Producing Acinetobacter baumannii Isolate.

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Al-Agamy, Mohamed H; Jeannot, Katy; El-Mahdy, Taghrid S; Shibl, Atef M; Kattan, Wael; Plésiat, Patrick; Courvalin, Patrice

2017-07-01

This study was conducted to investigate the molecular epidemiology of resistance in Acinetobacter baumannii isolates collected at a hospital in Riyadh, Saudi Arabia, from January through December 2010. Twenty-seven A. baumannii were highly resistant (MIC 90 > 256 μg/ml) to ceftazidime, cefepime, and aztreonam. Imipenem resistance was seen in 24 isolates, of which 18 had an minimum inhibitory concentration (MIC) >32 μg/mL. Ciprofloxacin, gentamicin, and amikacin resistance was found in 93%, 52%, and 37% of all the isolates, respectively. Moreover, 8 (30%) isolates showed colistin resistance, and 15 (56%) were found to have MICs ≥4 μg/mL for tigecycline. The frequency of ADC, GES-1, GES-11, and GES-5 were 96.3% (n = 26), 18.5% (n = 5), 11% (n = 3), and 3.7% (n = 1), respectively. OXA-23 was found in 63% (n = 17) of the isolates; ISAba1 was found upstream of OXA-23 in 16. OXA-24/40 was detected in only one strain. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis revealed that the 27 strains were distributed in 8 sequence types (STs) and 16 clonal pulsotypes (A-P). Five singleton STs were identified, including ST15 and ST113-ST116. The emergence of multidrug-resistant A. baumannii is becoming a major concern in Saudi Arabia. Metallo-β-lactamases have no role in carbapenem resistance in this collection. The spread of OXA-23 in our strains occurred across different STs and pulsotypes, unlike what has been observed in many other countries. PFGE typing was more discriminatory than MLST. The high frequency of colistin and tigecycline resistance found in the isolates calls for continuous monitoring. This study describes the first identification of GES-5 conferring carbapenem resistance in A. baumannii.

Characterization of a novel plasmid type and various genetic contexts of bla OXA-58 in Acinetobacter spp. from multiple cities in China.

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Yiqi Fu

Full Text Available BACKGROUND/OBJECTIVE: Several studies have described the epidemiological distribution of blaOXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of blaOXA-58-carrying plasmids and the genetic context surrounding blaOXA-58 in Acinetobacter spp. in China. METHODOLOGY/PRINCIPAL FINDINGS: Twelve non-duplicated blaOXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of blaOXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of blaOXA-58-harboring plasmids. The genetic structure surrounding blaOXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype, three Acinetobacter nosocomialis isolates (belong to two pulsotypes and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types. A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both blaOXA-58 and blaOXA-23. blaOXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in blaOXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of blaOXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15-ΔISAba3-like element-blaOXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element

Pasteurella multocida from outbreaks of avian cholera in wild and captive birds in Denmark

DEFF Research Database (Denmark)

Pedersen, Karl; Dietz, Hans-Henrik; Jørgensen, J.C.

2003-01-01

An outbreak of avian cholera was observed among wild birds in a few localities in Denmark in 2001. The highest mortalities were among breeding ciders (Somateria mollissima) and gulls (Larus spp.). Pulsed-field gel electrophoresis (PFGE) was conducted using ApaI and SmaI as restriction enzymes...... the outbreak strain. Among 68 isolates from wild birds, only one PFGE and one REA pattern were demonstrated, whereas among 23 isolates from domestic poultry, 14 different SmaI, 12 different ApaI, and 10 different HpaII patterns were found. The results suggest that a P. multocida strain has survived during...

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

DEFF Research Database (Denmark)

Feltrin, Fabiola; Alba, Patricia; Kraushaar, Britta

2016-01-01

by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most...... resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs......Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze...

Molecular epidemiology of community-onset methicillin-resistant Staphylococcus aureus infections in Israel.

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Biber, A; Parizade, M; Taran, D; Jaber, H; Berla, E; Rubin, C; Rahav, G; Glikman, D; Regev-Yochay, G

2015-08-01

Data on community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) in Israel are scarce. The objective of this study was to characterize the major CA-MRSA clones in Israel. All clinical MRSA isolates detected in the community during a period of 2.5 years (2011-2013) from individuals insured by a major health maintenance organization in Israel were collected, with additional data from medical records. Antibiotic susceptibility patterns and staphylococcal chromosomal cassette mec (SCCmec) typing were determined. SCCmec IV and V isolates were further typed by pulsed-field gel electrophoresis (PFGE), spa typing, and detection of a panel of toxin genes. MRSA were detected in 280 patients, mostly from skin infections. Patients with SCCmec IV (n = 120, 43 %) were younger (p Israel, approximately 20 % are typical CA-MRSA clones, mainly USA300 and a local clone, t991.

Prevalence and Characterization of Integrons in Multidrug Resistant Acinetobacter baumannii in Eastern China: A Multiple-Hospital Study

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Jing Chen

2015-08-01

Full Text Available Objective: The aim of this multiple-hospital study was to investigate the prevalence of integrons in multidrug-resistant Acinetobacter baumannii (MDRAB in Eastern China, and characterize the integron-integrase genes, so as to provide evidence for the management and appropriate antibiotic use of MDRAB infections. Methods: A total of 425 clinical isolates of A. baumannii were collected from 16 tertiary hospitals in 11 cities of four provinces (Fujian, Jiangsu, Zhejiang and Shandong from January 2009 to June 2012. The susceptibility of A. baumannii isolates to ampicillin/sulbactam, piperacillin/tazobactam, ceftazidime, ceftriaxone, cefepime, aztreonam, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, sulfamethoxazole/trimenthoprim, minocycline and imipenem was tested, and integrons and their gene cassettes were characterized in these isolates using PCR assay. In addition, integron-positive A. baumannii isolates were genotyped using pulsed-field gel electrophoresis (PFGE assay, and intI1 gene cassette was sequenced. Results: intI1 gene was carried in 69.6% of total A. baumannii isolates, while intI2 and intI3 genes were not detected. The prevalence of resistance to ampicillin/sulbactam, piperacillin/tazobactam, ceftazidime, ceftriaxone, cefepime, aztreonam, imipenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin and sulfamethoxazole/trimenthoprim was significantly higher in integron-positive A. baumannii isolates than in negative isolates (all p values <0.05, while no significant difference was observed in the prevalence of minocycline resistance (p > 0.05. PFGE assay revealed 27 PFGE genotypes and 4 predominant genotypes, P1, P4, P7 and P19. The PFGE genotype P1 contained 13 extensive-drug resistant and 89 non-extensive-drug resistant A. baumannii isolates, while the genotype P4 contained 34 extensive-drug resistant and 67 non-extensive-drug resistant isolates, appearing a significant

Use of genotypic identification by sodA sequencing in a prospective study to examine the distribution of coagulase-negative Staphylococcus species among strains recovered during septic orthopedic surgery and evaluate their significance.

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Sivadon, V; Rottman, M; Chaverot, S; Quincampoix, J-C; Avettand, V; de Mazancourt, P; Bernard, L; Trieu-Cuot, P; Féron, J-M; Lortat-Jacob, A; Piriou, P; Judet, T; Gaillard, J-L

2005-06-01

A total of 212 coagulase-negative Staphylococcus strains recovered prospectively during 119 surgeries for proven or suspected bone and joint infection (BJI) were identified by sodA sequencing. These strains were identified as 151 Staphylococcus epidermidis isolates, 15 S. warneri isolates, 14 S. capitis isolates, 9 S. hominis isolates, 6 S. lugdunensis isolates, 5 S. haemolyticus isolates, 4 S. caprae isolates, 4 S. pasteuri isolates, 3 S. simulans isolates, and 1 S. cohnii isolate. Only S. epidermidis, S. lugdunensis, S. capitis, and S. caprae were found to be infecting organisms and were involved, respectively, in 35 (81.4%), 3 (7.0%), 3 (7.0%), and 2 (4.6%) cases of BJI.

BACTERIA STAPHYLOCOCCUS SPP. IZOLATED FROM MASTITIS OF SHEEP AND THEIR ENTEROTOXIGENIC PROPERTIES

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František Zigo

2011-12-01

Full Text Available In our study was followed occurrence of staphylococcal mastitis in herd of 350 sheep during one lactation season. We found, the bacteria S. schleiferi was identified in 88 from all 204 isolates. In high number were identified also S. caprae (33, S. chromogenes (21, S. aureus (19, S. epidermidis (17, respectively. Important was occurrence S. intermedius, S. simulans, S. xylosus a S. warneri, too. The Staphylococcuss spp. caused latent and subclinical forms of mastitis predominantly, showed into subacute mastitis (26.5%. Acute mastitis was determined in 7.8 %. Eight bacteria S. aureus, two S. chromogenes, and two S. epidermidis produced staphylococcal enterotoxins.doi: 10.5219/171

Epidemiological characteristics of blaNDM-1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex in China from 2011 to 2012.

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Weimei Ou

Full Text Available The study aimed to investigate the prevalence and epidemiological characteristics of blaNDM-1 (encoding New Delhi metallo-β-lactamase 1 in Enterobacteriaceae and the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ABC in China from July 2011 to June 2012.PCR was used to screen for the presence of blaNDM-1 in all organisms studied. For blaNDM-1-positive strains, 16S rRNA analysis and Analytical Profile Index (API strips were used to identify the bacterial genus and species. The ABCs were reconfirmed by PCR detection of blaOXA-51-like. Antibiotic susceptibilities of the bacteria were assessed by determining minimum inhibitory concentration (MIC of them using two-fold agar dilution test, as recommended by the Clinical and Laboratory Standards Institute (CLSI. Molecular typing was performed using pulsed-field gel electrophoresis (PFGE. S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE and Southern blot hybridization were conducted to ascertain the gene location of blaNDM-1. Conjugation experiments were conducted to determine the transmission of blaNDM-1-positive strains.Among 2,170 Enterobacteriaceae and 600 ABCs, seven Enterobacteriaceae strains and two A. calcoaceticus isolates from five different cities carried the blaNDM-1 gene. The seven Enterobacteriaceae strains comprised four Klebsiella pneumoniae, one Enterobacter cloacae, one Enterobacter aerogen and one Citrobacter freundii. All seven were non-susceptible to imipenem, meropenem or ertapenem. Two A. calcoaceticus species were resistant to imipenem and meropenem. Three K. pneumoniae showed the same PFGE profiles. The blaNDM-1 genes of eight strains were localized on plasmids, while one was chromosomal.Compared with previous reports, the numbers and species containing the blaNDM-1 in Enterobacteriaceae have significantly increased in China. Most of them are able to disseminate the gene, which is cause for concern. Consecutive surveillance should be implemented and should

Competing failure analysis in phased-mission systems with functional dependence in one of phases

International Nuclear Information System (INIS)

Wang, Chaonan; Xing, Liudong; Levitin, Gregory

2012-01-01

This paper proposes an algorithm for the reliability analysis of non-repairable phased-mission systems (PMS) subject to competing failure propagation and isolation effects. A failure originating from a system component which causes extensive damage to other system components is a propagated failure. When the propagated failure affects all the system components, causing the entire system failure, a propagated failure with global effect (PFGE) is said to occur. However, the failure propagation can be isolated in systems subject to functional dependence (FDEP) behavior, where the failure of a component (referred to as trigger component) causes some other components (referred to as dependent components) to become inaccessible or unusable (isolated from the system), and thus further failures from these dependent components have no effect on the system failure behavior. On the other hand, if any PFGE from dependent components occurs before the trigger failure, the failure propagation effect takes place, causing the overall system failure. In summary, there are two distinct consequences of a PFGE due to the competition between the failure isolation and failure propagation effects in the time domain. Existing works on such competing failures focus only on single-phase systems. However, many real-world systems are phased-mission systems (PMS), which involve multiple, consecutive and non-overlapping phases of operations or tasks. Consideration of competing failures for PMS is a challenging and difficult task because PMS exhibit dynamics in the system configuration and component behavior as well as statistical dependencies across phases for a given component. This paper proposes a combinatorial method to address the competing failure effects in the reliability analysis of binary non-repairable PMS. The proposed method is verified using a Markov-based method through a numerical example. Different from the Markov-based approach that is limited to exponential distribution, the

Molecular epidemiology of carbapenem resistant gram-negative bacilli from infected pediatric population in tertiary - care hospitals in Medellín, Colombia: an increasing problem.

Science.gov (United States)

Vanegas, Johanna M; Parra, O Lorena; Jiménez, J Natalia

2016-09-01

Gram-negative bacilli are a cause of serious infections in the pediatric population. Carbapenem are the treatment of choice for infections caused by multidrug-resistant Gram-negative bacilli, but the emergence of carbapenem resistance has substantially reduced access to effective antimicrobial regimens. Children are a population vulnerable to bacterial infections and the emergence of resistance can worsen prognosis. The aim of this study is to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant Gram-negative bacilli in pediatric patients from five tertiary-care hospitals in Medellín, Colombia. A cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to June 2014. All pediatric patients infected by carbapenem-resistant Gram-negative bacilli were included. Clinical information for each patient was obtained from medical records. Molecular analyses included PCR for detection of bla VIM, bla IMP bla NDM, bla OXA-48 and bla KPC genes and PFGE and MLST for molecular typing. A total of 59 patients were enrolled, most of them less than 1 year old (40.7 % n = 24), with a previous history of antibiotic use (94.9 %; n = 56) and healthcare-associated infections - predominately urinary tract infections (31.0 %; n = 18). Klebsiella pneumoniae was the most frequent bacteria (47.4 %), followed by Enterobacter cloacae (40.7 %) and Pseudomonas aeruginosa (11.9 %). For K. pneumoniae, KPC was the predominant resistance mechanism (85.7 %; n = 24) and ST14 was the most common clone (39.3 % n = 11), which included strains closely related by PFGE. In contrast, E. cloacae and P. aeruginosa were prevailing non-carbapenemase-producing isolates (only KPC and VIM were detected in 1 and 3 isolates, respectively) and high genetic diversity according to PFGE and MLST was found in the majority of the cases. In recent years, increasing carbapenem-resistant bacilli in children has become in a matter

Antibiotic resistance and mecA characterization of coagulase-negative staphylococci isolated from three hotels in London, UK

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Zhen eXu

2015-09-01

Full Text Available Antibiotic resistance in bacteria isolated from non–healthcare environments, is a potential problem to public health. In our survey a total of 71 coagulase negative staphylococci belonging to 11 different species were isolated from 3 large hotels in London, UK. The most prevalent species was Staphylococcus haemolyticus, with S. hominis, S. warneri, S.cohnii and S. epidermidis commonly detected. Antimicrobial susceptibilities and carriage of the mecA gene were determined for all of these isolates. Most (85.9% staphylococci were resistant to multiple antibiotics with all displaying increased susceptibility towards penicillin, fusidic acid, erythromycin and cefepime. 21 (29.5% of the isolates were mecA positive, however MIC values to oxacillin, normally associated with the carriage of mecA, varied widely in this group (from 0.06 mg/L to 256 mg/L. 15 of the 21 mecA positive isolates carried SCCmec of these 7 were type V, 1 type I, 1 type II and 1 type IV. Additionally, five of these 15 isolates carried a previously unreported type, 1A, which involves an association between class A mec complex and ccr type 1. The remaining 6 of the 21 isolates were non-typeable and carried a combination of class A mec complex and ccrC. In addition to this, we also report on new MLST types which were assigned for 5 Staphylococcus epidermidis isolates. 4 out of these 5 isolates had MICs between 0.06 to 256 mg/L to oxacillin and would be regarded as clinically susceptible but one isolate had a high oxacillin MIC of 256 mg/L. We demonstrated widespread multiple drug resistance among different staphylococcal species isolated from non-healthcare environments highlighting the potential for these species to act as a reservoir for methicillin and other forms of drug resistance.

Antibiotic resistance and mecA characterization of coagulase-negative staphylococci isolated from three hotels in London, UK.

Science.gov (United States)

Xu, Zhen; Mkrtchyan, Hermine V; Cutler, Ronald R

2015-01-01

Antibiotic resistance in bacteria isolated from non-healthcare environments, is a potential problem to public health. In our survey a total of 71 coagulase negative staphylococci (CNS) belonging to 11 different species were isolated from three large hotels in London, UK. The most prevalent species was Staphylococcus haemolyticus, with S. hominis, S. warneri, S. cohnii, and Staphylococcus epidermidis commonly detected. Antimicrobial susceptibilities and carriage of the mecA gene were determined for all of these isolates. Most (85.9%) staphylococci were resistant to multiple antibiotics with all displaying increased susceptibility toward penicillin, fusidic acid, erythromycin, and cefepime. Twenty-one (29.5%) of the isolates were mecA positive, however MIC values to oxacillin, normally associated with the carriage of mecA, varied widely in this group (from 0.06 to 256 mg/L). Fifteen of the twenty-one mecA positive isolates carried SCCmec of these seven were type V, one type I, one type II, and one type IV. Additionally, five of these 15 isolates carried a previously unreported type, 1A, which involves an association between class A mec complex and ccr type 1. The remaining six of the 21 isolates were non-typeable and carried a combination of class A mec complex and ccrC. In addition to this, we also report on new MLST types which were assigned for five S. epidermidis isolates. Four out of these five isolates had MICs between 0.06 and 256 mg/L to oxacillin and would be regarded as clinically susceptible but one isolate had a high oxacillin MIC of 256 mg/L. We demonstrated widespread multiple drug resistance among different staphylococcal species isolated from non-healthcare environments highlighting the potential for these species to act as a reservoir for methicillin and other forms of drug resistance.

