CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

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Chong Seng Shueh

2012-10-01

Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

Encoded Extended Spectrum Beta-Lactamases Produced

African Journals Online (AJOL)

26.1 % Klebsiella spp were positive for extended spectrum beta-lactamases ... issue, and TEM, OXA and SHV type ESBL were the most common genotypes. ... mechanism of action. ..... and Multiplex PCR Screening of AmpC Genes from.

High beta-Lactamase Levels Change the Pharmacodynamics of beta-Lactam Antibiotics in Pseudomonas aeruginosa Biofilms

DEFF Research Database (Denmark)

Wang, Hengzhuang; Ciofu, Oana; Yang, Liang

2013-01-01

the role of beta-lactamase in the pharmacokinetics (PK) and pharmacodynamics (PD) of ceftazidime and imipenem on P. aeruginosa biofilms. P. aeruginosa PAO1 and its corresponding beta-lactamase-overproducing mutant, PA Delta DDh2Dh3, were used in this study. Biofilms of these two strains in flow chambers......, microtiter plates, and on alginate beads were treated with different concentrations of ceftazidime and imipenem. The kinetics of antibiotics on the biofilms was investigated in vitro by time-kill methods. Time-dependent killing of ceftazidime was observed in PAO1 biofilms, but concentration-dependent killing...... activity of ceftazidime was observed for beta-lactamase-overproducing biofilms of P. aeruginosa in all three models. Ceftazidime showed time-dependent killing on planktonic PAO1 and PA Delta DDh2Dh3. This difference is probably due to the special distribution and accumulation in the biofilm matrix of beta...

Lactam hydrolysis catalyzed by mononuclear metallo-beta-lactamases: A density functional study

DEFF Research Database (Denmark)

Hemmingsen, Lars Bo Stegeager; Olsen, L.; Antony, J.

2003-01-01

Two central steps in the hydrolysis of lactam antibiotics catalyzed by mononuclear metallo-beta-lactamases, formation of the tetrahedral intermediate and its breakdown by proton transfer, are studied for model systems using the density functional B3LYP method. Metallo-beta-lactamases have two metal...

The effect of a beta-lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study.

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Alaybeyoglu, Begum; Uluocak, Bilge Gedik; Akbulut, Berna Sariyar; Ozkirimli, Elif

2017-05-01

Co-administration of beta-lactam antibiotics and beta-lactamase inhibitors has been a favored treatment strategy against beta-lactamase-mediated bacterial antibiotic resistance, but the emergence of beta-lactamases resistant to current inhibitors necessitates the discovery of novel non-beta-lactam inhibitors. Peptides derived from the Ala46-Tyr51 region of the beta-lactamase inhibitor protein are considered as potent inhibitors of beta-lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell-penetrating peptides could guide the design of beta-lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta-lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell-penetrating peptide pVEC, our approach involved the addition of the N-terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta-lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta-lactam antibiotic ampicillin, and the beta-lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N-terminus of the beta-lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N-terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European

Molecular identification of TEM-116 beta-lactamase gene in isolates ...

African Journals Online (AJOL)

Keywords: P. aeruginosa, Clinical isolates, Sequencing, TEM-116, Antibiotic susceptibility. Tropical Journal of ... organisms produce a wide range of beta ... TEM-1 is the most commonly encountered beta-lactamase in Gram-negative bacteria.

Phenotypic detection of beta-lactamases production in enterobacteriaceae

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Ćirković Ivana

2014-01-01

Full Text Available Introduction. Beta-lactam antibiotics are the most commonly used antibacterial drugs. However, many bacteria have developed resistance to these antibiotics, and the most common form of resistance is the production of beta-lactamase enzymes. Many members of the Enterobacteriaceae family produce different types of these enzymes. Objective. The aim of this study was to perform phenotypic detection of production and identification of beta-lactamase type in Enterobacteriaceae isolated from different clinical specimens from patients hospitalized in the Clinical Center of Serbia. Methods. The strains of Enterobacteriaceae were collected between November 2011 and January 2012 in the laboratory of the Clinical Center of Serbia. The isolates were identified according to the standard microbiology procedures and confirmed by the Vitek2 automated system. Antimicrobial susceptibility testing was performed by the disk diffusion method, and the phenotypic detection of production and identification of betalactamases was performed according to previously described methodologies. Results. In this study, a total of 172 Enterobacteriaceae strains were isolated. Further testing was performed on 54/145 (37.2% strains showing decreased susceptibility to beta-lactam antibiotics: 13/85 (15.3% Escherichia coli, 31/46 (67.4% Klebsiella pneumoniae and 10/14 (71.4% Proteus mirabilis. Among them, 40/145 (27.6% strains produced extended spectrum betalactamases (ESBLs, 9/145 (6.2% - AmpC, 1/145 (0.7% - K1 beta-lactamase and 4/145 (2.8% - carbapenemases. Carbapenemases were predominantly detected in K. pneumoniae (75%. Conclusion. Enterobacteriaceae produce different types of betalactamases, and the most common type in our study was ESBLs. Production of carbapenemases detected in Enterobacteriaceae is also an associated problem. [Projekat Ministarstva nauke Republike Srbije, br. ON 175039

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

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Bruno F. Robles

2014-04-01

Full Text Available The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin. Results of all tests were not dependent of bacteria type (CNS or S. aureus. Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin.

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Tekiner, İsmail Hakkı; Özpınar, Haydar

2016-01-01

Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek(®) MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla-genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM &blaCTX-M (52.7%), blaTEM &blaSHV (20%), blaTEM &blaCTX-M &blaSHV (12.7%), and blaSHV &blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Antibiotic resistance patterns and beta-lactamase identification in ...

African Journals Online (AJOL)

Children acquire bacteria from their mother during birth,[3,4] and ... Our results revealed high resistance rates to co-trimoxazole (54.0%), penicillin .... the inclusion of a beta-lactamase inhibitor, clavulanic acid. .... Folate pathway inhibitor/.

Chromosome-encoded narrow-spectrum Ambler class A beta-lactamase GIL-1 from Citrobacter gillenii.

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Naas, Thierry; Aubert, Daniel; Ozcan, Ayla; Nordmann, Patrice

2007-04-01

A novel beta-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum beta-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A beta-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of beta-lactamases TEM-1 and SHV-1. The blaGIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C beta-lactamases was found upstream of the blaGIL-1 gene, which fits the noninducibility of beta-lactamase expression in C. gillenii. Rapid amplification of DNA 5' ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the d-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of beta-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.

Complete Sequence of p07-406, a 24,179-base-pair plasmid harboring the blaVIM-7 metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States.

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Li, Hongyang; Toleman, Mark A; Bennett, Peter M; Jones, Ronald N; Walsh, Timothy R

2008-09-01

An outbreak involving a Pseudomonas aeruginosa strain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-beta-lactamase (MBL) gene, designated bla(VIM-7), located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with a bla(VIM-7) gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB from Xanthomonas campestris pv. citri commonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.

Design, synthesis and bioactivity evaluation of tribactam beta lactamase inhibitors.

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Copar, Anton; Prevec, Tadeja; Anzic, Borut; Mesar, Tomaz; Selic, Lovro; Vilar, Mateja; Solmajer, Tom

2002-03-25

Known carbapenem compounds with inhibitory effect towards beta-lactamase enzymes are formed from bicyclical beta lactam structural scaffolds. On the basis of results from theoretical computational methods and molecular modelling we have designed and developed a synthetic route towards novel, biologically active tricyclic derivatives of carbapenems.

Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli.

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Ishii, Y; Ohno, A; Taguchi, H; Imajo, S; Ishiguro, M; Matsuzawa, H

1995-01-01

Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed beta-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to beta-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. The sequence of the gene for Toho-1 was determined, and the open reading frame of the gene consisted of 873 or 876 bases (initial sequence, ATGATG). The nucleotide sequence of the gene (DDBJ accession number D37830) was found to be about 73% homologous to the sequence of the gene encoding a class A beta-lactamase produced by Klebsiella oxytoca E23004. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 290 or 291 amino acid residues, which contained amino acid motifs common to class A beta-lactamases (70SXXK, 130SDN, and 234KTG). Toho-1 was about 83% homologous to the beta-lactamase mediated by the chromosome of K. oxytoca D488 and the beta-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated beta-lactamase Toho-1 produced by E. coli TUH12191 is similar to beta-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes

Phenotypic and Genotypic Detection of Metallo-beta-lactamases among Imipenem-Resistant Gram Negative Isolates

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Mohammad Mohammadzadeh

2016-08-01

Full Text Available Background:   Imipenem-resistant gram negative bacteria, resulting from metallo-beta-lactamase (MBLs-producing strains have been reported to be among the important causes of nosocomial infections and of serious therapeutic problem worldwide. Because of their broad range, potent carbapenemase activity and resistance to inhibitors, these enzymes can confer resistance to almost all beta-lactams. The prevalence of metallo-beta-lactamase among imipenem-resistant Acinetobacter spp., Pseudomonas spp. and Enerobacteriaceae isolates is determined.Methods:   In this descriptive study 864 clinical isolates of Acinetobacter spp., Pseudomonas spp. and Enterobacteriaceae, were initially tested for imipenem susceptibility. The metallo-beta-lactamase production was detected using combined disk diffusion, double disk synergy test, and Hodge test. Then all imipenem resistant isolates were tested by PCR for imp, vim and ndm genes. Results:   Among 864 isolates, 62 (7.17 % were imipenem-resistant. Positive phonetypic test for metallo-beta-lactamase was 40 (64.5%, of which 24 (17.1% and 16 (9.2% isolates were Acinetobacter spp. and Pseudomonas spp., respectively. By PCR method 30 (48.4% of imipenem resistant Acinetobacter, and Pseudomonas isolates were positive for MBL-producing genes. None of the Enterobacteriaceae isolates were positive for metallo-beta-lactamase activity. Conclusion:   The results of this study are indicative of the growing number of nosocomial infections associated with multidrug-resistant gram negative bacteria in this region leading to difficulties in antibiotic therapy. Thereby, using of phenotypic methods can be helpful for management of this problem.

Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark

DEFF Research Database (Denmark)

Olesen, Inger; Hasman, Henrik; Aarestrup, Frank Møller

2004-01-01

The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different...... leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla(TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two...

Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response

DEFF Research Database (Denmark)

Ciofu, Oana

2003-01-01

the development of resistance to beta-lactam antibiotics and the occurrence of high beta-lactamase producing strains and between the MIC of the beta-lactams and the levels of beta-lactamase expression. Partially derepressed mutants, characterized by high basal levels of beta-lactamase with the possibility...... of induction to even higher levels during treatment with beta-lactam antibiotics, were the most frequent phenotype found among resistant Danish P. aeruginosa CF isolates. We have also shown that the high alginate producing P. aeruginosa isolates, that characterize the chronic lung infection in CF patients......, are more susceptible to antibiotics and produce less beta-lactamase than the non-mucoid paired isolates. We propose that the non-mucoid isolates are exposed to a relatively higher antibiotic pressure than the mucoid isolates and therefore, they become easily antibiotic resistant and in consequence produce...

[Extended-spectrum beta-lactamase detection in Enterobacteriaceae and antibiotic susceptibility analysis].

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Cao, Wei; Tong, Ming-hua; Wang, Ji-gui

2002-02-28

To detect the extended-spectrum beta-lactamases (ESBLs) in family Enterobacteriaceae and analyze the antibiotic susceptibility of those ESBLs-producing strains. ESBLs were determined by the double-disk confirmatory test and 8 antibiotic susceptibilities were tested with the disk disffusion method in those strains producing ESBLs. Forty-seven ESBLs-producing strains comprised of 25 of E. coli, 14 of K. pneumoniae, 5 of E. cloacae, 1 of K. oxytoca, 1 of K. rhinoscleromatis, and 1 of S. liquefaciens. The susceptibility rates of those strains were: 100% for imipenem and meropenem, 89.4% for piperacillin/tazobactam, 72.4% for cefoxitin and 65.9% for cefotetan. E. coli and K. pneumoniae are the prime strains producing ESBLs in Enterobacteriaceae. Imipenem and meropenem are the best drugs to deal with those ESBLs-producing strains. Piperacillin/tazobactam is better than cephamycins and other beta-lactama/beta-lactamase inhibitor combination.

Family disintegration: one fusarium verticillioides beta-lactamase at a time

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Fusarium verticillioides is a mycotoxigenic fungus found commonly on maize, where it primarily exhibits asymptomatic endophytic growth. The F. verticillioides genome possesses approximately 30 regions that potentially encode beta-lactamase enzymatic domains. These enzymes are classically involved ...

SME-3, a novel member of the Serratia marcescens SME family of carbapenem-hydrolyzing beta-lactamases.

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Queenan, Anne Marie; Shang, Wenchi; Schreckenberger, Paul; Lolans, Karen; Bush, Karen; Quinn, John

2006-10-01

Imipenem-resistant Serratia marcescens isolates were cultured from a lung transplant patient given multiple antibiotics over several months. The strains expressed SME-3, a beta-lactamase of the rare SME carbapenem-hydrolyzing family. SME-3 differed from SME-1 by a single amino acid substitution of tyrosine for histidine at position 105, but the two beta-lactamases displayed similar hydrolytic profiles.

SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains.

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Queenan, A M; Torres-Viera, C; Gold, H S; Carmeli, Y; Eliopoulos, G M; Moellering, R C; Quinn, J P; Hindler, J; Medeiros, A A; Bush, K

2000-11-01

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two beta-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 beta-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U. S. isolates had beta-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing beta-lactamases first described in London has now been identified in S. marcescens isolates across the United States.

Raw pet food as a risk factor for shedding of extended-spectrum beta-lactamase-producing Enterobacteriaceae in household cats

NARCIS (Netherlands)

Baede, V.O. (Valérie O.); E.M. Broens; Spaninks, M.P. (Mirlin P.); Timmerman, A.J. (Arjen J.); Graveland, H. (Haitske); J.A. Wagenaar (Jaap); B. Duim; Hordijk, J. (Joost)

2017-01-01

textabstractBackground: Close contact between pets and owners provides the opportunity for transmission of antimicrobial resistant organisms like extended-spectrum beta-lactamase (ESBL)/AmpC beta-lactamase (AmpC)-producing Enterobacteriaceae, posing a risk to public health.

A fitness cost associated with the antibiotic resistance enzyme SME-1 beta-lactamase.

Science.gov (United States)

Marciano, David C; Karkouti, Omid Y; Palzkill, Timothy

2007-08-01

The bla(TEM-1) beta-lactamase gene has become widespread due to the selective pressure of beta-lactam use and its stable maintenance on transferable DNA elements. In contrast, bla(SME-1) is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of bla(SME-1) via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, bla(SME-1) was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 beta-lactamase. Taken together, these results suggest that fitness costs associated with some beta-lactamases may limit their dissemination.

Raw pet food as a risk factor for shedding of extended-spectrum beta-lactamase-producing Enterobacteriaceae in household cats

NARCIS (Netherlands)

Baede, Valérie O; Broens, Els M|info:eu-repo/dai/nl/314627723; Spaninks, Mirlin P; Timmerman, Arjen J; Graveland, Haitske|info:eu-repo/dai/nl/304841838; Wagenaar, Jaap A|info:eu-repo/dai/nl/126613354; Duim, Birgitta|info:eu-repo/dai/nl/143855352; Hordijk, Joost|info:eu-repo/dai/nl/314839542

2017-01-01

BACKGROUND: Close contact between pets and owners provides the opportunity for transmission of antimicrobial resistant organisms like extended-spectrum beta-lactamase (ESBL)/AmpC beta-lactamase (AmpC)-producing Enterobacteriaceae, posing a risk to public health. OBJECTIVES: To investigate whether

Raw pet food as a risk factor for shedding of extended-spectrum beta-lactamase-producing Enterobacteriaceae in household cats

NARCIS (Netherlands)

Baede, Valérie O.; Broens, Els M.; Spaninks, Mirlin P.; Timmerman, Arjen J.; Graveland, Haitske; Wagenaar, Jaap A.; Duim, Birgitta; Hordijk, Joost

2017-01-01

Background: Close contact between pets and owners provides the opportunity for transmission of antimicrobial resistant organisms like extended-spectrum beta-lactamase (ESBL)/AmpC beta-lactamase (AmpC)-producing Enterobacteriaceae, posing a risk to public health. Objectives: To investigate whether

Molecular characterization of Vulmar1, a complete mariner transposon of sugar beet and diversity of mariner- and En/Spm-like sequences in the genus Beta.

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Jacobs, Gunnar; Dechyeva, Daryna; Menzel, Gerhard; Dombrowski, Cora; Schmidt, Thomas

2004-12-01

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.

Novel Ambler class A beta-lactamase LAP-1 and its association with the plasmid-mediated quinolone resistance determinant QnrS1.

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Poirel, Laurent; Cattoir, Vincent; Soares, Ana; Soussy, Claude-James; Nordmann, Patrice

2007-02-01

The plasmid-mediated quinolone resistance determinant QnrS1 was identified in non-clonally related Enterobacter cloacae isolates in association with a transferable narrow-spectrum beta-lactam resistance marker. Cloning experiments allowed the identification of a novel Ambler class A beta-lactamase, named LAP-1. It shares 62 and 61% amino acid identity with the most closely related beta-lactamases, TEM-1 and SHV-1, respectively. It has a narrow-spectrum hydrolysis of beta-lactams and is strongly inhibited by clavulanic acid and sulbactam and, to a lesser extent, by tazobactam. Association of the blaLAP-1 gene with the qnrS1 gene was identified in E. cloacae isolates from France and Vietnam. These genes were plasmid located and associated with similar insertion sequences but were not associated with sul1-type class 1 integrons, as opposed to the qnrA genes.

Kinetic studies on the inhibition of Proteus vulgaris beta-lactamase by imipenem.

OpenAIRE

Hashizume, T; Yamaguchi, A; Hirata, T; Sawai, T

1984-01-01

Imipenem was found to inhibit Proteus vulgaris beta-lactamase in a progressive manner. Kinetic experiments confirmed that the inactivated enzyme was not completely recovered after intact imipenem had been exhausted.

Effects of beta-lactamases and omp mutation on susceptibility to beta-lactam antibiotics in Escherichia coli.

Science.gov (United States)

Hiraoka, M; Okamoto, R; Inoue, M; Mitsuhashi, S

1989-01-01

Four types of beta-lactamases consisting of a penicillinase type I (TEM-1), a penicillinase type II (OXA-1), a cephalosporinase of Citrobacter freundii, and a cephalosporinase of Proteus vulgaris were introduced into Escherichia coli MC4100 and its omp mutants, MH1160 (MC4100 ompR1) and MH760 (MC4100 ompR2), by transformation. Effects of the combination of the omp mutations and these beta-lactamases on the susceptibility of E. coli strains were studied with 15 beta-lactam antibiotics including cephalosporins, cephamycins, penicillins, imipenem, and aztreonam. The ompR1 mutant, MH1160, lacks OmpF and OmpC, and it showed reduced susceptibility to 11 of the 15 beta-lactam agents. The reduction in susceptibility to cefoxitin, moxalactam, and flomoxef was much greater than reduction in susceptibility to the other agents. When the ompR1 mutant produced the cephalosporinase of C. freundii, the susceptibility of the mutant to 12 of the 15 beta-lactam antibiotics decreased. The reduction in susceptibility of MH1160 to 10 of the 12 agents affected by the enzyme was two- to fourfold greater than that observed in MC4100. Such a synergistic effect was also observed with the cephalosporinase of P. vulgaris and ompR1 mutation against six cephalosporins, moxalactam, and aztreonam. Images PMID:2658786

ICU Acquisition Rate, Risk Factors, and Clinical Significance of Digestive Tract Colonization With Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae: A Systematic Review and Meta-Analysis.

Science.gov (United States)

Detsis, Marios; Karanika, Styliani; Mylonakis, Eleftherios

2017-04-01

To evaluate the acquisition rate, identify risk factors, and estimate the risk for subsequent infection, associated with the colonization of the digestive tract with extended-spectrum beta-lactamase-producing Enterobacteriaceae during ICU-hospitalization. PubMed, EMBASE, and reference lists of all eligible articles. Included studies provided data on ICU-acquired colonization with extended-spectrum beta-lactamase-producing Enterobacteriaceae in previously noncolonized and noninfected patients and used the double disk synergy test for extended-spectrum beta-lactamase-producing Enterobacteriaceae phenotypic confirmation. Studies reporting extended-spectrum beta-lactamase-producing Enterobacteriaceae outbreaks or data on pediatric population were excluded. Two authors independently assessed study eligibility and performed data extraction. Thirteen studies (with 15,045 ICUs-patients) were evaluated using a random-effect model and a meta-regression analysis. The acquisition rate of digestive tract colonization during ICU stay was 7% (95% CI, 5-10) and it varies from 3% (95% CI, 2-4) and 4% (95% CI, 2-6) in the Americas and Europe to 21% (95% CI, 9-35) in the Western Pacific region. Previous hospitalization (risk ratio, 1.57 [95% CI, 1.07-2.31]) or antibiotic use (risk ratio, 1.65 [95% CI, 1.15-2.37]) and exposure to beta-lactams/beta-lactamase inhibitors (risk ratio, 1.78 [95% CI, 1.24-2.56]) and carbapenems (risk ratio, 2.13 [95% CI, 1.49-3.06]) during the ICU stay were independent risk factors for ICU-acquired colonization. Importantly, colonized patients were more likely to develop an extended-spectrum beta-lactamase-producing Enterobacteriaceae infection (risk ratio, 49.62 [95% CI, 20.42-120.58]). The sensitivity and specificity of prior colonization to predict subsequent extended-spectrum beta-lactamase-producing Enterobacteriaceae infection were 95.1% (95% CI, 54.7-99.7) and 89.2% (95% CI, 77.2-95.3), respectively. The ICU acquisition rate of extended-spectrum beta-lactamase

Molecular epidemiology of outbreak-related pseudomonas aeruginosa strains carrying the novel variant blaVIM-17 metallo-beta-lactamase gene.

Science.gov (United States)

Siarkou, Victoria I; Vitti, Danai; Protonotariou, Efthimia; Ikonomidis, Alexandros; Sofianou, Danai

2009-04-01

A study was designed to investigate the molecular epidemiological characteristics of multidrug-resistant outbreak-related Pseudomonas aeruginosa isolates collected in a university hospital in northern Greece. Of 29 nonreplicate P. aeruginosa isolates resistant to carbapenems and ceftazidime, 14 were positive for metallo-beta-lactamase production. PCR analyses with primers specific for bla(VIM) and bla(IMP) revealed that 13 isolates carried a novel bla(VIM-2) gene variant, designated bla(VIM-17), and only 1 isolate carried bla(VIM-2), a gene predominant among P. aeruginosa strains in Greek hospitals. Pulsed-field gel electrophoresis of XbaI-digested genomic DNAs showed a close genetic relationship for 12 of 13 bla(VIM-17)-carrying outbreak-related isolates, which were of the O11 serotype; the clonally unrelated isolate carrying bla(VIM-17) was of the O12 serotype. PCR mapping strategies for the detection of class 1 integrons and sequencing approaches revealed the presence of integrons containing one bla(VIM) cassette flanked by two aacA29 cassettes. These integrons were similar but not identical to In59 (GenBank accession number AF263519) initially described in France. All isolates carrying bla(VIM-17), regardless of their genetic profile, had an identical integron, named In59.3, indicating that although the hospital outbreak was mainly due to clonal dissemination, the horizontal transmission of the bla(VIM-17)-containing integron among P. aeruginosa isolates should also have occurred. An outbreak-related isolate and a control strain, both of which carried the bla(VIM-2) gene but which were clonally distinct, had an identical integron, named In59.2, which differed only at the level of the bla(VIM) gene from In59.3 integrons, suggesting a common ancestry. The spread of the bla(VIM-17)-containing integron in clonally unrelated P. aeruginosa isolates without any evidence of plasmid carriage is probably associated with a transposon.

Amino acid residues that contribute to substrate specificity of class A beta-lactamase SME-1.

Science.gov (United States)

Majiduddin, Fahd K; Palzkill, Timothy

2005-08-01

Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by beta-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 beta-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A beta-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of beta-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

Evaluation of different phenotypic methods for detection of amp c beta-lactamase producing bacteria in clinical isolates

International Nuclear Information System (INIS)

Hassan, A.; Usman, J.; Kalim, F.; Gill, M.M.; Khalid, A.; Iqbal, M.; Ingram, P.

2013-01-01

To compare the sensitivity and specificity of different phenotypic methods for detection of Amp C betalactamase producing bacteria. Study Design: Analytical study. Place and Duration of Study: Department of Microbiology, Army Medical College / National University of Sciences and Technology (NUST), Islamabad, Pakistan, from June 2010 to December 2010. Methodology: A total of 150 clinical isolates were screened for presence of Amp C beta-lactamase by using the cefoxitin disc. The confirmatory methods evaluated were inhibitor based assay (boronic acid), Amp C disc test and Amp C Etest. Three dimensional enzyme extract assay was used as the reference method for determining the sensitivity and specificity. Results: Among the total isolates tested, 62.8% bacteria showed the presence of Amp C beta-lactamase by standard three dimensional enzyme extract assay. Among the three methods compared, boronic acid disk test found out to be highly sensitive (88%) and specific (92%) for the detection of Amp C beta-lactamase producing bacteria. Conclusion: Detection of Amp C production is crucial in order to establish the antibiotic therapy and to attain the favourable clinical outcomes. Implementation of simple tests like boronic acid disk tests in the laboratories will help to alleviate the spread of Amp C beta-lactamase harboring organisms. (author)

Overcoming resistance to beta-lactamase inhibitors: comparing sulbactam to novel inhibitors against clavulanate resistant SHV enzymes with substitutions at Ambler position 244.

Science.gov (United States)

Thomson, Jodi M; Distler, Anne M; Bonomo, Robert A

2007-10-09

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> beta-lactamase was unmodified at 15 min. A parallel experiment with R244S demonstrated 70 and 88 +/- 3 Da fragments of sulbactam covalently attached to the beta-lactamase. We also observed that the Ki values of penems 1 and 2 were increased for R244 variants compared to those for SHV; however, these inhibitors effectively restored ampicillin susceptibility in vitro. Compared to that of sulbactam, the kcat/kinact values of penems for SHV-1 and R244S were low (beta-lactamase inactivators differently, but resistance can be overcome by designing penem inhibitors with strategic chemical properties that improve affinity and impair turnover.

Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases.

Science.gov (United States)

Vecoli, C; Prevost, F E; Ververis, J J; Medeiros, A A; O'Leary, G P

1983-08-01

Plasmid-mediated beta-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observed for HMS-1 and PSE-1 beta-lactamases. The HMS-1 sample produced two highly resolvable enzyme bands in agarose gels rather than the single faint enzyme band observed on polyacrylamide gels. The PSE-1 sample showed an isoelectric point shift of 0.2 pH unit between polyacrylamide and agarose gel (pI 5.7 and 5.5, respectively). The short focusing time, lack of toxic hazard, and ease of formulation make agarose a practical medium for the characterization of beta-lactamases.

Plasmid-mediated AmpC-type beta-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum beta-lactams, including moxalactam.

Science.gov (United States)

Horii, T; Arakawa, Y; Ohta, M; Ichiyama, S; Wacharotayankun, R; Kato, N

1993-01-01

Klebsiella pneumoniae NU2936 was isolated from a patient and was found to produce a plasmid-encoded beta-lactamase (MOX-1) which conferred resistance to broad spectrum beta-lactams, including moxalactam, flomoxef, ceftizoxime, cefotaxime, and ceftazidime. Resistance could be transferred from K. pneumoniae NU2936 to Escherichia coli CSH2 by conjugation with a transfer frequency of 5 x 10(-7). The structural gene of MOX-1 (blaMOX-1) was cloned and expressed in E. coli HB101. The MIC of moxalactam for E. coli HB101 producing MOX-1 was > 512 micrograms/ml. The apparent molecular mass and pI of this enzyme were calculated to be 38 kDa and 8.9, respectively. Hg2+ and Cu2+ failed to block enzyme activity, and the presence of EDTA in the reaction buffer did not reduce the enzyme activity. However, clavulanate and cloxacillin, serine beta-lactamase inhibitors, inhibited the enzyme activity competitively (Kis = 5.60 and 0.35 microM, respectively). The kinetic study of MOX-1 suggested that it effectively hydrolyzed broad-spectrum beta-lactams. A hybridization study confirmed that blaMOX-1 is encoded on a large resident plasmid (pRMOX1; 180 kb) of strain NU2936. By deletion analysis, the functional region was localized within a 1.2-kb region of the plasmid. By amino acid sequencing, 18 of 33 amino acid residues at the N terminus of MOX-1 were found to be identical to those of Pseudomonas aeruginosa AmpC. These findings suggest that MOX-1 is a plasmid-mediated AmpC-type beta-lactamase that provides enteric bacteria resistance to broad-spectrum beta-lactams, including moxalactam. Images PMID:8517725

ORF Alignment: NC_003384 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available [Zymomonas mobilis] gb|AAQ67239.1| ... Bla [Transposon delivery vector pUT-miniTn5-gfp-tet] ... ... ... 3.5.2.6) precursor - Escherichia coli plasmids ... gb|AAC79082.1| beta lactamase [Transposon delivery...AG47772.1| beta-lactamase ... TEM-1 [Klebsiella pneumoniae] gb|AAG37887.1| ampicillin ... resistance [Tn10 delivery... vector pHV1249] gb|AAG37882.1| ... ampicillin resistance [Tn10 delivery

The 1.4 Å Crystal Structure of the Class D [beta]-Lactamase OXA-1 Complexed with Doripenem

Energy Technology Data Exchange (ETDEWEB)

Schneider, Kyle D.; Karpen, Mary E.; Bonomo, Robert A.; Leonard, David A.; Powers, Rachel A.; (Grand Valley); (Case Western U.-Med)

2010-01-12

The clinical efficacy of carbapenem antibiotics depends on their resistance to the hydrolytic action of {beta}-lactamase enzymes. The structure of the class D {beta}-lactamase OXA-1 as an acyl complex with the carbapenem doripenem was determined to 1.4 {angstrom} resolution. Unlike most class A and class C carbapenem complexes, the acyl carbonyl oxygen in the OXA-1-doripenem complex is bound in the oxyanion hole. Interestingly, no water molecules were observed in the vicinity of the acyl linkage, providing an explanation for why carbapenems inhibit OXA-1. The side chain amine of K70 remains fully carboxylated in the acyl structure, and the resulting carbamate group forms a hydrogen bond to the alcohol of the 6{alpha}-hydroxyethyl moiety of doripenem. The carboxylate attached to the {beta}-lactam ring of doripenem is stabilized by a salt bridge to K212 and a hydrogen bond with T213, in lieu of the interaction with an arginine side chain found in most other {beta}-lactamase-{beta}-lactam complexes (e.g., R244 in the class A member TEM-1). This novel set of interactions with the carboxylate results in a major shift of the carbapenem's pyrroline ring compared to the structure of the same ring in meropenem bound to OXA-13. Additionally, bond angles of the pyrroline ring suggest that after acylation, doripenem adopts the {Delta}{sup 1} tautomer. These findings provide important insights into the role that carbapenems may have in the inactivation process of class D {beta}-lactamases.

Involvement of AmpG in mediating a dynamic relationship between serine beta-lactamase induction and biofilm-forming ability of Escherichia coli.

Science.gov (United States)

Mallik, Dhriti; Pal, Shilpa; Ghosh, Anindya S

2018-04-01

AmpG permease is implicated both in beta-lactamase induction and peptidoglycan recycling in enterobacterial isolates. Here, physiological studies using molecular genetics show that deletion of AmpG permease dramatically increases beta-lactam susceptibility even in the presence of AmpC, TEM-1 and OXA beta-lactamases. Also, there is an appreciable decrease in the biofilm-forming ability of strains lacking this protein. Expression of this permease in excess probably compromises the integrity of the bacterial cells, leading to cell lysis. Based on these results, we propose that AmpG permease may be used as a potential antibiotic target and its suppression could efficiently inhibit both beta-lactamase induction and biofilm formation.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in hospital food: a risk assessment

NARCIS (Netherlands)

Stewardson, A.J.; Renzi, G.; Maury, N.; Vaudaux, C.; Brossier, C.; Fritsch, E.; Pittet, D.; Heck, M.; Zwaluw, K. van der; Reuland, E.A.; Laar, T. van; Snelders, E.; Vandenbroucke-Grauls, C.; Kluytmans, J.; Edder, P.; Schrenzel, J.; Harbarth, S.