Genetic diversity, anti-microbial resistance, plasmid profile and frequency of the Vi antigen in Salmonella Dublin strains isolated in Brazil.

Science.gov (United States)

Vilela, F P; Frazão, M R; Rodrigues, D P; Costa, R G; Casas, M R T; Fernandes, S A; Falcão, J P; Campioni, F

2018-02-01

Salmonella Dublin is strongly adapted to cattle causing enteritis and/or systemic disease with high rates of mortality. However, it can be sporadically isolated from humans, usually causing serious disease, especially in patients with underlying chronic diseases. The aim of this study was to molecularly type S. Dublin strains isolated from humans and animals in Brazil to verify the diversity of these strains as well as to ascertain possible differences between strains isolated from humans and animals. Moreover, the presence of the capsular antigen Vi and the plasmid profile was characterized in addition to the anti-microbial resistance against 15 drugs. For this reason, 113 S. Dublin strains isolated between 1983 and 2016 from humans (83) and animals (30) in Brazil were typed by PFGE and MLVA. The presence of the capsular antigen Vi was verified by PCR, and the phenotypic expression of the capsular antigen was determined serologically. Also, a plasmid analysis for each strain was carried out. The strains studied were divided into 35 different PFGE types and 89 MLVA-types with a similarity of ≥80% and ≥17.5%, respectively. The plasmid sizes found ranged from 2 to >150 kb and none of the strains studied presented the capsular antigen Vi. Resistance or intermediate resistance was found in 23 strains (20.3%) that were resistant to ampicillin, ciprofloxacin, chloramphenicol, imipenem, nalidixic acid, piperacillin, streptomycin and/or tetracycline. The majority of the S. Dublin strains studied and isolated over a 33-year period may descend from a common subtype that has been contaminating humans and animals in Brazil and able to cause invasive disease even in the absence of the capsular antigen. The higher diversity of resistance phenotypes in human isolates, as compared with animal strains, may be a reflection of the different anti-microbial treatments used to control S. Dublin infections in humans in Brazil. © 2017 Blackwell Verlag GmbH.

Molecular epidemiology and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level.

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Mahmmod, Y S; Klaas, I C; Katholm, J; Lutton, M; Zadoks, R N

2015-10-01

Host-adaptation of Streptococcus agalactiae subpopulations has been described whereby strains that are commonly associated with asymptomatic carriage or disease in people differ phenotypically and genotypically from those causing mastitis in dairy cattle. Based on multilocus sequence typing (MLST), the most common strains in dairy herds in Denmark belong to sequence types (ST) that are also frequently found in people. The aim of this study was to describe epidemiological and diagnostic characteristics of such strains in relation to bovine mastitis. Among 1,199 cattle from 6 herds, cow-level prevalence of S. agalactiae was estimated to be 27.4% based on PCR and 7.8% based on bacteriological culture. Quarter-level prevalence was estimated at 2.8% based on bacteriological culture. Per herd, between 2 and 26 isolates were characterized by pulsed-field gel electrophoresis (PFGE) and MLST. Within each herd, a single PFGE type and ST predominated, consistent with a contagious mode of transmission or point source infection within herds. Evidence of within-herd evolution of S. agalactiae was detected with both typing methods, although ST belonged to a single clonal complex (CC) per herd. Detection of CC23 (3 herds) was associated with significantly lower approximate count (colony-forming units) at the quarter level and significantly lower cycle threshold value at the cow level than detection of CC1 (2 herds) or CC19 (1 herd), indicating a lower bacterial load in CC23 infections. Median values for the number of infected quarters and somatic cell count (SCC) were numerically but not significantly lower for cows infected with CC23 than for cows with CC1 or CC19. For all CC, an SCC threshold of 200,000 cells/mL was an unreliable indicator of infection status, and prescreening of animals based on SCC as part of S. agalactiae detection and eradication campaigns should be discouraged. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy.

Science.gov (United States)

Feltrin, Fabiola; Alba, Patricia; Kraushaar, Britta; Ianzano, Angela; Argudín, María Angeles; Di Matteo, Paola; Porrero, María Concepción; Aarestrup, Frank M; Butaye, Patrick; Franco, Alessia; Battisti, Antonio

2016-02-01

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming

High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large foodborne outbreak

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Trout-Yakel Keri M

2010-02-01

Full Text Available Abstract Background A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE. Results The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. Conclusions High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

Science.gov (United States)

Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

2009-11-01

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Correlation of chromosome patterns in human leukemic cells with exposure to chemicals and/or radiation: Comprehensive progress report, January 1986--June 1988

International Nuclear Information System (INIS)

Rowley, J.D.

1988-06-01

I purchased one of the few available prototypes of the pulse field gel electrophoresis (PFGE) apparatus. We used PFGE and its various modifications to map the human Abelson protooncogene (ABL) and to show that the two alternative first exons (Ia and Ib) are separated by at least 200 kilobases (kb). This has provided the first evidence that alternative splicing from exon Ib to the common splice acceptor site (exon II) could occur over such very large distances. We are actively using vertical field gel electrophoresis, a modification of PFGE, for mapping various DNA probes on chromosome 5. Another major advance has been the development of the polymerase chain reaction (PCR). We are currently using this to define the breakpoints in the BCR gene in the 9; 22 translocation in chronic myeloid leukemia (CML) and in Ph 1 -positive acute lymphoblastic leukemia (ALL). I had expected to be able to describe major progress in cloning the chromosome translocation breakpoints in ANLL, and this has not occurred. Our laboratory knows how to solve the problem. We successfully cloned a new translocation breakpoint in B cell chronic lymphatic leukemia involving Nos. 14 and 19. 22 refs., 2 figs., 5 tabs

Bacterial pathogen spectrum of acute diarrheal outpatients in an urbanized rural district in Southwest China

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Yongming Zhou

2018-05-01

Full Text Available Objectives: To conduct a one-year pathogen surveillance of acute diarrheal disease based on outpatient clinics in township hospitals in rural Hongta District of Yunnan Province, China. Methods: Fecal specimens of acute diarrhea cases and relevant epidemiological information were collected. Salmonella, Shigella, Vibrio, Aeromonas, Plesiomonas shigelloides and diarrheogenic Escherichia coli (DEC were examined. Results: Among the 797 stool specimens sampled, 198 samples (24.8% were positive in pathogen isolation, and 223 strains were isolated. The order of isolation rates from high to low were DEC, Aeromonas, P. shigelloides, Salmonella, Shigella and Vibrio. The overall positive rate in middle school students and preschool children was relatively high; while the overall positive rate of less than 1-year-old infants and above 55 years olds was relatively low. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE. Some cases had the same or very close onset time, and the isolates had similar PFGE patterns, suggesting a possible outbreak once occurred but was not detected by the current infectious disease reporting system. Conclusions: Pathogen infection and transmission in rapidly urbanized rural areas is a serious issue. There is a great need for a more sensitive and accurate mode of monitoring, reporting and outbreak identification of diarrheal disease. Keywords: Diarrheal disease, Diarrheogenic pathogen, Molecular typing, Surveillance, Bacterial pathogen

Rapid molecular identification and characteristics of Lactobacillus strains.

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Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

2010-09-01

Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

Spread of Extended Spectrum Cephalosporinase-Producing Escherichia coli Clones and Plasmids from Parent Animals to Broilers and to Broiler Meat in a Production Without Use of Cephalosporins

DEFF Research Database (Denmark)

Agersø, Yvonne; Jensen, Jacob Dyring; Hasman, Henrik

2014-01-01

Objectives: This study investigated the occurrence of extended spectrum cephalosporinase (ESC)–producing Escherichia coli in a broiler production with no cephalosporin use and a low use of antimicrobials in general. Furthermore, it investigated whether the current consumption of aminopenicillins....... Isolates with blaCMY-2 were subtyped by pulsed-field gel electrophoresis (PFGE), phylotyping, and antimicrobial susceptibility testing. Selected isolates were used as donors in filter-mating experiments, multilocus sequence typing (MLST), and plasmid replicons were typed. Aminopenicillin use at the farm...... (not flock) level was obtained from VetStat, a database for mandatory registration of veterinary prescriptions in Denmark. Results: ESC-producing E. coli occurred in 93% (27/29) of broiler parent farms in 2011, 27% (53/197) of broiler flocks in 2010, and 3.3% (4/121) of Danish retail broiler meat...

An outbreak of borderline oxacillin-resistant Staphylococcus aureus (BORSA) in a dermatological unit

DEFF Research Database (Denmark)

Balslev, Ulla; Bremmelgaard, Annie; Svejgaard, E

2005-01-01

(PFGE), and spa genotyping that confirmed that they were indistinguishable from one another. In May, 2000, an intervention was initiated focusing on infection control. In-patients with BORSA were discharged and antibiotic treatment was stopped, if possible. Emphasis was put on disinfection of shared......From February through July, 2000, there was an accumulation of mecA-negative borderline resistant Staphylococcus aureus (BORSA) in the Department of Dermatology. BORSA was isolated in 37 samples from 11 patients. The isolates were typed by antibiogram, phage type, pulsed-field gel electrophoresis...... utensils and individualization of previously shared creams and lotions. After the intervention, BORSA was isolated from another 3 patients before the outbreak was terminated. A case-control study was undertaken to identify possible risk factors for being a BORSA patient. Compared to the controls...

Protracted Regional Dissemination of GIM-1-Producing Serratia marcescens in Western Germany.

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Wendel, Andreas F; Kaase, Martin; Autenrieth, Ingo B; Peter, Silke; Oberhettinger, Philipp; Rieber, Heime; Pfeffer, Klaus; MacKenzie, Colin R; Willmann, Matthias

2017-03-01

The metallo-beta-lactamase GIM-1 has been found in various bacterial host species nearly exclusively in western Germany. However, not much is known about the epidemiology of GIM-1-positive Serratia marcescens Here we report on a surprisingly protracted regional dissemination. In-hospital transmission was investigated by using conventional epidemiological tools to identify spatiotemporal links. Strain typing was performed using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS). Bayesian phylogeny was used to infer the time axis of the observed occurrence. Thirteen S. marcescens strains from 10 patients from 6 different German hospitals were investigated. Suspected in-hospital transmissions were confirmed by molecular typing at a higher resolution by WGS than by PFGE. A detailed sequence analysis demonstrated the spread of one predominant strain variant but also provided evidence for transfer of the bla GIM-1 gene cassette between different strains. A Bayesian phylogenetic analysis showed that the most recent common ancestor of the identified clonal cluster could be dated back to April 1993 (95% highest posterior density interval, January 1973 to March 2003) and that this strain might have already harbored the bla GIM-1 at that time and, therewith, years before the first detection of this resistance gene in clinical specimens. This study shows a long-standing clonal and plasmid-mediated expansion of GIM-1-producing S. marcescens that might have gone unnoticed in the absence of a standardized and effective molecular screening for carbapenemases. The systematic and early detection of resistance is thus highly advisable, especially for the prevention of potentially long-term dissemination that may progress beyond control. Copyright © 2017 American Society for Microbiology.

Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan.

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Kanamori, Hajime; Yano, Hisakazu; Hirakata, Yoichi; Hirotani, Ayako; Arai, Kazuaki; Endo, Shiro; Ichimura, Sadahiro; Ogawa, Miho; Shimojima, Masahiro; Aoyagi, Tetsuji; Hatta, Masumitsu; Yamada, Mitsuhiro; Gu, Yoshiaki; Tokuda, Koichi; Kunishima, Hiroyuki; Kitagawa, Miho; Kaku, Mitsuo

2012-01-01

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.

Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonella koehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

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Chomel, Bruno B.; Molia, Sophie; Kasten, Rickie W.; Borgo, Gina M.; Stuckey, Matthew J.; Maruyama, Soichi; Chang, Chao-chin; Haddad, Nadia; Koehler, Jane E.

2016-01-01

Domestic cats are the natural reservoir of Bartonella henselae, B. clarridgeiae and B. koehlerae. To determine the role of wild felids in the epidemiology of Bartonella infections, blood was collected from 14 free-ranging California mountain lions (Puma concolor) and 19 bobcats (Lynx rufus). Bartonella spp. were isolated from four (29%) mountain lions and seven (37%) bobcats. These isolates were characterized using growth characteristics, biochemical reactions, molecular techniques, including PCR-RFLP of selected genes or interspacer region, pulsed-field gel electrophoresis (PFGE), partial sequencing of several genes, and DNA-DNA hybridization. Two isolates were identical to B. henselae genotype II. All other isolates were distinguished from B. henselae and B. koehlerae by PCR-RFLP of the gltA gene using endonucleases HhaI, TaqI and AciI, with the latter two discriminating between the mountain lion and the bobcat isolates. These two novel isolates displayed specific PFGE profiles distinct from B. henselae, B. koehlerae and B. clarridgeiae. Sequences of amplified gene fragments from the three mountain lion and six bobcat isolates were closely related to, but distinct from, B. henselae and B. koehlerae. Finally, DNA-DNA hybridization studies demonstrated that the mountain lion and bobcat strains are most closely related to B. koehlerae. We propose naming the mountain lion isolates B. koehlerae subsp. boulouisii subsp. nov. (type strain: L-42-94), and the bobcat isolates B. koehlerae subsp. bothieri subsp. nov. (type strain: L-17-96), and to emend B. koehlerae as B. koehlerae subsp. koehlerae. The mode of transmission and the zoonotic potential of these new Bartonella subspecies remain to be determined. PMID:26981874

Significant spread of extensively drug-resistant Acinetobacter baumannii genotypes of clonal complex 92 among intensive care unit patients in a university hospital in southern Iran.

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Saffari, Fereshteh; Monsen, Tor; Karmostaji, Afsaneh; Azimabad, Fahimeh Bahadori; Widerström, Micael

2017-11-01

Infections associated with Acinetobacter baumannii represent an increasing threat in healthcare settings. Therefore, we investigated the epidemiological relationship between clinical isolates of A. baumannii obtained from patients in a university hospital in Bandar Abbas in southern Iran. Sixty-four consecutive non-duplicate clinical isolates collected during 2014-2015 were subjected to susceptibility testing, clonal relationship analysis using PFGE, multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST), and examined for the presence of carbapenemases and integrons. Almost all A. baumannii isolates were extensively drug-resistant (XDR; 98 %) and carried an OXA carbapenemase gene (blaOXA-23-like; 98 %) and class 1 integrons (48 %). PFGE and MLST analysis identified three major genotypes, all belonging to clonal complex 92 (CC92): sequence type 848 (ST848) (n=23), ST451 (n=16) and ST195 (n=8). CC92 has previously been documented in the hospital setting in northern Iran, and ST195 has been reported in Arab States of the Persian Gulf. These data suggest national and global transmission of A. baumannii CC92. This report demonstrates the occurrence and potential spread of closely related XDR genotypes of A. baumannii CC92 within a university hospital in southern Iran. These genotypes were found in the majority of the investigated isolates, showed high prevalence of blaOXA-23 and integron class 1, and were associated with stay in the intensive care unit. Very few treatment options remain for healthcare-adapted XDR A. baumannii, and hence effective measures are desperately needed to reduce the spread of these strains and resultant infections in the healthcare setting.

Carbapenem-resistant Klebsiella pneumoniae infections in a Greek intensive care unit: Molecular characterisation and treatment challenges.

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Katsiari, Maria; Panagiota, Giakkoupi; Likousi, Sophia; Roussou, Zoi; Polemis, Michalis; Alkiviadis Vatopoulos, C; Evangelia Platsouka, D; Maguina, Asimina

2015-06-01

Acquisition of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) strains poses a major threat to critically ill patients. The objectives of this study were to describe the epidemiology of CP-Kp isolates as well as the clinical outcome associated with the corresponding infections and to identify risk factors for mortality of intensive care unit (ICU) patients in a Greek hospital. A prospective, observational study was conducted in a nine-bed general ICU over a 2-year period (April 2010-March 2012). Imipenem-resistant K. pneumoniae isolates recovered from clinical samples of ICU patients were prospectively collected and studied for the presence of carbapenemases. Isolates were submitted to molecular typing using pulsed-field gel electrophoresis (PFGE). In total, 61 CP-Kp isolates (48 KPC-producers and 13 VIM-producers) were recovered from 58 ICU patients. The majority of KPC-producers were classified into a single PFGE type, indicating potent clonal dissemination. Among the 32 infected patients, bacteraemia was diagnosed in 16. Tigecycline+colistin was the most common combination antimicrobial regimen. Infection-attributable mortality was 43.8%. Regarding mortality risk factors, non-survivors were older (P=0.080), all of them presented with septic shock (P=0.010) and they had higher Sepsis-related Organ Failure Assessment (SOFA) scores at infection onset (P=0.004) compared with survivors. Appropriate definitive treatment and combination regimens were not associated with patient survival. In conclusion, CP-Kp infections are associated with limited treatment options and high in-hospital mortality. Effective measures for preventing dissemination of respective isolates in the hospital setting are required. Copyright © 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Molecular Characterization of Extended-Spectrum Cephalosporinase-Producing Salmonella enterica Serovar Choleraesuis Isolates from Patients in Thailand and Denmark

DEFF Research Database (Denmark)

Sirichote, P.; Hasman, Henrik; Pulsrikarn, C.

2010-01-01

isolates were recovered from 21 Thai patients during 2003, 2007, or 2008 and one isolate was recovered from a Danish traveler to Thailand. ESC production was confirmed in 13 out of the 24 isolates by MIC testing. Microarray and plasmid profiling (replicon typing and restriction fragment length polymorphism...... on susceptibility patterns, the ESC-producing isolates were more closely related than non-ESC-producing isolates. Microarray, PCR, plasmid profiling, and replicon typing revealed that the 13 ESC-producing isolates harbored either bla(CMY-2) containing incA/C or bla(CTX-M-14) containing incFIIA, inc......FrepB, and an unknown replicon located on plasmids ranging in size from 75 to 200 kb. The RFLP and replicon typing clustered the isolates into four distinct groups. PFGE revealed 16 unique patterns and five clusters; each cluster contained two or three of the 24 isolates. The isolate from the Danish patient...

The effectiveness of lysostaphin therapy for experimental coagulase-negative Staphylococcus endophthalmitis.

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McCormick, Clare C; Dajcs, Joseph J; Reed, Julian M; Marquart, Mary E; O'Callaghan, Richard J

2006-03-01

To quantitatively determine the effectiveness of lysostaphin therapy for experimental endophthalmitis mediated by coagulase-negative Staphylococcus species, the leading cause of postsurgical endophthalmitis. Minimal inhibitory concentrations (MIC) of lysostaphin were determined for 54 isolates representing the following species: S. epidermidis, S. warneri, S. haemolyticus, S. cohnii, S. simulans, and S. capitis. The effectiveness of lysostaphin therapy was tested in an experimental model of endophthalmitis by intravitreally injecting log phase bacteria (100 colony-forming units; cfu) into rabbit eyes (n = 3 eyes per group). At 8 hr postinfection (PI), lysostaphin (250 microg) was injected intravitreally, and the number of cfu/ml of vitreous was determined at 24 hr PI. Average MIC for S. epidermidis was 0.7 microg/ml for 90% of the 33 strains tested. Six methicillin-resistant strains of S. epidermidis (MRSE) had an average MIC of 0.74 micro g/ml. All other species had MIC values of =1.1 microg/ml, except for one strain of S. capitis (MIC = 15.6 microg/ml) and one S. haemolyticus (MIC = 1.41 microg/ml). At 24 hr PI, all untreated eyes had between 5.7 and 8.08 log cfu/ml vitreous humor. Treatment with lysostaphin significantly reduced the cfu/ml as compared with untreated eyes for 13 strains tested in vivo (p = 0.020), but not for two S. haemolyticus strains (p = 0.13), two MRSE strains (p = 0.544), or one S. cohnii strain (p = 0.1366). Treatment with lysostaphin reduced the cfu/ml of methicillin-sensitive S. epidermidis strains by 6 logs; for S. warneri, there was a 2 log reduction; and for the other species a 4 log reduction in cfu/ml relative to untreated eyes. Lysostaphin was mostly effective in treating coagulase-negative staphylococcal experimental endophthalmitis.

Prevalence and antibiotic resistance of 15 minor staphylococcal species colonizing orthopedic implants.

Science.gov (United States)

Arciola, C R; Campoccia, D; An, Y H; Baldassarri, L; Pirini, V; Donati, M E; Pegreffi, F; Montanaro, L

2006-04-01

Several species belonging to Staphylococcus genus (non Sau/ non Sep species) exhibit increasing abilities as opportunistic pathogens in colonisation of periprosthesis tissues. Here we report on antibiotic resistance of 193 strains, belonging to non Sau/ non Sep species, consecutively collected from orthopedic implant infections in a period of about 40 months. The 193 strains (representing 17% of all staphylococci isolated) were analysed for their antibiotic resistance to 16 different drugs. Five species turned out more prevalent, ranging from 1 to 5%: S. hominis (4.2%), S. haemolyticus (3.7%), S. capitis (2.7%), S. warneri (2.6%), and S. cohnii (1.6%). Among these, the prevalence of antibiotic resistance to penicillins was similar, ranging from 51% to 66%. Conversely, significant differences were observed for all the remaining antibiotics. For S. haemolyticus the resistances to oxacillin and imipenem, the four aminoglycosides and erythromycin were at least twice that of the other three species which were compared. S. warneri was on the contrary the species with the lowest occurrence of resistant strains. Ten species appeared only rarely at the infection sites: S. lugdunensis, S. caprae, S. equorum, S. intermedius, S. xylosus, S. simulans, S. saprophyticus, S. pasteuri, S. sciuri, and S. schleiferi. The behaviours of these species, often resistant to penicillins, were individually analysed. Differences in both the frequencies and the panels of antibiotic resistances observed among the non Sau/ non Sep species: i) suggest that horizontal spreading of resistance factors, if acting, was not sufficient per se to level their bio-diversities; ii) highlight and confirm the worrisome appearance within the Staphylococcus genus of emerging "new pathogens", not homogeneous for their virulence and antibiotic resistance prevalence, which deserve to be recognised and treated individually.

Longitudinal study of the excretion patterns of thermophilic Campylobacter spp. in young pet dogs in Denmark

DEFF Research Database (Denmark)

Hald, Birthe; Pedersen, Karl; Wainø, Michael

2004-01-01

on the Campylobacter-selective media CAT and modified CCDA agar at 37 and 42 C. This study comprised 366 fecal swab samples, of which 278 (76.2%) were found to be Campylobacter positive, with the following distribution of species: 75.0% Campylobacter upsaliensis, 19.4% Campylobacter jejuni, 2.1% Campylobacter lari, 0......% carriers at 1 year of age, whereafter the carriage rate decreased to 67% at 24 months of age. The PFGE types showed that individual dogs were often colonized by unique strains of C. upsaliensis for several months, up to 21 months or longer. These C. upsaliensis strains were either clonal (or underwent...

CARACTERIZAÇÃO DO STAPHYLOCOCCUS COAGULASE NEGATIVO ISOLADOS DE QUARTOS MAMÁRIOS DE VACAS COM MASTITE SUBCLÍNICA

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Marlice Teixeira Ribeiro

1994-01-01

Full Text Available RESUMO O presente trabalho foi desenvolvido na Zona da Mata - MG, durante três meses. De quatro rebanhos utilizados, trabalhou-se com 16 animais mestiços e 19 holandeses. O objetivo foi caracterizar Staphylococcus coagulase negativos, isolados de quartos mamários de vacas, que inicialmente reagiam à prova do "Califórnia Mastitis Test" (CMT, em quatro testes consecutivos, a intervalo de uma semana. A caracterização das 116 amostras encontradas, correspondentes a Staphylococcus coagulase negativo, foram: S. epidermidis (71,55%, S. hyicus subsp. chromogenes (6,03%, S. simulans (6,03%, S. warneri (4,31%, S. hominis (3,45%, S. cohnii (3,45%, S. sciuri (3,45% e S. haemolyticus (1,73%.

Clonal Diversity of Chilean Isolates of Enterohemorrhagic Escherichia coli from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products

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Rios, Maritza; Prado, Valeria; Trucksis, Michele; Arellano, Carolina; Borie, Consuelo; Alexandre, Marcela; Fica, Alberto; Levine, Myron M.

1999-01-01

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx1+ stx2+ hly+ eae+ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx1 and stx2 toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx1+ stx2+, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx1+ stx2+ eae+ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile. PMID:9986852

cfr-mediated linezolid-resistant clinical isolates of methicillin-resistant coagulase-negative staphylococci from China.

Science.gov (United States)

Song, Yunjia; Lv, Yuan; Cui, Lanqing; Li, Yun; Ke, Qian; Zhao, Yixuan

2017-03-01

Three linezolid-resistant coagulase-negative staphylococci (LR-CoNS), including two Staphylococcus cohnii and one Staphylococcus capitis, were isolated from 1104 clinical staphylococcal isolates across China in 2013-2014. Antibiotic susceptibilities of the bacteria were determined by the agar dilution method. PCR and DNA sequencing were performed to determine the potential molecular mechanism of linezolid resistance. The two linezolid-resistant S. cohnii isolates were subjected to pulsed-field gel electrophoresis (PFGE) to investigate their genetic relatedness. Primer walking, S1 nuclease PFGE and Southern blot hybridisation were conducted to ascertain the location and environment of the cfr gene. All three isolates were positive for the cfr gene. Amino acid mutations S158F and S158Y in the ribosomal protein L3 were identified in S. cohnii 13B289 and 13L105, respectively, both of which also had an additional substitution (D159Y) in L3. PFGE indicated that the two S. cohnii isolates belonged to diverse clonal strains. S1 nuclease PFGE and Southern blotting experiments indicated that the cfr gene of the three isolates resided on plasmids of similar size (ca. 35.4kb). The cfr-harbouring segments of S. capitis 13G350 and S. cohnii 13L105 were identical to plasmid pSS-01 reported previously. The cfr-carrying fragment of S. cohnii 13B289 was indistinguishable from the formerly described plasmid pSS-02. In conclusion, the presence of the cfr gene located on a plasmid was the main mechanism contributing to resistance to linezolid in the three staphylococcal isolates. Hence, timely detection and judicious use of antibiotics are essential to prevent further transmission of this resistance mechanism. Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

The Prevalence of ESBL Isolates of Acinetobacter baumannii Using Pulsed-Field Gel Electrophoresis

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Parviz Mohajeri

2014-12-01

Full Text Available Background: Antibiotics such as fluoroquinolones are used for treating infections caused by Gram-negative bacteria, including Acinetobacter baumannii strains some time have extended-spectrum β-lactamase (ESBL, but ESBL production is rather rare. Resistance to fluoroquinolones antibiotics is mediated by lactamases and other mechanisms of resistance. The aim of the present study was to investigate of the prevalence of ESBL production and clonal relatedness of A. baumannii in Iran. Materials and Methods: A. baumannii isolates identified from patients at hospitals in Kermanshah, Iran, were studied. The double disk method was used for detection of ESBL production. The susceptibility to different antibiotics was determined by the disk diffusion method (CLSI. Clonal relatedness was determined by pulsed-field gel electrophoresis (PFGE and processed by Bionumerics 7.0 software. Statistical analyses were performed using SPSS-16.0. Results: This study showed high prevalence of resistance to ampicillin and cefpodoxim (98.1 and 92.3%. Fifty-two of the 84 isolates were identified as ESBL producers. Only colistin and tigecycline remained active against all isolates tested. The PFGE identified eight distinct pulsotypes: A (N=9, B (N=10, C (N=2, D (N=5, E (N=9, F (N=15, G (N=1 and H (N=1. The PFGE profiles A, B and F were believed to be endemic (specially clone F that was dominant across different wards of the hospitals and appeared to be endemic in the ICU, emergency, pediatric and infection area throughout the years. Conclusion: Early and timely detection of ESBL-producing A. baumannii clones is useful for preventing their spread within the hospital. PFGE analysis is helpful for detection of common strains in different wards and prevention of further spread of these pulsotypes to other hospital environment.

Multifocal diffusion of KPC-3-producing ST512 Klebsiella pneumoniae in Italy

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Aurora Piazza

2012-03-01

Full Text Available Introduction. The dissemination of carbapenemase-producing Klebsiella pneumoniae strains is an increasing problem worldwide. KPC ß-lactamases are Ambler class A enzymes mostly plasmid-encoded; their global spread represents a threat to clinical patients care and public health. Multi locus sequence type (ST258 is currently the most spread K. pneumoniae clone associated with KPC enzymes. Here we report the first identification and multifocal spread of KPC-3 producing K. pneumoniae clinical strains belonging to ST512 in Italy. Materials and Methods. Fifty six carbapenem-resistant K. pneumoniae isolates were collected from 7 Italian hospitals during the period June 2009-May 2011. Isolates were obtained from different wards (spinal unit, medicine, hematology, etc. and biological samples (mostly rectal swabs, urine and blood. Species identification and antimicrobial susceptibilities were obtained by NBC46/NM40 Microscan panels (Siemens. MICs values were interpreted according to EUCAST 2011 breakpoints. Modified Hodge test and combined disk test with phenyl-boronic acid (BOR and EDTA were performed.The presence of blaKPC genes were confirmed by PCR and sequencing. A complete characterization of the produced ß-lactamases (BLs was obtained by IEF followed by PCR experiments using primers specific for the detection of blaCTX-M-, blaTEM- and blaSHV type genes. PFGE and multilocus sequence typing (MLST were both used to investigate clonal isolates relatedness. Results. All 56 isolates resulted positive for the presence of KPC-type carbapenemases by both phenotypical and molecular analysis. Fifteen isolates, chosen as representative, were further investigated. Ten out of 15 isolates harboring the blaKPC-2 gene clustered with the known ST258, while the remaining 5/10 belonged to the newly described ST512 and harbored the blaKPC-3 gene. ST512 isolates, from 3/7 hospitals, were collected from rectal swabs (40%, blood (20%, endotracheal aspirate (20% and

Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages

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Emanuella Francisco Fajardo

2016-06-01

Full Text Available Abstract: INTRODUCTION This work shows that 3% (v/v human urine (HU in semisolid Liver Infusion Tryptose (SSL medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU. Isolate DNA was analyzed using polymerase chain reaction (PCR and pulsed-field gel electrophoresis (PFGE. RESULTS SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines. CONCLUSIONS SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages.

An association of genotypes and antimicrobial resistance patterns among Salmonella isolates from pigs and humans in Taiwan.

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Hung-Chih Kuo

Full Text Available We collected 110 Salmonella enterica isolates from sick pigs and determined their serotypes, genotypes using pulsed-field gel electrophoresis (PFGE, and antimicrobial susceptibility to 12 antimicrobials and compared the data with a collection of 18,280 isolates obtained from humans. The pig isolates fell into 12 common serovars for human salmonellosis in Taiwan; S. Typhimurium, S. Choleraesuis, S. Derby, S. Livingstone, and S. Schwarzengrund were the 5 most common serovars and accounted for a total of 84% of the collection. Of the 110 isolates, 106 (96% were multidrug resistant (MDR and 48 (44% had PFGE patterns found in human isolates. S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund were among the most highly resistant serovars. The majority of the 3 serovars were resistant to 8-11 of the tested antimicrobials. The isolates from pigs and humans sharing a common PFGE pattern displayed identical or very similar resistance patterns and Salmonella strains that caused severe infection in pigs were also capable of causing infections in humans. The results indicate that pigs are one of the major reservoirs to human salmonellosis in Taiwan. Almost all of the pig isolates were MDR, which highlights the necessity of strictly regulating the use of antimicrobials in the agriculture sector in Taiwan.

Molecular epidemiology of Acinetobacter baumannii in central intensive care unit in Kosova teaching hospital

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Lul Raka

Full Text Available Infections caused by bacteria of genus Acinetobacter pose a significant health care challenge worldwide. Information on molecular epidemiological investigation of outbreaks caused by Acinetobacter species in Kosova is lacking. The present investigation was carried out to enlight molecular epidemiology of Acinetobacterbaumannii in the Central Intensive Care Unit (CICU of a University hospital in Kosova using pulse field gel electrophoresis (PFGE. During March - July 2006, A. baumannii was isolated from 30 patients, of whom 22 were infected and 8 were colonised. Twenty patients had ventilator-associated pneumonia, one patient had meningitis, and two had coinfection with bloodstream infection and surgical site infection. The most common diagnoses upon admission to the ICU were politrauma and cerebral hemorrhage. Bacterial isolates were most frequently recovered from endotracheal aspirate (86.7%. First isolation occurred, on average, on day 8 following admission (range 1-26 days. Genotype analysis of A. baumannii isolates identified nine distinct PFGE patterns, with predominance of PFGE clone E represented by isolates from 9 patients. Eight strains were resistant to carbapenems. The genetic relatedness of Acinetobacter baumannii was high, indicating cross-transmission within the ICU setting. These results emphasize the need for measures to prevent nosocomial transmission of A. baumannii in ICU.

An association of genotypes and antimicrobial resistance patterns among Salmonella isolates from pigs and humans in Taiwan.

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Kuo, Hung-Chih; Lauderdale, Tsai-Ling; Lo, Dan-Yuan; Chen, Chiou-Lin; Chen, Pei-Chen; Liang, Shiu-Yun; Kuo, Jung-Che; Liao, Ying-Shu; Liao, Chun-Hsing; Tsao, Chi-Sen; Chiou, Chien-Shun

2014-01-01

We collected 110 Salmonella enterica isolates from sick pigs and determined their serotypes, genotypes using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility to 12 antimicrobials and compared the data with a collection of 18,280 isolates obtained from humans. The pig isolates fell into 12 common serovars for human salmonellosis in Taiwan; S. Typhimurium, S. Choleraesuis, S. Derby, S. Livingstone, and S. Schwarzengrund were the 5 most common serovars and accounted for a total of 84% of the collection. Of the 110 isolates, 106 (96%) were multidrug resistant (MDR) and 48 (44%) had PFGE patterns found in human isolates. S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund were among the most highly resistant serovars. The majority of the 3 serovars were resistant to 8-11 of the tested antimicrobials. The isolates from pigs and humans sharing a common PFGE pattern displayed identical or very similar resistance patterns and Salmonella strains that caused severe infection in pigs were also capable of causing infections in humans. The results indicate that pigs are one of the major reservoirs to human salmonellosis in Taiwan. Almost all of the pig isolates were MDR, which highlights the necessity of strictly regulating the use of antimicrobials in the agriculture sector in Taiwan.