2014-01-01

OBJECTIVE: Determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) contamination of food and colonization of food handlers in a hospital kitchen and compare retrieved ESBL-PE strains with patient isolates. DESIGN: Cross-sectional study. SETTING: A

Burden of different beta-lactamase classes among clinical isolates of AmpC-producing Pseudomonas aeruginosa in burn patients: A prospective study

OpenAIRE

Kumar, V.; Sen, M. R.; Nigam, C.; Gahlot, R.; Kumari, S.

2012-01-01

Background: Pseudomonas aeruginosa is one of the most common pathogens causing infections in burns, and shows increasing resistance to β-lactam antibiotics by producing different classes of beta-lactamases. It is also not unusual to find a single isolate that expresses multiple β-lactamase enzymes, further complicating the treatment options. Thus, in this study, we aimed to determine the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa in the burn ward. Ma...

Sensitivity and specificity of various beta-lactam antibiotics and phenotypical methods for detection of TEM, SHV and CTX-M extended-spectrum beta-lactamases.

Science.gov (United States)

Bedenic, B; Vranes, J; Mihaljevic, Lj; Tonkic, M; Sviben, M; Plecko, V; Kalenic, S

2007-04-01

The aim of this study was to compare the sensitivity and specificity of six different beta-lactam antibiotics using five phenotypical tests for detection of extended spectrum beta-lactamases (ESBLs) based on synergism of beta-lactam antibiotics and clavulanate. Experiments were performed on a set of 80 Klebsiella pneumoniae strains and 105 Escherichia coli strains with previously characterized ESBLs (SHV, TEM and CTX-M). ESBLs were detected by five different phenotypical methods: MIC (minimum inhibitory concentration) determination of beta-lactam antibiotics with and without clavulanate, double-disk synergy test (DDST), inhibitor-potentiated disk-diffusion test (IPDDT), CLSI-Clinical and Laboratory Standard Institution (former NCCLS) combined-disk-test, and modified MAST-disk-diffusion test (MAST-DD-test). Seven antibiotics were tested as indicators of ESBL production: ceftazidime, cefotaxime, ceftriaxone, aztreonam, ceftibuten, cefpodoxime and cefepime. Ceftazidime and aztreonam were the best indicators for SHV-5, SHV-12 and TEM beta-lactamases whereas cefotaxime and ceftriaxone were the most sensitive in detection of SHV-2 and CTX-M beta-lactamases in DDST, IPDDT and CLSI test. MIC determination of beta-lactam antibiotics with and without clavulanate was the most sensitive method. DDST was the least sensitive test. Double-disk synergy test, which is the most frequently used test for detection of ESBLs in routine laboratories, was the least sensitive independently of the indicator antibiotic. Since MIC determination is a very laborious and time consuming method, we would recommend the NCCLS combined disk test or IPDD test for detection of ESBLs in routine laboratories with 5 mm zone augmentation breakpoint.

Extended Spectrum Beta-lactamases: Definition, Classification and Epidemiology.

Science.gov (United States)

Ghafourian, Sobhan; Sadeghifard, Nourkhoda; Soheili, Sara; Sekawi, Zamberi

2015-01-01

Extended spectrum beta-lactamases (ESBLs) are defined as enzymes produced by certain bacteria that are able to hydrolyze extended spectrum cephalosporin. They are therefore effective against beta-lactam antibiotics such as ceftazidime, ceftriaxone, cefotaxime and oxyimino-monobactam. The objective of the current review is to provide a better understanding of ESBL and the epidemiology of ESBL producing organisms which are among those responsible for antibiotic resistant strains. Globally, ESBLs are considered to be problematic, particularly in hospitalized patients. There is an increasing frequency of ESBL in different parts of the world. The high risk patients are those contaminated with ESBL producer strains as it renders treatment to be ineffective in these patients. Thus, there an immediate needs to identify EBSL and formulate strategic policy initiatives to reduce their prevalence.

Antibiogram Studies and Extended Spectrum Beta-lactamase Activity Profile of Salmonella-like Species Isolated from Poultry Soil of the University of Uyo, Nigeria

Directory of Open Access Journals (Sweden)

Ikpeme, E. M.

2012-01-01

Full Text Available Aims: The contribution of beta-lactamase activity of various bacterial species to the increased antimicrobial resistance being experienced worldwide is very scanty in the literature. This study was undertaken to investigate the antibiotic resistance pattern (antibiogram of Salmonella-like bacterial species against some antibiotics, and the role beta-lactamase assumably produced by the Salmonella-like species, played in producing resistance.Methodology and Results: The antimicrobial sensitivity test and the beta-lactamase test of the Salmonella-like species were carried out using the methods of Kirby Bauer sensitivity test and the Double Disk Synergy test respectively, following isolation and identification of the organisms from poultry soil. Results revealed that Salmonella-like species were most highly resistant to Nalidixic acid (20, 66.66%, followed by Tetracycline (19, 63.33%, Cotrimoxazole, Amoxicillin and Augmentin (18, 60%, while the least was Ofloxacin (8, 26.66%. Multiple resistance of 4 or more antibiotics among the isolates from the soil outside the broilers enclosure was observed, while there was a significant difference (P <0.05 between poultry soil and control soil. This implied that the antibiotics with the highest resistance were most often applied to the birds, the droppings of which contaminated the soil. The resistant pattern of the isolates from the control soil is lower than that from the poultry soil. Extended Spectrum Beta-lactamase activity was expressed by all the isolates against Cefotazime, while the least resistance was against mostly Cefotazime.Conclusion, significance and impact of study: It is concluded that there is a widespread Beta-lactamase activity causing antibiotic resistance by many species of bacteria as well as poultry Salmonella, thus exacerbating the global problem of antibiotic resistance and a serious health related implication for antibiotic use in poultry.

Substrate-induced inactivation of the OXA2 beta-lactamase.

Science.gov (United States)

Ledent, P; Frère, J M

1993-01-01

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. 'Burst' kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two 'bursts', and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product. PMID:8240304

Prevalence of extended-spectrum beta-lactamases among Enterobacteriaceae isolated from blood culture in a tertiary care hospital

International Nuclear Information System (INIS)

El-Khizzi, Noura A.; Bakheshwain, S. M.

2006-01-01

To determine the prevalence of extended spectrum beta-lactamase among Enterobacteriaceae isolated from blood culture in a tertiary care hospital. We carried out this study at the Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia during the period between January 2003 - December 2004. We tested a total of 601 isolates of the family Enterobacteriaceae from blood culture for the prevalence of extended spectrum beta-lactamase (ESBL) production by the standardized disc diffusion method and confirmed by the ESBL E test strips. Ninety-five (15.8%) of the isolates were ESBL producers. Among these, 48.4% were Klebsiella pneumoniae (K. pneumoniae) followed by15.8% of both Escherichia coli (E. coli) and Enterobacter cloacae (Ent. cloacae). Other isolates produced ESBL in low numbers. Klebsiella pneumoniae produced ESBL in significant numbers. Extended spectrum beta-lactamase gram-negative bacilli present significant diagnostic and therapeutic challenges to the management of infections due to these organisms. Microbiology laboratories should start reporting ESBL producing Enterobacteriaceae organism due to their importance in respect to antibiotic therapy and infection control aspects. (author)

Assessment of AmpC Beta-Lactamase Genes among Clinical Escherichia coli Isolates

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HedrooshaMolla Agha-Mirzaeie

2015-11-01

Full Text Available Background: AmpC bta lactamases play a significant role in creating resistance to third generation cephalosporins worldwide. They mostly express on chromosome of Enterobacteriaceae especially Escherichia coli and cause consequential problem inclinical treatment and lead to failure in diagnosis and phenotypic test recommended byClinical and Laboratory Standards Institute.Methods:Totally 200 E. coli isolates from different hospitals of Tehran were collected. The isolates were screened by disk diffusion method according to the CLSI guidelines. The profiles and prevalence surveys of AmpC (Dha, CITM, Mox and FOX-type β-lactamase genes in clinical isolates of E. coli by phenotypic and molecular methods.  Results:Out of 200 Ecoli isolated, 115 (89.8% and 13 (10.2% isolates were identified as ESBL- and AmpC- beta-lactamase producers, respectively. Among mpC producers, 13 (100% and 5 (38.5% isolates was reported by PCR assay as bla-CITM and Dha respectively. Mox and FOX genes were not detected in any sample.Conclusions:Our results highlight the importance of using molecular detection methods to identify β-lactamase-producer that have resistance to antibiotics. 

Evaluation of the MicroScan ESBL plus confirmation panel for detection of extended-spectrum beta-lactamases in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria.

Science.gov (United States)

Stürenburg, Enno; Lang, Melanie; Horstkotte, Matthias A; Laufs, Rainer; Mack, Dietrich

2004-11-01

We aimed to assess the performance of the MicroScan ESBL plus confirmation panel using a series of 87 oxyimino-cephalosporin-resistant Gram-negative bacilli of various species. Organisms tested included 57 extended-spectrum beta-lactamase (ESBL) strains comprising Enterobacter aerogenes (3), Enterobacter cloacae (10), Escherichia coli (11), Klebsiella pneumoniae (26), Klebsiella oxytoca (3) and Proteus mirabilis (4). Also included were 30 strains resistant to oxyimino cephalosporins but lacking ESBLs, which were characterized with other resistance mechanisms, such as inherent clavulanate susceptibility in Acinetobacter spp. (4), hyperproduction of AmpC enzyme in Citrobacter freundii (2), E. aerogenes (3), E. cloacae (3), E. coli (4), Hafnia alvei (1) and Morganella morganii (1), production of plasmid-mediated AmpC beta-lactamase in K. pneumoniae (3) and E. coli (3) or hyperproduction of K1 enzyme in K. oxytoca (6). The MicroScan MIC-based clavulanate synergy correctly classified 50 of 57 ESBL strains as ESBL-positive and 23 of 30 non-ESBL strains as ESBL-negative (yielding a sensitivity of 88% and a specificity of 76.7%, respectively). False negatives among ESBL producers were highest with Enterobacter spp. due to masking interactions between ESBL and AmpC beta-lactamases. False-positive classifications occurred in two Acinetobacter spp., one E. coli producing plasmid-mediated AmpC beta-lactamase and two K. oxytoca hyperproducing their chromosomal K1 beta-lactamase. The MicroScan clavulanate synergy test proved to be a valuable tool for ESBL confirmation. However, this test has limitations in detecting ESBLs in Enterobacter spp. and in discriminating ESBL-related resistance from the K1 enzyme and from inherent clavulanate susceptibility in Acinetobacter spp.

Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana.

Science.gov (United States)

Rasmussen, Mette Marie; Opintan, Japheth A; Frimodt-Møller, Niels; Styrishave, Bjarne

2015-01-01

The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC) were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%). Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3). The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype blaCTX-M-15 (50%; 10/20) was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats are potential

Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana.

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Mette Marie Rasmussen

Full Text Available The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%. Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3. The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype blaCTX-M-15 (50%; 10/20 was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats

The revolving door between hospital and community: extended-spectrum beta-lactamase-producing Escherichia coli in Dublin.

LENUS (Irish Health Repository)

Burke, L

2012-07-01

Escherichia coli that produce extended-spectrum beta-lactamases (ESBLs) are an increasing cause of healthcare-associated infection, and community healthcare facilities may be a reservoir for important epidemic clones.

Extended-spectrum beta-lactamases in urinary tract infections caused by Enterobacteria: understanding and guidelines for action.

Science.gov (United States)

García-Tello, A; Gimbernat, H; Redondo, C; Arana, D M; Cacho, J; Angulo, J C

2014-12-01

Beta-lactamases are bacterial enzymes that protect microorganisms from the lethal effects of β-lactam antibiotics. The production of beta-lactamases is the most important mechanism of resistance to these antibiotics, especially in Gram-negative bacteria. Review the magnitude of the problem of extended-spectrum beta-lactamases (ESBL) in the urological setting and present the fundamental action guidelines on the issue, the main risk factors and the prevention strategies. A structured search strategy for patient, problem, intervention, comparison and result was conducted in the PubMed-Medline database to identify the most relevant studies related to the management of patients with urinary tract infection by ESBL-producing microorganisms. We also present a caseload analysis of our center on this issue. ESBL are found in Enterobacteria, mainly Klebsiella sp. and Escherichia coli and are characterized by their hydrolytic ability compared with beta-lactam antibiotics, which entails resistance to penicillin, cephalosporin and aztreonam. They are also associated with resistance to other antibiotics. There is a high risk of infection and colonization by ESBL producers in patients with prolonged hospital stays or who required invasive devices. The prior use of antibiotics and stays in residential care are also risk factors. Prevention programs should focus on preventing nosocomial infection. It is essential that a restrictive policy on the use of antibiotics be implemented. The therapy of choice for severe infections is focused on carbapenems, although their indiscriminate use should be avoided. In uncomplicated lower urinary tract infections, fosfomycin and nitrofurantoin are the best treatment alternatives. ESBL-producing strains constitute a true global health problem. Prevention strategies should focus on nosocomial infection. We should not forget, however, that the appearance of these pathogens in community-acquired infections is increasingly frequent. Therapeutic

Novel ambler class A carbapenem-hydrolyzing beta-lactamase from a Pseudomonas fluorescens isolate from the Seine River, Paris, France.

Science.gov (United States)

Girlich, Delphine; Poirel, Laurent; Nordmann, Patrice

2010-01-01

A Pseudomonas fluorescens isolate (PF-1) resistant to carbapenems was recovered during an environmental survey performed with water from the Seine River (Paris). It expressed a novel Ambler class A carbapenemase, BIC-1, sharing 68 and 59% amino acid identities with beta-lactamases SFC-1 from Serratia fonticola and the plasmid-encoded KPC-2, respectively. beta-Lactamase BIC-1 hydrolyzed penicillins, carbapenems, and cephalosporins except ceftazidime and monobactams. The bla(BIC-1) gene was chromosomally located and was also identified in two other P. fluorescens strains isolated from the Seine River 3 months later.

Seagulls of the Berlengas natural reserve of Portugal as carriers of fecal Escherichia coli harboring CTX-M and TEM extended-spectrum beta-lactamases.

Science.gov (United States)

Poeta, Patricia; Radhouani, Hajer; Igrejas, Gilberto; Gonçalves, Alexandre; Carvalho, Carlos; Rodrigues, Jorge; Vinué, Laura; Somalo, Sergio; Torres, Carmen

2008-12-01

Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.

Susceptibility pattern of extended spectrum beta-lactamase producing isolates in various clinical specimens

International Nuclear Information System (INIS)

Roshan, M.

2011-01-01

Objective: To determine the susceptibility pattern of extended spectrum beta-lactamase (ESBL) producing Gram negative isolates from various clinical specimens. Study Design: Descriptive study. Place and Duration of Study: Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi, from January 2008 to January 2009. Methodology: A total of 308 ESBL producing isolates from various clinical specimens sent to AFIP for culture and sensitivity were identified using standard microbiological techniques and tested for antimicrobial susceptibility. At the same time screening for ESBL production was also done. ESBL production was confirmed by combination disc synergy method. The susceptibility pattern of isolates was then recorded in frequency percentages. Results: Out of the 308 ESBL producing isolates more than 99% were susceptible to carbapenems, 84% to tazobactam/ piperacillin, 81% to sulbactam/cefoperazone, 12% to fluoroquinolones, 13% to cotrimoxazole, 59% to amikacin and 18% to gentamicin. Among the urinary isolates 49% were susceptible to Nitrofurontoin and only 5% to Pipemidic acid. Conclusion: Antibiotic choices in case of ESBL producing isolates are limited and at present only carbapenems can be regarded as treatment of choice. As empirical agents, beta-lactam/beta lactamase inhibitor combinations should be used cautiously for serious infections. Fluoroquinolones showed very poor efficacy. Amikacin can be used alternatively in such cases. Nitrofurantoin is still a good oral agent for treating UTI. (author)

Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes

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Dwivedi Mayank

2009-07-01

Full Text Available Context: Ventilator-associated pneumonia (VAP is a leading nosocomial infection in the intensive care unit (ICU. Members of Enterobacteriaceae are the most common causative agents and carbapenems are the most commonly used antibiotics. Metallo-beta-lactamase (MBL production leading to treatment failure may go unnoticed by routine disc diffusion susceptibility testing. Moreover, there is not much information on association of MBL-producing Enterobacteriaceae with ICU-acquired VAP. Therefore, a study was undertaken to find out the association of MBL-producing Enterobacteriaceae with VAP. Settings: This study was conducted in a large tertiary care hospital of North India with an eight-bed critical care unit. Materials and Methods: The respiratory samples (bronchoalveolar lavage, protected brush catheter specimens and endotracheal or transtracheal aspirates obtained from VAP patients (during January 2005-December 2006 were processed, isolated bacteria identified and their antibiotic susceptibilities tested as per standard protocols. The isolates of Enterobacteriaceae resistant to carbapenem were subjected to phenotypic and genotypic tests for the detection of MBLs. Results: Twelve of 64 isolates of Enterobacteriaceae were detected as MBL producers, bla IMP being the most prevalent gene. Additionally, in three strains, simultaneous coexistence of multiple MBL genes was detected. Conclusion: The coexistence of multiple MBL genes in Enterobacteriaceae is an alarming situation. As MBL genes are associated with integrons that can be embedded in transposons, which in turn can be accommodated on plasmids thereby resulting in a highly mobile genetic apparatus, the further spread of these genes in different pathogens is likely to occur.

Detection of Beta-lactamase gene in the culturable bacteria isolated from agricultural, pasture and mining soils around mines in Hamedan, Iran

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Nayereh Younessi

2017-09-01

Full Text Available Introduction: Growing evidence exists that agriculture affects antibiotic resistance in human pathogens. Beta-lactam antibiotics are the most commonly used antimicrobial agents in many countries. The abundance of beta-lactamase encoding genes can be used as an indicator of antibiotic resistance in the environment. So, to determine the beta-lactamase resistance genes, the abundance of culturable bacteria having bla-TEM genesin the soils under different land uses wasexamined. Materials and methods: 44 Gram-positive and 34 Gram-negative bacteria plated on nutrient agar were isolated from agricultural, pasture and mining soils and selected to study the presence of TEM-class gene using PCR amplification. Antibiotic sensitivity test of bla-TEM+isolateswas done adopting the Kirby-Bauer disk diffusion method and antibiotic discs used were: ampicillin, amoxicillin, vancomicin, streptomycin, tetracycline and gentamicin. Finally, five multi-drug resistant and bla-TEM+ isolates were identified using universal primers. Results: The highest level of beta-lactamase genes was observed in the Gram-positive and Gram-negative isolates from the pasture soils. In the agricultural and mining soils, a high abundance of bla-TEM+ isolateswasfound which also showed resistance to beta-lactam antibiotics. The identified multi-drug resistant and bla-TEM+ isolates were from these genera: Achromobacter, Bacillus, Brevibacillus, Aminobacter and Brevundimonas. Discussion and conclusion: The high number of bla-TEM+ bacteria in all the soils may be attributed to the other important feature of bla genes which is their capability to extrude toxic compounds like heavy metals in contaminated environments. Sensitivity of some bla-TEM+ bacteria to beta-lactam antibiotics was interesting. This result shows that bla-TEM genes confer resistance to beta-lactamase inhibitors in a different degree. Some of the identified isolates were pathogen. These pathogens in soils can transfer to plants

Emergence of colistin resistance in extended-spectrum beta lactamase producing Enterobacteriaceae isolated from food animals and its public health implication: A review

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Asinamai Athliamai Bitrus

2018-03-01

Full Text Available Antimicrobial resistance as a result of emergence of extended-spectrum beta lactamase producing Enterobacteriaceae is a major health problem of human and animal that requires an intensive global attention. The production of beta lactamase enzymes remains as one of the major factors contributing to the development of resistance to beta lactams. These enzymes hydrolyze the beta lactam ring of the antibiotic and render it ineffective. Extended-spectrum beta lactamase producing bacteria have the ability to develop resistance to a number of antibiotics including the carbapenem and other third generation cephalosporins. In addition, the recent emergence and dissemination of the colistin resistance determinants mcr-1, mcr-2 and mcr-3 poses a serious threat to colistin as a drug of last resort in human medicine. In this review, we utilized words such as “colistin resistance and Escherichia coli”, “Klebsiella and colistin resistance”, “colistin resistance and Salmonella” as well as “detection of mcr-1 genes in Salmonella and E. coli”. The extended-spectrum beta lactamase producing bacteria under Enterobacteriaceae that are resistant to colistin possess the ability to be transferred resistant determinants to other susceptible cells at a higher frequency. In this paper, the role of manure from food animals and how air travel contributes to the dissemination of mcr-1 haboring bacteria, resistance determinants and other metabolites that constitute a public health problem was also reviewed. It is concluded that these pathogens have significant consequences to the control of infection and plays key roles in treatment failure with colistin. [J Adv Vet Anim Res 2018; 5(1.000: 1-11

beta-Lactamases among extended-spectrum beta-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands

DEFF Research Database (Denmark)

Hasman, Henrik; Mevius, D.; Veldman, K.

2005-01-01

Objectives: The purpose of this work was to study the genetic determinants responsible for extended-spectrum beta-lactamase (ESBL) resistance of Salmonella isolated from Dutch poultry, poultry meat and hospitalized humans. Methods: Thirty-four ESBL-resistant Salmonella isolates from The Netherlands...... were tested towards 21 antimicrobial agents. PCR and sequencing were used to determine the underlying genetic determinants responsible for the ESBL phenotypes. The transferability of the ESBL phenotypes was tested by conjugation to a susceptible Salmonella enterica serovar Dublin and plasmid....... Finally, the bla(ACC-1) gene was cloned from a S. Bareilly isolate and was found to be present on indistinguishable plasmids in all S. Bareilly isolates examined as well as in a S. Braenderup isolate and a S. Infantis isolate. Conclusions: Our data underscore the diversity of ESBL genes in Salmonella...

Binding of benzylpenicillin to metallo-ß-lactamase

DEFF Research Database (Denmark)

Olsen, Lars; Rasmussen, T; Hemmingsen, L

2004-01-01

Metallo-beta-lactamases are bacterial enzymes that may function with either one or two zinc ions bound in the active site. In this work, the binding of benzylpenicillin to mono-zinc metallo-beta-lactamase from Bacillus cereus has been investigated in a docking procedure applying a combined quantu...

In vitro Efficacy of Meropenem, Colistin and Tigecycline Against the Extended Spectrum Beta-Lactamase Producing Gram Negative Bacilli

International Nuclear Information System (INIS)

Gill, M. M.; Usman, J.; Hassan, A.; Kaleem, F.; Anjum, R.

2015-01-01

Objective:To compare the in vitroefficacy of meropenem, colistin and tigecycline against extended spectrum Betalactamase producing Gram negative bacilli by minimal inhibitory concentration. Study Design:Cross-sectional descriptive study. Place and Duration of Study: Department of Microbiology, Army Medical College, National University of Sciences and Technology, Rawalpindi, from June to December 2010. Methodology: Routine clinical specimens were subjected to standard microbiological procedures and the isolates were identified to species level. Extended spectrum beta-lactamase producing Gram negative bacilli were detected by Jarlier disc synergy method and confirmed by ceftazidime and ceftazidime-clavulanate Etest. Minimum Inhibitory Concentration (MIC90) of meropenem, colistin and tigecycline was determined by Etest (AB BIOMERIUX) and the results were interpreted according to the manufacturer's instructions and Clinical and Laboratory Standards Institute guidelines and Food and Drug Authority recommendations. Results were analyzed by using Statistical Package for the Social Sciences version 20. Results: A total of 52 non-duplicate extended spectrum Beta-lactamase-producing Gram negative bacilli were included in the study. The MIC90 of tigecycline (0.75 micro g/ml) was lowest as compared to the meropenem (2 micro g/ml) and colistin (3 micro g/ml). Conclusion: Tigecycline is superior in efficacy against the extended spectrum Beta-lactamase producing Gram negative bacilli as compared to colistin and meropenem. (author)

Method for phenotypic detection of extended-spectrum beta-lactamases in enterobacter species in the routine clinical setting

NARCIS (Netherlands)

Stuart, J.C.; Diederen, B.; Al Naiemi, N.; Fluit, A.; Arents, N.; Thijsen, S.; Vlaminckx, B.; Mouton, J.W.; Leverstein-van Hall, M.

2011-01-01

In 271 Enterobacter blood culture isolates from 12 hospitals, extended-spectrum beta-lactamase (ESBL) prevalence varied between 0% and 30% per hospital. High prevalence was associated with dissemination, indicating the potential relevance of infection control measures. Screening with cefepime or

Survey of metallo-beta-lactamase-producing Enterobacteriaceae colonizing patients in European ICUs and rehabilitation units, 2008-11

NARCIS (Netherlands)

Papagiannitsis, C. C.; Izdebski, R.; Baraniak, A.; Fiett, J.; Herda, M.; Hrabak, J.; Derde, L. P. G.; Bonten, M. J. M.; Carmeli, Y.; Goossens, H.; Hryniewicz, W.; Brun-Buisson, C.; Gniadkowski, M.

Objectives: The objective of this study was to perform a multinational survey of patients' colonization by metallo-beta-lactamase (MBL)-producing Enterobacteriaceae, including their molecular characterization. Methods: Patients in 18 hospital units across Europe and Israel (n = 17945) were screened

Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae

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M Shanthi

2012-01-01

Full Text Available Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR. Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25 and K. pneumoniae (n = 52 were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI guidelines. Minimum inhibitory concentration (MIC to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL. Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test. Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii, DHA (Dhahran Hospital, Saudi Arabia, ACC (Ambler class C, EBC (Amp C origin - Enterobacter cloacae groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

Restriction of cephalosporins and control of extended spectrum beta-lactamase producing gram negative bacteria in a neonatal intensive care unit.

Science.gov (United States)

Murki, Srinivas; Jonnala, Sravanthi; Mohammed, Faheemuddin; Reddy, Anupama

2010-09-01

This interventional study with historical controls was conducted to study the effect of cephalosporin restriction on the incidence of extended spectrum beta-lactamase (ESBL) gram negative infections in neonates admitted to intensive care unit. All gram negative isolates from the blood were evaluated for beta lactamase production. The incidence of ESBL production was compared before (year 2007) and after cephalosporin restriction (year 2008). Thirty two neonates (3% of NICU admissions) in the year 2007 and fifty six (5.2%) in the year 2008, had gram negative septicemia. The incidence of ESBL gram negatives decreased by 22% (47% to 25%, P=0.03). Restriction of all class of cephalosporins significantly decreased the incidence of ESBL gram negative infections.

Utility of the ceftazidime-imipenem antagonism test (CIAT to detect and confirm the presence of inducible AmpC beta-lactamases among enterobacteriaceae

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Vlademir Vicente Cantarelli

Full Text Available Detection of AmpC beta-lactamase production by enterobacteria has been problematic. Contrary to ESBLs, no specific guidelines are available for detection and confirmation of AmpC production by clinical relevant microorganisms. Moreover, some bacterial species may produce inducible AmpC beta-lactamases that can be easily overlooked by routine susceptibility tests. We reported here a new test based on the strong inducible effect of imipenem on AmpC genes and the consequent antagonism with ceftazidime. This test is very simple and proved to be helpful in detecting AmpC-inducible enzymes among several species of clinical isolates.

Pharmacodynamics of Imipenem in Combination with beta-Lactamase Inhibitor MK7655 in a Murine Thigh Model

NARCIS (Netherlands)

Mavridou, E.; Melchers, M.J.B.; Mil, A.C. van; Mangin, E.; Motyl, M.R.; Mouton, J.W.

2015-01-01

MK7655 is a newly developed beta-lactamase inhibitor of class A and class C carbapenemases. Pharmacokinetics (PK) of imipenem-cilastatin (IMP/C) and MK7655 were determined for intraperitoneal doses of 4 mg/kg to 128 mg/kg of body weight. MIC and pharmacodynamics (PD) studies of MK7655 were performed

In-vitro activity and beta-lactamase stability of methicillin, isoxazolyl penicillins and cephalothin against coagulase-negative staphylococci

DEFF Research Database (Denmark)

Jarløv, J O; Rosdahl, V T; Mortensen, I

1988-01-01

-level of the isoxazolyl penicillins showed a high degree of uniformity. However more strains were resistant to cloxacillin and oxacillin than to dicloxacillin and flucloxacillin. Only a weak correlation was found between beta-lactamase production, and resistance to the six antibiotics. Methicillin was the most stable...

Development of SYN-004, an oral beta-lactamase treatment to protect the gut microbiome from antibiotic-mediated damage and prevent Clostridium difficile infection.

Science.gov (United States)

Kaleko, Michael; Bristol, J Andrew; Hubert, Steven; Parsley, Todd; Widmer, Giovanni; Tzipori, Saul; Subramanian, Poorani; Hasan, Nur; Koski, Perrti; Kokai-Kun, John; Sliman, Joseph; Jones, Annie; Connelly, Sheila

2016-10-01

The gut microbiome, composed of the microflora that inhabit the gastrointestinal tract and their genomes, make up a complex ecosystem that can be disrupted by antibiotic use. The ensuing dysbiosis is conducive to the emergence of opportunistic pathogens such as Clostridium difficile. A novel approach to protect the microbiome from antibiotic-mediated dysbiosis is the use of beta-lactamase enzymes to degrade residual antibiotics in the gastrointestinal tract before the microflora are harmed. Here we present the preclinical development and early clinical studies of the beta-lactamase enzymes, P3A, currently referred to as SYN-004, and its precursor, P1A. Both P1A and SYN-004 were designed as orally-delivered, non-systemically available therapeutics for use with intravenous beta-lactam antibiotics. SYN-004 was engineered from P1A, a beta-lactamase isolated from Bacillus licheniformis, to broaden its antibiotic degradation profile. SYN-004 efficiently hydrolyses penicillins and cephalosporins, the most widely used IV beta-lactam antibiotics. In animal studies, SYN-004 degraded ceftriaxone in the GI tract of dogs and protected the microbiome of pigs from ceftriaxone-induced changes. Phase I clinical studies demonstrated SYN-004 safety and tolerability. Phase 2 studies are in progress to assess the utility of SYN-004 for the prevention of antibiotic-associated diarrhea and Clostridium difficile disease. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Neutron Diffraction Studies of a Class A beta-Lactamase Toho-1 E166A/R274N/R276N Triple Mutant

International Nuclear Information System (INIS)

Blakeley, Matthew P.; Chen, Yu; Afonine, Pavel

2010-01-01

beta-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the beta-lactam antibiotics is the production of beta-lactamase enzymes that inactivate beta-lactams by rapidly hydrolyzing the amide group of the beta-lactam ring. Specifically, the class A extended-spectrum beta-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a beta-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.

Multidrug resistance found in extended-spectrum beta-lactamase-producing Enterobacteriaceae from rural water reservoirs in Guantao, China

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Hongna eZhang

2015-03-01

Full Text Available Extended-spectrum beta-lactamase (ESBL-producing Enterobacteriaceae have been isolated from humans and animals across the world. However, data on prevalence of ESBL-producing Enterobacteriaceae from rural water reservoirs is limited. This study aimed to isolate and characterize ESBL-producing Enterobacteriaceae in rural water reservoirs in Guantao, China. ESBL-producing Enterobacteriaceae were found in 5 (16.7％ of 30 sampled rural water reservoirs. 66 individual isolates expressing an ESBL phenotype were obtained in the present study. Species identification showed that 42 representatives of Escherichia coli, 17 Klebsiella pneumoniae, 4 Raoultella planticola, and 3 Enterobacter cloacae. 20 isolates contained a single bla gene, including CTX-M (17 strains, TEM (2 strains, and SHV (1 strain. 46 isolates contained more than one type of beta-lactamase genes. ESBL-producing Enterobacteriaceae isolated in this study were all multidrug resistant. These findings indicated that the seroius contamination of ESBL-producing Enterobacteriaceae in rural water resevoirs existed in Guantao, China.