Molecular Analysis of Vancomycin-Resistant Enterococci Isolated from Regional Hospitals in Trinidad and Tobago

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Patrick E. Akpaka

2016-01-01

Full Text Available Geographic spread of vancomycin-resistant enterococci (VRE clones in cities, countries, or even continents has been identified by molecular techniques. This study aimed at characterizing virulent genes and determining genetic relatedness of 45 VRE isolates from Trinidad and Tobago using molecular tools, including polymerase chain reaction, pulsed-field gel electrophoresis (PFGE, and Random Amplification Polymorphic DNA (RAPD. The majority (84% of the isolates were Enterococcus faecium possessing vanA gene while the rest (16% were Enterococcus faecalis possessing vanB. The esp gene was found in all 45 VRE isolates while hyl genes were found only in E. faecium species. The E. faecium species expressed five distinct PFGE patterns. The predominant clones with similar or common patterns belonged to clones one and three, and each had 11 (29% of the VRE isolates. Plasmid content was identified in representative isolates from each clonal group. By contrast, the E. faecalis species had one PFGE pattern suggesting the presence of an occult and limited clonal spread. The emergence of VRE in the country seems to be related to intra/interhospital dissemination of an epidemic clone carrying the vanA element. Therefore, infection control measures will be warranted to prevent any potential outbreak and spread of VRE in the country.

Clonal spread of highly successful ST15-CTX-M-15 Klebsiella pneumoniae in companion animals and horses

DEFF Research Database (Denmark)

Ewers, Christa; Stamm, Ivonne; Pfeifer, Yvonne

2014-01-01

OBJECTIVES: To investigate the clinical relevance and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Klebsiella species in animals. METHODS: Antimicrobial susceptibilities and presence of ESBLs were examined among Klebsiella spp. (n = 1519) from clinical samples (>1200...... senders from Germany and other European countries) mainly from companion animals and horses from October 2008 to March 2010. Multilocus sequence typing (MLST) and PFGE were performed including human isolates for comparative purposes. RESULTS: The overall ESBL rate was 8% for Klebsiella pneumoniae subsp....... pneumoniae. Most K. pneumoniae subsp. pneumoniae ESBL producers were isolated from soft tissue infections (29.3%) and urinary tract infections (14.9%). The major ESBL type was CTX-M-15 (85.4%), located on different plasmid scaffolds (HI2, I1, FIA, FIB, FII, A/C, R and N). Other ESBL genes, such as bla...

Intracellular persisting Staphylococcus aureus is the major pathogen in recurrent tonsillitis.

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Andreas E Zautner

Full Text Available BACKGROUND: The two major indications for tonsillectomy are recurrent tonsillitis (RT and peritonsillar abscess (PTA. Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. METHODS: In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE, spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS, fluorescent in situ hybridization (FISH and immunohistochemistry. FINDINGS: S. aureus was the predominant species (57.7% in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2% in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. CONCLUSIONS: Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1.

Molecular characterization and genetic diversity of ESBL-producing Escherichia coli colonizing the migratory Franklin's gulls (Leucophaeus pipixcan) in Antofagasta, North of Chile.

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Báez, John; Hernández-García, Marta; Guamparito, Constanza; Díaz, Sofía; Olave, Abdon; Guerrero, Katherine; Cantón, Rafael; Baquero, Fernando; Gahona, Joselyne; Valenzuela, Nicomedes; Del Campo, Rosa; Silva, Juan

2015-02-01

The role of wild animals, particularly migratory birds, in the dissemination of antibiotic-resistant bacteria between geographically distant ecosystems is usually underestimated. The aim of this work was to characterize the Escherichia coli population from Franklin's gull feces, focusing on the extended-spectrum β-lactamase (ESBL)-producing strains. In the summer of 2011, 124 fecal swabs from seagulls (1 of each) migrating from the United States and Canada to the coast of Antofagasta, north of Chile, were collected. Samples were seeded on MacConkey agar supplemented with 2 μg/ml of cefotaxime and a single colony from each plate was tested for ESBL production by the double-disk ESBL synergy test. Antibiotic susceptibility was determined by the disk diffusion method and blaESBL genes were amplified and sequenced. The genetic diversity of isolates was explored by pulsed-field gel electrophoresis (PFGE)-XbaI and multilocus sequence typing. A total of 91 E. coli isolates with high rates of antibiotic resistance were identified. Carbapenemase production was not detected, whereas 67 of the 91 (54%) isolates exhibited an ESBL phenotype due to the presence of CTX-M-15 (61.3%), CTX-M-2 (19.3%), CTX-M-22 (16.1%), and CTX-M-3 (1.6%) coding genes. High genetic diversity was observed, with 30 PFGE patterns and 23 sequence types (STs), including ST131 (18%), ST44 (15%), ST617 (9%), and ST10 (9%). Results presented here are complementary to those previously reported by Hernández et al. in the same gull species, but located in the Central Region of Chile. Differences observed between gulls from both areas lead us to hypothesize that gulls from the northern location retain, as gut carriers, those resistant bacteria acquired in the United States and/or Canada.

Fifteen years of successful spread of Salmonella enterica serovar Mbandaka clone ST413 in Poland and its public health consequences

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Andrzej Hoszowski

2016-06-01

Full Text Available In the 1990s, [i]Salmonella enterica[/i] serovar (S. Mbandaka occurred in feed and poultry in Poland. In the following years, the serovar also gained epidemiological importance in other EU countries. The objectives of current study were to evaluate the genetic relationship of contemporary S. Mbandaka with isolates originating from the beginning of the epidemics, and to assess the contribution of poultry as the source of infections in humans. Seventy S. Mbandaka isolated mainly in 2009 – 2010 from humans, poultry, food, and feed were typed with API ID32 [sup]®[/sup], MIC, plasmid profiling, PFGE, and MLST. PCR and sequencing were used to identify plasmid mediated quinolone and cephalosporin resistance mechanisms. Six biochemical profiles were identified and 59 of S. Mbandaka proved to be susceptible to the applied antimicrobials. Eight strains carried plasmids and a few of them were positive for [i]bla[/i][sub]CMY-2[/sub] and [i]qnr[/i]S1 genes. Two clusters of 15 [i]XbaI[/i]-PFGE profiles with similarity of 77.5% were found. The first cluster, gathered 7 profiles involving historical isolates and several contemporary non-human S. Mbandaka. The predominant profile in the second cluster consisted of 28 human and 1 broiler isolate. MLST analysis showed sequence type ST413 occurring among all tested isolates. The identification of close genetic relationships between S. Mbandaka of human and poultry origin indicates animals as a primal human infection route. Despite [i]Salmonella [/i]control programmes, the S. Mbandaka ST413 clone has been circulating for several years in Poland. [i]Salmonella[/i] control polices in food production chain should be continuously updated to target serovars of major epidemiological importance. Resistance noted in S. Mbandaka to such antimicrobials as fluoroquinolones and cephalosporins may hinder public health.

Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

International Nuclear Information System (INIS)

Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

2003-01-01

Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

Clinical epidemiology and resistance mechanisms of carbapenem-resistant Acinetobacter baumannii, French Guiana, 2008-2014.

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Mahamat, Aba; Bertrand, Xavier; Moreau, Brigitte; Hommel, Didier; Couppie, Pierre; Simonnet, Christine; Kallel, Hatem; Demar, Magalie; Djossou, Felix; Nacher, Mathieu

2016-07-01

This study investigated the clinical epidemiology and resistance mechanisms of Acinetobacter baumannii and characterised the clonal diversity of carbapenem-resistant A. baumannii (CRAB) during an ICU-associated outbreak at Cayenne Hospital, French Guiana. All non-duplicate A. baumannii isolates from 2008 to 2014 were tested for antibiotic susceptibility by disk diffusion. Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on CRAB. Of the 441 A. baumannii isolates, most were from males (54.0%) and were detected mainly from the ICU (30.8%) and medicine wards (21.8%). In the ICU, strains were mainly isolated from the respiratory tract (44.1%) and bloodstream (14.0%), whereas in medicine wards they mainly were from wound/drainage (36.5%) and bloodstream (25.0%). A. baumannii showed the greatest susceptibility to piperacillin/tazobactam (92.7%), imipenem (92.5%), colistin (95.6%) and amikacin (97.2%), being lower in the ICU and medicine wards compared with other wards. An outbreak of OXA-23-producing CRAB occurred in the 13-bed ICU in 2010. CRAB strains were more co-resistant to other antimicrobials compared with non-CRAB. Molecular genetics analysis revealed five sequence types [ST78, ST107 and ST642 and two new STs (ST830 and ST831)]. Analysis of PFGE profiles indicated cross-transmissions of CRAB within the ICU, between the ICU and one medicine ward during transfer of patients, and within that medicine ward. This study provides the first clinical and molecular data of A. baumannii from French Guiana and the Amazon basin. The ICU was the highest risk unit of this nosocomial outbreak of OXA-23-producing CRAB, which could subsequently disseminate within the hospital. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

OXA-23 and ISAba1-OXA-66 class D β-lactamases in Acinetobacter baumannii isolates from companion animals.

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Ewers, Christa; Klotz, Peter; Leidner, Ursula; Stamm, Ivonne; Prenger-Berninghoff, Ellen; Göttig, Stephan; Semmler, Torsten; Scheufen, Sandra

2017-01-01

Acinetobacter baumannii is recognised as a major pathogen of nosocomial infections that frequently show resistance to last-resort antimicrobials. To investigate whether A. baumannii from companion animals harbour carbapenem resistance mechanisms, 223 clinical isolates obtained from veterinary clinics between 2000 and 2013 in Germany were screened for carbapenem-non-susceptibility employing meropenem-containing Mueller-Hinton agar plates. Minimum inhibitory concentration (MIC) data were obtained using the VITEK ® 2 system. Assignment to international clones (ICs) was done by multiplex PCR or repetitive sequence-based PCR employing the DiversiLab system. Clonality was studied using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Genes encoding carbapenemases and aminoglycoside-modifying enzymes were detected by PCR. In three samples from dogs, carbapenem-resistant A. baumannii carrying the bla OXA-23 gene on plasmids and located on transposon Tn2008 were identified. The isolates belonged to sequence type ST1 P (clonal complex CC1/IC1/pulsotype II) and ST10 P (CC10/IC8/pulsotype IV) according to the Pasteur MLST scheme, and to ST231 Ox (CC109) and ST585 Ox (CC447) following the Oxford scheme. Insertion sequence ISAba1 was identified upstream of bla OXA-66 in 58 A. baumannii isolates. MLST referred them to ST2 P (CC2/IC2/pulsotypes I and III), ST208 Ox , ST350 Ox and ST556 Ox (all CC118), respectively. PFGE suggested nosocomial spread of these highly related strains, which frequently demonstrated a multidrug-resistant phenotype, in one veterinary clinic. These data show that A. baumannii from companion animals reveal resistance determinants and clonal lineages of strains globally emerging in humans. This suggests an interspecies transmission and warrants molecular surveillance of A. baumannii in veterinary clinics to mitigate its further spread. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights

Genetic Characterization of Carbapenem-Resistant Enterobacteriaceae and the Spread of Carbapenem-Resistant Klebsiella pneumonia ST340 at a University Hospital in Thailand

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Netikul, Thidarat; Kiratisin, Pattarachai

2015-01-01

Carbapenem-resistant Enterobacteriaceae (CRE) has increasingly spread worldwide in the past decade. The prevalence and characteristics of CRE in Thailand are unknown. In this study, we conducted a 2-year surveillance of CRE among 12,741 clinical isolates of Enterobacteriaceae at the largest university hospital in Thailand with molecular characterization of beta-lactamase (bla) genes, including carbapenemase genes. The CRE prevalence was 1.4%. blaKPC-13 and blaIMP-14a were the only carbapenemase genes detected among these CRE isolates. blaKPC-13 gene was found in a single isolate of Escherichia coli, Enterobacter cloacae and Citrobacter freundii, and blaIMP-14a was found in four isolates of Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) isolates were resistant to multiple carbapenems at a higher ratio than other CRE species, and thus were further characterized for resistance phenotypes, bla genotypes and molecular epidemiology. Most CRKP isolates harboured multiple bla genes, especially those related to extended-spectrum beta-lactamases. Seven CRKP isolates were resistant to all tested carbapenems, and showed decreased ompK35 and/or ompK36 porin gene expression. Molecular typing of CRKP based on pulsed-field gel electrophoresis (PFGE) demonstrated several unrelated clones. Multilocus sequence typing (MLST) was partially concordant with PFGE results and revealed that ST340, a member of drug-resistant K. pneumoniae clonal complex 258, was the most predominant clone, followed by ST48, ST11 and ST273. The novel ST1645 was identified from this study. ST340 has neither been shown to be predominated among CRKP from other studies, nor been reported in Thailand. Therefore, it emphases a critical concern to monitor and control the spread of CRKP. PMID:26407326

Comparative Phenotypic and Molecular Genetic Profiling of Wild Lactococcus lactis subsp. lactis Strains of the L. lactis subsp. lactis and L. lactis subsp. cremoris Genotypes, Isolated from Starter-Free Cheeses Made of Raw Milk▿

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Fernández, Elena; Alegría, Ángel; Delgado, Susana; Martín, M. Cruz; Mayo, Baltasar

2011-01-01

Twenty Lactococcus lactis strains with an L. lactis subsp. lactis phenotype isolated from five traditional cheeses made of raw milk with no added starters belonging to the L. lactis subsp. lactis and L. lactis subsp. cremoris genotypes (lactis and cremoris genotypes, respectively; 10 strains each) were subjected to a series of phenotypic and genetic typing methods, with the aims of determining their phylogenetic relationships and suitability as starters. Pulsed-field gel electrophoresis (PFGE) analysis of intact genomes digested with SalI and SmaI proved that all strains were different except for three isolates of the cremoris genotype, which showed identical PFGE profiles. Multilocus sequence typing (MLST) analysis using internal sequences of seven loci (namely, atpA, rpoA, pheS, pepN, bcaT, pepX, and 16S rRNA gene) revealed considerable intergenotype nucleotide polymorphism, although deduced amino acid changes were scarce. Analysis of the MLST data for the present strains and others from other dairy and nondairy sources showed that all of them clustered into the cremoris or lactis genotype group, by using both independent and combined gene sequences. These two groups of strains also showed distinctive carbohydrate fermentation and enzyme activity profiles, with the strains in the cremoris group showing broader profiles. However, the profiles of resistance/susceptibility to 16 antibiotics were very similar, showing no atypical resistance, except for tetracycline resistance in three identical cremoris genotype isolates. The numbers and concentrations of volatile compounds produced in milk by the strains belonging to these two groups were clearly different, with the cremoris genotype strains producing higher concentrations of more branched-chain, derived compounds. Together, the present results support the idea that the lactis and cremoris genotypes of phenotypic Lactococcus lactis subsp. lactis actually represent true subspecies. Some strains of the two subspecies

Microbiological and molecular characterization of Staphylococcus hominis isolates from blood.

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Soraya Mendoza-Olazarán

Full Text Available BACKGROUND: Among Coagulase-Negative Staphylococci (CoNS, Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec type, and genetic relatedness of clinical S. hominis isolates. METHODOLOGY: S. hominis blood isolates (n = 21 were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus. RESULTS: Of the S. hominis isolates screened, 47.6% (10/21 were categorized as strong biofilm producers and 23.8% (5/21 as weak producers. Furthermore, 81% (17/21 of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in >70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0-95%. Among mecA-positive isolates, 14 (82% harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III. CONCLUSIONS: The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor

A Novel IncA/C1 Group Conjugative Plasmid, Encoding VIM-1 Metallo-Beta-Lactamase, Mediates the Acquisition of Carbapenem Resistance in ST104 Klebsiella pneumoniae Isolates from Neonates in the Intensive Care Unit of V. Monaldi Hospital in Naples.

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Esposito, Eliana P; Gaiarsa, Stefano; Del Franco, Mariateresa; Crivaro, Valeria; Bernardo, Mariano; Cuccurullo, Susanna; Pennino, Francesca; Triassi, Maria; Marone, Piero; Sassera, Davide; Zarrilli, Raffaele

2017-01-01

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim-sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase bla SHV -12 and carbapenem-hydrolyzing metallo-β-lactamase bla V IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with bla V IM-1 into a class I integron, bla SHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2'-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1 , and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying bla V IM-1 and bla SHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples.

A Novel IncA/C1 Group Conjugative Plasmid, Encoding VIM-1 Metallo-Beta-Lactamase, Mediates the Acquisition of Carbapenem Resistance in ST104 Klebsiella pneumoniae Isolates from Neonates in the Intensive Care Unit of V. Monaldi Hospital in Naples

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Eliana P. Esposito

2017-11-01

Full Text Available The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE and multi-locus sequence typing (MLST identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim–sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-β-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg ST12.1 using the IncA/C plasmid MLST (PMLST scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2′-phosphotransferase clusters, ant(3″, aph(3″, aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples.

Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins.

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Hammerum, Anette M; Larsen, Jesper; Andersen, Vibe D; Lester, Camilla H; Skovgaard Skytte, Timmy S; Hansen, Frank; Olsen, Stefan S; Mordhorst, Hanne; Skov, Robert L; Aarestrup, Frank M; Agersø, Yvonne

2014-10-01

To compare and characterize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-β-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection.

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Luis A Flores-Páez

Full Text Available Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62, and another from healthy conjunctiva (n = 45. All isolates were genotyped by pulsed field gel electrophoresis (PFGE, multilocus sequence typing (MLST, staphylococcal cassette chromosome mec (SCCmec, detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%, while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%. By means of a principal coordinates analysis (PCoA and a discriminant analysis (DA it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256+. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66% had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva

Clinical and molecular epidemiology of hospital Enterococcus faecium isolates in eastern France. Members of Réseau Franc-Comtois de Lutte contr les Infections Nosocomiales.

Science.gov (United States)

Bertrand, X; Thouverez, M; Bailly, P; Cornette, C; Talon, D

2000-06-01

We carried out a surveillance study of Enterococcus faecium isolates in the Franche-Comtéregion of France over three years. Clinical and epidemiological strains were characterized by antibiotype and genotype (pulsed field gel electrophoresis, PFGE). Three case-control studies were performed to identify risk factors for colonization/infection with three defined resistant phenotypes (amoxycillin, high-level gentamicin and high-level kanamycin). The crude incidence of colonization/infection was 0.156%, and 68.8% of cases were classified as hospital-acquired. Incidence did not differ according to the type of hospitalization (middle term or acute care). The urinary tract was the major site of infection. Resistance rates were: 45.8% (amoxycillin), 18.7% (high-level gentamicin), 61.4% (high-level kanamycin) and 3.1% (vancomycin). No isolate produced b-lactamase and one isolate carried the vanA gene. PFGE revealed two major epidemic patterns each including resistant strains isolated in different hospitals and during different periods in the study. Previous antimicrobial treatment was not identified as a risk factor for colonization/infection with any resistant phenotype. Despite the low frequency of vancomycin-resistant isolates in this study, resistant strains were widely disseminated and had characteristics enabling them to persist and spread. If these strains acquired the vanA gene, the risk of an outbreak would be large. So, the prevalence of vancomycin-resistant E. faecium in hospitals should be carefully monitored in the future. Copyright 2000 The Hospital Infection Society.

Limited prevalence of gaffkaemia (Aerococcus viridans var. homari) isolated from wild-caught European lobsters Homarus gammarus in England and Wales.

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Stebbing, P D; Pond, M J; Peeler, E; Small, H J; Greenwood, S J; Verner-Jeffreys, D

2012-08-27

Gaffkaemia, caused by Aerococcus viridans var. homari, causes fatal infections in Homarus spp. (clawed lobsters). Despite its high economic significance to the lobster fisheries in the USA and northern Europe, data on its prevalence in captured and wild populations, particularly in Europe, is scarce. Following an outbreak of gaffkaemia in a European lobster holding facility in South Wales (UK), a base-line survey was conducted for gaffkaemia in wild populations of European lobster Homarus gammarus around the coast of England and Wales. In addition, isolates recovered from the original outbreak and the survey were typed using pulsed-field gel electrophoresis (PFGE) and compared with previously characterised isolates from the USA, UK and Canada. Locally caught H. gammarus were sampled at 30 sites from around the coast of England and Wales between March 2006 and October 2008. Results confirmed that the prevalence of gaffkaemia in populations of H. gammarus was low, with only 9 positive isolates recovered from 952 samples examined. PFGE analysis showed that the isolates from the outbreak investigation shared the same pulsotype as A. viridans var. homari isolates from the USA, Norway and Canada, as well as an isolate (NCIMB 1119) reportedly recovered from an outbreak of European lobsters in England in the 1960s. This confirms earlier studies that suggest virulent strains of A. viridans var. homari show very limited geographical or temporal genetic variation and were introduced into the UK with American lobsters H. americanus.

Staphylococcus aureus epidemic in a neonatal nursery: a strategy of infection control.

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Bertini, Giovanna; Nicoletti, PierLuigi; Scopetti, Franca; Manoocher, Pourshaban; Dani, Carlo; Orefici, Graziella

2006-08-01

The risk of nosocomial infection due to Staphylococcus aureus in fullterm newborns is higher under hospital conditions where there are overcrowded nurseries and inadequate infection control techniques. We report on an outbreak of skin infection in a Maternity Nursery (May 21, 2000) and the measures undertaken to bring the epidemic under control. These measures included: separating neonates already present in the nursery on August 23, 2000 from ones newly arriving by creating two different cohorts, one of neonates born before this date and one of neonates born later; restricting healthcare workers caring for S. aureus- infected infants from working with non-infected infants; disallowing carrier healthcare workers from caring for patients; introducing contact and droplet precautions (including the routine use of gowns, gloves, and mask); ensuring appropriate disinfection of potential sources of contamination. A representative number of isolates were typed by genomic DNA restriction length polymorphism analysis by means of pulsed-field gel electrophoresis (PFGE). Among the 227 cases of skin lesions, microbiological laboratory analyses confirmed that 175 were staphylococcal infections. The outbreak showed a gradual reduction in magnitude when the overcrowding of the Nursery was reduced by separating the newborns into the two different Nurseries (two cohorts). The genotyping of the strains by PFGE confirmed the nurse-to-newborn transmission of S. aureus. The measures adopted for controlling the S. aureus outbreak can, in retrospect, be assessed to have been very effective.

Distribution of the ACME-arcA gene among meticillin-resistant Staphylococcus haemolyticus and identification of a novel ccr allotype in ACME-arcA-positive isolates.

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Pi, Borui; Yu, Meihong; Chen, Yagang; Yu, Yunsong; Li, Lanjuan

2009-06-01

The aim of this study was to investigate the prevalence and characteristics of ACME (arginine catabolic mobile element)-arcA-positive isolates among meticillin-resistant Staphylococcus haemolyticus (MRSH). ACME-arcA, native arcA and SCCmec elements were detected by PCR. Susceptibilities to 10 antimicrobial agents were compared between ACME-arcA-positive and -negative isolates by chi-square test. PFGE was used to investigate the clonal relatedness of ACME-arcA-positive isolates. The phylogenetic relationships of ACME-arcA and native arcA were analysed using the neighbour-joining methods of mega software. A total of 42 (47.7 %) of 88 isolates distributed in 13 PFGE types were positive for the ACME-arcA gene. There were no significant differences in antimicrobial susceptibility between ACME-arcA-positive and -negative isolates. A novel ccr allotype (ccrAB(SHP)) was identified in ACME-arcA-positive isolates. Among 42 ACME-arcA-positive isolates: 8 isolates harboured SCCmec V, 8 isolates harboured class C1 mec complex and ccrAB(SHP); 22 isolates harbouring class C1 mec complex and 4 isolates harbouring class C2 mec complex were negative for all known ccr allotypes. The ACME-arcA-positive isolates were first found in MRSH with high prevalence and clonal diversity, which suggests a mobility of ACME within MRSH. The results from this study revealed that MRSH is likely to be one of the potential reservoirs of ACME for Staphylococcus aureus.

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

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Ana Beatriz de Almeida Corrêa

2011-12-01

Full Text Available Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE, serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.

Prevalence of Staphylococcus aureus Isolated From Various Foods of Animal Origin in Kırşehir, Turkey and Their Enterotoxigenicity

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M. Dilek Avşaroğlu

2016-12-01

Full Text Available The aim of this study was to detect Staphylococcus aureus contamination to different types of animal origin foods collected in the Kırşehir province of Turkey and to examine their enterotoxin production ability. Out of 120 food samples 38 suspected colonies were obtained and 23 of them were identified as S. aureus by biochemical and molecular analyses. Other species detected were S. chromogenes, S. cohnii ssp. cohnii, S. hominis, S. lentus, S. warneri, and S. xylosus. The isolates were also analysed with regard to carry mecA gene. None of them was found to have mecA gene indicating susceptibility to methicillin. To determine the enterotoxigenic ability of the isolates phenotypically, reversed-passive-latex-agglutination test against SEA-SED was used. Six out of 23 S. aureus isolates were determined to produce SEA, SEC and SED. Three of them had only one enterotoxin production, whereas others had SEA and SED production together. The results of phenotypic analyses were confirmed by PCR based examination. None of the coagulase-negative staphylococci were found to be enterotoxigenic by both phenotypical and PCR-based analyses. In conclusion, enterotoxigenic S. aureus is a risk in foods of animal origin in Kırşehir and its counties.

Antimicrobial Resistance and Molecular Investigation of H2S-Negative Salmonella enterica subsp. enterica serovar Choleraesuis Isolates in China.

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Jing Xie

Full Text Available Salmonella enterica subsp. enterica serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 S. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H2S-negative S. Choleraesuis isolates and two H2S-positive isolates. This is the first report of H2S-negative S. Choleraesuis isolated from humans. The majority of H2S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the gyrA and parC genes and identified two new mutations in the parC gene. Pulsed-field gel electrophoresis (PFGE, multilocus sequence typing (MLST, and clustered regularly interspaced short palindromic repeat (CRISPR analysis were employed to investigate the genetic relatedness of H2S-negative and H2S-positive S. Choleraesuis isolates. PFGE revealed two groups, with all 19 H2S-negative S. Choleraesuis isolates belonging to Group I and H2S-positive isolates belonging to Group II. By MLST analysis, the H2S-negative isolates were all found to belong to ST68 and H2S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H2S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H2S-negative isolates also possessed a frame-shift mutation at position 760 of phsA gene compared with H2S-positive isolates, which may be responsible for the H2S-negative phenotype. Moreover, the 19 H2S-negative isolates have similar PFGE patterns and same mutation site in the phsA gene, these

Antimicrobial Resistance and Molecular Investigation of H2S-Negative Salmonella enterica subsp. enterica serovar Choleraesuis Isolates in China.

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Xie, Jing; Yi, Shengjie; Zhu, Jiangong; Li, Peng; Liang, Beibei; Li, Hao; Yang, Xiaoxia; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Wu, Zhihao; Qiu, Shaofu; Song, Hongbin

2015-01-01

Salmonella enterica subsp. enterica serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 S. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H2S-negative S. Choleraesuis isolates and two H2S-positive isolates. This is the first report of H2S-negative S. Choleraesuis isolated from humans. The majority of H2S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the gyrA and parC genes and identified two new mutations in the parC gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H2S-negative and H2S-positive S. Choleraesuis isolates. PFGE revealed two groups, with all 19 H2S-negative S. Choleraesuis isolates belonging to Group I and H2S-positive isolates belonging to Group II. By MLST analysis, the H2S-negative isolates were all found to belong to ST68 and H2S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H2S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H2S-negative isolates also possessed a frame-shift mutation at position 760 of phsA gene compared with H2S-positive isolates, which may be responsible for the H2S-negative phenotype. Moreover, the 19 H2S-negative isolates have similar PFGE patterns and same mutation site in the phsA gene, these results indicated

Atypical epidemiology of CTX-M-15 among Enterobacteriaceae from a high diversity of non-clinical niches in Angola.

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Ribeiro, T G; Novais, Â; Peixe, L; Machado, E

2016-05-01

The objective of this study was to investigate the distribution and molecular epidemiology of ESBLs, acquired AmpCs and carbapenemases in Enterobacteriaceae from non-clinical niches in Angola, an under-researched sub-Saharan country. Eighty-one samples were recovered from healthy persons (n = 18), healthy animals (n = 33) and their environments (n = 10) or aquatic settings (n = 20) in south Angola (2013). Samples were plated onto CHROMagar™ Orientation with/without antibiotics. Standard methods were used for bacterial identification, characterization of bla genes, antibiotic susceptibility testing and conjugation assays. Clonal analysis (XbaI-PFGE, MLST and Escherichia coli phylogroups), location of bla and plasmid characterization (S1-PFGE, I-CeuI-PFGE, replicon typing and hybridization) were also performed. ESBLs (almost exclusively CTX-M-15, 98%) were detected in 21% (45/216) of the isolates, recovered from diverse non-clinical niches and belonging to different Enterobacteriaceae species (mainly E. coli). Acquired AmpCs or carbapenemases were not found. The pandemic B2-ST131 E. coli clone was not identified, but some widespread clonal complexes (CCs) from A (CC10 and CC168), B1 (CC156) or D (CC38) phylogroups were detected. blaCTX-M-15 was variably identified on typeable (29%; 100-335 kb; IncFII, IncFIIK6, IncHI2 and IncY) or non-typeable (16%; 70-330 kb) plasmids or on the chromosome (14%), while for 41% of the isolates its specific location was not determined. This study reports, for the first time in Angola, an unexpected high occurrence of CTX-M-15 in diverse non-clinical niches and Enterobacteriaceae species, and uncovers novel plasmid replicons in under-researched geographical regions. The diffusion of blaCTX-M-15 through such a high diversity of genetic backgrounds (clones, typeable/non-typeable plasmids and genetic environments) unveils an extraordinary ability for blaCTX-M-15 acquisition and mobilization favoured by unrecognized

blaGES carrying Pseudomonas aeruginosa isolates from a public hospital in Rio de Janeiro, Brazil

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Flávia L. P. C. Pellegrino

Full Text Available Previous analysis of Pseudomonas aeruginosa class-1 integrons from Rio de Janeiro, Brazil, revealed the blaGES gene in one isolate. We screened isolates of two widespread PFGE genotypes, A and B, at a public hospital in Rio, for the presence of blaGES. The gene was detected in all seven P. aeruginosa isolates belonging to genotype B. Three of the seven genotype-B isolates were resistant to amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin-tazobactam and ticarcillin-clavulanic acid. The other four isolates were resistant to all these agents, except gentamicin, imipenem, meropenem and piperacillin-tazobactam. A synergistic effect between ceftazidime and imipenem or clavulanic acid suggested the production of GES-type ESBL.

Bovine-associated MRSA ST398 in The Netherlands

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Tavakol Mehri

2012-05-01

Full Text Available Abstract During routinely screening (50.000 milk samples on an annual basis 14 MRSA ST398 strains were identified in the period of January 2008 to September 2008 in 14 different dairy herds located in the provinces Overijssel and Gelderland, The Netherlands. Molecular analysis was performed by Cfr9I PFGE, ST398-specific diagnostic PCR, spa typing, SCCmec typing and Panton-Valentine Leukocidin (PVL gene PCR. The molecular analyses of 14 MRSA (one MRSA strain per herd strains revealed that all strains belong to ST398 with 3 closely related spa types (t011, t108 and t889, all commonly found in pigs and carry 2 different SCCmec types, IVa and V. All MRSA strains were resistant to two or more classes of antibiotics and also PVL negative. The majority of farms (n = 9, 64% harboured combined livestock with both cows and pigs present. Our study contributes to the growing evidence that MRSA ST398 is transmitted among various animal species and can be considered as an etiological agent of mastitis in dairy cows.

Foods confiscated from non-EU flights as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

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Rodríguez-Lázaro, David; Ariza-Miguel, Jaime; Diez-Valcarce, Marta; Fernández-Natal, Isabel; Hernández, Marta; Rovira, Jordi

2015-09-16

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food-producing animals has provoked a great concern in the presence of MRSA in associated foodstuff. In this study, we have assessed for the first time the presence of MRSA in food confiscated from non-EU flights. We performed a search for MRSA among 195 food samples confiscated from passengers on flights from twenty-one non-EU countries in 2012 and 2013. One hundred and seventeen meat samples of diverse animal origin (including antelope, beef, chicken, duck, guinea pig, pork, rodents, and turkey), 75 dairy products (74 cheeses and 1 butter) and 3 eggs were analyzed. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST), SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 66 food samples were positive for S. aureus (33.9%). Six S. aureus strains were MRSA (9.1%), all of them in flights from Bolivia (and 5 from the same passenger). Among methicillin-sensitive S. aureus (MSSA) (60 out of 66S. aureus strains), 44.1% were resistant to penicillin, 10.2% to tetracycline, 8.5% were resistant to aminoglycosides (amikacin and tobramycin) and 3.4% exhibited the M phenotype. MRSA isolates were sensitive to all non-β-lactam antibiotics tested. SmaI-PFGE analysis provided 40 genotypes among the S. aureus isolates (three genotypes among the six MRSA). Five MRSA isolates belonged to ST8 and harboured SCCmec type IVc as well as PVL genes. One isolate belonged to ST1649, harboured SCCmec type IVc and tested negative for the presence of the PVL genes. In conclusion, in this study, we report for the first time the presence of CA-MRSA in food confiscated from non-EU flights: ST8/ST1649-MRSA-IV. These results confirm the illegal entrance of food as a neglected route of transmission as well as the dissemination of successful CA

A correlation between residual radiation-induced DNA double-strand breaks in cultured fibroblasts and late radiotherapy reactions in breast cancer patients

International Nuclear Information System (INIS)

Kiltie, A.E.; Ryan, A.J.; Swindell, R.; Barber, J.B.P.; West, C.M.L.; Magee, B.; Hendry, J.H.