Chromobacterium spp. harbour Ambler class A beta-lactamases showing high identity with KPC

DEFF Research Database (Denmark)

Gudeta, Dereje Dadi; Bortolaia, Valeria; Jayol, Aurelie

2016-01-01

Objectives: The origin of KPC is unknown. The aim of this study was to detect progenitors of KPC in silico and to functionally verify their beta-lactam hydrolysis activity. Methods: The sequence of KPC-2 was used to mine the NCBI protein sequence database. The best non-KPC hits were analysed by a......-lactamases with up to 76% aa identity to KPC from distinct Chromobacterium species is highly indicative of the role played by this genus in the evolution of KPC....

Covalent docking of selected boron-based serine beta-lactamase inhibitors

Science.gov (United States)

Sgrignani, Jacopo; Novati, Beatrice; Colombo, Giorgio; Grazioso, Giovanni

2015-05-01

AmpC β-lactamase is a hydrolytic enzyme conferring resistance to β-lactam antibiotics in multiple Gram-negative bacteria. Therefore, identification of non-β-lactam compounds able to inhibit the enzyme is crucial for the development of novel antibacterial therapies. In general, AmpC inhibitors have to engage the highly solvent-exposed catalytic site of the enzyme. Therefore, understanding the implications of ligand-protein induced-fit and water-mediated interactions behind the inhibitor-enzyme recognition process is fundamental for undertaking structure-based drug design process. Here, we focus on boronic acids, a promising class of beta-lactamase covalent inhibitors. First, we optimized a docking protocol able to reproduce the experimentally determined binding mode of AmpC inhibitors bearing a boronic group. This goal was pursued (1) performing rigid and flexible docking calculations aiming to establish the role of the side chain conformations; and (2) investigating the role of specific water molecules in shaping the enzyme active site and mediating ligand protein interactions. Our calculations showed that some water molecules, conserved in the majority of the considered X-ray structures, are needed to correctly predict the binding pose of known covalent AmpC inhibitors. On this basis, we formalized our findings in a docking and scoring protocol that could be useful for the structure-based design of new boronic acid AmpC inhibitors.

Varying occurrence of extended-spectrum beta-lactamase bacteria among three produce types

KAUST Repository

Toh, Benjamin E. W.; Bokhari, Osama Mohammed; Kutbi, Abdullah; Haroon, Mohamed; Mantilla-Calderon, David; Zowawi, Hosam; Hong, Pei-Ying

2017-01-01

Three types of vegetables were sampled and evaluated over 1.5 years to determine differences in their associated bacterial isolates. Particular emphasis was placed on identifying pathogenic strains that were positive for extended spectrum beta-lactamase (ESBL). Quantitative estimates of the microbial risk associated with the ESBL-positive pathogens showed that different produce types may incur varying levels of ingestion risk. Most of the currently reported ESBL-positive bacterial isolates have been identified in nosocomial environments. However, the carriage of such drug-resistant bacteria in vegetables suggests a possible connection between our daily diet and human health.

Biofilm and metallo beta-lactamase production among the strains of Pseudomonas aeruginosa and Acinetobacter spp. at a Tertiary Care Hospital in Kathmandu, Nepal

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Bandana Baniya

2017-11-01

Full Text Available Abstract Introduction Pseudomonas aeruginosa and Acinetobacter spp. are found to be associated with biofilm and metallo-β-lactamase production and are the common causes of serious infections mainly in hospitalized patients. So, the main aims of this study were to determine the rates of biofilm production and metallo beta-lactamase production (MBL among the strains of Pseudomonas aeruginosa and Acinetobacter spp. isolated from hospitalized patients. Methods A total of 85 P. aeruginosa isolates and 50 Acinetobacter spp. isolates isolated from different clinical specimens from patients admitted to Shree Birendra Hospital, Kathmandu, Nepal from July 2013 to May 2014 were included in this study. The bacterial isolates were identified with the help of biochemical tests. Modified Kirby-Bauer disc diffusion technique was used for antimicrobial susceptibility testing. Combined disc diffusion technique was used for the detection of MBL production, while Congo red agar method and tube adherence method were used for detection of biofilm production. Results Around 16.4% of P. aeruginosa isolates and 22% of the strains of Acinetobacter spp. were metallo β-lactamase producers. Out of 85 P. aeruginosa isolates, 23 (27.05% were biofilm producers according to tube adherence test while, only 13 (15.29% were biofilm producers as per Congo red agar method. Similarly, out of 50 Acinetobacter spp. 7 (14% isolates were biofilm producers on the basis of tube adherence test, while only 5 (10% were positive for biofilm production by Congo red agar method. Highest rates of susceptibility of P. aeruginosa as well as Acinetobacter spp. were seen toward colistin. Conclusion In our study, biofilm production and metallo beta-lactamase production were observed among Pseudomonas aeruginosa and Acinetobacter spp. However, no statistically significant association could be established between biofilm production and metallo beta-lactamase production.

Meta-analysis of proportion estimates of Extended-Spectrum-Beta-Lactamase-producing Enterobacteriaceae in East Africa hospitals

DEFF Research Database (Denmark)

Sonda, Tolbert; Kumburu, Happiness; van Zwetselaar, Marco

2016-01-01

Background: A high proportion of Extended-Spectrum-Beta-Lactamase (ESBL) producing Enterobacteriaceae is causing common infections in all regions of the world. The burden of antibiotic resistance due to ESBL in East Africa is large but information is scarce and thus it is unclear how big the prob......Background: A high proportion of Extended-Spectrum-Beta-Lactamase (ESBL) producing Enterobacteriaceae is causing common infections in all regions of the world. The burden of antibiotic resistance due to ESBL in East Africa is large but information is scarce and thus it is unclear how big...... the problem really is. To gain insight into the magnitude and molecular epidemiology of ESBL-producing Enterobacteriaceae in East Africa a literature search was performed in PubMed on 31 July 2015 to retrieve articles with relevant information on ESBL. Methods and results: Meta-analysis was performed...... to determine overall proportion estimate of ESBL-producing Enterobacteriaceae. A total of 4076 bacterial isolates were included in the analysis. The overall pooled proportion of ESBL-producing Enterobacteriaceae among included surveys done in East African hospitals was found to be 0. 42 (95 % CI: 0...

lactamase in clinical isolates of Escherichia coli

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... The beta lactamase enzyme producing E. coli, resistant to β-lactam antibiotics, created many problems ... Key words: Escherichia coli, β-lactamase enzymes, TEM-type extended spectrum ... difficulties in treatment using antibiotics that are currently ... and chloramphenicol (30 µg) (Mast Diagnostics Ltd., UK).

Estudo da produção de beta -lactamase e sensibilidade às drogas em linhagens de estafilococos coagulase-negativos isolados de recém-nascidos Study of production of beta-lactamase and drugs susceptibility in strains of coagulase-negative staphylococci isolated of neonates

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Maria de Lourdes Ribeiro de Souza da Cunha

2002-01-01

Full Text Available Os estafilococos coagulase-negativos (ECN, embora reconhecidos como saprófitas por muito tempo, têm emergido como agentes etiológicos de uma série de infecções, sendo atualmente os principais responsáveis por sepse em UTI neonatal. Tendo em vista estas características, este estudo objetivou a identificação de estafilococos coagulase-negativos isolados de processos infecciosos em recém-nascidos, bem como a determinação da produção de beta-lactamase e sensibilidade às drogas pelas linhagens isoladas. O Staphylococcus epidermidis foi a espécie mais freqüentemente isolada (77,8%. O estudo da produção de beta-lactamase revelou esta característica na maioria das linhagens de ECN isoladas (71,8%. As linhagens de ECN mostraram, ainda, resistência múltipla aos antibióticos utilizados, com 63,2% dos isolados apresentando resistência a cinco ou mais drogas. A elevada transmissibilidade de plasmídios entre estas linhagens e o uso abusivo de drogas antimicrobianas têm-se constituído em importantes fatores na seleção de amostras multirresistentes e na transferência de genes de resistência.Although coagulase-negative staphylococci (CNS have been recognized as saprophytes for a long time, they had emerged as etiologic agents of infections. They have currently been the most frequently isolated pathogen in sepsis in neonatal intensive care unit (NICU. This study aimed the identification of CNS strains isolated from newborns' infections and to determination of beta-lactamase and drugs susceptibility. Staphylococcus epidermidis was the most frequently isolated species (77,8%. The study of the beta-lactamase production revealed this characteristic in the most of the strains of CNS isolated (71,8%. The strains isolated in this study presented multiple resistance to the antibiotics tested, with 63,2% of isolates presenting resistance to five or more drugs. The high transmissibility of plasmids among those strains and the abusive use of

Characterization of Multidrug Resistant Extended-Spectrum Beta-Lactamase-Producing Escherichia coli among Uropathogens of Pediatrics in North of Iran

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Mohammad Sadegh Rezai

2015-01-01

Full Text Available Escherichia coli remains as one of the most important bacteria causing infections in pediatrics and producing extended-spectrum beta-lactamases (ESBLs making them resistant to beta-lactam antibiotics. In this study we aimed to genotype ESBL-producing E. coli isolates from pediatric patients for ESBL genes and determine their association with antimicrobial resistance. One hundred of the E. coli isolates were initially considered ESBL producing based on their MIC results. These isolates were then tested by polymerase chain reaction (PCR for the presence or absence of CTX, TEM, SHV, GES, and VEB beta-lactamase genes. About 30.5% of isolated E. coli was ESBL-producing strain. The TEM gene was the most prevalent (49% followed by SHV (44%, CTX (28%, VEB (8%, and GES (0% genes. The ESBL-producing E. coli isolates were susceptible to carbapenems (66% and amikacin (58% and showed high resistance to cefixime (99%, colistin (82%, and ciprofloxacin (76%. In conclusion, carbapenems were the most effective antibiotics against ESBl-producing E. coli in urinary tract infection in North of Iran. The most prevalent gene is the TEM-type, but the other resistant genes and their antimicrobial resistance are on the rise.

[Investigation of OXA type beta-lactamases and PFGE patterns in Acinetobacter baumannii strains resistant to carbapenems].

Science.gov (United States)

Keyik, Serafettin; Arslan, Uğur; Türk Dağı, Hatice; Seyhan, Tuba; Fındık, Duygu

2014-10-01

Acinetobacter baumannii is an important opportunistic and multidrug-resistant pathogen leading to nosocomial infections. Over the last 10 years, a significant and threatening increase in resistance to carbapenems, mainly due to the dissemination of class D beta-lactamases, has been reported in A.baumannii worldwide. The most common types of beta-lactamases causing carbapenem resistance in A.baumannii are the OXA-23, OXA-24, OXA-40, OXA-58 and OXA-143 type serine beta-lactamases. The aim of this study was to investigate the presence of OXA type beta-lactamases in carbapenem-resistant A.baumannii strains and the clonal relationship between the strains. A total of 105 non-duplicate carbapenem-resistant A.baumannii strains isolated from various clinical samples (68 blood, 18 bronchoalveolar lavage, 13 drainage, 3 urine, 2 cerebrospinal fluid and 1 catheter samples) in the Microbiology Laboratories of Selcuk University, Meram (2009-2012) and Selcuklu (2007-2008) Medical School Hospitals, were included in the study. The isolates were identified by conventional methods and Phoenix 100 BD (BD Diagnostic, USA) and Vitek II (bioMerieux, France) automated systems. Carbapenem susceptibility test was performed by Kirby-Bauer disk diffusion method according to the CLSI standards. bla(OXA 23-like), bla(OXA 24-like), bla(OXA 58-like) and bla(OXA 51-like) genes were amplified by multiplex PCR assay and clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE) using ApaI enzyme. The bla(OXA 51-like) gene was determined in all carbapenem-resistant A.baumannii isolates, while the bla(OXA 23-like) and bla(OXA 58-like) genes were detected in 46.6% and 53.3% of isolates, respectively. However bla(OXA 24-like) gene was not demonstrated in any isolates. bla(OXA 23-like) gene was determined in both Meram and Selcuklu Medical School hospitals, but bla(OXA 58-like) gene was detected only in Meram Medical School hospital. PFGE analysis of the isolates revealed 32 different

Expression, purification, crystallization and preliminary X-ray analysis of Aeromonas hydrophilia metallo-[beta]-lactamase

Energy Technology Data Exchange (ETDEWEB)

Sharma, N.; Toney, J.H.; Fitzgerald, P.M.D. (Merck)

2010-07-20

The CphA metallo-{beta}-lactamase from Aeromonas hydrophilia has been expressed, purified and crystallized by the hanging-drop vapor-diffusion method using ammonium sulfate as the precipitant. The crystals exhibit orthorhombic symmetry (P2{sub 1}2{sub 1}2), with unit-cell parameters a = 40.75, b = 42.05, c = 128.88 {angstrom}. There is one monomer in the asymmetric unit and the solvent content is estimated to be 44% by volume. A data set extending to 1.8 {angstrom} has been measured.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

Science.gov (United States)

Bansal, Ankita; Kar, Debasish; Murugan, Rajagopal A; Mallick, Sathi; Dutta, Mouparna; Pandey, Satya Deo; Chowdhury, Chiranjit; Ghosh, Anindya S

2015-05-01

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities. © 2015 The Authors.

beta-lactamase producing bacteria in the subgingival microflora of adult patients with periodontitis. A comparison between Spain and The Netherlands

NARCIS (Netherlands)

Herrera, D; van Winkelhoff, AJ; Dellemijn-Kippuw, N; Winkel, EG; Sanz, M

Background/aims: Countries with a high per capita antibiotic use frequently demonstrate a high level of drug resistance. The aim of this study was to compare the prevalence and levels of beta-lactamase producing bacteria in the subgingival microflora in adult patients with periodontitis in Spain and

Varying occurrence of extended-spectrum beta-lactamase bacteria among three produce types

KAUST Repository

Toh, Benjamin E. W.

2017-07-07

A monitoring effort that spanned across 1.5 years was conducted to examine three types of produce-associated microbiota. The average amount of antibiotic-resistant bacteria recovered from lettuce, tomato, and cucumber was 1.02 × 1010, 2.05 × 107, and 4.78 × 109 cells per 50 g of each produce, respectively. A total of 480 bacterial isolates were obtained and identified from their 16S rRNA genes, revealing isolates that were ubiquitously recovered from all three types of produce. However, sporadic presence of Klebsiella pneumoniae and Acinetobacter baumannii was detected on lettuce and cucumbers but not tomatoes. End-point PCR revealed that the K. pneumoniae and A. baumannii isolates were positive for genes encoding extended spectrum beta-lactamase. Whole genome sequencing of two of the K. pneumoniae isolates further suggested the presence of the blaCTX-M-15 gene in a conjugative plasmid, as well as other antibiotic resistance genes and virulence-associated traits in either conjugative plasmids or the chromosomal genome. Quantitative microbial risk assessment indicated varying levels of ingestion risk associated with different types of produce. In particular, the risk arising from ESBL-positive K. pneumoniae in lettuce, but not in cucumbers or tomatoes, was higher than the acceptable annual risk of 10−4. Practical applications Three types of vegetables were sampled and evaluated over 1.5 years to determine differences in their associated bacterial isolates. Particular emphasis was placed on identifying pathogenic strains that were positive for extended spectrum beta-lactamase (ESBL). Quantitative estimates of the microbial risk associated with the ESBL-positive pathogens showed that different produce types may incur varying levels of ingestion risk. Most of the currently reported ESBL-positive bacterial isolates have been identified in nosocomial environments. However, the carriage of such drug-resistant bacteria in vegetables suggests a possible connection

Loop-Mediated Isothermal Amplification untuk Mendeteksi Gen blaTEM sebagai Penyandi Extended-Spectrum Beta-Lactamase pada Isolat Enterobacteriaceae

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Bayu A. P. Wilopo

2015-12-01

Full Text Available Extended-spectrum beta-lactamase (ESBL is a beta-lactamase enzyme that is capable of hydrolyzing penicillin, cephalosporin, and monobactam, and can be inhibited by clavulanic acid. This enzyme is encoded by multiple genes, one of them is blaTEM. Polymerase chain reaction (PCR is one of the DNA amplification methods that are frequently used; however, there are other methods that can be used including, among others, loop-mediated isothermal amplification (LAMP. LAMP requires simple equipment with quicker and easy-to-read results compared to PCR. This study was a diagnostic test to explore the sensitivity and specificity of LAMP method compared to PCR in detecting blaTEM gene. Furthermore, the concordance between LAMP and PCR methods was assessed. A total of 92 Enterobacteriaceae isolates were examined by PCR and LAMP methods and compared. The result showed that the LAMP method had a sensitivity of 91.4% and a specificity of 91.2% with a concordance value (kappa of 85.4%. In conclusion, LAMP method has a good validity and a very good conformity compared to the PCR method. Therefore, LAMP method can be used as an alternative diagnostic test, especially in limited settings.

Looking for the new preparations for antibacterial therapy. II. Clinical trials; new beta-lactam antibiotics and beta-lactamase inhibitors.

Science.gov (United States)

Karpiuk, Izabela; Tyski, Stefan

2013-01-01

To obtain a status of a medicinal product, a compound possessing potential antimicrobial activity and displaying no cytotoxicity, must undergo three phases of clinical trials to prove its therapeutic efficacy, safety and quality. Properties of the compound should be based on the results of studies meeting specific criteria. Studies should be: randomized, double-blind, involving sufficient number of volunteers, concerning the infections localized in strictly defined area and caused by identified microorganisms. After the medicinal product is authorized to be on the market, clinical trials of the fourth phase are carried out to detect adverse effects, overdose symptoms, interactions of the new drug with other medicinal products and to establish characteristic of activity among groups such as children, elderly, women in pregnancy and patients suffering from other diseases, but only if the benefits of receiving treatment outweigh the risks. This article is a second part of the series associated with searching for new antibacterial agents and it relates to performance of clinical trials and the new compounds belonging to the class of beta-lactams. Among the 9 presented compounds, candidates to become medicinal products, two belong to the cephalosporins (CXA-101, S-649266), one to carbapenems (razupenem), three to monobactams (BAL30072, BAL30376, MC-1) and three to beta-lactamase inhibitors (NXL-104, MK-7655, ME1071).

Characterization of the Extended-Spectrum beta-Lactamase Producers among Non-Fermenting Gram-Negative Bacteria Isolated from Burnt Patients

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Mojdeh Hakemi Vala

2013-09-01

Full Text Available Please cite this article as: Hakemi Vala M, Hallajzadeh M, Fallah F, Hashemi A, Goudarzi H. Characterization of the extended-spectrum beta-lactamase producers among non-fermenting gram-negative bacteria isolated from burnt patients. Arch Hyg Sci 2013;2(1:1-6. Background & Aims of the Study: Extended-spectrum beta-Lactamases (ESBLs represent a major group of beta-lactamases which are responsible for resistance to oxyimino-cephalosporins and aztreonam and currently being identified in large numbers throughout the world. The objective of this study was to characterize ESBL producers among non-fermenter gram-negative bacteria isolated from burnt patients. Materials & Methods: During April to July 2012, 75 non-fermenter gram-negative bacilli were isolated from 240 bacterial cultures collected from wounds of burnt patients admitted to the Burn Unit at Shahid Motahari Hospital (Tehran, Iran. Bacterial isolation and identification was done using standard methods. Antimicrobial susceptibility testing was performed by disk diffusion method for all strains against selected antibiotics and minimum inhibitory concentration was determined by microdilution test. The ability to produce ESBL was detected through double disk synergy test among candidate strains. Results: Of 75 non-fermenter isolates, 47 Pseudomonas aeruginosa and 28 Acinetobacter baumannii were identified. The resistance of P. aeruginosa isolates to tested antibiotics in antibiogram test were 100% to cefpodoxime, 82.98% to ceftriaxone, 78.73% to imipenem, 75% to meropenem, 72.72% to gentamicin, 69.23% to ciprofloxacin and aztreonam, 67.57% to cefepime, 65.95% to ceftazidime, and 61.53% to piperacillin. The results for Acinetobacter baumannii were 100% to ceftazidime, cefepime, ciprofloxacin, imipenem, meropenem, cefpodoxime, and cefotaxim, 96.85% to gentamicin, 89.65% to ceftriaxone, 65.51% to aztreonam, and 40% to piperacillin. Double disk synergy test showed that 21 (28% of non

Plasmid-mediated AmpC beta-lactamase-producing Escherichia coli causing urinary tract infection in the Auckland community likely to be resistant to commonly prescribed antimicrobials.

Science.gov (United States)

Drinkovic, Dragana; Morris, Arthur J; Dyet, Kristin; Bakker, Sarah; Heffernan, Helen

2015-03-13

To estimate the prevalence and characterise plasmid-mediated AmpC beta-lactamase (PMACBL)- producing Escherichia coli in the Auckland community. All cefoxitin non-susceptible (NS) E. coli identified at the two Auckland community laboratories between 1 January and 31 August 2011 were referred to ESR for boronic acid double-disc synergy testing, to detect the production of AmpC beta-lactamase, and polymerase chain reaction (PCR) to identify the presence of PMACBL genes. PMACBL-producing isolates were typed using pulsed-field gel electrophoresis (PFGE), and PCR was used to determine their phylogenetic group and to identify multilocus sequence type (ST)131. Antimicrobial susceptibility testing and detection of extended-spectrum beta-lactamases (ESBLs) were performed according to the Clinical and Laboratory Standards Institute recommendations. 101 (51%) and 74 (37%) of 200 non-duplicate cefoxitin-NS E. coli were PMACBL producers or assumed hyper-producers of chromosomal AmpC beta-lactamase, respectively. The prevalence of PMACBL-producing E. coli was 0.4%. PMACBL-producing E. coli were significantly less susceptible to norfloxacin, trimethoprim and nitrofurantoin than E. coli that produced neither a PMACBL nor an ESBL. Very few (4%) PMACBL-producing E. coli co-produced an ESBL. Most (88%) of the PMACBL-producing isolates had a CMY-2-like PMACBL. The PMACBL-producing E. coli isolates were diverse based on their PFGE profiles, 44% belonged to phylogenetic group D, and only four were ST131. 100 of the 101 PMACBL-producing E. coli were cultured from urine, and were causing urinary tract infection (UTI) in the majority of patients. The median patient age was 56 years and most (94%) of the patients were women. A greater proportion of patients with community-acquired UTI caused by PMACBL-producing E. coli received a beta-lactam antimicrobial than patients with community-acquired UTI caused by other non-AmpC, non-ESBL-producing E. coli. Thirty-six (43%) patients with community

Detection of Healthcare-Related Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Transmission Events Using Combined Genetic and Phenotypic Epidemiology.

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Anne F Voor In 't Holt

Full Text Available Since the year 2000 there has been a sharp increase in the prevalence of healthcare-related infections caused by extended-spectrum beta-lactamase (ESBL-producing Escherichia coli. However, the high community prevalence of ESBL-producing E. coli isolates means that many E. coli typing techniques may not be suitable for detecting E. coli transmission events. Therefore, we investigated if High-throughput MultiLocus Sequence Typing (HiMLST and/or Raman spectroscopy were suitable techniques for detecting recent E. coli transmission events.This study was conducted from January until December 2010 at Erasmus University Medical Center, Rotterdam, the Netherlands. Isolates were typed using HiMLST and Raman spectroscopy. A genetic cluster was defined as two or more patients carrying identical isolates. We used predefined definitions for epidemiological relatedness to assess healthcare-related transmission.We included 194 patients; strains of 112 patients were typed using HiMLST and strains of 194 patients were typed using Raman spectroscopy. Raman spectroscopy identified 16 clusters while HiMLST identified 10 clusters. However, no healthcare-related transmission events were detected. When combining data from both typing techniques, we identified eight clusters (n = 34 patients, as well as 78 patients with a non-cluster isolate. However, we could not detect any healthcare-related transmission in these 8 clusters.Although clusters were genetically detected using HiMLST and Raman spectroscopy, no definite epidemiological relationships could be demonstrated which makes the possibility of healthcare-related transmission events highly unlikely. Our results suggest that typing of ESBL-producing E. coli using HiMLST and/or Raman spectroscopy is not helpful in detecting E. coli healthcare-related transmission events.

A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

Institute of Scientific and Technical Information of China (English)

Qingshu Meng; Kaifu Chen; Lina Ma; Songnian Hu; Jun Yu

2011-01-01

Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ～ 160 Mb) has been sequenced and is composed of ～65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.

Method for Phenotypic Detection of Extended-Spectrum Beta-Lactamases in Enterobacter Species in the Routine Clinical Setting ▿

Science.gov (United States)

Stuart, James Cohen; Diederen, Bram; al Naiemi, Nashwan; Fluit, Ad; Arents, Niek; Thijsen, Steven; Vlaminckx, Bart; Mouton, Johan W.; Leverstein-van Hall, Maurine

2011-01-01

In 271 Enterobacter blood culture isolates from 12 hospitals, extended-spectrum beta-lactamase (ESBL) prevalence varied between 0% and 30% per hospital. High prevalence was associated with dissemination, indicating the potential relevance of infection control measures. Screening with cefepime or Vitek 2, followed by a cefepime/cefepime-clavulanate Etest, was an accurate strategy for ESBL detection in Enterobacter isolates (positive predictive value, 100%; negative predictive value, 99%). PMID:21562100

Prevalence of AmpC type extended spectrum beta lactamases genes in clinical Samples of E.coli Isolated from Poultry and Humans

Directory of Open Access Journals (Sweden)

Elham Farrokhnazar

2016-07-01

Full Text Available Emergence of antibiotic resistance among pathogens, particularly in health centers and hospitals, has become a major public health problem. This study identified AmpC-type beta-lactamase against the antibiotic ceftazidime, cefotaxime and cefpodoxime in E.coli isolated from human and poultry and types of producing genes were studied by PCR. In this study, 500 clinical human samples of urine from hospitals of Tehran during 5 months as well as 300 poultry samples were collected and transferred to the microbiology laboratory. Biochemical tests such as TSI, Urea and IMViC were performed on suspected colonies with E.coli. To identify ESBL producing strains, beta-lactamase samples were cultured on Mueller-Hinton agar through antimicrobial susceptibility test by disk agar diffusion based on the standard CLSI for initial screening. PCR reactions were done using specific primers CITM, EBCM, DHAM and MOXM to identify the beta-lactamase AmpC. A number of 200 human and 55 poultry E.coli samples were screened. In human samples, 105 (52.5% were resistant and potential producers of ESBL and AmpC; out of those, 102 (51% produced ESBL and 3 (1.5% potentially produced AmpC. In the study on 55 E.coli isolates from poultry samples based on the results of disk agar diffusion test, 4 (7.2% samples were resistant and potential producers of ESBL. None of the samples were AmpC producers. Through PCR, 2 human samples (1% were CITM positive and one sample (0.5% was DHAM positive. Through the PCR carried out on poultry samples, there were no bands with 4 primers. There was AmpC in human samples; while further studies are required for poultry samples, because poultry significantly contribute in production of food for humans and can be an important source for dissemination of antibiotic resistance. Given the significance of Ampc in providing high levels of beta-lactam antibiotic resistance, particularly third generation cephalosporins which are very common treatments, more

TEM and CTX-M extended-spectrum beta-lactamase in Klebsiella spp and Escherichia coli isolates from inanimate surfaces of hospital environments

OpenAIRE

Rivera-Jacinto, Marco; Facultad de Ciencias de la Salud, Universidad Nacional de Cajamarca. Cajamarca, Perú.; Rodríguez-Ulloa, Claudia; Facultad de Ciencias de la Salud, Universidad Nacional de Cajamarca. Cajamarca, Perú.; Flores Clavo, René; Hospital Regional de Lambayeque. Lambayeque, Perú.; Serquén López, Luis; Hospital Regional de Lambayeque. Lambayeque, Perú.; Arce Gil, Zhandra; Universidad Católica Santo Toribio de Mogrovejo. Lambayeque, Perú.

2015-01-01

The aim of the study was to determine the genotype of 15 ESBL strains of Enterobacteriaceae resistant to beta-lactams, isolated from inanimate surfaces and phenotypically characterized as producing extended-spectrum beta-lactamase. After evaluation and screening of the bacterial strains, a PCR was conducted to amplify fragments of 1078 bp and 544 bp corresponding to type TEM and CTX-M ESBL. Eleven strains presented both fragments at the time and only three had blaCTX-M. In conclusion, the pre...

Evolving beta-lactamase epidemiology in Enterobacteriaceae from Italian nationwide surveillance, October 2013: KPC-carbapenemase spreading among outpatients.

Science.gov (United States)

Giani, Tommaso; Antonelli, Alberto; Caltagirone, Mariasofia; Mauri, Carola; Nicchi, Jessica; Arena, Fabio; Nucleo, Elisabetta; Bracco, Silvia; Pantosti, Annalisa; Luzzaro, Francesco; Pagani, Laura; Rossolini, Gian Maria

2017-08-03

Extended-spectrum beta-lactamases (ESBLs), AmpC-type beta-lactamases (ACBLs) and carbapenemases are among the most important resistance mechanisms in Enterobacteriaceae. This study investigated the presence of these resistance mechanisms in consecutive non-replicate isolates of Escherichia coli (n = 2,352), Klebsiella pneumoniae (n = 697), and Proteus mirabilis (n = 275) from an Italian nationwide cross-sectional survey carried out in October 2013. Overall, 15.3% of isolates were non-susceptible to extended-spectrum cephalosporins but susceptible to carbapenems (ESCR-carbaS), while 4.3% were also non-susceptible to carbapenems (ESCR-carbaR). ESCR-carbaS isolates were contributed by all three species, with higher proportions among isolates from inpatients (20.3%) but remarkable proportions also among those from outpatients (11.1%). Most ESCR-carbaS isolates were ESBL-positive (90.5%), and most of them were contributed by E. coli carrying bla CTX-M group 1 genes. Acquired ACBLs were less common and mostly detected in P. mirabilis. ESCR-carbaR isolates were mostly contributed by K. pneumoniae (25.1% and 7.7% among K. pneumoniae isolates from inpatients and outpatients, respectively), with bla KPC as the most common carbapenemase gene. Results showed an increasing trend for both ESBL and carbapenemase producers in comparison with previous Italian surveys, also among outpatients. This article is copyright of The Authors, 2017.

Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307.

Science.gov (United States)

Long, S Wesley; Olsen, Randall J; Eagar, Todd N; Beres, Stephen B; Zhao, Picheng; Davis, James J; Brettin, Thomas; Xia, Fangfang; Musser, James M

2017-05-16

treat because many strains are resistant to multiple antibiotics. Clonal group 258 (CG258) organisms have caused outbreaks in health care settings worldwide. Using a comprehensive population-based sample of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae strains, we show that a relatively uncommon clonal type, CG307, caused the plurality of ESBL-producing K. pneumoniae infections in our patients. We discovered that CG307 strains have been abundant in Houston for many years. As assessed by experimental mouse infection, CG307 strains were as virulent as pandemic CG258 strains. Our results may portend the emergence of an especially successful clonal group of antibiotic-resistant K. pneumoniae . Copyright © 2017 Long et al.

Detection of metallo-beta-lactamase producing Pseudomonas ...

African Journals Online (AJOL)

sunny t

2016-02-24

Feb 24, 2016 ... Since the increasing prevalence of carbapenem-resistant Pseudomonas aeruginosa spp., accurate detection of metallo-β-lactamase (MBL) such as blaVIM type enzyme producing isolates became very important. However, phenotypic MBL detection methods previously reported are not highly sensitive or.