1999-01-01

Background and purpose: Prediction of late normal tissue reactions to radiotherapy would permit tailoring of dosage to each patient. Measurement of residual DNA double strand breaks using pulsed field gel electrophoresis (PFGE) shows promise in this field. The aim of this study was to test the predictive potential of PFGE in a group of retrospectively studied breast cancer patients.Materials and methods: Thirty nine patients, treated uniformly for breast cancer 9-15 years previously, with excision of the tumour and radiotherapy to the breast and drainage areas, were assessed clinically using the LENT SOMA scale, and a 5-mm punch biopsy taken from the buttock. Fibroblast cell strains were established and used to study residual DNA double strand breaks, using PFGE.Results: There were significant correlations between the DNA assay results and the fibrosis score (r s =0.46; P=0.003), the combined fibrosis and retraction score (r s =0.45, P=0.004) and the overall LENT score (r s =0.43; P=0.006). Using polychotomous logistic regression, the fibroblast DNA assay result was an independent prognostic factor for fibrosis severity.Conclusions: There is a relationship between residual radiation-induced DNA damage in fibroblasts and the severity of the late normal tissue damage seen in the patients from whom the cells were cultured. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

[Etiological and molecular characteristics of diarrhea caused Proteus mirabilis].

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Shi, Xiaolu; Hu, Qinghua; Lin, Yiman; Qiu, Yaqun; Li, Yinghui; Jiang, Min; Chen, Qiongcheng

2014-06-01

To analyze the etiological characteristics, virulence genes and plasmids that carrying diarrhea-causing Proteus mirabilis and to assess their relationship with drug resistance and pathogenicity. Proteus mirabilis coming from six different sources (food poisoning, external environment and healthy people) were analyzed biochemically, on related susceptibility and pulsed-field gel electrophoresis (PFGE). Virulence genes were detected by PCR. Plasmids were extracted and sequenced after gel electrophoresis purification. The biochemical characteristics of Proteus mirabilis from different sources seemed basically the same, and each of them showed having common virulence genes, as ureC, rsmA, hpmA and zapA. However, the PFGE patterns and susceptibility of these strains were different, so as the plasmids that they carried. Plasmid that presented in the sequenced strain showed that the 2 683 bp length plasmid encodes qnrD gene was associated with the quinolone resistance. Etiological characteristics and molecular characteristics of Proteus mirabilis gathered from different sources, were analyzed. Results indicated that traditional biochemical analysis and common virulence gene identification might be able to distinguish the strains with different sources. However, PFGE and plasmids analysis could distinguish the sources of strains and to identify those plasmids that commonly carried by the drug-resistant strains. These findings also provided theoretical basis for further study on the nature of resistance and pathogenicity in Proteus mirabilis.

1 original article molecular identification of methicillin-resistant

African Journals Online (AJOL)

boaz

University of Benin Teaching Hospital between July-September, 2007. The MRSA strains were ... evaluating the effectiveness of infection control and antimicrobial ... (PCR) and Pulsed Field Gel Electrophoresis (PFGE) to identify MRSA from ...

Colonization of butchers with livestock-associated methicillin-resistant Staphylococcus aureus

DEFF Research Database (Denmark)

Boost, Maureen; Ho, J.; Guardabassi, Luca

2013-01-01

contamination levels of 10% of retail pork were reported from the Netherlands and Canada, there are limited data of contamination rates of workers handling raw meat. We investigated the rates of MRSA contamination of local butchers working in wet markets, where recently slaughtered pigs are cut up. Nasal swabs...... collected from 300 pork butchers at markets throughout Hong Kong were enriched in brain heart infusion broth with 5% salt and cultured on MRSASelect(®) . Isolates were confirmed as Staphylococcus aureus and susceptibility testing performed. The presence of mecA was confirmed, SCCmec and spa type determined...... and relatedness investigated by PFGE. Subjects completed a questionnaire on MRSA carriage risk factors. Seventeen samples (5.6%) yielded MRSA, 15 harbouring SCCmec IVb. Ten strains were t899 (CC9), previously reported from local pig carcasses. Five strains were healthcare associated: SCCmec type II, t701(CC6...

Molecular characterization of the first leptospires isolated from goats in Brazil

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Walter Lilenbaum

2014-12-01

Full Text Available Two Leptospira sp. isolates were obtained by the first time from goats in Brazil and characterized by sequencing rrs, rpoB and secY genes, PFGE and typing with monoclonal antibodies. Both isolates are identical and belong to Leptospira santarosai. Analysis of the rrs and the rpoB genes sequences revealed 100% identity between the goat isolates and the Bananal reference strain. When secY sequences of the two isolates were compared to each other, it was observed that they had identical sequences. However, when compared to that of the Bananal reference strain, there were 15 mismatches along the 549 bp secY sequence. In conclusion, molecular methods are increasingly useful for the characterization of leptospires and allowed to identify those isolates of caprine origin as closely related but not identical to serovar Bananal, and constitute a new type named Carioca.

Microbiological Features of KPC-Producing Enterobacter Isolates Identified in a U.S. Hospital System

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Ahn, Chulsoo; Syed, Alveena; Hu, Fupin; O’Hara, Jessica A.; Rivera, Jesabel I.; Doi, Yohei

2014-01-01

Microbiological data regarding KPC-producing Enterobacter spp. are scarce. In this study, 11 unique KPC-producing Enterobacter isolates were identified among 44 ertapenem-non-susceptible Enterobacter isolates collected between 2009 and 2013 at a hospital system in Western Pennsylvania. All cases were healthcare-associated and occurred in medically complex patients. While pulsed-field gel electrophoresis (PFGE) showed diverse restriction patterns overall, multilocus sequence typing (MLST) identified Enterobacter cloacae isolates with sequence types (STs) 93 and 171 from two hospitals each. The levels of carbapenem minimum inhibitory concentrations were highly variable. All isolates remained susceptible to colistin, tigecycline, and the majority to amikacin and doxycycline. A blaKPC-carrying IncN plasmid conferring trimethoprim-sulfamethoxazole resistance was identified in three of the isolates. Spread of blaKPC in Enterobacter spp. appears to be due to a combination of plasmid-mediated and clonal processes. PMID:25053203

Molecular characterization and functional analysis of pteridine reductase in wild-type and antimony-resistant Leishmania lines.

Science.gov (United States)

de Souza Moreira, Douglas; Ferreira, Rafael Fernandes; Murta, Silvane M F

2016-01-01

Pteridine reductase (PTR1) is an NADPH-dependent reductase that participates in the salvage of pteridines, which are essential to maintain growth of Leishmania. In this study, we performed the molecular characterization of ptr1 gene in wild-type (WTS) and SbIII-resistant (SbR) lines from Leishmania guyanensis (Lg), Leishmania amazonensis (La), Leishmania braziliensis (Lb) and Leishmania infantum (Li), evaluating the chromosomal location, mRNA levels of the ptr1 gene and PTR1 protein expression. PFGE results showed that the ptr1 gene is located in a 797 kb chromosomal band in all Leishmania lines analyzed. Interestingly, an additional chromosomal band of 1070 kb was observed only in LbSbR line. Northern blot results showed that the levels of ptr1 mRNA are increased in the LgSbR, LaSbR and LbSbR lines. Western blot assays using the polyclonal anti-LmPTR1 antibody demonstrated that PTR1 protein is more expressed in the LgSbR, LaSbR and LbSbR lines compared to their respective WTS counterparts. Nevertheless, no difference in the level of mRNA and protein was observed between the LiWTS and LiSbR lines. Functional analysis of PTR1 enzyme was performed to determine whether the overexpression of ptr1 gene in the WTS L. braziliensis and L. infantum lines would change the SbIII-resistance phenotype of transfected parasites. Western blot results showed that the expression level of PTR1 protein was increased in the transfected parasites compared to the non-transfected ones. IC50 analysis revealed that the overexpression of ptr1 gene in the WTS L. braziliensis line increased 2-fold the SbIII-resistance phenotype compared to the non-transfected counterpart. Furthermore, the overexpression of ptr1 gene in the WTS L. infantum line did not change the SbIII-resistance phenotype. These results suggest that the PTR1 enzyme may be implicated in the SbIII-resistance phenotype in L. braziliensis line. Copyright © 2015 Elsevier Inc. All rights reserved.

Research

African Journals Online (AJOL)

abp

2017-09-12

Sep 12, 2017 ... from porin or penicillin-binding protein modifications [5]. The aim of ... Methods. Bacterial isolates: This study analyzed 27 unduplicated .... including PFGE of chromosomal DNA restriction fragments have been used to ...

DNA gel electrophoresis: the reptation model(s).

Science.gov (United States)

Slater, Gary W

2009-06-01

DNA gel electrophoresis has been the most important experimental tool to separate DNA fragments for several decades. The introduction of PFGE in the 1980s and capillary gel electrophoresis in the 1990s made it possible to study, map and sequence entire genomes. Explaining how very large DNA molecules move in a gel and why PFGE is needed to separate them has been an active field of research ever since the launch of the journal Electrophoresis. This article presents a personal and historical overview of the development of the theory of gel electrophoresis, focusing on the reptation model, the band broadening mechanisms, and finally the factors that limit the read length and the resolution of electrophoresis-based sequencing systems. I conclude with a short discussion of some of the questions that remain unanswered.

Identification of Staphylococcus species and subspecies with the MicroScan Pos ID and Rapid Pos ID panel systems.

Science.gov (United States)

Kloos, W E; George, C G

1991-01-01

The accuracies of the MicroScan Pos ID and Rapid Pos ID panel systems (Baxter Diagnostic Inc., MicroScan Division, West Sacramento, Calif.) were compared with each other and with the accuracies of conventional methods for the identification of 25 Staphylococcus species and 4 subspecies. Conventional methods included those used in the original descriptions of species and subspecies and DNA-DNA hybridization. The Pos ID panel uses a battery of 18 tests, and the Rapid Pos ID panel uses a battery of 42 tests for the identification of Staphylococcus species. The Pos ID panel has modified conventional and chromogenic tests that can be read after 15 to 48 h of incubation; the Rapid Pos ID panel has tests that use fluorogenic substrates or fluorometric indicators, and test results can be read after 2 h of incubation in the autoSCAN-W/A. Results indicated that both MicroScan systems had a high degree of congruence (greater than or equal to 90%) with conventional methods for the species S. capitis, S. aureus, S. auricularis, S. saprophyticus, S. cohnii, S. arlettae, S. carnosus, S. lentus, and S. sciuri and, in particular, the subspecies S. capitis subsp. capitis and S. cohnii subsp. cohnii. The Rapid Pos ID panel system also had greater than or equal to 90% congruence with conventional methods for S. epidermidis, S. caprae, S. warneri subsp. 2, S. xylosus, S. kloosii, and S. caseolyticus. For both MicroScan systems, congruence with conventional methods was 80 to 90% for S. haemolyticus subsp. 1, S. equorum, S. intermedius, and S. hyicus; and in addition, with the Rapid Pos ID panel system congruence was 80 to 89% for S. capitis subsp. ureolyticus, S. warneri subsp. 1, S. hominis, S. cohnii subsp. urealyticum, and S. simulans. The MicroScan systems identified a lower percentage (50 to 75%) of strains of S. lugdunensis, S. gallinarum, S. schleiferi, and S. chromogenes, although the addition of specific tests to the systems might increase the accuracy of identification

Faecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae among humans in Java, Indonesia, in 2001-2002.

Science.gov (United States)

Severin, Juliëtte A; Lestari, Endang Sri; Kloezen, Wendy; Lemmens-den Toom, Nicole; Mertaniasih, Ni Made; Kuntaman, Kuntaman; Purwanta, Marijam; Duerink, D Offra; Hadi, Usman; van Belkum, Alex; Verbrugh, Henri A; Goessens, Wil H

2012-04-01

To characterise commensal Escherichia coli and other Enterobacteriaceae with reduced susceptibility to cefotaxime that were collected in a large survey carried out among 3995 patients and healthy persons in two urban regions on Java, Indonesia, in 2001-2002. The putative extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae were analysed using double-disk synergy tests, isoelectric focusing, PCR assays, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). On the day of discharge after five or more days of hospitalisation, at least 95 of 999 (9.5%) patients carried ESBL-positive Enterobacteriaceae as dominant faecal flora. Six patients were simultaneously colonised with E. coli and Klebsiella pneumoniae isolates with ESBL activity. On admission, only 6 of 998 (0.6%) patients were colonised. Faecal carriage of ESBL-producing Enterobacteriaceae among healthy persons or persons visiting a public health centre was not detected. The 107 ESBL-positive strains included 68 E. coli, 35 K. pneumoniae, and four other Enterobacteriaceae. bla(CTX-M-15) was the most prevalent ESBL in both E. coli (47.1%) and K. pneumoniae (45.7%), but the E. coli O25b-ST131 clone was virtually absent. Other ESBL types found were: SHV-2, -2a, -5, -12, CTX-M-3, -9, -14, and TEM-19. PFGE revealed extensive genetic diversity among the isolates. In 2001-2002, faecal carriage of ESBL-producing Enterobacteriaceae as dominant flora in Indonesia was almost exclusively hospital-associated. The presence of various bla(ESBL) genes and the extensive genetic diversity among isolates argue against a single/dominant strain outbreak. © 2012 Blackwell Publishing Ltd.

Methicillin-resistant Staphylococcus aureus transmission and infections in a neonatal intensive care unit despite active surveillance cultures and decolonization: challenges for infection prevention.

Science.gov (United States)

Popoola, Victor O; Budd, Alicia; Wittig, Sara M; Ross, Tracy; Aucott, Susan W; Perl, Trish M; Carroll, Karen C; Milstone, Aaron M

2014-04-01

To characterize the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) transmission and infections in a level IIIC neonatal intensive care unit (NICU) and identify barriers to MRSA control. Retrospective cohort study in a university-affiliated NICU with an MRSA control program including weekly nares cultures of all neonates and admission nares cultures for neonates transferred from other hospitals or admitted from home. Medical records were reviewed to identify neonates with NICU-acquired MRSA colonization or infection between April 2007 and December 2011. Compliance with hand hygiene and an MRSA decolonization protocol were monitored. Relatedness of MRSA strains were assessed using pulsed-field gel electrophoresis (PFGE). Of 3,536 neonates, 74 (2.0%) had a culture grow MRSA, including 62 neonates with NICU-acquired MRSA. Nineteen of 74 neonates (26%) had an MRSA infection, including 8 who became infected before they were identified as MRSA colonized, and 11 of 66 colonized neonates (17%) developed a subsequent infection. Of the 37 neonates that underwent decolonization, 6 (16%) developed a subsequent infection, and 7 of 14 (50%) that remained in the NICU for 21 days or more became recolonized with MRSA. Using PFGE, there were 14 different strain types identified, with USA300 being the most common (31%). Current strategies to prevent infections-including active identification and decolonization of MRSA-colonized neonates-are inadequate because infants develop infections before being identified as colonized or after attempted decolonization. Future prevention efforts would benefit from improving detection of MRSA colonization, optimizing decolonization regimens, and identifying and interrupting reservoirs of transmission.

A case of familial transmission of community-acquired methicillin-resistant Staphylococcus aureus carrying the lnu(A gene in Santa Fe city, Argentina Caso de transmisión familiar de Staphylococcus aureus resistente a la meticilina adquirido en la comunidad portador del gen lnu(A en la ciudad de Santa Fe, Argentina

Directory of Open Access Journals (Sweden)

Emilce de los A Méndez

2012-12-01

Full Text Available Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA is increasingly recognized as an important pathogen causing skin and soft tissue infections as well as necrotizing pneumonia. We describe a case of familial transmission of CA-MRSA between a 6-month-old boy and his mother in Santa Fe City, Argentina. Both isolates showed an identical antimicrobial susceptibility profile, carried type IV SCCmec and harboured the pvl and the lnu(A genes. Isolates showed indistinguishable SmaI-PFGE patterns confirming their genetic relationship. These results corroborate the intrafamilial transmission of CA-MRSA and might associate this strain with the repetitive events of furunculosis within the family.Staphylococcus aureus resistente a la meticilina adquirido en la comunidad (SARM-AC es reconocido como un patógeno importante que causa infecciones de piel y partes blandas y neumonía necrotizante. Describimos un caso de transmisión familiar de SARM-AC entre un nino de 6 meses de edad y su madre en la ciudad de Santa Fe, Argentina. Ambos aislamientos mostraron idéntico perfil de sensibilidad a los antimicrobianos, tenían el SCCmec tipo IV, y contenían los genes pvl y lnu(A. Los aislamientos presentaron patrones de SmaI-PFGE indistinguibles entre sí, lo cual confirmó su relación genética. Estos resultados corroboran la transmisión intrafamiliar de SARM-AC; asimismo, este aislamiento podría asociarse con los eventos repetitivos de furunculosis en la familia.