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

Synthesis and processing of escherichia-coli tem-beta-lactamase and bacillus-licheniformis alpha-amylase in escherichia-coli : The role of signal peptidase-i

NARCIS (Netherlands)

van Dijl, J M; Smith, H; Bron, Sierd; Venema, Gerard

A mutant of Escherichia coli, in which signal peptidase I synthesis can be regulated, was constructed. The mutant was used to study the effects of signal peptidase I limitation on the synthesis and efficiency of processing of two proteins: the periplasmic E. coli TEM-beta-lactamase and Bacillus

SYNTHESIS AND PROCESSING OF ESCHERICHIA-COLI TEM-BETA-LACTAMASE AND BACILLUS-LICHENIFORMIS ALPHA-AMYLASE IN ESCHERICHIA-COLI : THE ROLE OF SIGNAL PEPTIDASE-I

NARCIS (Netherlands)

van Dijl, J M; SMITH, H; BRON, S; VENEMA, G

A mutant of Escherichia coli, in which signal peptidase I synthesis can be regulated, was constructed. The mutant was used to study the effects of signal peptidase I limitation on the synthesis and efficiency of processing of two proteins: the periplasmic E. coli TEM-beta-lactamase and Bacillus

Bovine intestinal bacteria inactivate and degrade ceftiofur and ceftriaxone with multiple beta-lactamases.

Science.gov (United States)

Wagner, R Doug; Johnson, Shemedia J; Cerniglia, Carl E; Erickson, Bruce D

2011-11-01

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinder-plate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatography-mass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific β-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum β-lactamases and metallo-β-lactamases. Seven isolates of Bacteroides spp. produced multiple β-lactamases, including possibly CepA, and metallo-β-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading β-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that β-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple β-lactamases from bacteria in the large intestines of cattle.

Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase producing urinary isolates of Escherichia coli in outpatients

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Marković Tatjana

2013-01-01

Full Text Available Introduction. In Gram-negative bacteria, the production of beta-lactamases is the most important mechanism of resistance to beta-lactam antibiotics. In the Banja Luka region, there were no extensive researches on the prevalence and antimicrobial resistance of the extended-spectrum beta-lactamase (ESBL producing Escherichia coli (E. coli isolates. Objective. The aim of the present study was to determine the presence of ESBL producing E. coli isolates as the cause of the urinary tract infections in outpatients, the distribution of these ESBL isolates according to age and gender of patients and their susceptibility to antimicrobials. Methods. Urine specimens obtained from outpatients were cultured on chromogenic CPS-ID3 media. All plates showing significant (>105 cfu/ml growth of E. coli in pure culture were further processed. Antimicrobial susceptibility testing was performed on VITEK TWO Compact using AST-GN27 cards for testing Gram negative bacteria and detection of ESBL producers. Results. Out of 2,195 isolates, 177 (8.1% were ESBL producers. Ninety-two isolates were obtained from female patients (5% of E. coli isolated from women and 85 isolates from male patients (23% of E. coli isolated from men. High percentage of ESBL isolates was detected in the infant age group under one year (36.7% and in the age group over 60 years (28.8%. All ESBL isolates were susceptible to imipenem and resistant to ampicillin, piperacillin, cefazolin, cefotaxime, ceftazidime and cefepime. There was a significant resistance to amikacin (79.1%, gentamicin (76.8%, amoxicillin/clavulanate (54.8% and trimethoprim/sulphamethoxazole (45.8%. Resistance to nutrofurantoin was 13.6%. Conclusion. This study has demonstrated the presence of ESBL producing E. coli urinary isolates in outpatients, and their extensive susceptibility to imipenem and nitrofurantoin.

Antibiotic resistance profile and phenotypic detection of beta ...

African Journals Online (AJOL)

Abstract. Background: Cockroaches are carriers of numerous microorganisms. However, there is paucity of information on their role as potential reservoir for beta-lactamase producers. Objectives: This research determined the antibiotics susceptibility profile of Beta-lactamase producing Gram-negative bacteria isolated from ...

Virulence of a Klebsiella pneumoniae strain carrying the New Delhi metallo-beta-lactamase-1 (NDM-1)

DEFF Research Database (Denmark)

Fuursted, Kurt; Schøler, Lone; Hansen, Frank

2011-01-01

, and in vitro virulence by assessing various virulence factors. The NDM-1 carrying K. pneumoniae isolate was the most virulent in the murine sepsis model but there was no clear cut correlation to in vitro virulence factors or killing in C. elegans. It is concluded that K. pneumoniae carrying NDM-1 have......The aim of the study was to compare and evaluate virulence in five strains of Klebsiella pneumoniae, including an isolate carrying New Delhi metallo-beta-lactamase-1 (NDM-1). In vivo virulence was assessed using a murine sepsis model and using the nematode Caenorhabditis elegans killing model...

OXA β-Lactamases

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Evans, Benjamin A.

2014-01-01

SUMMARY The OXA β-lactamases were among the earliest β-lactamases detected; however, these molecular class D β-lactamases were originally relatively rare and always plasmid mediated. They had a substrate profile limited to the penicillins, but some became able to confer resistance to cephalosporins. From the 1980s onwards, isolates of Acinetobacter baumannii that were resistant to the carbapenems emerged, manifested by plasmid-encoded β-lactamases (OXA-23, OXA-40, and OXA-58) categorized as OXA enzymes because of their sequence similarity to earlier OXA β-lactamases. It was soon found that every A. baumannii strain possessed a chromosomally encoded OXA β-lactamase (OXA-51-like), some of which could confer resistance to carbapenems when the genetic environment around the gene promoted its expression. Similarly, Acinetobacter species closely related to A. baumannii also possessed their own chromosomally encoded OXA β-lactamases; some could be transferred to A. baumannii, and they formed the basis of transferable carbapenem resistance in this species. In some cases, the carbapenem-resistant OXA β-lactamases (OXA-48) have migrated into the Enterobacteriaceae and are becoming a significant cause of carbapenem resistance. The emergence of OXA enzymes that can confer resistance to carbapenems, particularly in A. baumannii, has transformed these β-lactamases from a minor hindrance into a major problem set to demote the clinical efficacy of the carbapenems. PMID:24696435

Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution

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Lewandowski, Eric M. [Department of Molecular Medicine, University of South Florida College of Medicine, Tampa FL USA; Lethbridge, Kathryn G. [Department of Molecular Medicine, University of South Florida College of Medicine, Tampa FL USA; Sanishvili, Ruslan [GMCA@APS, X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, IL USA; Skiba, Joanna [Department of Organic Chemistry, Faculty of Chemistry, University of Lodz, Poland; Kowalski, Konrad [Department of Organic Chemistry, Faculty of Chemistry, University of Lodz, Poland; Chen, Yu [Department of Molecular Medicine, University of South Florida College of Medicine, Tampa FL USA

2017-11-20

The beta-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A beta-lactamases, the beta-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A beta-lactamase with a ruthenocene-conjugated penicillin-a 0.85 angstrom resolution structure of E166A mutant complexed with the penilloate product, a 1.30 angstrom resolution complex structure of the same mutant with the penicilloate product, and a 1.18 angstrom resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A beta-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A beta-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine.

Crystal Structures of KPC-2[beta]-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

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Ke, Wei; Bethel, Christopher R.; Papp-Wallace, Krisztina M.; Pagadala, Sundar Ram Reddy; Nottingham, Micheal; Fernandez, Daniel; Buynak, John D.; Bonomo, Robert A.; van den Akker, Focco (Case Western); (Stokes); (SMU)

2012-08-01

Class A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by {beta}-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two {beta}-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-{angstrom} resolution. 3-NPBA demonstrated a Km value of 1.0 {+-} 0.1 {micro}M (mean {+-} standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-{angstrom} resolution. PSR-3-226 displayed a K{sub m} value of 3.8 {+-} 0.4 {micro}M for KPC-2, and the inactivation rate constant (kinact) was 0.034 {+-} 0.003 s{sup -1}. When covalently bound to S70, PSR-3-226 forms a trans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first {beta}-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.

Classification of Beta-lactamases and penicillin binding proteins using ligand-centric network models.

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Hakime Öztürk

Full Text Available β-lactamase mediated antibiotic resistance is an important health issue and the discovery of new β-lactam type antibiotics or β-lactamase inhibitors is an area of intense research. Today, there are about a thousand β-lactamases due to the evolutionary pressure exerted by these ligands. While β-lactamases hydrolyse the β-lactam ring of antibiotics, rendering them ineffective, Penicillin-Binding Proteins (PBPs, which share high structural similarity with β-lactamases, also confer antibiotic resistance to their host organism by acquiring mutations that allow them to continue their participation in cell wall biosynthesis. In this paper, we propose a novel approach to include ligand sharing information for classifying and clustering β-lactamases and PBPs in an effort to elucidate the ligand induced evolution of these β-lactam binding proteins. We first present a detailed summary of the β-lactamase and PBP families in the Protein Data Bank, as well as the compounds they bind to. Then, we build two different types of networks in which the proteins are represented as nodes, and two proteins are connected by an edge with a weight that depends on the number of shared identical or similar ligands. These models are analyzed under three different edge weight settings, namely unweighted, weighted, and normalized weighted. A detailed comparison of these six networks showed that the use of ligand sharing information to cluster proteins resulted in modules comprising proteins with not only sequence similarity but also functional similarity. Consideration of ligand similarity highlighted some interactions that were not detected in the identical ligand network. Analysing the β-lactamases and PBPs using ligand-centric network models enabled the identification of novel relationships, suggesting that these models can be used to examine other protein families to obtain information on their ligand induced evolutionary paths.

Risk Factor Analysis of Ciprofloxacin-Resistant and Extended Spectrum Beta-Lactamases Pathogen-Induced Acute Bacterial Prostatitis in Korea.

Science.gov (United States)

Lee, Young; Lee, Dong Gi; Lee, Sang Hyub; Yoo, Koo Han

2016-11-01

The objectives of this study were to investigate risk factors and the incidence of ciprofloxacin resistance and extended-spectrum beta-lactamases (ESBL) in patients with acute bacterial prostatitis (ABP). We reviewed the medical records of 307 patients who were diagnosed with ABP between January 2006 and December 2015. The etiologic pathogens and risk factors for ciprofloxacin-resistant E. coli and ESBL-producing microbes, susceptibility to ciprofloxacin, and the incidence of ESBL in patients with ABP were described. History of prior urologic manipulation was an independent risk factor for ciprofloxacin-resistant (P = 0.005) and ESBL-producing microbes (P = 0.005). Advanced age (over 60 years) was an independent risk factor for ciprofloxacin-resistant microbes (P = 0.022). The ciprofloxacin susceptibility for Escherichia coli in groups without prior manipulation was documented 85.7%. For groups with prior manipulation, the susceptibility was 10.0%. Incidence of ESBL-producing microbes by pathogen was 3.8% for E. coli and 1.0% for Klebsiella pneumonia in the absence of manipulation group, and 20% and 33.3% in the presence of manipulation group, respectively. Initial treatment of ABP must consider patient's age and the possibility of prior manipulation to optimize patient treatment. With the high rate of resistance to fluoroquinolone, cephalosporins with amikacin, or carbapenems, or extended-spectrum penicillin with beta lactamase inhibitor should be considered as the preferred empirical ABP treatment in the patients with history of prior urologic manipulation.

Incidence of temonera, sulphuhydryl variables and cefotaximase genes associated with β-lactamase producing escherichia coli in clinical isolates

Science.gov (United States)

Isaiah, Ibeh Nnana; Nche, Bikwe Thomas; Nwagu, Ibeh Georgina; Nwagu, Ibeh Isaiah

2011-01-01

Background: the occurrence of the different types of Extended spectrum beta Lactamase producing Escherichia coli with the, Sulphurhydryl variable, Temonera and the Cefotaximase have been on the rise Aim: The study was to determine the prevalence of extended spectrum beta lactamase gene resistance across the clinical isolates of hospitalized patients. Materials and Method: Three hundred and fifty isolates of Escherichia coli were received from different clinical specimens. The susceptibility profile of the isolates against 10 different antibiotics was examined, the MICs (Minimum Inhibitory Concentration) for ceftazidime were also determined using micro-broth dilution assay. Isolates showing MIC ≥ 6 μg/ml for ceftazidime were screened for ESBL (PCT)phenotypic confirmatory test and subjected to PCR (polymerase chain reaction) to further. Results: By disk diffusion test, there was resistance to ceftazidime and cefotaxime were 180(51.4%) and 120 (34.2%) respectively. However, all strains were susceptible to imipenem. 250 isolates showed MICs≥ 6 μg/ml for ceftazidime of which 180 (72%) were positive for extended spectrum beta lactamase. The prevalence of Sulphurhydryl variable, Temonera and the Cefotaximase among these isolates were 17.1%, 6.6% and 17%, respectively. Conclusion: For the identification of extended spectrum beta lactamase producing isolates it is recommended that clinical laboratories adopt simple test based on Cinical laboratory standard institute recommendation for confirming extended spectrum beta lactamase production in enterobacteriacea species. PMID:22363078

Expression, purification, crystallization, and preliminary X-ray crystallographic analysis of OXA-17, an extended-spectrum {beta}-lactamase conferring severe antibiotic resistance

Energy Technology Data Exchange (ETDEWEB)

Lee, J. H., E-mail: msgjhlee@mju.ac.kr; Sohn, S. G., E-mail: sgsohn@mju.ac.kr; Jung, H. I., E-mail: jhinumber1@hanmail.net; An, Y. J., E-mail: anyj0120@hanmail.net; Lee, S. H., E-mail: sangheelee@mju.ac.kr [Myongji University, Drug Resistance Proteomics Laboratory, Department of Biological Sciences (Korea, Republic of)

2013-07-15

OXA-17, an extended-spectrum {beta}-lactamase (ESBL) conferring severe antibiotic resistance, hydrolytically inactivates {beta}-lactam antibiotics, inducing a lack of eradication of pathogenic bacteria by oxyimino {beta}-lactams and not helping hospital infection control. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing ESBLs. OXA-17 was purified and crystallized at 298 K. X-ray diffraction data from OXA-17 crystal have been collected to 1.85 A resolution using synchrotron radiation. The crystal of OXA-17 belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 48.37, b = 101.12, and c = 126.07 A. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 54.6%.

Beta Lactamase Producing Clostridium perfringens Bacteremia in an Elderly Man with Acute Pancreatitis

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Rashmi Mishra

2016-01-01

Full Text Available Clostridium perfringens bacteremia is associated with adverse outcomes. Known risk factors include chronic kidney disease, malignancy, diabetes mellitus, and gastrointestinal disease. We present a 74-year-old man admitted with confusion, vomiting, and abdominal pain. Exam revealed tachycardia, hypotension, lethargy, distended abdomen, and cold extremities. He required intubation and aggressive resuscitation for septic shock. Laboratory data showed leukocytosis, metabolic acidosis, acute kidney injury, and elevated lipase. CT scan of abdomen revealed acute pancreatitis and small bowel ileus. He was started on vancomycin and piperacillin-tazobactam. Initial blood cultures were positive for C. perfringens on day five. Metronidazole and clindamycin were added to the regimen. Repeat CT (day 7 revealed pancreatic necrosis. The patient developed profound circulatory shock requiring multiple vasopressors, renal failure requiring dialysis, and bacteremia with vancomycin-resistant enterococci. Hemodynamic instability precluded surgical intervention and he succumbed to multiorgan failure. Interestingly, our isolate was beta lactamase producing. We review the epidemiology, risk factors, presentation, and management of C. perfringens bacteremia. This case indicates a need for high clinical suspicion for clostridial sepsis and that extended spectrum beta lactam antibiotic coverage may be inadequate and should be supplemented with use of clindamycin or metronidazole if culture is positive, until sensitivities are known.

The potential role of microbiota for controlling the spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE in neonatal population [version 1; referees: 2 approved

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Thibaud Delerue

2017-07-01

Full Text Available The spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE in the hospital and also the community is worrisome. Neonates particularly are exposed to the risk of ESBL-PE acquisition and, owing to the immaturity of their immune system, to a higher secondary risk of ESBL-PE-related infection. Reducing the risk of acquisition in the hospital is usually based on a bundle of measures, including screening policies at admission, improving hand hygiene compliance, and decreasing antibiotic consumption. However, recent scientific data suggest new prevention opportunities based on microbiota modifications.

Ertapenem susceptibility of extended spectrum beta-lactamase-producing organisms

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Selby Edward B

2007-06-01

Full Text Available Abstract Background Infections caused by multiply drug resistant organisms such as extended spectrum beta-lactamase (ESBL-producing Escherichia coli and Klebsiella pneumoniae are increasing. Carbapenems (imipenem and meropenem are the antibiotics commonly used to treat these agents. There is limited clinical data regarding the efficacy of the newest carbapenem, ertapenem, against these organisms. Ertapenem susceptibility of ESBL-producing E. coli and K. pneumoniae clinical isolates were evaluated and compared to imipenem to determine if imipenem susceptibility could be used as a surrogate for ertapenem susceptibility. Methods 100 ESBL isolates (n = 34 E. coli and n = 66 K. pneumoniae collected from 2005–2006 clinical specimens at WRAMC were identified and tested for susceptibility by Vitek Legacy [bioMerieux, Durham, NC]. Ertapenem susceptibility was performed via epsilometer test (E-test [AB Biodisk, Solna, Sweden]. Results 100% of ESBL isolates tested were susceptible to ertapenem. 100% of the same isolates were also susceptible to imipenem. Conclusion These results, based on 100% susceptibility, suggest that ertapenem may be an alternative to other carbapenems for the treatment of infections caused by ESBL-producing E. coli and K. pneumoniae. Clinical outcomes studies are needed to determine if ertapenem is effective for the treatment of infection caused by these organisms. However, due to lack of resistant isolates, we are unable to conclude whether imipenem susceptibility accurately predicts ertapenem susceptibility.

The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV beta-lactamase: a study of ambler position Asn276.

Science.gov (United States)

Drawz, Sarah M; Bethel, Christopher R; Hujer, Kristine M; Hurless, Kelly N; Distler, Anne M; Caselli, Emilia; Prati, Fabio; Bonomo, Robert A

2009-06-02

Inhibitor-resistant class A beta-lactamases of the TEM and SHV families that arise by single amino acid substitutions are a significant threat to the efficacy of beta-lactam/beta-lactamase inhibitor combinations. To better understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis to examine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only the Asn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2 --> 50/8 microg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 microg/mL). Steady-state kinetic analyses of Asn276Asp revealed slightly diminished k(cat)/K(m) for all substrates tested. In contrast, we observed a 5-fold increase in K(i) for clavulanate (7.4 +/- 0.9 microM for Asn276Asp vs 1.4 +/- 0.2 microM for SHV-1) and a 40% reduction in k(inact)/K(I) (0.013 +/- 0.002 microM(-1 )s(-1) for Asn276Asp vs 0.021 +/- 0.004 microM(-1) s(-1) for SHV-1). Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Asp showed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modeling shows that novel electrostatic interactions are formed between Arg244Neta2 and both 276AspOdelta1 and Odelta2; these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C(3)/C(4) carboxylate of beta-lactam substrates and inhibitors. Testing the functional consequences of this interaction, we noted considerable free energy costs (+DeltaDeltaG) for substrates and inhibitors. A rigid carbapenem (meropenem) was most affected by the Asn276Asp substitution (46-fold increase in K(i) vs SHV-1). We conclude that residue 276 is an important second-shell residue in class A beta-lactamase-mediated resistance to substrates and inhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required for successful

Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase.

Science.gov (United States)

Ferreira, Adriano Martison; Martins, Katheryne Benini; Silva, Vanessa Rocha da; Mondelli, Alessandro Lia; Cunha, Maria de Lourdes Ribeiro de Souza da

Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase

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Adriano Martison Ferreira

Full Text Available Abstract Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28 mm (penicillin resistant and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29 mm, 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%, followed by MIC determination (85.5%, but the specificity of the former was low (40.0%. The nitrocefin test was the least sensitive (28.9%. However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%. The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli

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Catherine Ludden

2014-09-01

Full Text Available Objectives: E. coli O25b-ST131 has disseminated worldwide in hospitals and the community. The objective of this study was to determine the extent to which E. coli O25b-ST131 accounts for extended-spectrum beta-lactamase (ESBLproducing E. coli from clinical samples from all sources in this region. Methods: Between January and June 2010 ESBL-producing E. coli were collected from 94 routine samples including 47 from residents of 25 nursing homes, 15 categorized as hospital acquired and 32 others. PCR was performed for detection of bla CTX-M, bla OXA-1, bla TEM, bla SHV and for the identification of members of the E. coli O25b:ST131 clonal group. PFGE was carried out using Xba I in accordance with PulseNet protocols. Results: The majority (97% of isolates harbored a bla CTX-M gene.E. coli O25b-ST131 accounted for 87% of all ESBL-producing E. coliand for 96% of isolates from nursing home residents. Conclusion:The E. coli O25b-ST131 clonal group predominated in the collection of ESBL-producing E. coli, particularly in nursing home isolates. J Microbiol Infect Dis 2014; 4(3: 92-96

Incidence of temonera, sulphuhydryl variables and cefotaximase genes associated with ?-lactamase producing escherichia coli in clinical isolates

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Isaiah, Ibeh Nnana; Nche, Bikwe Thomas; Nwagu, Ibeh Georgina; Nwagu, Ibeh Isaiah

2011-01-01

Background: The occurrence of the different types of Extended spectrum beta Lactamase producing Escherichia coli with the, Sulphurhydryl variable, Temonera and the Cefotaximase have been on the rise Aim: The study was to determine the prevalence of extended spectrum beta lactamase gene resistance across the clinical isolates of hospitalized patients. Materials and Method: Three hundred and fifty isolates of Escherichia coli were received from different clinical specimens. The susceptibility p...

A trial with IgY chicken antibodies to eradicate faecal carriage of Klebsiella pneumoniae and Escherichia coli producing extended-spectrum beta-lactamases

OpenAIRE

Jonsson, Anna-Karin; Larsson, Anders; Tängdén, Thomas; Melhus, Åsa; Lannergård, Anders

2015-01-01

Background: Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is an emerging therapeutic challenge, especially in the treatment of urinary tract infections. Following an outbreak of CTX-M-15 Klebsiella pneumoniae in Uppsala, Sweden, an orphan drug trial on IgY chicken antibodies was undertaken in an attempt to eradicate faecal carriage of ESBL-producing K. pneumoniae and Escherichia coli.Methods: Hens were immunised with epitopes from freeze-dried, whole-cell bacteria (ESBL...

Molecular characteristics of travel-related extended-spectrum-beta-lactamase-producing Escherichia coli isolates from the Calgary Health Region.

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Pitout, Johann D D; Campbell, Lorraine; Church, Deirdre L; Gregson, Daniel B; Laupland, Kevin B

2009-06-01

Extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli has recently emerged as a major risk factor for community-acquired, travel-related infections in the Calgary Health Region. Molecular characterization was done on isolates associated with infections in returning travelers using isoelectric focusing, PCR, and sequencing for bla(CTX-M)s, bla(TEM)s, bla(SHV)s, bla(OXA)s, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequence typing (MLST). A total of 105 residents were identified; 6/105 (6%) presented with hospital-acquired infections, 9/105 (9%) with health care-associated community-onset infections, and 90/105 (86%) with community-acquired infections. Seventy-seven of 105 (73%) of the ESBL-producing E. coli isolates were positive for bla(CTX-M) genes; 55 (58%) produced CTX-M-15, 13 (14%) CTX-M-14, six (6%) CTX-M-24, one (1%) CTX-M-2, one (1%) CTX-M-3, and one (1%) CTX-M-27, while 10 (10%) produced TEM-52, three (3%) TEM-26, 11 (11%) SHV-2, and four (4%) produced SHV-12. Thirty-one (30%) of the ESBL-producing E. coli isolates were positive for aac(6')-Ib-cr, and one (1%) was positive for qnrS. The majority of the ESBL-producing isolates (n = 95 [90%]) were recovered from urine samples, and 83 (87%) were resistant to ciprofloxacin. The isolation of CTX-M-15 producers belonging to clone ST131 was associated with travel to the Indian subcontinent (India, Pakistan), Africa, the Middle East, and Europe, while clonally unrelated strains of CTX-M-14 and -24 were associated with travel to Asia. Our study suggested that clone ST131 coproducing CTX-M-15, OXA-1, TEM-1, and AAC(6')-Ib-cr and clonally unrelated CTX-M-14 producers have emerged as important causes of community-acquired, travel-related infections.

Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana

DEFF Research Database (Denmark)

Rasmussen, Mette Marie; Opintan, Japheth A; Frimodt-Møller, Niels

2015-01-01

whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified...... phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported......The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate...

Escherichia coli-producing extended-spectrum beta-lactamase CTX-M-15 in a captive South American tapir (Tapirus terrestris).

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Klimes, Jiri; Machalkova, Marketa; Dolejska, Monika; Cizek, Alois; Janoszowska, Dagmar; Alexa, Pavel; Albrechtova, Katerina; Vojtech, Jiri; Literak, Ivan

2013-03-01

Only a few reports exist on the occurrence of resistant bacteria in zoo animals. Therefore, an isolation of multiresistant Escherichia coli from the lungs of a captive South American tapir (Tapirus terrestris) lead to its characterization and further investigation of samples from animals inhabiting the same paddock and from the shared environment. The tapir suffered from an intermandibular abscess and pneumonia and was euthanatized after unsuccessful therapy, including administration of antibiotics. The authors performed selective isolation of extended-spectrum beta-lactamase (ESBL)-positive E. coli strains and identification of resistance genes using polymerase chain reaction. Seven multiresistant, ESBL-producing E. coli isolates were obtained, all belonging to the B2 phylogenetic group and showing identical profile on pulsed-field gel electrophoresis. These isolates carried several resistance genes, including the gene bla(CTX-M-15). This case demonstrates the transmission of related epidemiologically important E. coli isolates whose potential transmission to other animals and zoo staff can be assumed.

Resistance Pattern and Detection of Metallo-beta-lactamase Genes ...

African Journals Online (AJOL)

2018-02-23

Feb 23, 2018 ... Background: Acquired metallo-β-lactamases (MBLs) pose serious problem both in terms of ... P. aeruginosa from clinical samples submitted to the Medical Microbiology ... pan-drug-resistant .... Phenotypically confirmed MBL producers were stored ... 103 (51.5%); ear swab 32 (16%); urine 27 (13.5%); and.

Global mapping of transposon location.

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Abram Gabriel

2006-12-01

Full Text Available Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.

Differential Binding of Co(II) and Zn(II) to Metallo-beta-Lactamase Bla2 from Bacillus anthracis

Energy Technology Data Exchange (ETDEWEB)

Hawk, M.; Breece, R; Hajdin, C; Bender, K; Hu, Z; Costello, A; Bennett, B; Tierney, D; Crowder, M

2009-01-01

In an effort to probe the structure, mechanism, and biochemical properties of metallo-{beta}-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV-vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.

Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307

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S. Wesley Long

2017-05-01

Full Text Available Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae, it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K. pneumoniae strains cultured from patients in the 2,000-bed Houston Methodist Hospital system between September 2011 and May 2015, representing a comprehensive, population-based strain sample. Strains of largely uncharacterized clonal group 307 (CG307 caused more infections than those of well-studied epidemic CG258. Strains varied markedly in gene content and had an extensive array of small and very large plasmids, often containing antimicrobial resistance genes. Some patients with multiple strains cultured over time were infected with genetically distinct clones. We identified 15 strains expressing the New Delhi metallo-beta-lactamase 1 (NDM-1 enzyme that confers broad resistance to nearly all beta-lactam antibiotics. Transcriptome sequencing analysis of 10 phylogenetically diverse strains showed that the global transcriptome of each strain was unique and highly variable. Experimental mouse infection provided new information about immunological parameters of host-pathogen interaction. We exploited the large data set to develop whole-genome sequence-based classifiers that accurately predict clinical antimicrobial resistance for 12 of the 16 antibiotics tested. We conclude that analysis of large, comprehensive, population-based strain samples can assist understanding of the molecular diversity of these organisms and contribute to enhanced translational research.

Extended spectrum beta lactamase (ESBL) producing bacteria urinary tract infections and complex pediatric urology.

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Wragg, Ruth; Harris, Anna; Patel, Mitul; Robb, Andrew; Chandran, Harish; McCarthy, Liam

2017-02-01

Extended spectrum beta lactamase (ESBL) producing bacteria are resistant to most beta-lactam antibiotics including third-generation cephalosporins, quinolones and aminoglycosides. This resistance is plasmid-borne and can spread between species. Management of ESBL is challenging in children with recurrent urinary tract infections (UTIs) and complex urological abnormalities. We aim to quantify the risk in children and specifically in urological patients. Retrospective review of a microbiology database (April 2014 to November 2015). This identified urine isolates, pyuria, ESBL growth and patient demographics. Data analysis was by Chi square, Mann-Whitney U-test and ANOVA. A P value of 10×10 6 WC/L). 136 urine cultures (n=79 patients) grew purely ESBL. Overall, 5.2% of urine isolates were ESBL and 9.5% isolates with pyuria (>100×10 6 WC/L) had ESBL, whereas only 22/1032 (2.1%) with no pyuria, (Pantibiotics). Over the study period, there was no significant rise of the monthly incidence between 2014 and 2015 (ANOVA P=0.1). This study is the first to document the incidence of ESBL in children (5%), and estimate the frequency of possible plasmid transmission between bacterial species in children. This quantifies the risk of ESBL, especially to urology patients, and mandates better antibiotic stewardship. Level IIc. Copyright © 2017. Published by Elsevier Inc.

Extended-spectrum beta-lactamase orthopedic wound infections in Nigeria

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Olusolabomi J Idowu

2011-01-01

Full Text Available Background: Extended-spectrum beta-lactamase (ESBL-producing Gram-negative bacteria are emerging and impacting significantly on the management of patients and hospital costs. Besides, they are not being routinely sought after in diagnostic laboratories thus contributing to treatment failure. Materials and Methods: Bacterial isolates from wounds of 45 patients were identified using commercial identification kits and antibiotic susceptibility was evaluated by the Bauer-Kirby method. Screening and phenotypic confirmation of ESBL production were done as prescribed by the Clinical and Laboratory Standards Institute. The conjugation experiment was performed by the mating assay in broth between the ESBL producers and E. coli ATCC 25922 as the recipient. Results: Out of 102 Gram-negative bacteria isolated, 36 were positive for ESBL mainly of the Enterobacteriaceae family (33 and the rest were oxidase-positive bacilli (3. The predominant bacteria were Klebsiella spp. and E. coli. Others were Serratia rubidae, Citrobacter freundii, Morganella morgannii, Proteus spp., Providencia stuartii, and Enterobacter spp. There was a significant association between treatment with third-generation cephalosporins (3GCs and isolation of ESBLs ( p=0.0020 . The ESBL producers were multiply resistant and moderately sensitive to colistin. The conjugation experiment showed that the ESBL gene was transferred horizontally and tetracycline, cotrimoxazole, nitrofurantoin, gentamicin, and aztreonam resistance genes were co-transferred. No mortality was recorded but the mean length of stay in the hospital was 82 days. Conclusion: The development and spread of ESBL among Gram-negative bacteria and possible horizontal transfer calls for concern, especially in view of treatment failure, high treatment cost, and consequent discomfort to patients.

Nitrofurantoin and Fosfomycin for Extended Spectrum Beta-lactamases Producing Escherichia coli and Klebsiella pneumoniae

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Neeraj Kumar Tulara

2018-01-01

Full Text Available Urinary tract infection (UTI is a common and painful human illness that, unfortunately not responsive to commonly used antibiotics in current practice. The role of fosfomycin and nitrofurantoin in the era of growing bacteria resistance has been widely discussed. In this study, we aimed to know the local antimicrobial susceptibilities, fosfomycin and nitrofurantoin susceptibility in particular, for urinary extended-spectrum-beta-lactamase-producing Escherichia coli and Escherichia pneumoniae (ESBL-EC and ESBL-KP isolates in our hospital. We collected 464 urine isolates, including 384 ESBL-EC and 80 ESBL-KP isolates. Of 464 urine isolates culture positive ESBL-UTIs, EC caused 384 (82.75%, followed by Klebsiella in 80 (17.24%. Carbapenems and Colistin seems to remain as the first line therapy for the majority of ESBL-UTIs in the local setting. Colistin and fosfomycin remains the most sensitive antibiotic while nitrofurantoin still preserves the good sensitivity against ESBL and found to be an only oral sensitive antibiotic.

PEDIATRIC URINARY INFECTIONS, CAUSED BY EXTENDED-SPECTRUM BETA-LACTAMASE - PRODUCING MICROORGANISMS IN VARNA, BULGARIA

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Neli M. Ermenlieva

2016-05-01

Full Text Available Background: Extended-spectrum beta-lactamase (ESBLs producing bacteria are microorganisms which have the ability to hydrolyze β-lactame ring of a large part of the antibiotics, commonly used to treat bacterial infections including urinary tract infections. Purpose: The aim of this study is present the epidemiology of childhood urinary tract infections caused by ESBL-producing strains in Varna, Bulgaria. Material/methods: A total of 3895 urine samples of children patients (aged 0 to 18 years were examined during the period 2010-2012 for presence of ESBL-producing bacteria. Results: Six percent of the tested urinary samples were positive for ESBL production. All of the isolates were resistant to ampicillin, piperacillin, cephalothin, cefprozil, cefuroxime, ceftriaxone, ceftazidime, levofloxacin, cefaclor, but were were sensitive to meropenem and imipenem. Conclusions: Cephalosporins and penicillins are the most used antibiotics in Bulgaria, but they should be very precisely prescribed in medical practice, because otherwise preconditions for maintaining high share of ESBLs are created.

Characteristics of bacteremia caused by extended-spectrum beta-lactamase-producing Proteus mirabilis.

Science.gov (United States)

Kurihara, Yoko; Hitomi, Shigemi; Oishi, Tsuyoshi; Kondo, Tsukasa; Ebihara, Tsugio; Funayama, Yasunori; Kawakami, Yasushi

2013-10-01

Although Proteus mirabilis is a common human pathogen, bacteremia caused by the organism, especially strains producing extended-spectrum beta-lactamase (ESBL), has rarely been investigated. We examined 64 cases of P. mirabilis bacteremia identified in the Minami Ibaraki Area, Japan, between 2001 and 2010 and compared the characteristics of cases with ESBL-producing and ESBL-non-producing strains (13 and 51 cases, respectively). All ESBL-producing strains with the gene encoding the CTX-M-2-group were genetically nonidentical. Isolation of ESBL-producing strains was significantly associated with onset in a hospital (p = 0.030), receiving hemodialysis (p = 0.0050), and previous antibiotic use within 1 month (p = 0.036; especially penicillin and/or cephalosporin (p = 0.010) and fluoroquinolone (p = 0.0069)). Isolation was also associated with inappropriate antibiotic therapy on the 1st and 4th days (p = 0.011 and 0.032, respectively) but not with mortality on the 30th day. These findings indicate that, for P. mirabilis bacteremia, isolation of ESBL-producing strains causes delay of initiating appropriate antimicrobial therapy but may not be associated with mortality.

Development of a multiplex real-time PCR for the rapid detection of the predominant beta-lactamase genes CTX-M, SHV, TEM and CIT-type AmpCs in Enterobacteriaceae.

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Nicole Roschanski

Full Text Available Beta-lactamase resistant bacteria and especially ESBL producing Enterobacteriaceae are an increasing problem worldwide. For this reason a major interest in efficient and reliable methods for rapid screening of high sample numbers is recognizable. Therefore, a multiplex real-time PCR was developed to detect the predominant class A beta-lactamase genes blaCTX-M, blaSHV, blaTEM and CIT-type AmpCs in a one-step reaction. A set of 114 Enterobacteriaceae containing previously identified resistance gene subtypes and in addition 20 undefined animal and environmental isolates were used for the validation of this assay. To confirm the accessibility in variable settings, the real-time runs were performed analogous in two different laboratories using different real-time cyclers. The obtained results showed complete accordance between the real-time data and the predetermined genotypes. Even if sequence analyses are further necessary for a comprehensive characterization, this method was proofed to be reliable for rapid screening of high sample numbers and therefore could be an important tool for e. g. epidemiological purposes or support infection control measures.

Prevalence and antimicrobial susceptibility patterns of extended spectrum beta-lactamase producing Entrobacteriaceae in the University of Gondar Referral Hospital environments, northwest Ethiopia.

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Engda, Tigist; Moges, Feleke; Gelaw, Aschalew; Eshete, Setegn; Mekonnen, Feleke

2018-05-22

This study aimed at assessing the magnitude, distribution, and the antimicrobial susceptibility of the extended spectrum beta-lactamase producing Entrobacteriaceae in the University of Gondar Referral Hospital environments. Out of a total of 384 samples, 14.8% were ESBL producing Entrobacteriaceae, where 42.10% Klebsiella pneumoniae, 35.09% Escherchia coli and 7.01% Proteus mirabilis were the predominant isolates. Most ESBL producing isolates, that is, 24.56, 22.8, and 22.8% were found from waste water, sinks and bedside tables respectively. All ESBL producing Entrobacteriaceae were found to be resistant to ceftriaxone, ceftazidime, cefpirome, cefpodoxime, and amoxicillin with Clavulanic acid. Resistance rate was also high for non-beta-lactam antimicrobials, like chloramphenicol (70.18%), cotrimoxazole (64.91%), norfloxacin (42.10%), ciprofloxacin (43.86%), and gentamicin (19.30%).

Genomic Dissection of Travel-Associated Extended-Spectrum-Beta-Lactamase-Producing Salmonella enterica Serovar Typhi Isolates Originating from the Philippines: a One-Off Occurrence or a Threat to Effective Treatment of Typhoid Fever?

DEFF Research Database (Denmark)

Hendriksen, Rene S.; Leekitcharoenphon, Pimlapas; Mikoleit, Matthew

2015-01-01

One unreported case of extended-spectrum-beta-lactamase (ESBL)-producing Salmonella enterica serovar Typhi was identified, whole-genome sequence typed, among other analyses, and compared to other available genomes of S. Typhi. The reported strain was similar to a previously published strain harbo...

Improved Detection of Extended Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in Input and Output Samples of German Biogas Plants by a Selective Pre-Enrichment Procedure

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Schauss, Thorsten; Glaeser, Stefanie P.; Gütschow, Alexandra; Dott, Wolfgang; Kämpfer, Peter

2015-01-01

The presence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli was investigated in input (manure from livestock husbandry) and output samples of six German biogas plants in 2012 (one sampling per biogas plant) and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU) per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%), few SHV-type beta lactamase (6%). Sixty-four bla CTX-M genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85%) and 9 (13%), and one to group 2. Phylogenetic grouping of 80 E. coli isolates showed that most were assigned to group A (71%) and B1 (27%), only one to group D (2%). Genomic fingerprinting and multilocus sequence typing (MLST) showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing E. coli revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT) genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing E. coli in

Improved detection of extended spectrum beta-lactamase (ESBL-producing Escherichia coli in input and output samples of German biogas plants by a selective pre-enrichment procedure.

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Thorsten Schauss

Full Text Available The presence of extended-spectrum beta-lactamase (ESBL-producing Escherichia coli was investigated in input (manure from livestock husbandry and output samples of six German biogas plants in 2012 (one sampling per biogas plant and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%, few SHV-type beta lactamase (6%. Sixty-four blaCTX-M genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85% and 9 (13%, and one to group 2. Phylogenetic grouping of 80 E. coli isolates showed that most were assigned to group A (71% and B1 (27%, only one to group D (2%. Genomic fingerprinting and multilocus sequence typing (MLST showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing E. coli revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing E

First report on extended-spectrum beta-lactamase (ESBL) producing Escherichia coli from European free-tailed bats (Tadarida teniotis) in Portugal: A one-health approach of a hidden contamination problem.

Science.gov (United States)

Garcês, Andreia; Correia, Susana; Amorim, Francisco; Pereira, José Eduardo; Igrejas, Gilberto; Poeta, Patrícia

2017-12-23

The main aim of this study was to characterize the diversity of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates from European free tailed-bats (Tadarida teniotis) in Portugal. ESBL-producing E. coli isolates were recovered from 14 of 146 faecal samples (9.6%) and a total of 19 isolates were completely characterized. The more prevalent beta-lactamase genes detected were bla CTX-M-1 (57.9%) and bla CTX-M-3 (36.8%), followed by bla SHV (31.6%), bla TEM (21.1%), bla OXA (10.5%) and bla CTX-M-9 (10.5%). Among other associated resistance genes studied, tet(A) and tet(B) were predominant and fimA was the main virulence factor detected. Phylogroups D (47.4%) and A (31.6%) were the more prevalent, followed by group B2 (21.1%). Bats are reservoirs of antimicrobial-resistant bacteria and resistance determinants and is important in further studies to identify the main sources of pollution in the environment, such as water or insects that may contain these multiresistant organisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative analysis of the IgG and IgG subclass immune responses to chromosomal Pseudomonas aeruginosa beta-lactamase in serum from patients with cystic fibrosis by western blotting and laser scanning densitometry

DEFF Research Database (Denmark)

Petersen, T D; Ciofu, O; Pressler, T

1996-01-01

BACKGROUND: Antibodies against chromosomal beta-lactamase of Pseudomonas aeruginosa (a beta ab) are markers of the development of resistance of P aeruginosa to beta-lactam antibiotics in patients with cystic fibrosis and chronic lung infection. The role of these antibodies in patients with chronic...... of the chronic infection the a beta ab titres were higher in patients with good lung function than in those with poor lung function. CONCLUSIONS: The association of a weak IgG3 and a strong IgG4 a beta ab response suggests that the contribution of a beta ab antibodies to lung diseases mediated by immune...... complexes might be less important than other antipseudomonal antibodies. A beneficial neutralising effect of the a beta ab antibodies on the antibiotic destroying enzymes may be an additional factor....

In vitro activity of fosfomycin tromethamine against extended spectrum beta-lactamase producing urinary tract bacteria

International Nuclear Information System (INIS)

Khan, I.U.; Mirza, I.A.; Ali, S.; Hussain, A.

2014-01-01

To determine the in vitro activity of Fosfomycin tromethamine against extended spectrum beta-lactamase producing uropathogens. Study Design: Experimental study. Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi, from October 2011 to October 2012. Methodology: A total of 381 culture positive ESBL producing isolates from 2400 urine samples submitted over a period of one year were included in this study. Identification of isolates was done by standard biochemical profile of the organisms. The antimicrobial susceptibility of culture positive isolates was performed by disk diffusion method as recommended by Clinical Laboratory Standard Institute guidelines (CLSI). Results: The antimicrobial activity of Fosfomycin to various isolates revealed that 93% of E. coli, 64% Klebsiella spp. 50% Proteus spp. 75% Enterobacter cloacae, 100% Citrobacter freundii, 100% Burkholderia spp. 100% Serratia spp. and 50% Stenotrophomonas maltophilia were susceptible to this chemical compound. Conclusion: Fosfomycin showed excellent effectiveness to most of the common ESBL producing bacteria such as E. coli, Klebsiella and Proteus spp. (author)

Role of ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A beta-lactamase Sme-1.

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Sougakoff, W; Naas, T; Nordmann, P; Collatz, E; Jarlier, V

1999-08-17

The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine. The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1. By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine. In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme. Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1.

Occurrence of qnr-positive clinical isolates in Klebsiella pneumoniae producing ESBL or AmpC-type beta-lactamase from five pediatric hospitals in China.

Science.gov (United States)

Wang, Aihua; Yang, Yonghong; Lu, Quan; Wang, Yi; Chen, Yuan; Deng, Li; Ding, Hui; Deng, Qiulian; Wang, Li; Shen, Xuzhuang

2008-06-01

The plasmid-mediated quinolone resistance qnr genes in clinical isolates in adults have been described in different countries; however, the frequency of their occurrence has not been detected in pediatric patients. A total of 410 clinical isolates of Klebsiella pneumoniae, identified as producers of an extended-spectrum beta-lactamase (ESBL), or AmpC beta-lactamase, were collected from five children's hospitals in China during 2005-2006. The isolates were screened for the presence of the qnrA, qnrB, and qnrS genes, and then the harboring qnr gene isolates were detected for a bla gene coding for the TEM, SHV, CTX-M, and plasmid-mediated ampC gene by a PCR experiment. Ninety-two isolates (22.7%) were positive for the qnr gene, including 10 of qnrA (2.4%), 25 of qnrB (6.1%), and 62 of qnrS (15.1%). Eighty-one of the 92 (88.0%) qnr-positive isolates carried at least one bla gene for TEM, SHV, CTX-M, or DHA-1. The ciprofloxacin resistance increased 16-256-fold and oflaxacin resistance increased 2-32-fold in transconjugants, respectively. These results indicated that the plasmid-mediated qnr quinolone resistance gene was qnrS, followed by qnrB and qnrA. Most of the isolates also carried a bla gene coding ESBL or ampC gene coding DHA-1 among Klebsiella pneumoniae isolated from Chinese pediatric patients.

[Differential expression of DTSsa4 Tc1-like transposons in closely related populations of Baikal ciscoes].

Science.gov (United States)

Bychenko, O S; Sukhanova, L V; Azhikina, T L; Sverdlov, E D

2009-01-01

Two representatives of Baikal ciscoes - lake cisco and omul - diverged from a common ancestor as recently as 10-20 thousand years ago. We have found an increasing expression level of DTSsa4 Tc1-like DNA transposons in cisco and omul brains. The mapping of the sequences of these transposons from Salmo salar and Danio rerio genomes has shown that in some cases, these transposons are located in the 5' and 3' regions, as well as in the promoter regions of various genes. Probably, Tc1-like transposons affect the activity of neighboring genes, providing the adaptive divergence of the cisco population.

Phage transposon mutagenesis.

Science.gov (United States)

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

PREVALENCE AND SUSCEPTIBILITY OF EXTENDED SPECTRUM BETA-LACTAMASES IN URINARY ISOLATES OF ESCHERICHIA COLI IN A TERTIARY CARE HOSPITAL, CHENNAI-SOUTH INDIA

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Dr. Anbumani Narayanaswamy MD PhD

2011-01-01

Full Text Available Extended spectrum beta – lactamases (ESBLs are on the rise in hospital settings across the globe. The presence of ESBLs significantly affects the outcome of an infection and poses a challenge to the management of infection worldwide. Therefore, the aim of the present study is to determine the prevalence and susceptibility of extended spectrum beta – lactamase in urinary isolates of Escherichia coli (E.coli in a tertiary care hospital, Chennai-South India. A total of 450 urinary isolates of E.coli were collected over a period of six months from April 2008 to September 2008. Antimicrobial susceptibility testing was determined to commonly used antibiotics using the modified Kirby-Bauer’s disc diffusion method. ESBL detection was done by the screening method of double disc synergy test and then confirmed by the phenotypic confirmatory test with combination disc as recommended by the Clinical Laboratory Standards Institute (CLSI and the minimum inhibitory concentration (MIC method using the E test strips (AB Biodisk,Sweden - as per manufacturer’s instructions. The prevalence of E.coli ESBL was 60%. The ESBL producing isolates were significantly resistant (p < 0.01 to ampicillin, trimethoprim / sulfamethoxazole, norfloxacin and nalidixic acid as compared to non-ESBL producers. Multidrug resistance was significantly (p < 0.01 higher (69% in ESBL positive isolates than non-ESBL isolates (21%. Knowledge of the prevalence of ESBL and resistance pattern of bacterial isolates in a geographical area will help the clinicians to formulate the guidelines for antibiotic therapy to avoid inappropriate use of extended spectrum cephalosporins.

Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase (blaNDM)-producing Escherichia coli in the piglets of organized farms in India.

Science.gov (United States)

Pruthvishree, B S; Vinodh Kumar, O R; Sinha, D K; Malik, Y P S; Dubal, Z B; Desingu, P A; Shivakumar, M; Krishnaswamy, N; Singh, B R

2017-06-01

A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. In this study, fecal sample (n = 673) from non-diarrheic (n = 501) and diarrheic (n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate (n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase (blaNDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1, stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E. coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with SaTScan helped in identification of carbapenem-resistant clusters. The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. 1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India. © 2017

Prevalence of metallo-beta-lactamase among Pseudomonas aeruginosa and Acinetobacter baumannii isolated from burn wounds and in vitro activities of antibiotic combinations against these isolates.

Science.gov (United States)

Altoparlak, Ulku; Aktas, Ferda; Celebi, Demet; Ozkurt, Zulal; Akcay, Mufide N

2005-09-01

The prevalence of metallo-beta-lactamases (MBLs) produced by isolates of Pseudomonas aeruginosa and Acinetobacter baumannii and the activities of various antmicrobial combinations against MBL producer strains were investigated. During the period from June 2003 till July 2004, 120 P. aeruginosa and 9 A. baumannii nonduplicate isolates were obtained from burn wounds. Forty strains (37 P. aeruginosa, 3 A. baumannii) were selected because of resistance to carbapenems. Screening for MBL production was performed in the latter isolates by the combined disk method which depends on comparing the zones given by disks containing imipenem with and without ethylenediaminetetraacetic acid (EDTA). Of imipenem resistant P. aeruginosa strains, 21 and 1 of A. baumannii were found metallo-beta-lactamase producers. Disk approximation studies were then performed to test for in vitro activities of various antimicrobial combinations. For a total of 21 P. aeruginosa strains, synergy was demonstrated predominantly by ciprofloxacin in combination with ceftazidime and imipenem, by ofloxacin in combination with astreonam. Against MBL producer A. baumannii strain, synergy was detected only with imipenem-ofloxacin combination. None of the combinations were antagonistic. These results suggest that MBL producing P. aeruginosa and A. baumanni strains have been introduced into burn centers, and to prevent the further spread of MBL producers, it is essential for carbapenem resistant isolates to be screened for MBLs.

Antibacterial activities of multi drug resistant Myroides odoratimimus bacteria isolated from adult flesh flies (Diptera: Sarcophagidae are independent of metallo beta-lactamase gene Atividades antibacterianas de Myroides odoratimimus isolada de moscas varejeiras adultas (Diptera: Sarcophagidae são independentes do gene metalo beta lactamase

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M.S. Dharne

2008-06-01

� avaliação de parâmetros nutricionais pelo sistema BIOLOG GN, ao sequenciamento genético 16S rRNA, à sensibilidade a vários antimicrobianos pelo método de difusão de discos e à detecção dos genes de metalo beta lactamases (TUS/MUS. Os efeitos antagonistas foram testados contra bactérias Gram negativas e Gram positivas isoladas de material clínico humano, amostras ambientais e intestino do inseto. As espécies bacterianas incluíram Aeromonas hydrophila, A. culicicola, Morganella morganii subsp sibonii, Ochrobactrum anthropi, Weissella confusa, Escherichia coli, Ochrobactrum sp, Serratia sp, Kestersia sp, Ignatzschineria sp e Bacillus sp. A cepa Myroides sp foi resistente à penicilina G, eritromicina, estreptomicina, amicacina, canamicina, gentamicina, ampicilina, trimetoprim e tobramicina. Esta cepa apresentou atividade antimicrobiana contra todas as cepas exceto W.confusa, Ignatzschineria sp, A. hydrophila e M. morgani subsp sibonii. A resistência múltipla da cepa foi semelhante à de isolados clínicos, inibindo bactérias das amostras clínicas, ambientais e do intestino do inseto. Os genes de metalo beta lactamases (TUS/MUS estavam ausentes, excluindo-se a resistência mediada por esses genes, o que indica o envolvimento de um mecanismo alternativo de secreção.

Predicament in detection and reporting of extended spectrum beta lactamase production in routine antibiotic susceptibility testing

International Nuclear Information System (INIS)

Butt, T.; Butt, E.; Raza, S.

2017-01-01

This descriptive and cross-sectional study was planned to determine the dilemma of inadvertent detection of extended spectrum beta lactamase (ESBL) production in Enterobacteriaceaewhen using inhibition zone size of antibiotic disks of Cefotaxime or Aztreonam in routine antibiotic susceptibility testing as recommended by Clinical Laboratory Standards Institute (CLSI). Screening and double disk tests were adopted as per CLSI. Escherichia coli ATCC 25922 was used as control strain. Among total specimens of 5346, there were 348 isolates of Escherichia coli(n=235), Klebsiella pneumonia (n=92), Klebsiella oxytoca(n=3) or Proteus mirabilus(n=18). The screening method recommended by CLSI significantly falsely detected ESBL production in 79 (32.3%) isolates (p<0.0001). ESBL detection is important as its frequency is high and treatment of the infection varies with the presence and absence of ESBL. To avoid false reporting, proper phenotypic detection of ESBL confirmatory method-like double-disk synergy test, should be used routinely. (author)

Split Beta-Lactamase Complementation Assay

Indian Academy of Sciences (India)

IAS Admin

A Search for the Molecular Better Half! Vaishali Verma ... These assays comprise of a protein molecule, ... ciferase, beta-galactosidase, GFP, g3p of M13 filamentous ph- .... sensors of protein–protein interactions, Nature Biotechnology, Vol.20,.

A mariner transposon vector adapted for mutagenesis in oral streptococci

DEFF Research Database (Denmark)

Nilsson, Martin; Christiansen, Natalia; Høiby, Niels

2014-01-01

This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication rep...... 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen....

Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae isolated from blood cultures in Africa.

Science.gov (United States)

Sangare, S A; Maiga, A I; Guindo, I; Maiga, A; Camara, N; Savadogo, S; Diallo, S; Bougoudogo, F; Armand-Lefevre, L; Andremont, A; Maiga, I I

2015-09-01

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have been isolated from many regions of the world. Epidemiological studies are being conducted in Europe, North America, and Asia. No study has however been conducted in Africa to determine the prevalence and distribution of ESBLs on the continent. This literature review aimed at describing the prevalence of ESBL-producing Enterobacteriaceae isolated from blood cultures, as well as the ESBL genes involved at the international level. Our focus was mainly on Africa. We conducted a literature review on PubMed. Articles related to our study field and published between 1996 and 2014 were reviewed and entirely read for most of them, while we only focused on the abstracts of some other articles. Relevant articles to our study were then carefully reviewed and included in the review. The prevalence of ESBL-producing Enterobacteriaceae differs from one country to another. The results of our literature review however indicate that class A ESBLs prevail over the other types. We took into consideration articles focusing on various types of samples to assess the prevalence of ESBL-producing Enterobacteriaceae, but information on isolates from blood cultures is limited. The worldwide prevalence of ESBL-producing Enterobacteriaceae has increased over time. Evidence of ESBL-producing Enterobacteriaceae can be found in all regions of the world. Studies conducted in Africa mainly focused on the Northern and Eastern parts of the continent, while only rare studies were carried out in the rest of the continent. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

The distribution of beta lactamase genes in Escherichia coli phylotypes isolated from diarrhea and UTI cases in northwest Iran.

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Hemati, Zahra; Ghanbarpour, Reza; Alizade, Hesam

2014-01-01

Pathogenic Escherichia coli strains are a common cause of intestinal and extra-intestinal infections, especially in developing countries. Extended spectrum beta-lactamases (ESBLS), a heterogeneous group of plasmid-encoded beta-lactamases, are common throughout the world. The aim of the present study was to determine the phenotypic and genotypic characteristics of ESBLS produced by E. coli isolates taken from patients with diarrhea and urinary tract infections (UTI) in northwest Iran. A total of 132 E. coli isolates (92 isolates from UTI and 40 isolates from diarrheic cases) were recovered and confirmed by biochemical tests. The isolates were examined for blaTEM and blaSHV genes and phylogenetic background by two multiplex PCR assays. The isolates were tested for antibiotic susceptibility against nine antibiotic agents by the disk diffusion method. The phylogenetic analysis showed that the UTI isolates mostly fell into phylo-group B2, followed by D, while the diarrheic isolates belonged to phylo-groups D and A. Out of 92 UTI isolates, 29.3% and 17.4% possessed blaTEM and blaSHV genes, respectively. Ten diarrheic isolates were positive for blaTEM, two isolates possessed the blaSHV gene, and one isolate was positive for both genes. The UTI isolates that were positive for blaTEM and blaSHV genes mostly belonged to phylo-groups D and B2, whereas the diarrhea isolates were in phylo-groups D and A. Phylogenetic group D isolates have an accumulation of ESBLS genes in the diarrheic and UTI isolates. In both the UTI and diarrhea isolates, the maximum rate of resistance was against cefazolin, and the minimum rate of resistance was against nitrofurantoin. Twenty-four antibiotic resistance patterns were observed among the isolates. The amikacin, ciprofloxacin, cefotaxime, cefuroxime, cefazolin, gentamicin, nalidixic acid and trimethoprim/sulfamethoxazole resistance pattern was the most prevalent in the isolates that belonged to phylo-group D. The correct choice of effective

Determinação da produção de metalo-beta-lactamases em amostras de Pseudomonas aeruginosa isoladas em João Pessoa, Paraíba

OpenAIRE

Santos Filho,Lauro; Santos,Isabele Beserra; Assis,Alexandro Mangueira Lima de; Xavier,Danilo Elias

2002-01-01

Bactérias produtoras de metalo-beta-lactamases (MBLs) são em grande parte resistentes aos betalactâmicos de largo espectro, incluindo oximino-aminotiazol cefalosporinas e também aos carbapenens. O objetivo deste trabalho foi detectar cepas de Pseudomonas aeruginosa resistentes ao imipenem e à ceftazidima, assim como identificar aquelas produtoras de MBLs. Foram estudadas 198 linhagens não-repetitivas isoladas de diversas amostras clínicas, hospitalares e comunitárias, identificadas bioquimica...

Identification of DHA-23, a Novel Plasmid-mediated and Inducible AmpC beta-Lactamase from Enterobacteriaceae in Northern Taiwan

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Wen-Shyang eHsieh

2015-05-01

Full Text Available Objectives: AmpC β-lactamases are classified as Amber Class C and Bush Group 1. AmpC β-lactamases can hydrolyze broad and extended-spectrum cephalosporins, and are not inhibited by β-lactamase inhibitors such as clavulanic acid. This study was conducted to identify DHA-23, a novel plasmid-mediated and inducible AmpC β-lactamase obtained from Enterobacteriaceae. Methods: A total of 210 carbapenem-resistant Enterobacteriaceae isolates were collected from a medical center (comprising 2 branches in Northern Taiwan during 2009–2012. AmpC β-lactamase genes were analyzed through a polymerase chain reaction using plasmid DNA templates and gene sequencing. The genetic relationships of the isolates were typed using pulsed-field gel electrophoresis following the digestion of intact genomic DNA by using XbaI. Results: Three enterobacterial isolates (one Escherichia coli and 2 Klebsiella pneumoniae were obtained from 3 hospitalized patients. All 3 isolates were resistant or intermediately susceptible to all β-lactams, and exhibited reduced susceptibility to carbapenems. These 3 isolates expressed a novel AmpC β-lactamase, designated DHA-23, approved by the curators of the Lahey website. DHA-23 differs from DHA-1 and DHA-6 by one amino acid substitution (Ser245Ala, exhibiting 2 amino acid changes compared with DHA-7 and DHA-Morganella morganii; 3 amino acid changes compared with DHA-3; 4 amino acid changes compared with DHA-5; and 8 amino acid changes compared with DHA-2 (> 97% identity. This AmpC β-lactamase is inducible using a system involving ampR. Conclusion: This is the first report to address DHA-23, a novel AmpC β-lactamase. DHA-type β-lactamases are continuous threat in Taiwan.

Detection of cfxA2, cfxA3, and cfxA6 genes in beta-lactamase producing oral anaerobes

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Buhle BINTA

2016-04-01

Full Text Available ABSTRACT Purpose The aim of this study was to identify β-lactamase-producing oral anaerobic bacteria and screen them for the presence of cfxA and BlaTEM genes that are responsible for β-lactamase production and resistance to β-lactam antibiotics. Material and Methods Periodontal pocket debris samples were collected from 48 patients with chronic periodontitis and anaerobically cultured on blood agar plates with and without β-lactam antibiotics. Presumptive β-lactamase-producing isolates were evaluated for definite β-lactamase production using the nitrocefin slide method and identified using the API Rapid 32A system. Antimicrobial susceptibility was performed using disc diffusion and microbroth dilution tests as described by CLSI Methods. Isolates were screened for the presence of the β-lactamase-TEM (BlaTEM and β-lactamase-cfxA genes using Polymerase Chain Reaction (PCR. Amplified PCR products were sequenced and the cfxA gene was characterized using Genbank databases. Results Seventy five percent of patients carried two species of β-lactamase-producing anaerobic bacteria that comprised 9.4% of the total number of cultivable bacteria. Fifty one percent of β-lactamase-producing strains mainly Prevotella, Porphyromonas, and Bacteroides carried the cfxA gene, whereas none of them carried blaTEM. Further characterization of the cfxA gene showed that 76.7% of these strains carried the cfxA2 gene, 14% carried cfxA3, and 9.3% carried cfxA6. The cfxA6 gene was present in three Prevotella spp. and in one Porphyromonas spp. Strains containing cfxA genes (56% were resistant to the β-lactam antibiotics. Conclusion This study indicates that there is a high prevalence of the cfxA gene in β-lactamase-producing anaerobic oral bacteria, which may lead to drug resistance and treatment failure.

Prevalence of AmpC and other beta-lactamases in enterobacteria at a large urban university hospital in Brazil

OpenAIRE

Dias, Rubens Clayton da Silva; Borges-Neto, Armando Alves; Ferraiuoli, Giovanna Ianini D’Almeida; de-Oliveira, Márcia P.; Riley, Lee W.; Moreira, Beatriz Meurer

2007-01-01

Production of extended-spectrum β-lactamases (ESBL) has been reported in virtually all species of Enterobacteriaceae, which greatly complicates the therapy of infections caused by these organisms. However, the frequency of isolates producing AmpC β-lactamases, especially plasmid mediated AmpC (pAmpC), is largely unknown. These β-lactamases confer resistance to extended spectrum cephalosporins and aztreonam, a multidrug-resistant (MDR) profile. The aim of the present study was to determine the...

Transposon domestication versus mutualism in ciliate genome rearrangements.

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Alexander Vogt

Full Text Available Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling.

Extended-spectrum beta-lactamase (ESBL)-positive Enterobacteriaceae in municipal sewage and their emission to the environment.

Science.gov (United States)

Korzeniewska, Ewa; Harnisz, Monika

2013-10-15

The spread of Gram-negative bacteria with plasmid-borne extended-spectrum beta-lactamases (ESBLs) has become a worldwide problem. Their prevalence is increasing, both in hospitals and in the environment. The aim of this study was to investigate the presence of ESBL-positive Enterobacteriaceae in municipal sewage and their emission to the ambient air and the river receiving effluent from wastewater treatment plant (WWTP). In the group of 455 isolated strains, up to 19.8% (90 isolates) were phenotypic ESBL-producers. They were detected in the 63 (100%) of sewage samples analyzed, 7 (33.3%) of river water and in 10 (23.8%) of air samples collected at the WWTP area. The plasmid-mediated genes encoding beta-lactams resistance were detected in almost 10% out of bacteria of the WWTP's final effluents and in above 32% out of bacteria of air at the WWTP area. It confirms that those genes are released into the environment, which might facilitate further dissemination among environmental bacteria. Moreover, genes encoding antibiotic resistance were shown to be transferrable to an Escherichia coli recipient strain, which indicates a high possibility of horizontal gene transfer among strains of different genera within the sewage and environmental samples. This study demonstrated that despite the treatment, the municipal sewage may be a reservoir of antibiotic-resistant microorganisms and plasmid-mediated antibiotic resistance genes. This may pose a public health risk, which requires future evaluation and control. Copyright © 2013 Elsevier Ltd. All rights reserved.

High diversity of beta-lactamases in the General Hospital Vienna verified by whole genome sequencing and statistical analysis.