Prevalence and Characterization of Staphylococcus aureus Cultured From Raw Milk Taken From Dairy Cows With Mastitis in Beijing, China

Directory of Open Access Journals (Sweden)

Wei Wang

2018-06-01

Full Text Available The colonization of dairy herds and subsequent contamination of raw milk by Staphylococcus aureus (S. aureus, especially those expressing a multi-drug resistance (MDR, biofilm and toxins producing ability, remains an important issue for both the dairy producer and public health. In this study, we investigated the prevalence, antimicrobial resistance, virulence, and genetic diversity of S. aureus in raw milk taken from 2 dairy farms in Beijing, China. Ninety (46.2%, 90/195 samples were positive for S. aureus. Resistant to penicillin (PEN (31.3%, ciprofloxacin (18.8% and enrofloxacin (15.6% were the most often observed. Isolates cultured from farm B showed significantly higher resistance to penicillin (73.9%, ciprofloxacin (34.8%, enrofloxacin (34.8%, tilmicosin (17.4%, and erythromycin (17.4% than those from farm A (p < 0.05. Totally, 94.8% S. aureus harbored at least one virulence gene and the pvl (93.8%, sec (65.6%, and sea (60.4% genes were the most frequently detected. The pvl and sec genes were more often detected in isolates from farm A (97.3% and 84.9% respectively than those from farm B (p < 0.05. Of all 77 staphylococcus enterotoxin (SE-positive isolates, more than 90% could produce enterotoxins and 70.1% could produce two types. Biofilm related genes (icaA/D, clf/B, can, and fnbA were detected in all96 isolates. All 96 isolates could produce biofilm with 8.3, 70.8, and 18.8% of the isolates demonstrating weak, moderate and strong biofilm formation, respectively. A total of 5 STs, 7 spa types (1 novel spa type t17182, 3agr types (no agrII, and 14 SmaI-pulso-types were found in this study. PFGE cluster II-CC1-ST1-t127-agr III was the most prevalent clone (56.3%. Isolates of agr III (PFGE Cluster I/II-CC1-ST1-t127/2279 had higher detection of virulence genes than those of agr I and agr IV. TheMSSA-ST398-t1456-agr I clone expressed the greatest MDRbut with no virulence genes and weakly biofilm formation. Our finding indicated a relatively

PFGE, Lior Serotype, and Antimicrobial Resistance Patterns Among Campylobacter jejuni Isolated from Travelers and US Military Personnel with Acute Diarrhea in Thailand, 1998-2003

Science.gov (United States)

2010-01-01

dlstllb!Jtlon. and reproduction 1n any med1um, prOVIded the 01191nal w01k is propeliy Cited Report Documentation Page Form ApprovedOMB No. 0704-0188...Clin Microbial 1998, 36:1787-1789. 18. PeacockS . de Silva G. Justice A. Cowland A. Moore C. Winearls C. Day N: Comparison of multilocus sequence typing

Speciation and antibiotic susceptibility patterns of coagulase-negative staphylococci.

Science.gov (United States)

Smith, D J; Kaplan, R L; Landau, W; Trenholme, G M

1982-08-01

During a six month period, 191 isolates of coagulase-negative staphylococci from blood, urine, cerebrospinal fluid and heart valves were identified to species level and tested for antimicrobial susceptibility. Seventy-one percent of isolates were Staphylococcus epidermidis, 8% Staphylococcus warneri, 7% Staphylococcus hominis, 7% Staphylococcus haemolyticus, 4% Staphylococcus capitis, 2% Staphylococcus saprophyticus and 1% Staphylococcus cohnii. Approximately 4% of isolates were felt to be associated with infection. Overall, 18% of isolates were susceptible to penicillin G, 61% oxacillin, 98% cephalothin, 98% cefamandole, 72% cefotaxime, 95% cefsulodin, 76% gentamicin, 64% clindamycin and 98% rifampicin. All isolates were susceptible to vancomycin. Vancomycin, rifampicin, cephalothin and cefamandole showed excellent activity against oxacillin-resistant isolates. With one exception, speciation was not helpful in determining whether or not an isolate was associated with infection.

Evaluation of different analysis and identification methods for Salmonella detection in surface drinking water sources

Energy Technology Data Exchange (ETDEWEB)

Hsu, Bing-Mu, E-mail: bmhsu@ccu.edu.tw [Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC (China); Huang, Kuan-Hao; Huang, Shih-Wei [Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC (China); Tseng, Kuo-Chih [Department of Internal Medicine, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan, ROC (China); Su, Ming-Jen [Department of Clinical Pathology, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan, ROC (China); Lin, Wei-Chen; Ji, Dar-Der [Research and Diagnostic Center, Centers for Disease Control, Taipei, Taiwan, ROC (China); Shih, Feng-Cheng; Chen, Jyh-Larng [Department of Environmental Engineering and Health, Yuanpei University of Science and Technology, HsinChu, Taiwan, ROC (China); Kao, Po-Min [Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC (China)

2011-09-15

The standard method for detecting Salmonella generally analyzes food or fecal samples. Salmonella often occur in relatively low concentrations in environmental waters. Therefore, some form of concentration and proliferation may be needed. This study compares three Salmonella analysis methods and develops a new Salmonella detection procedure for use in environmental water samples. The new procedure for Salmonella detection include water concentration, nutrient broth enrichment, selection of Salmonella containing broth by PCR, isolation of Salmonella strains by selective culture plates, detection of possible Salmonella isolate by PCR, and biochemical testing. Serological assay and pulsed-field gel electrophoresis (PFGE) can be used to identify Salmonella serotype and genotype, respectively. This study analyzed 116 raw water samples taken from 18 water plants and belonging to 5 watersheds. Of these 116, 10 water samples (8.6%) taken from 7 water plants and belonging to 4 watersheds were positive for a Salmonella-specific polymerase chain reaction targeting the invA gene. Guided by serological assay results, this study identified 7 cultured Salmonella isolates as Salmonella enterica serovar: Alnaby, Enteritidis, Houten, Montevideo, Newport, Paratyphi B var. Java, and Victoria. These seven Salmonella serovars were identified in clinical cases for the same geographical areas, but only one of them was 100% homologous with clinical cases in the PFGE pattern. - Research highlights: {yields} A new Salmonella detecting procedure for environmental water is developed. {yields} Salmonella isolates are identified by serological assay and PFGE. {yields} A total of seven Salmonella serovars is isolated from environmental water.

Further characterization of porcine Brachyspira hyodysenteriae isolates with decreased susceptibility to tiamulin.

Science.gov (United States)

Karlsson, M; Aspán, A; Landén, A; Franklin, A

2004-04-01

Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe diarrhoeal disease in pigs. Few drugs are available to treat the disease, owing to both antimicrobial resistance and withdrawal of drugs authorized for use in pigs. Tiamulin is the drug of choice in many countries, but isolates with decreased susceptibility have recently been reported. The mechanism of tiamulin resistance in B. hyodysenteriae is not known and this facet is essential to understand the dissemination of the trait. To study the resistance epidemiology of B. hyodysenteriae, further characterization of a set of isolates from Germany (n = 16) and the UK (n = 6) with decreased susceptibility to tiamulin was performed. The relatedness between the isolates was studied by comparing PFGE patterns, and the in vitro susceptibility to five other antimicrobials (aivlosin, doxycycline, salinomycin, chloramphenicol and avilamycin) was also determined. For comparison of the antimicrobial-susceptibility pattern, Swedish (n = 20) and British (n = 4) tiamulin-susceptible isolates were tested. The German isolates represented several different PFGE patterns, indicating that tiamulin usage has been sufficient to select clones with decreased tiamulin susceptibility at different farms in Germany. The PFGE pattern for the six British isolates with decreased tiamulin susceptibility was identical to that of the German isolates, and they had a similar antimicrobial-susceptibility pattern, except for resistance to aivlosin, which was only found in a few German isolates. No other co-resistance with tiamulin was found.

Prevalence of Extended-Spectrum β-Lactamases CTX-M-8 and CTX-M-2-Producing Salmonella Serotypes from Clinical and Nonhuman Isolates in Brazil.

Science.gov (United States)

Fernandes, Sueli Aparecida; Camargo, Carlos Henrique; Francisco, Gabriela Rodrigues; Bueno, Maria Fernanda Campagnari; Garcia, Doroti Oliveira; Doi, Yohei; Casas, Monique Ribeiro Tiba

2017-07-01

We characterized extended-spectrum β-lactamases (ESBL) enzymes among Salmonella strains isolated in Brazil from 2009 to 2014. Salmonella recovered from both clinical and nonhuman (food, poultry, and environment) sources were subjected to antimicrobial susceptibility testing. β-lactamases genes were detected by polymerase chain reaction/sequencing; plasmid profiles and transferability were assessed by S1-pulsed field gel electrophoresis (PFGE). Genetic diversity was evaluated by XbaI-PFGE. Out of 630 Salmonella strains screened, 46 displayed ESBL phenotype, distributed across 11 different serotypes. bla CTX-M-8 and bla CTX-M-2 genes were detected at frequencies of 47% and 41%, respectively. bla SHV-5 and bla SHV-2 were also detected but in lower frequencies (4%, 2%). bla TEM-1 gene was detected in 22% of the strains. Most of the ESBL genes were transferable by conjugation, and the respective bla ESBL gene was detected in the recipient strain, indicating the location of ESBL determinants on transferable plasmids. XbaI-PFGE revealed genomic diversity of Salmonella Typhimurium bearing bla CTX-M-2 , bla CTX-M-8 , bla TEM-1 , and bla SHV-2 genes. Salmonella Muenchen (harboring bla CTX-M-2 ) and Salmonella Corvallis (bla CTX-M-8 and bla SHV-5 ) showed clonal relatedness within respective serotypes. Our findings underscore the occurrence of diverse ESBL genes in several Salmonella serotypes, reinforcing the need for continuous surveillance of resistance genes circulating in human and nonhuman sources.

Evaluation of different analysis and identification methods for Salmonella detection in surface drinking water sources

International Nuclear Information System (INIS)

Hsu, Bing-Mu; Huang, Kuan-Hao; Huang, Shih-Wei; Tseng, Kuo-Chih; Su, Ming-Jen; Lin, Wei-Chen; Ji, Dar-Der; Shih, Feng-Cheng; Chen, Jyh-Larng; Kao, Po-Min

2011-01-01

The standard method for detecting Salmonella generally analyzes food or fecal samples. Salmonella often occur in relatively low concentrations in environmental waters. Therefore, some form of concentration and proliferation may be needed. This study compares three Salmonella analysis methods and develops a new Salmonella detection procedure for use in environmental water samples. The new procedure for Salmonella detection include water concentration, nutrient broth enrichment, selection of Salmonella containing broth by PCR, isolation of Salmonella strains by selective culture plates, detection of possible Salmonella isolate by PCR, and biochemical testing. Serological assay and pulsed-field gel electrophoresis (PFGE) can be used to identify Salmonella serotype and genotype, respectively. This study analyzed 116 raw water samples taken from 18 water plants and belonging to 5 watersheds. Of these 116, 10 water samples (8.6%) taken from 7 water plants and belonging to 4 watersheds were positive for a Salmonella-specific polymerase chain reaction targeting the invA gene. Guided by serological assay results, this study identified 7 cultured Salmonella isolates as Salmonella enterica serovar: Alnaby, Enteritidis, Houten, Montevideo, Newport, Paratyphi B var. Java, and Victoria. These seven Salmonella serovars were identified in clinical cases for the same geographical areas, but only one of them was 100% homologous with clinical cases in the PFGE pattern. - Research highlights: → A new Salmonella detecting procedure for environmental water is developed. → Salmonella isolates are identified by serological assay and PFGE. → A total of seven Salmonella serovars is isolated from environmental water.

Characteristics of Clusters of Salmonella and Escherichia coli O157 Detected by Pulsed-Field Gel Electrophoresis that Predict Identification of Outbreaks.

Science.gov (United States)

Jones, Timothy F; Sashti, Nupur; Ingram, Amanda; Phan, Quyen; Booth, Hillary; Rounds, Joshua; Nicholson, Cyndy S; Cosgrove, Shaun; Crocker, Kia; Gould, L Hannah

2016-12-01

Molecular subtyping of pathogens is critical for foodborne disease outbreak detection and investigation. Many clusters initially identified by pulsed-field gel electrophoresis (PFGE) are not confirmed as point-source outbreaks. We evaluated characteristics of clusters that can help prioritize investigations to maximize effective use of limited resources. A multiagency collaboration (FoodNet) collected data on Salmonella and Escherichia coli O157 clusters for 3 years. Cluster size, timing, extent, and nature of epidemiologic investigations were analyzed to determine associations with whether the cluster was identified as a confirmed outbreak. During the 3-year study period, 948 PFGE clusters were identified; 849 (90%) were Salmonella and 99 (10%) were E. coli O157. Of those, 192 (20%) were ultimately identified as outbreaks (154 [18%] of Salmonella and 38 [38%] of E. coli O157 clusters). Successful investigation was significantly associated with larger cluster size, more rapid submission of isolates (e.g., for Salmonella, 6 days for outbreaks vs. 8 days for nonoutbreaks) and PFGE result reporting to investigators (16 days vs. 29 days, respectively), and performance of analytic studies (completed in 33% of Salmonella outbreaks vs. 1% of nonoutbreaks) and environmental investigations (40% and 1%, respectively). Intervals between first and second cases in a cluster did not differ significantly between outbreaks and nonoutbreaks. Molecular subtyping of pathogens is a rapidly advancing technology, and successfully identifying outbreaks will vary by pathogen and methods used. Understanding criteria for successfully investigating outbreaks is critical for efficiently using limited resources.

Refinement Types for TypeScript

OpenAIRE

Vekris, Panagiotis; Cosman, Benjamin; Jhala, Ranjit

2016-01-01

We present Refined TypeScript (RSC), a lightweight refinement type system for TypeScript, that enables static verification of higher-order, imperative programs. We develop a formal core of RSC that delineates the interaction between refinement types and mutability. Next, we extend the core to account for the imperative and dynamic features of TypeScript. Finally, we evaluate RSC on a set of real world benchmarks, including parts of the Octane benchmarks, D3, Transducers, and the TypeScript co...

Wound and soft tissue infections of Serratia marcescens in patients receiving wound care: A health care-associated outbreak.

Science.gov (United States)

Us, Ebru; Kutlu, Huseyin H; Tekeli, Alper; Ocal, Duygu; Cirpan, Sevilay; Memikoglu, Kemal O

2017-04-01

We described a health care-associated Serratia marcescens outbreak of wound and soft tissue infection lasting approximately 11 months at Ankara University Ibni Sina Hospital. After identification of S marcescens strains from the clinical and environmental samples, and their susceptibility testing to antimicrobial agents, pulsed-field gel electrophoresis (PFGE) was performed to detect molecular epidemiologic relationships among these isolates. The strains which were isolated from the saline bottles used for wound cleansing in the wound care unit were found to be 100% interrelated by PFGE to the strains from the samples of the outbreak patients. Reuse of the emptied bottles has no longer been allowed since the outbreak occurred. Besides, more efficient and frequent infection control training for hospital staff has been conducted. Copyright © 2017 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Genomic relationships of Actinobacillus pleuropneumoniae serotype 2 strains evaluated by ribotyping, sequence analysis of ribosomal intergenic regions, and pulsed-field gel electrophoresis

DEFF Research Database (Denmark)

Fussing, V.

1998-01-01

The aim of the present study was to examine the genomic relationship among 112 Actinobacillus pleuropneumoniae serotype 2 strains obtained throughout Europe and North America. HindIII ribotyping of the strains resulted in five ribotypes of high similarity (87-98%). Sequence analysis of the riboso......The aim of the present study was to examine the genomic relationship among 112 Actinobacillus pleuropneumoniae serotype 2 strains obtained throughout Europe and North America. HindIII ribotyping of the strains resulted in five ribotypes of high similarity (87-98%). Sequence analysis...... of the ribosomal intergenic region of strains representing each ribotype and each country showed no differences. A common ribotype was further characterized by PFGE of 12 strains representing all countries. The resultant five PFGE patterns of European strains showed a similarity of more than 91%, to which the two...

Refining types using type guards in TypeScript

NARCIS (Netherlands)

de Wolff, Ivo Gabe; Hage, J.

2017-01-01

We discuss two adaptations of the implementation of type guards and narrowing in the TypeScript compiler. The first is an improvement on the original syntax-directed implementation, and has now replaced the original one in the TypeScript compiler. It is specifically suited for the scenario in which

Characterization of Carbapenem Nonsusceptible Pseudomonas aeruginosa in Denmark

DEFF Research Database (Denmark)

Hansen, Frank; Johansen, Helle Krogh; Østergaard, Claus

2014-01-01

with combination tablet-diffusion methodology and polymerase chain reaction. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay, and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing acquired β-lactamases were further investigated by serotyping and multi...

Amplified fragment length polymorphism and pulsed field gel electrophoresis for subspecies differentiation of Serpulina pilosicoli

DEFF Research Database (Denmark)

Møller, Kristian; Jensen, Tim Kåre; Boye, Mette

1999-01-01

Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due...

Type Classes for Lightweight Substructural Types

Directory of Open Access Journals (Sweden)

Edward Gan

2015-02-01

Full Text Available Linear and substructural types are powerful tools, but adding them to standard functional programming languages often means introducing extra annotations and typing machinery. We propose a lightweight substructural type system design that recasts the structural rules of weakening and contraction as type classes; we demonstrate this design in a prototype language, Clamp. Clamp supports polymorphic substructural types as well as an expressive system of mutable references. At the same time, it adds little additional overhead to a standard Damas-Hindley-Milner type system enriched with type classes. We have established type safety for the core model and implemented a type checker with type inference in Haskell.

Antimicrobial resistance of Enterococcus faecium strains isolated from commercial probiotic products used in cattle and swine.

Science.gov (United States)

Amachawadi, Raghavendra G; Giok, Felicia; Shi, Xiaorong; Soto, Jose; Narayanan, Sanjeev K; Tokach, Mike D; Apley, Mike D; Nagaraja, T G

2018-04-03

. faecium strains were positive for any of the virulence genes tested. The clonal relationships among the 22 E. faecium strains were determined by pulsed-field gel electrophoresis (PFGE) typing. A total of 10 PFGE patterns were observed with 22 strains and a few of the strains from different probiotic products had identical (100% Dice similarity) PFGE patterns. In conclusion, the E. faecium strains in a few commercial probiotics exhibited AMR to medically-important antimicrobials, but none contained virulence genes.