Science.gov (United States)

Barišić, Ivan; Mitteregger, Dieter; Hirschl, Alexander M; Noehammer, Christa; Wiesinger-Mayr, Herbert

2014-10-01

The detailed analysis of antibiotic resistance mechanisms is essential for understanding the underlying evolutionary processes, the implementation of appropriate intervention strategies and to guarantee efficient treatment options. In the present study, 110 β-lactam-resistant, clinical isolates of Enterobacteriaceae sampled in 2011 in one of Europe's largest hospitals, the General Hospital Vienna, were screened for the presence of 31 β-lactamase genes. Twenty of those isolates were selected for whole genome sequencing (WGS). In addition, the number of β-lactamase genes was estimated using biostatistical models. The carbapenemase genes blaKPC-2, blaKPC-3, and blaVIM-4 were identified in carbapenem-resistant and intermediate susceptible isolates, blaOXA-72 in an extended-spectrum β-lactamase (ESBL)-positive one. Furthermore, the observed high prevalence of the acquired blaDHA-1 and blaCMY AmpC β-lactamase genes (70%) in phenotypically AmpC-positive isolates is alarming due to their capability to become carbapenem-resistant upon changes in membrane permeability. The statistical analyses revealed that approximately 55% of all β-lactamase genes present in the General Hospital Vienna were detected by this study. In summary, this work gives a very detailed picture on the disseminated β-lactamases and other resistance genes in one of Europe's largest hospitals. Copyright © 2014 Elsevier B.V. All rights reserved.

Emergence of New Delhi metalo-β- lactamase (NDM-1) – producing ...

African Journals Online (AJOL)

The New Delhi metallo-beta- lactamase (NDM-1) gene is an emerging well acknowledged public health threats among human and animal pathogens worldwide. Since its first ... The Gram negative bacteria were. identified by conventional bacteriological procedures and with16s RNA PCR method. In all, 7.3% (4/55) of the ...

Multiplex PCR Study of Plasmid-Mediated AmpC Beta-Lactamases Genes in Clinical Isolates of Escherichia coli

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Maryam Dehghani

2017-02-01

Full Text Available Background:   AmpC β-lactamases are important cephalosporinases chromosomally encoded in many of Enterobacteriaceae and a few other organisms where they mediate resistance to cephalothin, cefazolin, cefoxitin and penicillins. The six different families of plasmid-mediated AmpC β-lactamases have been described, but no phenotypic test can discriminate among them. AmpC multiplex PCR has been successfully used to discriminate plasmid-mediated ampC specific families in organisms such as Klebsiella pneumonia and Escherichia coli. The aim of this study was to indicate the prevalence of AmpC β-lactamase genes by specifically designed primers through PCR test.Methods:   243 total clinical urine samples were collected, and 227 isolates were identified as Escherichia coli based on standard biochemical tests. Subsequently, the isolates were screened by disc diffusion and combined disc test for β-lactamase production. Resistant isolates were evaluated by PCR for ampC family determination. Results:  Antibiotic resistance pattern were observed as follows: cefepime (%25, ceftazidime (%31, ceftriaxone (%37, cefotaxime (%38. The ratio of isolates was detected as ESBLs and AmpC producers were 34% and 5.2%, respectively. PCR performed on 12 selected isolates via phenotypic tests and the results revealed that among 12 isolates, 11 contained blaCMY-42. Conclusion:  Unfortunately, antibiotic resistance has become an increasingly critical problem in many countries like Iran and occurrence of isolates co-expressing AmpC-β-lactamases and ESBLs can create serious problems in the future. As antibiotic options in the treatment of AmpC β-lactamases and ESBLs producing organisms are extremely limited, molecular screening by laboratories is suggested to reduce the risk of therapeutic defeat.

Biochemical and Structural Characterization of Mycobacterium tuberculosis beta-Lactamase with the Carbapenems Ertapenem and Doripenem

Energy Technology Data Exchange (ETDEWEB)

L Tremblay; F Fan; J Blanchard

2011-12-31

Despite the enormous success of {beta}-lactams as broad-spectrum antibacterials, they have never been widely used for the treatment of tuberculosis (TB) due to intrinsic resistance that is caused by the presence of a chromosomally encoded gene (blaC) in Mycobacterium tuberculosis. Our previous studies of TB BlaC revealed that this enzyme is an extremely broad-spectrum {beta}-lactamase hydrolyzing all {beta}-lactam classes. Carbapenems are slow substrates that acylate the enzyme but are only slowly deacylated and can therefore act also as potent inhibitors of BlaC. We conducted the in vitro characterization of doripenem and ertapenem with BlaC. A steady-state kinetic burst was observed with both compounds with magnitudes proportional to the concentration of BlaC used. The results provide apparent K{sub m} and k{sub cat} values of 0.18 {micro}M and 0.016 min{sup -1} for doripenem and 0.18 {micro}M and 0.017 min{sup -1} for ertapenem, respectively. FTICR mass spectrometry demonstrated that the doripenem and ertapenem acyl-enzyme complexes remain stable over a time period of 90 min. The BlaC-doripenem covalent complex obtained after a 90 min soak was determined to 2.2 {angstrom}, while the BlaC-ertapenem complex obtained after a 90 min soak was determined to 2.0 {angstrom}. The 1.3 {angstrom} diffraction data from a 10 min ertapenem-soaked crystal revealed an isomerization occurring in the BlaC-ertapenem adduct in which the original {Delta}2-pyrroline ring was tautomerized to generate the {Delta}1-pyrroline ring. The isomerization leads to the flipping of the carbapenem hydroxyethyl group to hydrogen bond to carboxyl O2 of Glu166. The hydroxyethyl flip results in both the decreased basicity of Glu166 and a significant increase in the distance between carboxyl O2 of Glu166 and the catalytic water molecule, slowing hydrolysis.

Relation between the 2{nu}{beta}{beta} and 0{nu}{beta}{beta} nuclear matrix elements

Energy Technology Data Exchange (ETDEWEB)

Vogel, Petr [Kellogg Radiation Laboratory, Caltech, Pasadena, CA 91125 (United States); Simkovic, Fedor [Department of Nuclear Physics and Biophysics, Comenius University, Mlynska dolina F1, SK-84248 Bratislava (Slovakia)

2011-12-16

A formal relation between the GT part of the nuclear matrix elements M{sub GT}{sup 0{nu}} of 0{nu}{beta}{beta} decay and the closure matrix elements M{sub cl}{sup 2{nu}} of 2{nu}{beta}{beta} decay is established. This relation is based on the integral representation of these quantities in terms of their dependence on the distance r between the two nucleons undergoing transformation. We also discuss the difficulties in determining the correct values of the closure 2{nu}{beta}{beta} decay matrix elements.

Detection of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Market-Ready Chickens in Zambia.

Science.gov (United States)

Chishimba, K; Hang'ombe, B M; Muzandu, K; Mshana, S E; Matee, M I; Nakajima, C; Suzuki, Y

2016-01-01

The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains. The objective of this study was to detect the presence of extended-spectrum β-lactamase- (ESBL-) producing Escherichia coli in poultry in Zambia. A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichia coli. The cultured E. coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detection of bla CTX-M, bla SHV, and bla TEM genes. Overall 20.1%, 77/384, (95% CI; 43.2-65.5%) of total samples analyzed contained ESBL-producing Escherichia coli. The antimicrobial sensitivity test revealed that 85.7% (66/77; CI: 75.7-92) of ESBL-producing E. coli isolates conferred resistance to beta-lactam and other antimicrobial agents. These results indicate that poultry is a potential reservoir for ESBL-producing Escherichia coli. The presence of ESBL-producing Escherichia coli in poultry destined for human consumption requires strengthening of the antibiotic administering policy. This is important as antibiotic administration in food animals is gaining momentum for improved animal productivity in developing countries such as Zambia.

Detection of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Market-Ready Chickens in Zambia

Directory of Open Access Journals (Sweden)

K. Chishimba

2016-01-01

Full Text Available The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains. The objective of this study was to detect the presence of extended-spectrum β-lactamase- (ESBL- producing Escherichia coli in poultry in Zambia. A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichia coli. The cultured E. coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detection of blaCTX-M, blaSHV, and blaTEM genes. Overall 20.1%, 77/384, (95% CI; 43.2–65.5% of total samples analyzed contained ESBL-producing Escherichia coli. The antimicrobial sensitivity test revealed that 85.7% (66/77; CI: 75.7–92 of ESBL-producing E. coli isolates conferred resistance to beta-lactam and other antimicrobial agents. These results indicate that poultry is a potential reservoir for ESBL-producing Escherichia coli. The presence of ESBL-producing Escherichia coli in poultry destined for human consumption requires strengthening of the antibiotic administering policy. This is important as antibiotic administration in food animals is gaining momentum for improved animal productivity in developing countries such as Zambia.

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

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Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad

2014-11-01

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. Copyright © 2014 Elsevier GmbH. All rights reserved.

Prevalence of beta-lactams resistance among Escherichia coli clinical isolates from a hospital in Algiers.

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Messai, Y; Benhassine, T; Naim, M; Paul, G; Bakour, R

2006-06-01

A high prevalence of beta-lactams resistance among Enterobacteriaceae have been reported worldwide; however, there are not sufficient data on this issue in Algeria. beta-Lactams susceptibility of 203 Escherichia coli clinical isolates was determined by agar diffusion method, and production of extended-spectrum beta-lactamases (ESBL) was screened by double-disk synergy test. This analysis showed five well-defined phenotypes: 1) 62 isolates (30.5%) were susceptible to all beta-lactams; 2) 135 isolates (66.5%) presented a broad-spectrum beta-lactamases phenotype (BSBL); 3) three isolates (1.5%) were defined as producing ESBLs; 4) two isolates (1%) were AmpC cephalosporinase producers; and 5) one isolate (0.5%) presented a phenotype of cell-decreased permeability to beta-lactams. Isoelectric focusing revealed beta-lactamases with isolectric points of 5.4 or 7.6 for isolates with BSBL phenotype; approximately 9.0 for two ESBL isolates; 5.4, 7.6 and approximately 9.0 for the remaining ESBL isolate; and 5.4 and approximately 9.0 for the AmpC isolates. The cefotaxime hydrolysis corresponds to the basic bands with an isoelectric point of approximately 9.0. Conjugation assay showed transfer of penicillinase and AmpC resistance phenotypes and their corresponding beta-lactamases to recipient E. coli BM21 in association with plasmids of 71.4 kb for the AmpC isolates and from 40-56 kb for penicillinase isolates. This result showed that the AmpC phenotype is plasmid mediated. ESBL isolates were found not to transfer their resistance through conjugation experiment. Polymerase chain reaction (PCR) experiments using primers specific to blaTEM, blaAmpC and blaCTX-M genes showed specific amplification with blaCTX-M primer for two ESBL isolates; blaTEM and blaCTX-M for the remaining ESBL isolate; and blaTEM and blaAmpC for the AmpC isolates and their corresponding transconjugants. The study showed a high rate of isolates producing penicillinase, and low frequencies of AmpC and ESBL

Nosocomial extended-spectrum beta-lactamase-producing Klebsiella pneumoniae bacteremia in hemodialysis patients and the implications for antibiotic therapy.

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Yang, Chih-Chao; Wu, Chien-Hsing; Lee, Chien-Te; Liu, Han-Tsung; Chen, Jin-Bor; Chiu, Chien-Hua; Chen, Chih-Hung; Chuang, Feng-Rong

2014-11-01

In the face of increasing treatment options for extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) hemodialysis (HD) access-related bacteremia, the difference in clinical effectiveness between ertapenem and flomoxef remains unclear. We conducted this retrospective study to determine their efficacies and treatment outcomes. Patients on maintenance HD with fistula-, graft-, or catheter-related ESBL-Kp bacteremia were enrolled. Data related to clinical features and antibiotic treatments were collected. Outcome was determined by mortality resulting from bacteremia during the 14-day period after the collection of the first positive blood culture for flomoxef-susceptible ESBL-Kp. The 64 patients studied had severe septicemia as determined by the Pitt bacteremia score; 50% (32/64) were in the intensive care unit (ICU) at the time of bacteremia. Old age (>65 years; 57.8%), malnutrition (albumin30 days; 75%) were also highly prevalent. The study population comprised nine fistula-, 10 graft-, and 45 HD catheter-related bacteremia cases, and the mortality rate was high (38/64, 59.4%). The mortality rate was significantly higher in the flomoxef treatment group than in the ertapenem treatment group (22/30, 73% vs. 16/34, 47%, pflomoxef use (odds ratio (OR) 2.52, 95% confidence interval (CI) 1.34-35.17) and Pitt bacteremia score (OR 4.37, 95% CI 1.28-5.26) were independently associated with mortality. In accordance with our previous study, our results have demonstrated the inferiority of flomoxef to carbapenems in the treatment of HD access-related ESBL-Kp bacteremia and provide an insight into the possibility of using ertapenem rather than flomoxef as an initial or de-escalating therapy for infections caused by ESBL-producing bacteria.

Biosynthesis of ketomycin. (II) biomimetic model for beta-lactamase catalysis: host-guest interactions in cyclodextrin-penicillin inclusion complex

International Nuclear Information System (INIS)

Mak, H.W.

1986-01-01

The antibiotic ketomycin is formed from shikimic acid via chorismic acid and prephenic acid. Phenylalanine and 2',5'-dihydrophenylalanine derived from shikimic acid are not intermediates in the biosynthesis. Degradation of ketomycin derived from [1,6- 14 C]shikimic acid showed that prephenic acid is converted into ketomycin with stereospecific discrimination between the two enantiotopic edges of the ring, the pro-S-R edge giving rise to the C-2', C-3' side of the cyclohexane ring of ketomycin. The resistance of pathogenic bacteria to the action of β-lactam antibiotics is mainly ascribed to their ability to produce β-lactamase to cleave the β-lactam ring. It is essential to understand the molecular nature of β-lactamase-penicillin recognition for designing and formulating more effective β-lactam antibiotics. A biomimetic study of β-lactamase is therefore initiated. To meet the requirements of hydrophobic and serine protease characteristics of β-lactamase, α-cyclodextrin is chosen as a biomimetic model for β-lactamase. The structural specificity and the chemical dynamics of α-cyclodextrin-phenoxymethyl penicillin inclusion complex in solid state and in solution have been determined by IR and NMR spectroscopy. The spectral results strongly indicate that the phenyl portion of the phenoxymethyl penicillin forms a stable inclusion complex with the hydrophobic cavity of α-cyclodextrin in solution as well as in the solid state. Kinetic studies followed by 1 HNMR and HPLC analyses under alkaline condition have shown that the α-cyclodextrin mimics the catalytic function of serine of β-lactamase in the stereospecific hydrolysis of the β-lactam ring of phenoxymethyl penicillin

Emergence of extended spectrum beta-lactamases-producing strains belonging to cefotaxime-M-1 class from intensive care units patients and environmental surfaces in Pakistan

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Aqsa Ashraf Bukhari

2016-11-01

Full Text Available Aim: The emergence of multidrug-resistant (MDR bacteria is the most dangerous threat for the treatment of infectious diseases. The aim of this study was to detect and characterize extended spectrum beta-lactamases (ESBLs and carbapenemase-producing Klebsiella pneumoniae and Escherichia coli among patients and environment of intensive care units (ICUs of three tertiary care hospitals in Pakistan. Materials and Methods: A total of 82 samples from ICU’s patients and inanimate environment (injection trays, wash basins, door handles, hand swabs of professionals, and ICU fridges were screened for ESBL by culturing on CHROMagar-ESBL. ESBL and carbapenemases production were confirmed by double disc synergy test and modified Hodge’s test, respectively. Polymerase chain reaction was used to detect ESBL encoding genes bla cefotaxime (CTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaTEM, blaSHV and carbapenemase genes blaKPC, bla New Delhi metallo-beta-lactamase-1, blaOXA-48 and blaVIM. Results: Overall, ESBL production was found high 30/82 (36.5% among isolates of which 15.8% K. pneumoniae and 20.7% E. coli were identified. All the K. pneumoniae and majority of E. coli isolates were MDR, i.e., resistance to three or more antimicrobial categories. Molecular characterization showed the blaCTX-M-1 as the predominant genotype found in 17/21 (80% of the isolates. None of the strains was found positive for carbapenemase-encoding genes. Conclusion: In conclusion, this study demonstrates the emergence of MDR ESBL producing strains among ICU patients and hospital environment, posing a serious threat for the control of nosocomial infections.

Longitudinal study of extended-spectrum-β-lactamase- and AmpC-Producing Enterobacteriaceae in household dogs

NARCIS (Netherlands)

Baede, V.O.; Wagenaar, J.A.; Broens, E.M.; Duim, Birgitta; Dohmen, Wietske; Nijsse, Rolf; Timmerman, Arjen J.; Hordijk, Joost

2015-01-01

A longitudinal study was performed to (i) investigate the continuity of shedding of extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceae in dogs without clinical signs, (ii) identify dominant plasmid-mediated ESBL genes, and (iii) quantify ESBL-producing Enterobacteriaceae in

[Antimicrobial activity of fosfomycin under various conditions against standard strains, beta-lactam resistant strains, and multidrug efflux system mutants].

Science.gov (United States)

Mikuniya, Takeshi; Hiraishi, Toru; Maebashi, Kazunori; Ida, Takashi; Takata, Toshihiko; Hikida, Muneo; Yamada, Sakuo; Gotoh, Naomasa; Nishino, Takeshi

2005-04-01

The purpose of this study was to evaluate the possible benefit of fosfomycin (FOM) as prophylactic antibiotic in terms of antimicrobial activity and the potential of inducibility of beta-lactamase, compared with cefazolin, cefotiam, cefmetazole, and piperacillin that are commonly used as perioperative agents. The in vitro activity of FOM against aerobic Gram-negative bacteria using Mueller-Hinton agar or nutrient agar supplemented with glucose-6-phosphate (G6P) as tested medium increased within a range from 2 to 256 times the activity in the medium without G6P. However, the susceptibility of Gram-positive bacteria to FOM remained largely unchanged with or without G6P. There was no aerobic- or anaerobic-bacteria which changed susceptibility against beta-lactam antibiotics under various tested medium conditions. FOM demonstrated strong bactericidal activity against Escherichia coli and Pseudomonas aeruginosa in a dose dependent manner, and decreased viable cell counts of Staphylococcus aureus. In the case of P. aeruginosa, transmission electron micrographs study revealed that numerous lysed cells were present 2 hours after treatment with FOM at four times the MIC. First and second generation cephalosporins induced AmpC-type beta-lactamase in a dose dependent manner among beta-lactamase inducible strains of P. aeruginosa and Enterobacter cloacae. On the other hand, inducible activity of FOM on beta-lactamase production was less than 1/25 to 1/65 compared with those of cephalosporins. In addition, FOM maintained strong antimicrobial activity for over then 20 years after marketing, because of the excellent stability against various types of beta-lactamase produced by plasmid-carrying bacteria and clinical isolates. FOM was not extruded by four types of efflux systems, such as MexAB-OprM, MexCD-OprJ, MexXY/ OprM and MexEF-OprN, however beta-lactam antibiotics were substrates of MexAB-OprM and MexCD-OprJ. In conclusion, FOM provides adequate coverage for both aerobic Gram

Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients

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Varaiya Ami

2008-04-01

Full Text Available Metallo-beta-lactamase (MBL-producing Pseudomonas aeruginosa strains have been reported to be an important cause of nosocomial infections. There is not enough information from India regarding their prevalence in diabetic and cancer patients. The present study was undertaken over a period of one year from January to December 2006 to study the incidence of MBL P. aeruginosa and the clinical outcome in diabetes and cancer patients admitted to S.L. Raheja Hospital, Mumbai. Two hundred and thirty isolates of P. aeruginosa were obtained from different samples of patients. These isolates were subjected to susceptibility testing to anti-pseudomonal drugs as per CLSI guidelines. They were further screened for the production of MBL by disc potentiation testing using EDTA-impregnated imipenem and meropenem discs. Of the 230 isolates of P. aeruginosa, 60 (26% isolates were found resistant to carbapenems (both imipenem and meropenem and 33 (14.3% were found to be MBL producers. Of the 33 MBL-producing isolates, 24 (72.7% were diabetic patients, six (18.1% were cancer patients and three (9% patients had both diabetes and cancer. Five (15.1% patients responded to the combination therapy of colistin, piperacillin with tazobactam and amikacin, while 28 (84.8% patients responded to the combination therapy of amikacin, piperacillin with tazobactam and gatifloxacin. Thus, the rapid dissemination of MBL producers is worrisome and necessitates the implementation of not just surveillance studies but also proper and judicious selection of antibiotics, especially carbapenems.

New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae: emergence and response in Europe.

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Struelens, M J; Monnet, D L; Magiorakos, A P; Santos O'Connor, F; Giesecke, J

2010-11-18

Acquired carbapenemases confer extensive antibiotic resistance to Enterobacteriaceae and represent a public health threat. A novel acquired carbapenemase, New Delhi metallo-beta-lactamase 1 (NDM-1), has recently been described in the United Kingdom and Sweden, mostly in patients who had received care on the Indian subcontinent. We conducted a survey among 29 European countries (the European Union Member States, Iceland and Norway) to gather information on the spread of NDM-1-producing Enterobacteriaceae in Europe, on public health responses and on available national guidance on detection, surveillance and control. A total of 77 cases were reported from 13 countries from 2008 to 2010. Klebsiella pneumoniae was the most frequently reported species with 54%. Among 55 cases with recorded travel history, 31 had previously travelled or been admitted to a hospital in India or Pakistan and five had been hospitalised in the Balkan region. Possible nosocomial acquisition accounted for 13 of 77 cases. National guidance on NDM-1 detection was available in 14 countries and on NDM-1 control in 11 countries. In conclusion, NDM-1 is spreading across Europe, where it is frequently linked to a history of healthcare abroad, but also to emerging nosocomial transmission. National guidance in response to the threat of carbapenemase-producing Enterobacteriaceae is available in approximately half of the surveyed European countries. Surveillance of carbapenemase- producing Enterobacteriaceae must be enhanced in Europe and effective control measures identified and implemented.

Extended-spectrum beta-lactamase- and carbapenemase-producing Enterobacteriaceae among Ethiopian children

Science.gov (United States)

Legese, Melese Hailu; Weldearegay, Gebru Mulugeta; Asrat, Daniel

2017-01-01

Background Infections by extended-spectrum beta-lactamase- (ESBL) and carbapenem-resistant Enterobacteriaceae (CRE) are an emerging problem in children nowadays. Hence, the aim of this study was to determine the prevalence of ESBL- and carbapenemase-producing Enterobacteriaceae among children suspected of septicemia and urinary tract infections (UTIs). Methods A cross-sectional study was conducted from January to March 2014. A total of 322 study participants suspected of septicemia and UTIs were recruited. All blood and urine samples were cultured on blood and MacConkey agar. All positive cultures were characterized by colony morphology, Gram stain, and standard biochemical tests. Antimicrobial susceptibility test was performed on Muller-Hinton agar using disk diffusion. ESBL was detected using combination disk and double-disk synergy methods, and the results were compared. Carbapenemase was detected by modified Hodge method using meropenem. Data were analyzed using SPSS version 20. Results The overall prevalence of ESBL- and carbapenemase-producing Enterobacteriaceae was 78.57% (n=22/28) and 12.12%, respectively. Among the Enterobacteriaceae tested, Klebsiella pneumoniae (84.2%, n=16/19), Escherichia coli (100%, n=5/5), and Klebsiella oxytoca (100%, n=1/1) were positive for ESBL. Double-disk synergy method showed 90.9% sensitivity, 66.7% specificity, 95.2% positive predictive value, and 50% negative predictive value. Carbapenemase-producing Enterobacteriaceae were K. pneumoniae (9.09%, n=3/33) and Morganella morganii (3.03%, n=1/33). Conclusion Screening Enterobacteriaceae for ESBL production is essential for better antibiotics selection and preventing its further emergence and spread. In resource-limited settings, double-disk synergy method can be implemented for screening and confirming ESBL production. Moreover, occurrence of CRE in countries where no carbapenems are sold is worrying microbiologists as well as clinicians. Hence, identifying factors that induce

Collateral damage of flomoxef therapy: in vivo development of porin deficiency and acquisition of blaDHA-1 leading to ertapenem resistance in a clinical isolate of Klebsiella pneumoniae producing CTX-M-3 and SHV-5 beta-lactamases.

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Lee, Chen-Hsiang; Chu, Chishih; Liu, Jien-Wei; Chen, Yi-Shung; Chiu, Chiung-Jung; Su, Lin-Hui

2007-08-01

The study aimed to characterize the genetic basis of flomoxef and collateral ertapenem resistance in a clinical isolate of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) after flomoxef exposure. Four ESBL-KP isolates (Lkp11-14) were recovered sequentially from four episodes of bacteraemia in an elderly patient. Laboratory investigations included genotyping by PFGE, resistance gene analysis by PCR and sequencing, and outer membrane protein analysis by SDS-PAGE. Plasmid analysis by DNA-DNA hybridization, electroporation and conjugation was also performed. Lkp14 was recovered after 21 days of flomoxef therapy. It demonstrated an indistinguishable PFGE pattern when compared with those produced by Lkp11-13. However, resistance to both flomoxef and ertapenem emerged in Lkp14. Molecular characterization revealed that, in addition to the pre-existing ESBL production (CTX-M-3 and SHV-5) and OmpK35 deficiency found in Lkp11-13, Lkp14 had acquired an extra plasmid-mediated AmpC beta-lactamase gene (blaDHA-1) and failed to express OmpK36, because of insertional inactivation by an insertion sequence IS5. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Conjugational transfer of the plasmid-mediated blaDHA-1 gene into Lkp11 resulted in a significant increase in the MICs of cephamycins and beta-lactamase inhibitors, but not in those of carbapenems. Lkp14 was apparently derived from the previously flomoxef-susceptible isolates, Lkp11-13. After flomoxef exposure, the in vivo acquisition of the plasmid-mediated blaDHA-1 gene has led to flomoxef resistance in Lkp14, and the concomitant depletion of OmpK36 expression has resulted in a collateral effect of ertapenem resistance and diminished susceptibilities to imipenem and meropenem.

Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

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Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

2013-01-01

Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

Dual beta-lactam therapy for serious Gram-negative infections: is it time to revisit?

Science.gov (United States)

Rahme, Christine; Butterfield, Jill M; Nicasio, Anthony M; Lodise, Thomas P

2014-12-01

We are rapidly approaching a crisis in antibiotic resistance, particularly among Gram-negative pathogens. This, coupled with the slow development of novel antimicrobial agents, underscores the exigency of redeploying existing antimicrobial agents in innovative ways. One therapeutic approach that was heavily studied in the 1980s but abandoned over time is dual beta-lactam therapy. This article reviews the evidence for combination beta-lactam therapy. Overall, in vitro, animal and clinical data are positive and suggest that beta-lactam combinations produce a synergistic effect against Gram-negative pathogens that rivals that of beta-lactam-aminoglycoside or beta-lactam-fluoroquinolone combination therapy. Although the precise mechanism of improved activity is not completely understood, it is likely attributable to an enhanced affinity to the diverse penicillin-binding proteins found among Gram negatives. The collective data indicate that dual beta-lactam therapy should be revisited for serious Gram-negative infections, especially in light of the near availability of potent beta-lactamase inhibitors, which neutralize the effect of problematic beta-lactamases. Copyright © 2014 Elsevier Inc. All rights reserved.

Immobilization of the enzyme {beta}-lactamase by self-assembly on thin films of a poly(phenyleneethynylene) sequenced with flexible segments containing sulfur atoms

Energy Technology Data Exchange (ETDEWEB)

Vazquez, Erika [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico); Aguilar, Abdieel Esquivel [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico); Facultad de Ciencias Quimicas, Universidad Autonoma de Coahuila, Blvd. V. Carranza and Ing. J. Cardenas, 25000 Saltillo (Mexico); Moggio, Ivana [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico)]. E-mail: imoggio@ciqa.mx; Arias, Eduardo [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico); Romero, Jorge [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico); Barrientos, Hector [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico); Torres, Jose Roman [Centro de Investigacion en Quimica Aplicada (CIQA), Blvd. Enrique Reyna 140, 25253, Saltillo (Mexico); Luz Reyes Vega, Maria de la [Facultad de Ciencias Quimicas, Universidad Autonoma de Coahuila, Blvd. V. Carranza and Ing. J. Cardenas, 25000 Saltillo (Mexico)

2007-05-16

A novel poly(phenyleneethynylene) sequenced in the main conjugated chain with flexible groups containing sulfur atoms has been synthesized by Heck-Sonogashira coupling reaction. Layer-by-layer films of the polymer have been prepared with a linear growth in thickness up to four layers as evidenced by UV-Vis spectroscopy and perfilometry. On the top of these multilayers, the enzyme {beta}-lactamase was deposited by self-assembly. The enzymatic activity was measured by a modified spectrophotometric standard assay method for penicillin G, ampicillin and amoxicillin. A higher and faster activity was obtained for penicillin G and thus preliminary study of the biosensor response by fluorescence was carried out for this antibiotic revealing a decrease in the polymer fluorescence as function of the penicillin G concentration.

Characterization of extended-spectrum β-lactamases (ESBLs)-producing Salmonella in retail raw chicken carcasses.

Science.gov (United States)

Qiao, Jing; Zhang, Qiang; Alali, Walid Q; Wang, Jiawei; Meng, Lingyuan; Xiao, Yingping; Yang, Hua; Chen, Sheng; Cui, Shenghui; Yang, Baowei

2017-05-02

Extended-spectrum β-lactamases (ESBLs)-producing Salmonella is considered a serious concern to public health worldwide. However, limited information is available on ESBLs-producing Salmonella in retail chicken products in China. The objective of this study was to characterize ESBLs-producing Salmonella isolates from retail chickens in China. A total of 890 Salmonella isolates from retail chicken carcasses collected from 4 provinces were firstly screened for ESBLs-production phenotype via the double-disk synergy test method. A total of 96 (10.8%, n=890) ESBLs-producing Salmonella were identified and subjected to PFGE analysis, characterization for the presence of ESBLs encoding genes, transposons, carbapenemase and virulence genes. A total of 59 PFGE profiles were detected in these 96 isolates, among which 57.3% were found to harbor bla TEM-1 , whereas 30.2%, 24.0%, 18.8% and 7.3% were carrying bla OXA-1 , bla CTX-M-15 , bla CTX-M-3 and bla PSE-1 genes, respectively. Moreover, 42 (43.8%) isolates co-carried 2 ESBLs-producing genes, and two (2.1%) isolates co-carried 3 genes. Furthermore, 24 (25.0%) ESBLs-producing isolates carried VIM and 10 (10.4%) carried KPC encoding genes that closely associated with carbapenems resistance. Eighty-eight isolates harbored transposons ranging from 4.2% for Tn903 to 76.0% for Tn21. Out of the 88 Salmonella that harbored transposons, 25%, 22.7%, 23.9%, 10.2% and 1.1% of isolates were found to carry 2, 3, 4, 5 and 6 transposons, respectively. The minimum inhibitory concentration (MIC) values for cephalosporins (ceftriaxone, cefoperazone and cefoxitin) to ESBLs-producing isolates were from 4 to 1024μg/mL, for nalidixic acid were from 64 to 512μg/mL, for fluoroquinolones (ciprofloxacin, levofloxacin and gatifloxacin) were from 4 to 256μg/mL. Twenty-nine virulence genes were detected in the 96 ESBLs-producing isolates with 2.1% harbored spvR (lowest) and 90.6% harbored marT and steB (highest). All isolates carried at least one

Detection of Ambler class A, B and D ß-lactamases among Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates from burn patients.

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Hakemi Vala, M; Hallajzadeh, M; Hashemi, A; Goudarzi, H; Tarhani, M; Sattarzadeh Tabrizi, M; Bazmi, F

2014-03-31

In this study, we evaluated the existence of classes A, B and D ß-lactamases among Pseudomonas aeruginosa (P.aeruginosa) and Acinetobacter baumannii (A.baumannii) strains isolated from burn patients in Tehran during the years 2012 and 2013. From these strains, the frequency of MBL (metallo-beta-lactamase) and ESBL (extended-spectrum beta-lactamase) producers were evaluated using CDDT (Combined Disk Diffusion Tests). The prevalence of some related genes, including blaIMP, blaVIM, blaSPM, blaKPC, blaGIM, blaDIM, blaBIC, blaOXA-48, blaCTX-M-15 and blaNDM genes, was evaluated using PCR and sequencing methods. Of the 75 non-fermenter isolates, 47 P.aeruginosa and 28 A.baumannii were isolated and identified. A high rate of resistance to common antibiotics was detected among A.baumannii isolates in particular, showing 100% resistance to 9 tested antibiotics. CDDT showed that 21 (28%) and 25 (34.25%) of the non-fermenter isolates were ESBL and MBL producers respectively. The prevalence of blaCTX-M-15 and blaIMP genes among the 75 non-fermenter isolates was 7 (9.3%) and 1 (1.3%), respectively. Fortunately, no other genes were detected in either of the non-fermenters. The mortality rate due to MBL-producing isolates was 5 (20%). This study showed specific resistance genes exist among some MBL and ESBL gram-negative non-fermenters which were isolated from burn patients in Tehran.

Investigations of multiresistance to antibiotics and chemotherapeutics and extended spectrum beta: Lactamase effect (ESBL test in strains E.coli and salmonella originating from domestic animals

Directory of Open Access Journals (Sweden)

Mišić Dušan

2006-01-01

Full Text Available The presence of multiresistance to the effects of antibiotics and chemotherapeutics and extended spectrum beta-lactamase were examined in 45 strains of E. coli and 35 strains of Salmonella. The strains of E. coli originated from several species of domestic animals: dogs, cats, poultry, and cattle, and 30 strains of Salmonella originated from poultry, 4 strains from cattle, and 1 strain from swine. The presence of the following serovarieties was established using serological examinations: Salmonella Enteritidis 17 strains, Salmonella Gallinarum 1 strain, Salmonella Hartford 5 strains, Salmonella Anatum 1 strain, Salmonella Typhimurium 4 strains, Salmonella Agona 1 strain, Salmonella Infantis 1 strain, Salmonella Thompson var. Berlin 1 strain, Salmonella Tennessee 1 strain, Salmonella Senftenberg 1 strain, Salmonella Glostrup 1 strain, and Salmonella Hadar 1 strain. In the examinations of the listed strains we used antibiogram discs of ampicillin, amoxicillin with clavulanic acid, cephalexin, cephtriaxon, cephotaxim, cephtazidime, aztreonam, gentamycin, chloramphenicol, tetracycline, cyprofloxacine, and a combination of sulphamethoxasole and trimethoprim. The lowest prevalence of multiresistance in E. Coli strains to 3 or more antibiotics was established in dogs 20%, and the highest in 60% strains originating from swine. In 62.88% strains of Salmonella we established sensitivity to all applied antibiotics. Resistance was also established in a small number of the examined strains to ampicillin (11 strains, to tetracycline (5 strains, to amoxicillin with clavulanic acid (5 strains, to sulphamethoxasole with trimethoprim (5 strains, to gentamycin (3 strains, and to cloramphenicol (1 strain. Of all the examined strains of Salmonella, 6 strains originating from poultry exhibited multiresistence. The presence of extended spectrum beta-lactamase effects examined using the ESBL test, was not established in strains of E. coli and Salmonella strains.

Antibiotic combinatorial approach utilized against extended spectrum beta-lactamase (ESBL bacteria isolates from Enugu, South Eastern Nigeria

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Ruth A. Afunwa

2014-04-01

Full Text Available Introduction: Antibiotic options in the treatment of extended spectrum beta-lactamase (ESBL producing bacteria are very limited. The purpose of this study was to analyze several commonly applied antibiotics in quite various novel combinations for use against ESBL-producing bacteria isolates.Methods: Total of 460 samples of urine, throat and anal swab were collected from volunteers and patients from nursery, primary and secondary schools and from other individuals in the community. Hospital and community isolates comprised of 65% and 35% respectively. The identification and characterization of the isolates were done by standard culturing and in vitro antibiotic sensitivity procedures.Results: The antibiotic combination studies showed that the combination of gentamicin with the other antibiotics had predominantly synergistic effects. The percentage synergistic effect for the combinations of gentamicin/pefloxacin was 69%, gentamicin/[Amoxicillin and clavulanic acid] 72%, gentamicin/ceftriaxone 68%, gentamicin/cefuroxime 81.9%, and gentamicin/ciprofloxacin 80.6%, against the community and hospital derived ESBL producing organisms of both Enterobacteriaceae and Pseudomonas species.Conclusion: Good antimicrobial monitoring exercise and corresponding antimicrobial screening activities should work towards a dynamic approach to generate effective treatment options using combination therapy.

Prevalence of AmpC and other beta-lactamases in enterobacteria at a large urban university hospital in Brazil

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Dias, Rubens Clayton da Silva; Borges-Neto, Armando Alves; Ferraiuoli, Giovanna Ianini D’Almeida; de-Oliveira, Márcia P.; Riley, Lee W.; Moreira, Beatriz Meurer

2010-01-01

Production of extended-spectrum β-lactamases (ESBL) has been reported in virtually all species of Enterobacteriaceae, which greatly complicates the therapy of infections caused by these organisms. However, the frequency of isolates producing AmpC β-lactamases, especially plasmid mediated AmpC (pAmpC), is largely unknown. These β-lactamases confer resistance to extended spectrum cephalosporins and aztreonam, a multidrug-resistant (MDR) profile. The aim of the present study was to determine the occurrence of ESBL and pAmpC β-lactamases in a hospital where MDR enterobacterial isolates recently emerged. A total of 123 consecutive enterobacterial isolates obtained from 112 patients at a university hospital in Rio de Janeiro, Brazil during March-June 2001 were included in the study. ESBL was detected by the addition of clavulanate to cephalosporin containing disks and by double diffusion. AmpC production was evaluated by a modified tridimensional test and a modified Hodge test. The presence of plasmid-mediated ampC β-lactamase genes was evaluated by multiplex-PCR. Sixty-five (53%) of 123 enterobacterial isolates were MDR, obtained from 56 patients. ESBL production was detected in 35 isolates; 5 clonal E. coli isolates exhibited high levels of chromosomal AmpC and ESBL production. However, no isolates contained pAmpC genes. Infection or colonization by MDR enterobacteria was not associated with any predominant resistant clones. A large proportion of hospital infections caused by ESBL-producing enterobacteria identified during the study period were due to sporadic infections rather than undetected outbreaks. This observation emphasizes the need to improve our detection methods for ESBL- and AmpC-producing organisms in hospitals where extended-spectrum cephalosporins are in wide use. PMID:17900845

Detection of Extended Spectrum Beta-Lactamases Resistance Genes among Bacteria Isolated from Selected Drinking Water Distribution Channels in Southwestern Nigeria.

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Adesoji, Ayodele T; Ogunjobi, Adeniyi A

2016-01-01

Extended Spectrum Beta-Lactamases (ESBL) provide high level resistance to beta-lactam antibiotics among bacteria. In this study, previously described multidrug resistant bacteria from raw, treated, and municipal taps of DWDS from selected dams in southwestern Nigeria were assessed for the presence of ESBL resistance genes which include bla TEM, bla SHV, and bla CTX by PCR amplification. A total of 164 bacteria spread across treated (33), raw (66), and municipal taps (68), belonging to α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Flavobacteriia, Bacilli, and Actinobacteria group, were selected for this study. Among these bacteria, the most commonly observed resistance was for ampicillin and amoxicillin/clavulanic acid (61 isolates). Sixty-one isolates carried at least one of the targeted ESBL genes with bla TEM being the most abundant (50/61) and bla CTX being detected least (3/61). Klebsiella was the most frequently identified genus (18.03%) to harbour ESBL gene followed by Proteus (14.75%). Moreover, combinations of two ESBL genes, bla SHV + bla TEM or bla CTX + bla TEM, were observed in 11 and 1 isolate, respectively. In conclusion, classic bla TEM ESBL gene was present in multiple bacterial strains that were isolated from DWDS sources in Nigeria. These environments may serve as foci exchange of genetic traits in a diversity of Gram-negative bacteria.

Silver nanoparticle production by Rhizopus stolonifer and its antibacterial activity against extended spectrum {beta}-lactamase producing (ESBL) strains of Enterobacteriaceae

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Banu, Afreen [Department of Microbiology, Gulbarga University, Gulbarga 585106, Karnataka (India); Rathod, Vandana, E-mail: drvandanarathod@rediffmail.com [Department of Microbiology, Gulbarga University, Gulbarga 585106, Karnataka (India); Ranganath, E. [Department of Microbiology, Gulbarga University, Gulbarga 585106, Karnataka (India)

2011-09-15

Highlights: {yields} Silver nanoparticle production by using Rhizopus stolonifer. {yields} Antibacterial activity of silver nanoparticles against extended spectrum {beta}-lactamase producing (ESBL) strains of Enterobacteriaceae. {yields} Synergistic effect of antibiotics with silver nanoparticles towards ESBL-strains. {yields} Characterization of silver nanoparticles made by UV-vis spectra, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transformed infrared (FTIR) spectroscopy, atomic force microscopy (AFM). -- Abstract: This report focuses on the synthesis of silver nanoparticles using the fungus, Rhizopus stolonifer and its antimicrobial activity. Research in nanotechnology highlights the possibility of green chemistry pathways to produce technologically important nanomaterials. Characterization of newly synthesized silver nanoparticles was made by UV-visible absorption spectroscopy, scanning electron microscope (SEM), transmission electron microscope (TEM), Fourier transform infrared (FTIR) spectroscopy and atomic force microscope (AFM). TEM micrograph revealed the formation of spherical nanoparticles with size ranging between 3 and 20 nm. The biosynthesized silver nanoparticles (AgNPs) showed excellent antibacterial activity against ESBL-strains which includes E. coli, Proteus. sp. and Klebsiella sp.

Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

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Yergeau Donald A

2011-11-01

Full Text Available Abstract Background The Sleeping Beauty (SB transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Results Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. Conclusions In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

Antibiotic Susceptibilities and Genetic Characteristics of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Stools of Pediatric Diarrhea Patients in Surabaya, Indonesia.

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Bagus Wasito, Eddy; Shigemura, Katsumi; Osawa, Kayo; Fardah, Alpha; Kanaida, Akiho; Raharjo, Dadik; Kuntaman, K; Hadi, Usman; Harijono, Sugeng; Marto Sudarmo, Subijanto; Nakamura, Tatsuya; Shibayama, Keigo; Fujisawa, Masato; Shirakawa, Toshiro

2017-07-24

The purpose of this study was to investigate extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates from pediatric (aged 0 to 3 years) diarrhea patients in Surabaya, Indonesia, where this kind of survey is rare; our study included assessment of their antibiotic susceptibilities, as well as ESBL typing, multilocus sequence typing (MLST), and diarrheagenic E. coli (DEC)-typing. ESBL-producing E. coli were detected in 18.8% of all the samples. Many ESBL-producing E. coli had significantly lower susceptibility to gentamicin (p ＜ 0.0001) and the quinolones nalidixic acid (p＝0.004) and ciprofloxacin (p ＜ 0.0001) than non-producers. In ESBL-producing E. coli, 84.0% of strains expressed CTX-M-15 alone or in combination with other ESBL types. MLST revealed that 24.0% of ESBL-producers had sequence type 617, all of which expressed the CTX-M-15 gene; we also detected expression of 3 DEC-related genes: 2 enteroaggregative E. coli genes and 1 enteropathogenic E. coli gene. In conclusion, CTX-M-15-type ESBL-producing E. coli ST617 appear to have spread to Indonesia.

An Enterobacter plasmid as a new genetic background for the transposon Tn1331

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Alavi MR

2011-11-01

Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into

Genome-wide analysis of Tol2 transposon reintegration in zebrafish.

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Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir

2009-09-08

Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Comparison of detection methods for extended-spectrum beta-lactamases in Escherichia coli strains

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Ewelina Kałużna

2014-06-01

Full Text Available Introduction: Detection of extended-spectrum beta-lactamases (ESBLs could be a major challenge for microbiologists – the difficulties arise mainly from the phenotypic differences among strains.Materials and Methods: Evaluation of ESBLs was performed on 42 strains of E. coli by: 1 DDST on MHA, 2 DDST on MHA with cloxacillin, 3 CT on MHA, according to CLSI, 4 CT on MHA with cloxacillin, 5 Etest ESBL (AB Biodisk, 6 CHROMagarTM ESBL (GRASO, 7 ChromID® ESBL (bioMérieux, and 8 automatic system VITEK2 ESBL test (bioMérieux.Result: Positive results were obtained for 20 strains using method 1, for 18 strains using method 2, 17 by method 3, 14 by method 4, 11 by method 5, 39 by method 6, 40 by method 7, and 15 by method 8. Using Etest ESBL 6.0 non-determinable results were obtained. The most consistent results were obtained when comparing the results of method 3 with results of method 2 (97.6%, and comparing the results obtained using methods 3 and 8 (95.2%.Conclusions: Based on our study we conclude that the chromogenic media can only be used as a screening method for the detection of ESBLs in E. coli rods. Etest is less useful compared to other phenotype methods, due to the impossibility of obtaining results for all the tested strains. Adding cloxacillin to MHA does not increase the frequency of detection of ESBLs in E. coli strains. DDST seems to be the most reliable among phenotypic methods for the detection of ESBLs in E. coli rods.

Seagulls of the Berlengas Natural Reserve of Portugal as Carriers of Fecal Escherichia coli Harboring CTX-M and TEM Extended-Spectrum Beta-Lactamases▿

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Poeta, Patricia; Radhouani, Hajer; Igrejas, Gilberto; Gonçalves, Alexandre; Carvalho, Carlos; Rodrigues, Jorge; Vinué, Laura; Somalo, Sergio; Torres, Carmen

2008-01-01

Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes. PMID:18835997

Gene-specific cell labeling using MiMIC transposons.

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Gnerer, Joshua P; Venken, Koen J T; Dierick, Herman A

2015-04-30

Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-wide analysis of Tol2 transposon reintegration in zebrafish

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Parinov Sergey

2009-09-01

Full Text Available Abstract Background Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. Results We performed a large-scale enhancer trap (ET screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Conclusion Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Rapid development in vitro and in vivo of resistance to ceftazidime in biofilm-growing Pseudomonas aeruginosa due to chromosomal beta-lactamase

DEFF Research Database (Denmark)

Bagge, N; Ciofu, O; Skovgaard, L T

2000-01-01

The aim of this study was to examine the development of resistance of biofilm-growing P. aeruginosa during treatment with ceftazidime. Biofilms were established in vitro using a modified Robbins device (MRD) and in vivo in the rat model of chronic lung infection. Three P. aeruginosa strains...... of ceftazidime to biofilms established in MDR, a statistically significant development of resistance to ceftazidime in PAO 579 or 19676A bacterial populations occurred. When ceftazidime was administered 4 h/day (200 mg/l) for 2 weeks, the frequency of resistant 19676A having MIC>25 mg/l was 4.4 10(-1) compared...... to 6.0-10(-5) in the control biofilm. The same trend was observed after continuous administration of ceftazidime. MICceftazidime of the more resistant variants was increased 500-fold for PAO 579 and 8-fold for 19676A, and the specific basal beta-lactamase activities from 19 to 1,400 units for PAO 579...

An amperometric penicillin biosensor with enhanced sensitivity based on co-immobilization of carbon nanotubes, hematein, and {beta}-lactamase on glassy carbon electrode

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Chen Bi; Ma Ming [Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081 (China); Su Xiaoli, E-mail: xsu@hunnu.edu.cn [Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081 (China)

2010-07-26

An amperometric penicillin biosensor with enhanced sensitivity was successfully developed by co-immobilization of multi-walled carbon nanotubes (MWCNTs), hematein, and {beta}-lactamase on glassy carbon electrode using a layer-by-layer assembly technique. Under catalysis of the immobilized enzyme, penicillin was hydrolyzed, decreasing the local pH. The pH change was monitored amperometrically with hematein as a pH-sensitive redox probe. MWCNTs were used as an electron transfer enhancer as well as an efficient immobilization matrix for the sensitivity enhancement. The effects of immobilization procedure, working potential, enzyme quantity, buffer concentration, and sample matrix were investigated. The biosensor offered a minimum detection limit of 50 nM (19 {mu}g L{sup -1}) for penicillin V, lower than those of the conventional pH change-based biosensors by more than two orders of magnitude. The electrode-to-electrode variation of the response sensitivity was 7.0% RSD.

IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.

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Kumar, Rajesh; Grover, Sunita; Kaushik, Jai K; Batish, Virender Kumar

2014-01-01

Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system. Copyright © 2013 Elsevier GmbH. All rights reserved.

Occurrence of Multidrug Resistant Extended Spectrum Beta-Lactamase-Producing Bacteria on Iceberg Lettuce Retailed for Human Consumption

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Natasha Bhutani

2015-01-01

Full Text Available Antibiotic resistance in bacteria is a global problem exacerbated by the dissemination of resistant bacteria via uncooked food, such as green leafy vegetables. New strains of bacteria are emerging on a daily basis with novel expanded antibiotic resistance profiles. In this pilot study, we examined the occurrence of antibiotic resistant bacteria against five classes of antibiotics on iceberg lettuce retailed in local convenience stores in Rochester, Michigan. In this study, 138 morphologically distinct bacterial colonies from 9 iceberg lettuce samples were randomly picked and tested for antibiotic resistance. Among these isolates, the vast majority (86% demonstrated resistance to cefotaxime, and among the resistant bacteria, the majority showed multiple drug resistance, particularly against cefotaxime, chloramphenicol, and tetracycline. Three bacterial isolates (2.17% out of 138 were extended spectrum beta-lactamase (ESBL producers. Two ESBL producers (T1 and T5 were identified as Klebsiella pneumoniae, an opportunistic pathogen with transferable sulfhydryl variable- (SHV- and TEM-type ESBLs, respectively. The DNA sequence analysis of the blaSHV detected in K. pneumoniae isolate T1 revealed 99% relatedness to blaSHV genes found in clinical isolates. This implies that iceberg lettuce is a potential reservoir of newly emerging and evolving antibiotic resistant bacteria and its consumption poses serious threat to human health.

Extended-spectrum beta-lactamase-producing Escherichia coli in common vampire bats Desmodus rotundus and livestock in Peru.

Science.gov (United States)

Benavides, J A; Shiva, C; Virhuez, M; Tello, C; Appelgren, A; Vendrell, J; Solassol, J; Godreuil, S; Streicker, D G

2018-06-01

Antibiotic resistance mediated by bacterial production of extended-spectrum beta-lactamase (ESBL) is a global threat to public health. ESBL resistance is most commonly hospital-acquired; however, infections acquired outside of hospital settings have raised concerns over the role of livestock and wildlife in the zoonotic spread of ESBL-producing bacteria. Only limited data are available on the circulation of ESBL-producing bacteria in animals. Here, we report ESBL-producing Escherichia coli in wild common vampire bats Desmodus rotundus and livestock near Lima, Peru. Molecular analyses revealed that most of this resistance resulted from the expression of bla CTX-M-15 genes carried by plasmids, which are disseminating worldwide in hospital settings and have also been observed in healthy children of Peru. Multilocus sequence typing showed a diverse pool of E. coli strains carrying this resistance that were not always host species-specific, suggesting sharing of strains between species or infection from a common source. This study shows widespread ESBL resistance in wild and domestic animals, supporting animal communities as a potential source of resistance. Future work is needed to elucidate the role of bats in the dissemination of antibiotic-resistant strains of public health importance and to understand the origin of the observed resistance. © 2018 Blackwell Verlag GmbH.

Occurrence and characteristics of extended-spectrum β-lactamase (ESBL producing Enterobacteriaceae in food producing animals, minced meat and raw milk

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Geser Nadine

2012-03-01

Full Text Available Abstract Background The impact of food animals as a possible reservoir for extended-spectrum beta-lactamase (ESBL producing Enterobacteriaceae, and the dissemination of such strains into the food production chain need to be assessed. In this study 334 fecal samples from pigs, cattle, chicken and sheep were investigated at slaughter. Additionally, 100 raw milk samples, representing bulk tank milk of 100 different dairy farms, 104 minced meat (pork and beef samples and 67 E. coli isolates from cattle E. coli mastitis were analyzed. Results As many as 15.3% of the porcine, 13.7% of the bovine, 8.6% of the sheep and 63.4% of the chicken fecal samples yielded ESBL producers after an enrichment step. In contrast, none of the minced meat, none of the bulk tank milk samples and only one of the mastitis milk samples contained ESBL producing strains. Of the total of 91 isolates, 89 were E. coli, one was Citrobacter youngae and one was Enterobacter cloacae. PCR analysis revealed that 78 isolates (85.7% produced CTX-M group 1 ESBLs while six isolates (6.6% produced CTX-M group 9 enzymes. Five detected ESBLs (5.5% belonged to the SHV group and 2 isolates (2.2% contained a TEM-type enzyme. A total of 27 CTX-M producers were additionally PCR-positive for TEM-beta-lactamase. The ESBL-encoding genes of 53 isolates were sequenced of which 34 produced CTX-M-1, 6 produced CTX-M-14, 5 produced CTX-M-15 and also 5 produced SHV-12. Two isolates produced TEM-52 and one isolate expressed a novel CTX-M group 1 ESBL, CTX-M-117. One isolate--aside from a CTX-M ESBL-- contained an additional novel TEM-type broad-spectrum beta-lactamase, TEM-186. Conclusions The relatively high rates of ESBL producers in food animals and the high genetic diversity among these isolates are worrisome and indicate an established reservoir in farm animals.

Discrepancy between effects of carbapenems and flomoxef in treating nosocomial hemodialysis access-related bacteremia secondary to extended spectrum beta-lactamase producing Klebsiella pneumoniae in patients on maintenance hemodialysis.

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Yang, Chih-Chao; Li, Shau-Hsuan; Chuang, Feng-Rong; Chen, Chih-Hung; Lee, Chih-Hsiung; Chen, Jin-Bor; Wu, Chien-Hsing; Lee, Chien-Te

2012-09-05

Hemodialysis (HD) patients are susceptible to extended spectrum beta-lactamase (ESBL)-producing bacterial infections. Because the optimal treatment and clinical significance of ESBL-producing Klebsiella pneumoniae (ESBL-Kp) HD access-related bacteremia remain unclear, we conducted this retrospective study to determine the clinical outcomes of patients treated with either flomoxef or a carbapenem. The eligibility criterion was fistula or graft- or catheter- related ESBL-Kp bacteremia in patients on maintenance HD. The clinical characteristics and antibiotic management were analyzed. Outcome was determined by mortality resulting from bacteremia during the 14-day period after the first positive blood culture for flomoxef-susceptible ESBL-Kp. The 57 patients studied were predominantly elderly, malnourished, with a history of severe illnesses and broad-spectrum antibiotic use before the onset of bacteremia, and with severe septicemia as determined by the Pitt bacteremia score (PBS). The study population comprised 7 fistula, 8 graft, and 42 HD catheter-related bacteremia (CRB) cases, and the mortality rate was high (36/57, 63.2%) in these 57 patients. Of 42 patients with CRB, those in the deceased group (27/42, 64.3%) had significantly lower levels of serum albumin, longer prior hospital stay and duration of catheter-dependent HD, and higher PBS than patients in the survived group. Failure to receive effective antibiotics (flomoxef or a carbapenem) within 5 days after onset of bacteremia and treatment with flomoxef both significantly contributed to higher mortality. Multivariate analyses revealed that flomoxef use, PBS, and catheter-dependent HD >30 days were independently associated with increased mortality (OR, 3.52; 95% CI, 1.19-58.17, OR, 2.92; 95% CI, 1.36-6.26 and OR, 5.73; 95% CI, 1.21-63.2, respectively). Considering the high mortality rate, ESBL-Kp should be recognized as a possible pathogen in patients on maintenance HD at high risk of acquiring HD access

Discrepancy between effects of carbapenems and flomoxef in treating nosocomial hemodialysis access-related bacteremia secondary to extended spectrum beta-lactamase producing klebsiella pneumoniae in patients on maintenance hemodialysis

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Yang Chih-Chao

2012-09-01

Full Text Available Abstract Background Hemodialysis (HD patients are susceptible to extended spectrum beta-lactamase (ESBL-producing bacterial infections. Because the optimal treatment and clinical significance of ESBL-producing Klebsiella pneumoniae (ESBL-Kp HD access-related bacteremia remain unclear, we conducted this retrospective study to determine the clinical outcomes of patients treated with either flomoxef or a carbapenem. Methods The eligibility criterion was fistula or graft- or catheter- related ESBL-Kp bacteremia in patients on maintenance HD. The clinical characteristics and antibiotic management were analyzed. Outcome was determined by mortality resulting from bacteremia during the 14‐day period after the first positive blood culture for flomoxef-susceptible ESBL-Kp. Results The 57 patients studied were predominantly elderly, malnourished, with a history of severe illnesses and broad-spectrum antibiotic use before the onset of bacteremia, and with severe septicemia as determined by the Pitt bacteremia score (PBS. The study population comprised 7 fistula, 8 graft, and 42 HD catheter-related bacteremia (CRB cases, and the mortality rate was high (36/57, 63.2% in these 57 patients. Of 42 patients with CRB, those in the deceased group (27/42, 64.3% had significantly lower levels of serum albumin, longer prior hospital stay and duration of catheter-dependent HD, and higher PBS than patients in the survived group. Failure to receive effective antibiotics (flomoxef or a carbapenem within 5 days after onset of bacteremia and treatment with flomoxef both significantly contributed to higher mortality. Multivariate analyses revealed that flomoxef use, PBS, and catheter-dependent HD >30 days were independently associated with increased mortality (OR, 3.52; 95% CI, 1.19–58.17, OR, 2.92; 95% CI, 1.36–6.26 and OR, 5.73; 95% CI, 1.21–63.2, respectively. Conclusions Considering the high mortality rate, ESBL-Kp should be recognized as a possible pathogen in

Risk factors for extended-spectrum b-lactamases-producing Escherichia coli urinary tract infections in a tertiary hospital

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María Dolores Alcántar-Curiel

2015-09-01

Full Text Available Objective. To assess the risks factors for urinary tract infections (UTIs caused by Extended-Spectrum Beta-Lactamases (ESBLs-producing E. coli and the molecular characterization of ESBLs. Materials and methods. A case-control study was performed to identify risk factors in consecutively recruited patients with UTIs caused by ESBLs or non-ESBLs-producing E. coli in a tertiary hospital in Mexico. Results. ESBLs-producing E. coli were isolated from 22/70 (31% patients with E. coli UTIs over a three month period. All isolates were resistant to cephalosporins and quinolones but susceptible to carbapenems, amikacin and nitrofurantoin. Prior antibiotic treatment with more than two antibiotic families (OR=6.86; 95%CI 1.06-157.70; p=0.028, recurrent symptomatic UTIs (OR=5.60; 95%CI 1.88-17.87; p=0.001 and previous hospitalization (OR=5.06; 95%CI 1.64-17.69;p=0.002 were significant risk factors. Sixteen isolates harbored the beta-lactamase (blaCTX-M-15 gene and five the blaTEM-1 gene. Conclusions. One of every three patients presented UTIs with ESBLs-producing beta-lactams and fluoroquinolone resistant E. coli. Risk factors and resistance patterns must be taken into account for developing antibiotic use policies in these settings

Insight into the effect of inhibitor resistant S130G mutant on physico-chemical properties of SHV type beta-lactamase: a molecular dynamics study.

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Mohd Hassan Baig

Full Text Available Bacterial resistance is a serious threat to human health. The production of β-lactamase, which inactivates β-lactams is most common cause of resistance to the β-lactam antibiotics. The Class A enzymes are most frequently encountered among the four β-lactamases in the clinic isolates. Mutations in class A β-lactamases play a crucial role in substrate and inhibitor specificity. SHV and TEM type are known to be most common class A β-lactamases. In the present study, we have analyzed the effect of inhibitor resistant S130G point mutation of SHV type Class-A β-lactamase using molecular dynamics and other in silico approaches. Our study involved the use of different in silico methods to investigate the affect of S130G point mutation on the major physico-chemical properties of SHV type class A β-lactamase. We have used molecular dynamics approach to compare the dynamic behaviour of native and S130G mutant form of SHV β-lactamase by analyzing different properties like root mean square deviation (RMSD, H-bond, Radius of gyration (Rg and RMS fluctuation of mutation. The results clearly suggest notable loss in the stability of S130G mutant that may further lead to decrease in substrate specificity of SHV. Molecular docking further indicates that S130G mutation decreases the binding affinity of all the three inhibitors in clinical practice.

Acquired Class D β-Lactamases

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Nuno T. Antunes

2014-08-01

Full Text Available The Class D β-lactamases have emerged as a prominent resistance mechanism against β-lactam antibiotics that previously had efficacy against infections caused by pathogenic bacteria, especially by Acinetobacter baumannii and the Enterobacteriaceae. The phenotypic and structural characteristics of these enzymes correlate to activities that are classified either as a narrow spectrum, an extended spectrum, or a carbapenemase spectrum. We focus on Class D β-lactamases that are carried on plasmids and, thus, present particular clinical concern. Following a historical perspective, the susceptibility and kinetics patterns of the important plasmid-encoded Class D β-lactamases and the mechanisms for mobilization of the chromosomal Class D β-lactamases are discussed.

Caracterização dos Genes de metalo-beta-lactamase blaNDM, blaSPM e blaIMP em Pseudomonas aeruginosa Resistentes a Carbapenens

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Carolina Jahn

2017-01-01

Full Text Available Justificativa e objetivo: P. aeruginosa é responsável por causar grande variedade de infecções agudas e crônicas. A resistência aos carbapenêmicos está se tornando um problema terapêutico mundial e a produção de metalo-beta-lactamases (MBL tem surgido como um dos mecanismos responsáveis por esta resistência. Objetivou-se identificar as cepas produtoras de MBL e verificar a presença dos genes codificador blaNDM, blaSPM e blaIMP em isolados de P. aeruginosa resistentes aos carbapenens. Métodos: Foram analisadas 16 cepas de P. aeruginosa, isoladas de diferentes sítios de indivíduos atendidos pelo Hospital do Vale do Rio Pardo-RS, no período de março 2014 a fevereiro de 2015. A identificação bioquímica foi realizada segundo protocolo da ANVISA e o antibiograma pelo método de ágar difusão em disco. A triagem fenotípica foi feita pelo teste modificado de aproximação de discos utilizando o EDTA como inibidor e a presença dos genes através da técnica de reação em cadeia da polimerase. Resultados: O setor clínico com maior prevalência de isolamento de P. aeruginosa resistente aos carbapenens é a unidade de terapia intensiva (50%. Em relação aos espécimes clínicos, 62,5% dos isolados foram identificados no trato respiratório inferior. Através do teste fenotípico, 50% das amostras analisadas mostraram ser produtora da enzima MBL. Através da técnica de reação em cadeia da polimerase foi possível identificar a presença de um dos três genes propostos, sendo eles blaSPM. Conclusão: Os isolados de P. aeruginosa mostraram serem produtores de MBL e apresentaram o gene de resistência blaSPM nesta espécie no interior do Rio Grande do Sul. Palavras Chaves: Pseudomonas aeruginosa; metalo-beta-lactamases; blaNDM, blaIMP, e blaSPM .

Rapid, low-cost fluorescent assay of β-lactamase-derived antibiotic resistance and related antibiotic susceptibility

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Erdem, S. Sibel; Khan, Shazia; Palanisami, Akilan; Hasan, Tayyaba

2014-10-01

Antibiotic resistance (AR) is increasingly prevalent in low and middle income countries (LMICs), but the extent of the problem is poorly understood. This lack of knowledge is a critical deficiency, leaving local health authorities essentially blind to AR outbreaks and crippling their ability to provide effective treatment guidelines. The crux of the problem is the lack of microbiology laboratory capacity available in LMICs. To address this unmet need, we demonstrate a rapid and simple test of β-lactamase resistance (the most common form of AR) that uses a modified β-lactam structure decorated with two fluorophores quenched due to their close proximity. When the β-lactam core is cleaved by β-lactamase, the fluorophores dequench, allowing assay speeds of 20 min to be obtained with a simple, streamlined protocol. Furthermore, by testing in competition with antibiotics, the β-lactamase-associated antibiotic susceptibility can also be extracted. This assay can be easily implemented into standard lab work flows to provide near real-time information of β-lactamase resistance, both for epidemiological purposes as well as individualized patient care.