Outbreak by ventilator-associated ST11 K. pneumoniae with co-production of CTX-M-24 and KPC-2 in SICU of a tertiary teaching hospital in central China

Directory of Open Access Journals (Sweden)

Longhua Hu

2016-08-01

Full Text Available The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP often responsible for numerous hospital-associated outbreaks has become an important public health problem. From January 2013 to February 2014, a total of 41 non-duplicate K. pneumoniae isolates with carbapenem resistance, were collected at a tertiary teaching hospital in Nanchang, central China. Among 41 K. pneumoniae isolates, 28 were isolated from hospitalized patients including 19 from the patients in surgery intensive care unit (SICU and 13 were isolated from ventilators. Twenty-four of 28 patients infected by CRKP have been submitted to mechanical ventilation using ventilator. More than 95% of the CRKP isolates were resistant to 13 antimicrobials tested.All CRKP isolates were confirmed as carbapenemase producer and were positive for blaKPC-2, with one positive for both blaKPC-2 and blaNDM-1. All carbapenemase-producing isolates harbored at least one of extended spectrum βlactamase genes tested, among which95.1% (39/41 of the tested isolates were found to harbor both blaCTX-M-24 and blaKPC-2, Of note, one isolate harbored simultaneously two carbapenemase genes (blaKPC-2 and blaNDM-1 and two ESBL genes (blaCTX-M-3 and blaTEM-104 . To the best of our knowledge, coexistence of blaKPC-2 and blaCTX-M-24 in one isolate is first reported. MLST results showed that 41 CRKP isolates belonged to 4 sequence types (STs including ST11, novel ST1854, novel ST1855 and ST1224. PFGE results displayed 3 PFGE clusters. Thirty-eight ST11 CRKP isolates (92.7%, 38/41 including all 13 isolates from ventilators and 25 isolates from patients from 7 wards (18 from是ICU belonged to same PFGE cluster, indicating these isolates were clonally related. Fifteen isolates have an identical undistinguished pattern (100% similarity forming a single clonal population. Moreover, this clone was exclusively linked to the cases attended in SICU and linked to the Ventilators. Additionally, the other SICU cases were

Dissemination and genetic support of broad-spectrum beta-lactam ...

African Journals Online (AJOL)

Methods: A total of 113 E.coli isolates obtained during the period 2004 through 2012 showed a ... to the PFGE analysis and characterization of the regions surrounding the blaCTX-M-15 ... 240-Gly which increases its catalytic efficiency against.

High Prevalence and Genetic Polymorphisms of Legionella in Natural and Man-Made Aquatic Environments in Wenzhou, China

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Leyi Zhang

2017-02-01

Full Text Available Natural and engineered water systems are the main sources of Legionnaires’ disease. It is essential from a public health perspective to survey water environments for the existence of Legionella. To analyze the main serogroups, genotypes and pathogenicity of the pathogen, a stratified sampling method was adopted to collect water samples randomly from shower water, cooling tower water, and local public hot springs in Wenzhou, China. Suspected strains were isolated from concentrated water samples. Serum agglutination assay and real-time PCR (Polymerase chain reaction were used to identify L. pneumophila. Sequence-based typing (SBT and pulsed-field gel electrophoresis (PFGE were used to elucidate the genetic polymorphisms in the collected isolates. The intracellular growth ability of the isolates was determined through their interaction with J774 cells and plating them onto BCYE (Buffered Charcoal Yeast Extract agar plates. Overall, 25.56% (46/180 of water samples were Legionella-positive; fifty-two strains were isolated and two kinds of serogroups were co-detected from six water samples from 2015 to 2016. Bacterial concentrations ranged from 20 CFU/100 mL to 10,720 CFU/100 mL. In detail, the Legionella-positive rates of shower water, cooling tower water and hot springs water were 15.45%, 13.33%, and 62.5%, respectively. The main serogroups were LP1 (30.69% and LP3 (28.85% and all strains carried the dot gene. Among them, 52 isolates and another 10 former isolates were analyzed by PFGE. Nineteen distinct patterns were observed in 52 strains isolated from 2015 to 2016 with three patterns being observed in 10 strains isolated from 2009 to 2014. Seventy-three strains containing 52 from this study and 21 former isolates were selected for SBT analysis and divided into 25 different sequence types in 4 main clonal groups belonging to 4 homomorphic types. Ten strains were chosen to show their abilities to grow and multiply in J744 cells. Taken together

Infection prevention and control interventions in the first outbreak of methicillin-resistant Staphylococcus aureus infections in an equine hospital in Sweden.

Science.gov (United States)

Bergström, Karin; Nyman, Görel; Widgren, Stefan; Johnston, Christopher; Grönlund-Andersson, Ulrika; Ransjö, Ulrika

2012-03-08

The first outbreak of methicillin-resistant Staphylococcus aureus (MRSA) infection in horses in Sweden occurred in 2008 at the University Animal Hospital and highlighted the need for improved infection prevention and control. The present study describes interventions and infection prevention control in an equine hospital setting July 2008 - April 2010. This descriptive study of interventions is based on examination of policy documents, medical records, notes from meetings and cost estimates. MRSA cases were identified through clinical sampling and telephone enquiries about horses post-surgery. Prospective sampling in the hospital environment with culture for MRSA and genotyping of isolates by spa-typing and pulsed-field gel electrophoresis (PFGE) were performed. Interventions focused on interruption of indirect contact spread of MRSA between horses via staff and equipment and included: Temporary suspension of elective surgery; and identification and isolation of MRSA-infected horses; collaboration was initiated between authorities in animal and human public health, human medicine infection control and the veterinary hospital; extensive cleaning and disinfection was performed; basic hygiene and cleaning policies, staff training, equipment modification and interior renovation were implemented over seven months.Ten (11%) of 92 surfaces sampled between July 2008 and April 2010 tested positive for MRSA spa-type 011, seven of which were from the first of nine sampling occasions. PFGE typing showed the isolates to be the outbreak strain (9 of 10) or a closely related strain. Two new cases of MRSA infection occurred 14 and 19 months later, but had no proven connections to the outbreak cases. Collaboration between relevant authorities and the veterinary hospital and formation of an infection control committee with an executive working group were required to move the intervention process forward. Support from hospital management and the dedication of staff were essential for

High Prevalence, Genetic Diversity and Intracellular Growth Ability of Legionella in Hot Spring Environments

Science.gov (United States)

Zhou, Haijian; Wang, Huanxin; Xu, Ying; Zhao, Mingqiang; Guan, Hong; Li, Machao; Shao, Zhujun

2013-01-01

Background Legionella is the causative agent of Legionnaires' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella. Methods Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates. Results Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (pLegionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (pLegionella pneumophila was the most frequently isolated species (98.9%), and the isolated serogroups included serogroups 3 (25.3%), 6 (23.4%), 5 (19.2%), 1 (18.5%), 2 (10.2%), 8 (0.4%), 10 (0.8%), 9 (1.9%) and 12 (0.4%). Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability. Conclusions Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control and prevention strategies are

Prevalence of Smqnr and plasmid-mediated quinolone resistance determinants in clinical isolates of Stenotrophomonas maltophilia from Japan: novel variants of Smqnr

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H. Kanamori

2015-09-01

Full Text Available Stenotrophomonas maltophilia is an important pathogen in healthcare-associated infections. S. maltophilia may contain Smqnr, a quinolone resistance gene encoding the pentapeptide repeat protein, which confers low-level quinolone resistance upon expression in a heterologous host. We investigated the prevalence of Smqnr and plasmid-mediated quinolone resistance (PMQR determinants in S. maltophilia isolates from Japan. A total of 181 consecutive and nonduplicate clinical isolates of S. maltophilia were collected from four areas of Japan. The antimicrobial susceptibility profiles for these strains were determined. PCR was conducted for Smqnr and PMQR genes, including qnrA, qnrB, qnrC, qnrS, aac(6′-Ib and qepA. PCR products for Smqnr and aac(6′-Ib were sequenced. For the S. maltophilia isolates containing Smqnr, pulsed-field gel electrophoresis (PFGE was performed using XbaI. Resistance rates to ceftazidime, levofloxacin, trimethoprim–sulfamethoxazole, chloramphenicol and minocycline were 67.4%, 6.1%, 17.7%, 8.8% and 0%, respectively. The minimum inhibitory concentration required to inhibit the growth of 50% and 90% of organisms were 0.5 and 2 mg/L for moxifloxacin but 1 and 4 mg/L for levofloxacin, respectively. Smqnr was detected in 104 of the 181 S. maltophilia isolates (57.5%, and the most frequent was Smqnr6, followed by Smqnr8 and Smqnr11. Eleven novel variants from Smqnr48 to Smqnr58 were detected. The 24 Smqnr-containing S. maltophilia isolates were typed by PFGE and divided into 21 unique types. Nine S. maltophilia isolates (5.0% carried aac(6′-Ib-cr. No qnr or qepA genes were detected. This study describes a high prevalence of Smqnr and novel variants of Smqnr among S. maltophilia from Japan. Continuous antimicrobial surveillance and further molecular epidemiological studies on quinolone resistance in S. maltophilia are needed.

High prevalence, genetic diversity and intracellular growth ability of Legionella in hot spring environments.

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Tian Qin

Full Text Available BACKGROUND: Legionella is the causative agent of Legionnaires' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella. METHODS: Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE and sequence-based typing (SBT were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates. RESULTS: Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (p<0.01. The Legionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (p<0.01. Legionella pneumophila was the most frequently isolated species (98.9%, and the isolated serogroups included serogroups 3 (25.3%, 6 (23.4%, 5 (19.2%, 1 (18.5%, 2 (10.2%, 8 (0.4%, 10 (0.8%, 9 (1.9% and 12 (0.4%. Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability. CONCLUSIONS: Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina

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C. Bidinost

2004-12-01

Full Text Available V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.

Shiga toxin-producing Escherichia coli in yaks (Bos grunniens from the Qinghai-Tibetan Plateau, China.

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Xiangning Bai

Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx 1 and stx 2 genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96% stx 1 and/or stx 2-positive samples, 85 (61.59% were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx 1 subtype (stx 1a and 5 stx 2 subtypes (stx 2a, stx 2b, stx 2c, stx 2d and stx 2g were present in these STEC isolates. Apart from lpfA O157/OI-141, lpfA O157/OI-154, lpfA O113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.

Molecular epidemiology and population structure of bovine Streptococcus uberis

DEFF Research Database (Denmark)

Rato, M G; Bexiga, R; Nunes, S F

2008-01-01

The molecular epidemiology and population structure of 30 bovine subclinical mastitis field isolates of Streptococcus uberis, collected from 6 Portuguese herds (among 12 farms screened) during 2002 and 2003, were examined by using pulsed-field gel electrophoresis (PFGE) for clustering of the isol...

Surveillance of circulatingstrains in Europe during 1998-2015

DEFF Research Database (Denmark)

Barkoff, Alex-Mikael; Mertsola, Jussi; Pierard, Denis

2018-01-01

EUpert I-III studies (1998-2009). The analyses included genotyping, serotyping and pulsed-field gel electrophoresis (PFGE) and multi-locus variable-number tandem repeat analysis (MLVA). Genotyping results showed only small variation among the common virulence genes ofB. pertussisFrequencies of serotypes...

Type inference for correspondence types

DEFF Research Database (Denmark)

Hüttel, Hans; Gordon, Andy; Hansen, Rene Rydhof

2009-01-01

We present a correspondence type/effect system for authenticity in a π-calculus with polarized channels, dependent pair types and effect terms and show how one may, given a process P and an a priori type environment E, generate constraints that are formulae in the Alternating Least Fixed......-Point (ALFP) logic. We then show how a reasonable model of the generated constraints yields a type/effect assignment such that P becomes well-typed with respect to E if and only if this is possible. The formulae generated satisfy a finite model property; a system of constraints is satisfiable if and only...... if it has a finite model. As a consequence, we obtain the result that type/effect inference in our system is polynomial-time decidable....

Genotypic profile of Listeria monocytogenes isolated in refrigerated chickens in southern Rio Grande do Sul, Brazil

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Karla Sequeira Mendonça

2016-01-01

Full Text Available ABSTRACT: Listeria monocytogenes is of notable concern to the food industry, due to its ubiquitous nature and ability to grow in adverse conditions. This study aimed to determine the genotypic profile of L. monocytogenes strains isolated from refrigerated chickens marketed in the southern part of Rio Grande do Sul, Brazil. The strains of L. monocytogenes isolated were characterized by serotyping and Pulsed Field Gel Electrophoresis (PFGE. Three different serotypes (1/2a, 1/2b and 4e were evaluated by PFGE, and the macrorestriction patterns utilizing enzymes AscI and ApaI, revealed five different pulsotypes. The presence of such varied genotypic profiles demonstrates the prevalence of L. monocytogenes contamination of chicken processing environments, which combined with ineffective cleaning procedures, allowing the survival, adaptation and proliferation of these pathogens, not only in the processing environment, but also in local grocery stores.

Tiamulin resistance in porcine Brachyspira pilosicoli isolates.

Science.gov (United States)

Pringle, M; Landén, A; Franklin, A

2006-02-01

There are few studies on antimicrobial susceptibility of Brachyspira pilosicoli, therefore this study was performed to investigate the situation among isolates from pigs. The tiamulin and tylosin susceptibility was determined by broth dilution for 93 and 86 porcine B. pilosicoli isolates, respectively. The isolates came from clinical samples taken in Swedish pig herds during the years 2002 and 2003. The tylosin minimal inhibitory concentration (MIC) was >16 microg/ml for 50% (n=43) of the isolates tested. A tiamulin MIC >2 microg/ml was obtained for 14% (n=13) of the isolates and these were also tested against doxycycline, salinomycin, valnemulin, lincomycin and aivlosin. For these isolates the susceptibility to salinomycin and doxycycline was high but the MICs for aivlosin varied. The relationship between the 13 tiamulin resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Among the 13 isolates 10 different PFGE patterns were identified.

DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing

International Nuclear Information System (INIS)

Loebrich, M.; Ikpeme, S.; Kiefer, J.

1994-01-01

DNA samples prepared from human SP 3 cells, which had not been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3±0.7) x 10 -3 /Mbp/Gy was deduced for 80 kV X-rays. (Author)

[Microbiological verification of a self control plan for a hospital food service].

Science.gov (United States)

Torre, I; Pennino, F; Crispino, M

2006-01-01

During the past years, it has been an increment of food related infectious diseases. In order to avoid micro biological food contamination, adherence to good manufacturing is required through control measures of food safety practices. Updated national and European regulations underline the need to apply the HACCP system, overcoming the old concept of sample control on the end user product. This work shows results of microbiological controls made along the whole productive chain. Measurements are made using biomolecular techniques (PFGE) in order to assess the management of the micro biological risk of the self control plan applied to a hospital food service of Naples. The use of the PFGE applied on some micro-organisms gram negative potentially pathogen, underlines the circulation, continued in time, of these micro-organisms within the cooking area. In addition, cross contamination between several matrixes of samples has been detected.

Yersinia ruckeri biotype 2 isolates from mainland Europe and the UK likely represent different clonal groups

DEFF Research Database (Denmark)

Wheeler, Richard W.; Davies, Robert L.; Dalsgaard, Inger

2009-01-01

the restriction enzyme NotI. Serotype O1 isolates responsible for ERM in rainbow trout in both the US and Europe, and including biotype 2 isolates, represented a distinct subgroup of similar pulsotypes. Biotype 2 isolates, responsible for outbreaks of the disease in rainbow trout in the UK, Denmark and Spain, had....... In contrast, US biotype 2 isolate YRNC10 had an identical pulsotype and OMP profile to UK biotype 2 isolates, suggesting that there had been exchange of these isolates between the UK and the US in the past. UK Atlantic salmon isolates were genetically and serologically diverse, with 12 distinct pulsotypes...... their likely origins and relationships, a geographically and temporally diverse collection of isolates were characterised by serotyping, biotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) profiling. A total of 44 pulsotypes were identified from 160 isolates by PFGE, using...

Personal hygiene and methicillin-resistant Staphylococcus aureus infection.

Science.gov (United States)

Turabelidze, George; Lin, Mei; Wolkoff, Barbara; Dodson, Douglas; Gladbach, Stephen; Zhu, Bao-Ping

2006-03-01

Methicillin-resistant Staphylococcus aureus (MRSA) infections outside the healthcare setting are an increasing concern. We conducted a case-control study to investigate an MRSA outbreak during 2002-2003 in a Missouri prison and focused on hygiene factors. Information on sociodemographic characteristics, medical history, and hygiene practices of study participants was collected by interview and medical record review. Logistic regression was used to evaluate MRSA infection in relation to hygiene factors individually and as a composite hygiene score; potential confounding factors were controlled. Selected MRSA isolates were analyzed by pulsed-field gel electrophoresis (PFGE). MRSA infection was significantly associated with a low composite hygiene score. Transmission among prison inmates appeared to be responsible for this outbreak. PFGE analysis showed that isolates were indistinguishable and associated with community-onset MRSA infections in other US prisons. Improving hygiene practices and environmental conditions may help prevent and interrupt future MRSA outbreaks in prison settings.

Outbreak of Pseudomonas aeruginosa bacteraemia in a haematology department