Distribution and Antimicrobial Susceptibility Pattern of Gram Negative Bacteria Causing Urinary Tract Infection (UTI and Detection New Delhi Metallo-beta-lactamase-1 (NDM-1 Producing Isolates in Ahwaz

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Parviz Afrugh

2016-04-01

Full Text Available Background: Urinary tract infection (UTI is the commonest bacterial infectious disease in worldwide (especially in developing countries with a high rate of morbidity and financial cost. The management of UTI infections has been jeopardized by increase in immergence of antimicrobial drug resistance. Knowledge of the local bacterial etiology and susceptibility patterns is required to trace any change that might have occurred in time so that updated recommendation for optimal empirical therapy of UTI can be made. The aim of this investigation was distribution and antimicrobial susceptibility pattern of gram negative bacteria causing urinary tract infection (UTI and detection NDM-1 (new-delhi-metallo-beta-lactamase-1 producing isolates in Ahwaz. Materials and Methods: This cross-sectional study was done during a period of one year from April 2013 to March 2014. Clean catch midstream urine samples were collected from suspected patients to UTI. The isolates were identified based on morphological and biochemical testes. Culture was performed on routine microbiological media. Susceptibility testing was performed according CLSI (2013 guidelines. Detection of carbapenemase producing isolates was performed by modified hodge test (MHT. Metallo-beta-lactamase isolates were detected by imipenem-EDTA combined disc test (CDT. Results: In this study 708 gram negative organisms were isolated from urine samples. E.coli was the most common isolated bacteria (67% followed by Klebsiella spp. (26.5% and Enterobacter spp. (2.5%. In antibiotic susceptibility testing more than 90% of isolates were sensitive to tetracycline, ceftazidime, meropenem, amikacin, cefotaxime, imipenem, and cefepime. Isolates were more resistant to cephalothin (32%, co-trimoxazol (30.5%, and nalidixic acid (25%. Conclusion: In our results isolated organisms from outpatients showed very high sensitivity to common antibiotics. Continuous and regular monitoring of susceptibility pattern of

OXA-48 and CTX-M-15 extended-spectrum beta-lactamases in raw milk in Lebanon: epidemic spread of dominant Klebsiella pneumoniae clones.

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Diab, Mohamad; Hamze, Monzer; Bonnet, Richard; Saras, Estelle; Madec, Jean-Yves; Haenni, Marisa

2017-11-01

Raw milk has recently been reported as a source of extended-spectrum beta-lactamase (ESBL) and carbapenemase genes. We thus investigated the prevalence of ESBL- and carbapenemase-producing Enterobacteriaceae in raw milk in Lebanon in order to assess the risk of transfer of these bacteria to humans. A high prevalence (30.2 %) of CTX-M-15-producing K. pneumoniae was detected in raw bovine milk. Three main K. pneumoniae clones were identified by PFGE and MLST typing. Southern blot experiments revealed that one of these clones carried the blaCTX-M-15 gene chromosomally. Moreover, one OXA-48-producing K. pneumoniae ST530 and seven CTX-M-15-producing Escherichia coli sharing the same ST were also detected. These findings highlight the spread of dominant CTX-M-15-producing K. pneumoniae clones and OXA-48-producing isolates in the food chain. Milk, which is mostly consumed raw in Lebanon, may be a source of human exposure to ESBLs and carbapenemases.

Molecular characterization of the extended-spectrum beta-lactamase (ESBL)-producing Shigella spp. in Shanghai.

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Li, J; Li, B; Ni, Y; Sun, J

2015-03-01

Shigellosis is a public health concern in China. We tested 216 Shigella isolates collected in Shanghai in 2007 for the production of extended-spectrum beta-lactamases (ESBLs). ESBL-producing isolates were characterized using polymerase chain reaction (PCR)-based genotyping, conjugation, pulsed-field gel electrophoresis (PFGE), and DNA sequence analysis of regions adjacent to bla genes. Plasmids containing genes encoding ESBLs were analyzed using plasmid replicon typing. ESBLs were produced by 18.1 % (39/216) of Shigella isolates, and all 39 ESBL-producing strains harbored bla CTX-M genes. CTX-M-14 was the most frequent variant (69.2 %, 27/39), followed by CTX-M-15 (15.4 %, 6/39). All bla CTX-M genes were transferable by conjugation, and the insertion sequence ISEcp1 was detected upstream of all bla CTX-M genes. The CTX-M-producing Shigella isolates showed high clonal diversity. IncI1, IncFII, IncN, and IncB/O replicons were respectively detected in 23 (58.9 %), 9 (23.1 %), 1 (2.6 %), and 1 (2.6 %) of the 39 transconjugants carrying bla CTX-M. The bla CTX-M-14 genes were most frequently carried by IncI1 (n = 13, 48.1 %) or IncFII (n = 9, 33.3 %) plasmids, and the bla CTX-M-15 genes were closely associated with IncI1 (n = 5, 83.3 %). Our findings demonstrate the high prevalence of ESBL-producing Shigella in Shanghai, the importance of plasmids and ISEcp1 as carriers of bla CTX-M genes, and the close association between certain bla CTX-M genes with a specific plasmid.

Identifying transposon insertions and their effects from RNA-sequencing data.

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de Ruiter, Julian R; Kas, Sjors M; Schut, Eva; Adams, David J; Koudijs, Marco J; Wessels, Lodewyk F A; Jonkers, Jos

2017-07-07

Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Microbial resistance and frequency of extended-spectrum beta-lactamase (ESBL in isolated from blood cultures

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Ruan Carlos Gomes da Silva

2014-12-01

Full Text Available Introduction:The emergence and spread of isolated carriers of extended-spectrum beta-lactamase (ESBL have complicated the treatment of nosocomial infections, since its production is not easily identified by the sensitivity tests, routinely performed in clinical laboratories, leading to difficulties in the hospital control of resistant microorganisms and antibiotics misuse.Objective:The objective of this study was to analyze the resistance profile and the frequency of ESBL in Gram-negative bacteria isolated from blood cultures. A hundred bacterial samples from blood cultures of adult patients were analyzed, which were phenotypically identified by biochemical tests of carbohydrates fermentation and submitted to determination of the resistance profile by disc diffusion test and ESBL screening by disc approximation and disc replacement methods.Results:Among the bacterial samples tested, 30 were identified as Gram-negative bacteria, predominantly by Proteus mirabilis, Pantoea agglomerans, and Escherichia coli. Of these, 73.33% were positive for the detection of ESBL by phenotypic tests, and was found mainly in Pantoea agglomerans, Proteus mirabilis, and Enterobacter cloacae.Conclusion:The increase in the occurrence of ESBL in different Enterobacteriaceae shows the importance of the amplification of detection in other species than Escherichia coli or Klebsiella sp., so that the assistance to the patient is not restrained, since these resistant bacteria cannot be detected by the laboratories. Considering the frequency of ESBL in this study, we highlight the importance of its detection, aiming to its contribution to the development of improvements in the health care policies of hospitals.

Prevalence and characterization of extended-spectrum beta-lactamases in Klebsiella pneumoniae in Algiers hospitals (Algeria).

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Messai, Y; Iabadene, H; Benhassine, T; Alouache, S; Tazir, M; Gautier, V; Arlet, G; Bakour, R

2008-07-01

To determine the prevalence and the diversity of extended-spectrum beta-lactamases (ESBLs) in 196 Klebsiella pneumoniae clinical isolates collected from three hospitals in Algiers. Antibiograms were done on Mueller-Hinton agar plates with the disc-diffusion method and MICs were determined by agar-dilution method. Mating experiments were performed in agar medium. Plasmid DNA was extracted by the alcalin-lysis method. Total DNA was extracted with a Qiagen mini kit and screened for bla(TEM) and bla(CTX-M) genes by PCR. Linkage of bla(CTX-M) genes with insertion sequence ISEcp1B and class 1 integrons was investigated by PCR. PCR products were sequenced by the Sanger method. The epidemiological relationships between ESBL-producing K. pneumoniae isolates were analyzed by ERIC-PCR. Thirty-nine (19.9%) isolates were found to produce ESBLs belonging to CTX-M-1 group and TEM penicillinases (CTX-M-3, CTX-M-15 and TEM-1). ERIC-PCR analysis showed that the isolates are genetically unrelated. The bla(TEM) and bla(CTX-M) genes as well as aminoglycosides and sulfonamides resistance determinants were found located in self-transferable plasmids of approximately 85 kb. The class 1 integrons and the insertion sequence ISEcp1B were present in the isolates and in their transconjugants. ISEcp1B was found genetically linked to the bla(CTX-M) genes and located 127bp upstream, with the presence of the V and W sequences. The study revealed a high rate of ESBL-producing K. pneumoniae in Algerian hospitals, resulting from horizontal dissemination of mobile bla(CTX-M) genes.

Detection of Extended Spectrum Beta-Lactamases Among Gram Negative Bacilli Recovered from Cattle Feces In Benin City, Nigeria

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Helen Oroboghae OGEFERE

2017-06-01

Full Text Available This study was carried out to determine the prevalence of extended spectrum beta-lactamase (ESBL among Gram negative bacteria isolated from cattle feces in Benin City, Nigeria. A total of 250 Gram negative bacteria isolates were recovered from cattle feces and were processed microbiologically using standard techniques. Emergent colonies were identified and antibacterial susceptibility tests were determined using Kirby-Bauer disk diffusion method. All bacterial isolates were screened for the presence of ESBL using the double-disc synergy method. A total of 37 (14.8% isolates were positive for ESBL, with 33 (13.2% indicated by ceftazidime, while only 4 (1.6% were indicated by both ceftazidime and cefotaxime (P < 0.0001. Of the Gram negative bacterial isolates recovered, Salmonella species was the most prevalent ESBL-producer with 55.0% prevalence (P = 0.0092, while no isolate of Pseudomonas aeruginosa produced ESBL. ESBL-positive isolates showed poor susceptibility to the tested antibacterial agents in comparison with non-ESBL-producers and imipenem was the most active antibiotic. The prevalence of ESBL among Gram negative bacilli recovered from cattle feces was 14.8%. The study advises prudent use of antibiotics in the treatment of cattle and harps on improved hygiene in managing cattle, as they are potential reservoirs of ESBL-producing organisms.

Prostatic Abscess after Stapled Hemorrhoidopexy Caused by ESBL Extended Spectrum Beta Lactamase Producing Klebsiella pneumoniae: An Additional Challenge to Postoperative Sepsis

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Asem Saleh

2017-01-01

Full Text Available Postoperative septic complications of hemorrhoids surgical interventions are rare, but very serious with high mortality rate. Early diagnosis and prompt therapy are essential to save patient’s life. There are a good number of articles and case reports about these septic complications. We are presenting a case report of a prostatic abscess caused by extended spectrum beta lactamase (ESBL producing Klebsiella pneumoniae after hemorrhoidopexy. Our patient was a healthy middle aged Saudi male who has no significant medical history apart from morbid obesity and recurrent urinary tract infections. ESBL producing K. pneumoniae could be detected only after aspiration of the prostatic abscess, but proper antibiotic was introduced intravenously on admission before culture of aspirate of the abscess was available. Antibiotic was continued for 30 days and abscess resolved completely. In our electronic search, we could not find any case report of prostatic abscess after stapled hemorrhoidopexy caused by ESBL producing organism. This is an additional challenge for treating physicians as these organisms are sensitive only to one group of antibiotics (carbapenem group.

In vitro susceptibility pattern of extended spectrum ?-lactamase producing gram negative bacilli against tetracyclines

International Nuclear Information System (INIS)

Gill, M.M.

2015-01-01

Extended Spectrum beta-lactamases (ESBLs) are emerging as common nosocomial pathogens and important cause of mortality and morbidity, if not treated properly. The need of the hour is to find effective treatment options for dealing with ESBL producing organisms. This study was aimed to evaluate in vitro susceptibility pattern of extended spectrum beta-lactamase producers against tetracyclines. Methods: This descriptive cross-sectional study was carried out in the department of Microbiology, Army Medical College, Rawalpindi, National University of Sciences and Technology over a period of 6 months. Seventy eight non-duplicate isolates were included in the study. ESBL detection was done using Jarlier et al method. In vitro susceptibility of tetracyclines like tetracycline, doxycycline, minocycline and tigecycline was then tested using Modified Kirby Bauer disc diffusion method. The zones of inhibition were measured after completion of incubation period and interpreted as per CLSI and FDA guidelines. Results: Approximately 56.4% of the isolates were Escherichia coli, 28.2% were Klebsiella pneumoniae, 10.26% were Enterobacter species, and 2.6% were each Klebsiella oxytoca and Acinetobacter species. ESBLs were found to be most sensitive to tigecycline, intermediate in susceptibility to minocycline while least sensitive to doxycycline and tetracycline. Conclusion: Among tetracyclines, tigecycline has best in vitro susceptibility against ESBL producing Gram negative rods. (author)

Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.

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Yongming Wang

2014-03-01

Full Text Available Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB and piggyBac (PB that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1, a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

TEM-145 and TEM-146 β-lactamases produced by Escherichia coli ...

African Journals Online (AJOL)

GREGO

2007-03-05

Mar 5, 2007 ... Key words: Escherichia coli, plasmid-mediated, TEM β-lactamase. ... Enterobacteriaceae, the most prevalent mechanism of resistance to ... the production of a relatively inhibitor-resistant OXA-type β-lactamase .... and 8.6 as transcripts of the E. coli chromosomal AmpC .... Mode of action and mechanisms of.

Killing curve activity of ciprofloxacin is comparable to synergistic effect of beta-lactam-tobramycin combinations against Haemophilus species endocarditis strains

DEFF Research Database (Denmark)

Westh, H; Frimodt-Møller, N; Gutschik, E

1992-01-01

Nine Haemophilus species strains, all beta-lactamase negative, isolated from patients with endocarditis were tested in killing curve experiments. Antibiotics used were penicillin, amoxicillin, aztreonam alone and in combination with tobramycin, as well as ciprofloxacin alone. Synergism between beta...

Novel sequence types of extended-spectrum and acquired AmpC beta-lactamase producing Escherichia coli and Escherichia clade V isolated from wild mammals.

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Alonso, Carla Andrea; Alcalá, Leticia; Simón, Carmen; Torres, Carmen

2017-08-01

The closer contact with wildlife due to the growing human population and the destruction of natural habitats emphasizes the need of gaining insight into the role of animals as source of antimicrobial resistance. Here, we aim at characterizing the antimicrobial resistance genes and phylogenetic distribution of commensal Escherichia coli from 62 wild mammals. Isolates exhibiting resistance to ≥1 antibiotic were detected in 25.8% of the animals and 6.4% carried an extended-spectrum beta-lactamase (ESBL)/AmpC-producing E. coli. Genetic mechanisms involved in third-generation cephalosporin resistance were as follows: (i) hyperproduction of chromosomal AmpC (hedgehog), (ii) production of acquired CMY-2 β-lactamase (hedgehog), (iii) production of SHV-12 and CTX-M-14 ESBLs (n = 2, mink and roe-deer). ESBL genes were transferable by conjugation, and blaCMY-2 was mobilized by a 95kb IncI1 plasmid. The distribution of the phylogenetic groups in the E. coli collection studied was B1 (44.6%), B2 (24.6%), E (15.4%), A (4.6%) and F (3.1%). Five isolates (7.7%) were cryptic Escherichia clades (clade IV, 4 mice; clade V, 1 mink). ESBL/AmpC-E. coli isolates showed different sequence types (STs): ST1128/B1, ST4564/B1 (new), ST4996/B1 (new) and a non-registered ST. This study contributes to better understand the E. coli population and antimicrobial resistance flow in wildlife and reports new AmpC-E. coli STs and a first described ESBL-producing Escherichia clade V isolate. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae in HIV-infected children in Zimbabwe.

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Wilmore, S M S; Kranzer, K; Williams, A; Makamure, B; Nhidza, A F; Mayini, J; Bandason, T; Metcalfe, J; Nicol, M P; Balakrishnan, I; Ellington, M J; Woodford, N; Hopkins, S; McHugh, T D; Ferrand, R A

2017-05-01

Antimicrobial resistance is an emerging global health issue. Data on the epidemiology of multidrug-resistant organisms are scarce for Africa, especially in HIV-infected individuals who often have frequent contact with healthcare. We investigated the prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in stool among HIV-infected children attending an HIV outpatient department in Harare, Zimbabwe. We recruited children who were stable on antiretroviral therapy (ART) attending a HIV clinic from August 2014 to June 2015. Information was collected on antibiotic use and hospitalization. Stool was tested for ESBL-E through combination disc diffusion. API20E identification and antimicrobial susceptibility was performed on the positive samples followed by whole genome sequencing. Stool was collected from 175/202 (86.6 %) children. Median age was 11 [inter-quartile range (IQR) 9-12] years. Median time on ART was 4.6 years (IQR 2.4-6.4). ESBL-Es were found in 24/175 samples (13.7 %); 50 % of all ESBL-Es were resistant to amoxicillin-clavulanate, 100 % to co-trimoxazole, 45.8 % to chloramphenicol, 91.6 % to ceftriaxone, 20.8 % to gentamicin and 62.5 % to ciprofloxacin. ESBL-Es variously encoded CTX-M, OXA, TEM and SHV enzymes. The odds of ESBL-E carriage were 8.5 times (95 % CI 2.2-32.3) higher in those on ART for less than one year (versus longer) and 8.5 times (95 % CI 1.1-32.3) higher in those recently hospitalized for a chest infection. We found a 13.7 % prevalence of ESBL-E carriage in a population where ESBL-E carriage has not been described previously. Antimicrobial resistance (AMR) in Africa merits further study, particularly given the high HIV prevalence and limited diagnostic and therapeutic options available.

Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.

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Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K

2017-01-01

Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

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Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

2016-08-01

β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

Extended-spectrum beta-lactamases producing E. coli in wildlife, yet another form of environmental pollution?

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Sebastian eGuenther

2011-12-01

Full Text Available Wildlife is normally not exposed to antimicrobial agents but can acquire antimicrobial resistant bacteria through contact with humans, domesticated animals and the environment, where water polluted with faeces seems to be the most important vector. E. coli, a ubiquitous commensal bacterial species colonizing the intestinal tract of mammals and birds, is also found in the environment. Extended-spectrum beta-lactamases producing E. coli (ESBL-E. coli represent a major problem in human and veterinary medicine, particular in nosocomial infections. Additionally an onset of community acquired ESBL-E. coli infections and an emergence in livestock farming has been observed in recent years, suggesting a successful transmission as well as persistence of ESBL-E. coli strains outside clinical settings. Another parallel worldwide phenomenon is the spread of ESBL-E. coli into the environment beyond human and domesticated animal populations, and this seems to be directly influenced by antibiotic practice. This might be a collateral consequence of the community onset of ESBL-E. coli infections but can result (a in a subsequent colonization of wild animal populations which can turn into an infectious source or even a reservoir of ESBL-E.coli, (b in a contribution of wildlife to the spread and transmission of ESBL-E. coli into fragile environmental niches, (c in new putative infection cycles between wildlife, domesticated animals and humans, and (d in problems in the medical treatment of wildlife. This review aims to summarize the current knowledge on ESBL-E. coli in wildlife, in turn underlining the need for more large scale investigations, in particular sentinel studies to monitor the impact of multiresistant bacteria on wildlife.

The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes

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Satoshi Kimura

2016-08-01

Full Text Available Transposon insertion sequencing (TIS; also known as TnSeq is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences.

Transposons As Tools for Functional Genomics in Vertebrate Models.

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Kawakami, Koichi; Largaespada, David A; Ivics, Zoltán

2017-11-01

Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles, and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice, and rats. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elaboration and evaluation of a new screening medium for detection and presumptive identification of extended-spectrum beta-lactamase-producing organisms (ESBL Elaboração e avaliação de um novo meio de triagem para a detecção e identificação presuntiva de enterobactérias produtoras de beta-lactamase de espectro estendido (ESBL

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Carlos Henrique Pessôa de Menezes e Silva

2000-10-01

Full Text Available The new ß-lactamases have arisen largely as a consequence of heavy use of new expanded spectrum ß-lactam antibiotics. Health professionals need to be aware of the resistance problems caused by the new enzymes, and know the correct procedures to detect, prevent and control such problems. In this study, a new screening medium called Ceftazidime-Inositol-Vancomycin-Amphotericin B Agar (CIVA was elaborated and the microbiological performance was evaluated for the detection and presumptive identification of ESBL-producing members of Enterobacteriaceae. It was performed in 126 stool samples from hospitalized patients at Santa Monica Hospital (Vila Velha, ES, Brazil, who had been heavily exposed to broad-spectrum antibiotic combinations. The bacteria were detected by the medium based on their colony colours (due to inositol fermentation. Additional tests were required for correct identification of these strains. No false positive rates were detected.As novas beta-lactamases têm aparecido, na maioria das vezes, como consequência do amplo uso de antibióticos beta-lactâmicos de largo espectro. Os profissionais de saúde precisam estar atentos acerca do problema da resistência bacteriana causado por estas novas enzimas e conhecer os corretos procedimentos para detectar, prevenir e controlar tais situações. Neste estudo, um novo meio de triagem foi desenvolvido no setor de Microbiologia do Marcos Daniel Laboratório - Vitória, ES (denominado CIVA - Ceftazidima-Inositol-Vancomicina-Anfotericina B e a sua performance microbiológica foi avaliada quanto à detecção e identificação presuntiva de enterobactérias produtoras de ESBL. Foram realizadas 126 culturas de amostras fecais de pacientes hospitalizados no Hospital Santa Monica (Vila Velha, ES, Brasil, previamente expostos a combinações de antibióticos de largo espectro. As bactérias foram detectadas pelo meio baseado nas cores das colônias desenvolvidas (devido à fermentação do

Empiric Piperacillin-Tazobactam versus Carbapenems in the Treatment of Bacteraemia Due to Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae.

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Ng, Tat Ming; Khong, Wendy X; Harris, Patrick N A; De, Partha P; Chow, Angela; Tambyah, Paul A; Lye, David C

2016-01-01

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are a common cause of bacteraemia in endemic countries and may be associated with high mortality; carbapenems are considered the drug of choice. Limited data suggest piperacillin-tazobactam could be equally effective. We aimed to compare 30-day mortality of patients treated empirically with piperacillin-tazobactam versus a carbapenem in a multi-centre retrospective cohort study in Singapore. Only patients with active empiric monotherapy with piperacillin-tazobactam or a carbapenem were included. A propensity score for empiric carbapenem therapy was derived and an adjusted multivariate analysis of mortality was conducted. A total of 394 patients had ESBL-Escherichia.coli and ESBL-Klebsiella pneumoniae bacteraemia of which 23.1% were community acquired cases. One hundred and fifty-one received initial active monotherapy comprising piperacillin-tazobactam (n = 94) or a carbapenem (n = 57). Patients who received carbapenems were less likely to have health-care associated risk factors and have an unknown source of bacteraemia, but were more likely to have a urinary source. Thirty-day mortality was comparable between those who received empiric piperacillin-tazobactam and a carbapenem (29 [30.9%] vs. 17 [29.8%]), P = 0.89). Those who received empiric piperacillin-tazobactam had a lower 30-day acquisition of multi-drug resistant and fungal infections (7 [7.4%] vs. 14 [24.6%]), Pcarbapenem.

Phenotypic Detection of Metallo-Beta-Lactamases in Carbapenem Resistant Acinetobacter baumannii Isolated from Pediatric Patients in Pakistan

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Muneeza Anwar

2016-01-01

Full Text Available Multidrug resistant A. baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections especially in neonates and immunocompromised patients because they have least capacity to fight against infections. Carbapenems are used as last resort antibiotics for treating these infections but currently resistance against carbapenems due to MBL production is on the rise. The objective of this study was to determine the frequency of antibiotic resistance in A. baumannii and also to compare the efficacy of combined disk test and double disk synergy test for detection of metallo-beta-lactamases. A total of 112 A. baumannii were identified from various clinical samples and antibiotic susceptibility profile was determined by Kirby-Bauer Disk Diffusion method. Out of 112, 66 (58.9% isolates were resistant to both imipenem and meropenem (OXOID. These resistant isolates were tested for carbapenemase production, and 55 (83.3% were carbapenemase producers by Modified Hodge Test. These isolates were further tested for MBL production by combined disk test and double disk synergy test. Out of 66, 49 isolates were positive by both methods, CDT and DDST, and only one isolate was detected as negative (with kappa value = 0.038. All MBL producing strains showed remarkable resistance to cephalosporins, fluoroquinolones, aminoglycosides, and piperacillin/tazobactam (OXOID. The antibiotic resistance was very high in A. baumannii which were isolated from children in Pakistan specially attending a nephrology unit.

Stochastic Predator-Prey Dynamics of Transposons in the Human Genome

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Xue, Chi; Goldenfeld, Nigel

2016-11-01

Transposable elements, or transposons, are DNA sequences that can jump from site to site in the genome during the life cycle of a cell, usually encoding the very enzymes which perform their excision. However, some transposons are parasitic, relying on the enzymes produced by the regular transposons. In this case, we show that a stochastic model, which takes into account the small copy numbers of the active transposons in a cell, predicts noise-induced predator-prey oscillations with a characteristic time scale that is much longer than the cell replication time, indicating that the state of the predator-prey oscillator is stored in the genome and transmitted to successive generations. Our work demonstrates the important role of the number fluctuations in the expression of mobile genetic elements, and shows explicitly how ecological concepts can be applied to the dynamics and fluctuations of living genomes.

Resistencia de la bacteria Escherichiacoli por la beta-lactamasas

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Armijos- Nieves, Bryan,

2017-09-01

Full Text Available E. coli bacterial infections are frequently present with multiple factors that make their treatment more expensive, in the health field at the national level. The objective of this work is to determine the influence of the beta-lactamase enzyme on E. coliresistance against antibiotics, recognizing the antibacterialsfrom the families of penicillins and cephalosporins inefficient in their treatment. Based on a documentary study of scientific articles on the subject under study, it is conclude that the presence of the enzyme beta-lactamase greatly influences the resistance developed by the bacterium E. coli towards antibiotics such as penicillins and cephalosporins first, second, third and fourth generation. In addition, it was determined that the antibiotics derived from penicillin, such as ampicillin and antibiotics of the cephalosporin family cefazolin, cephalothin, cefotaxime, cefepime and cefoptaxime by hydrolysis of these drugs, do not exert any antibacterial effect of resistant E. coli, representing a big problem that may increase the risk of patient mortality. It is recommended that, in case of suspected E. coli infection, an antibiogram be performed to detect the indicated treatment.

Bursts and horizontal evolution of DNA transposons in the speciation of pseudotetraploid salmonids

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Davidson William S

2007-11-01

Full Text Available Abstract Background Several genome duplications have occurred in the evolutionary history of teleost fish. In returning to a stable diploid state, the polyploid genome reorganized, and large portions are lost, while the fish lines evolved to numerous species. Large scale transposon movement has been postulated to play an important role in the genome reorganization process. We analyzed the DNA sequence of several large loci in Salmo salar and other species for the presence of DNA transposon families. Results We have identified bursts of activity of 14 families of DNA transposons (12 Tc1-like and 2 piggyBac-like families, including 11 novel ones in genome sequences of Salmo salar. Several of these families have similar sequences in a number of closely and distantly related fish, lamprey, and frog species as well as in the parasite Schistosoma japonicum. Analysis of sequence similarities between copies within the families of these bursts demonstrates several waves of transposition activities coinciding with salmonid species divergence. Tc1-like families show a master gene-like copying process, illustrated by extensive but short burst of copying activity, while the piggyBac-like families show a more random copying pattern. Recent families may include copies with an open reading frame for an active transposase enzyme. Conclusion We have identified defined bursts of transposon activity that make use of master-slave and random mechanisms. The bursts occur well after hypothesized polyploidy events and coincide with speciation events. Parasite-mediated lateral transfer of transposons are implicated.

Enzymatic engineering of the porcine genome with transposons and recombinases

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Carlson Daniel F

2007-07-01

Full Text Available Abstract Background Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs. Results Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons. Conclusion We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.

Should the patients colonized with extended-spectrum beta-lactamase-producing Gram-negative bacilli (E-GNB) coming to hospital from the community with pneumonia get anti-E-GNB active empirical treatment?

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Peterlin, Lara; Žagar, Mateja; Lejko Zupanc, Tatjana; Paladin, Marija; Beović, Bojana

2017-10-01

Extended-spectrum beta-lactamases are responsible for resistance of Gram-negative bacilli to several beta-lactam antibiotics, including those prescribed for treatment pneumonia. To evaluate the importance of colonization with E-GNB for the choice of empirical treatment we performed a retrospective case-control study including 156 patients, hospitalized for treatment of pneumonia from 2009 through 2013. Empirical treatment success and in-hospital survival were significantly lower in patients colonized with E-GNB compared to non-colonized (p = 0.002, p = 0.035). When comparing subgroups of colonized patients, treatment success was significantly lower in patients who were colonized with E-GNB resistant to empirical antibiotic (p = 0.010), but not in those colonized by E-GNB susceptible to empirically given antibiotic (p = 0.104). Difference in in-hospital mortality was insignificant in both subgroups (p = 0.056, p = 0.331). The results of study suggest that an anti-E-GNB active antibiotic should be used for empirical treatment of pneumonia in E-GNB colonized patients.

The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

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Boris Troyanovsky

2016-01-01

Full Text Available Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

The bandit, a new DNA transposon from a hookworm-possible horizontal genetic transfer between host and parasite.

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Thewarach Laha

2007-09-01

Full Text Available An enhanced understanding of the hookworm genome and its resident mobile genetic elements should facilitate understanding of the genome evolution, genome organization, possibly host-parasite co-evolution and horizontal gene transfer, and from a practical perspective, development of transposon-based transgenesis for hookworms and other parasitic nematodes.A novel mariner-like element (MLE was characterized from the genome of the dog hookworm, Ancylostoma caninum, and termed bandit. The consensus sequence of the bandit transposon was 1,285 base pairs (bp in length. The new transposon was flanked by perfect terminal inverted repeats of 32 nucleotides in length with a common target site duplication TA, and it encoded an open reading frame (ORF of 342 deduced amino acid residues. Phylogenetic comparisons confirmed that the ORF encoded a mariner-like transposase, which included conserved catalytic domains, and that the bandit transposon belonged to the cecropia subfamily of MLEs. The phylogenetic analysis also indicated that the Hsmar1 transposon from humans was the closest known relative of bandit, and that bandit and Hsmar1 constituted a clade discrete from the Tc1 subfamily of MLEs from the nematode Caenorhabditis elegans. Moreover, homology models based on the crystal structure of Mos1 from Drosophila mauritiana revealed closer identity in active site residues of the catalytic domain including Ser281, Lys289 and Asp293 between bandit and Hsmar1 than between Mos1 and either bandit or Hsmar1. The entire bandit ORF was amplified from genomic DNA and a fragment of the bandit ORF was amplified from RNA, indicating that this transposon is actively transcribed in hookworms.A mariner-like transposon termed bandit has colonized the genome of the hookworm A. caninum. Although MLEs exhibit a broad host range, and are identified in other nematodes, the closest phylogenetic relative of bandit is the Hsmar1 element of humans. This surprising finding suggests

Mariner and the ITm Superfamily of Transposons.

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Tellier, Michael; Bouuaert, Corentin Claeys; Chalmers, Ronald

2015-04-01

The IS630-Tc1-mariner (ITm) family of transposons is one of the most widespread in nature. The phylogenetic distribution of its members shows that they do not persist for long in a given lineage, but rely on frequent horizontal transfer to new hosts. Although they are primarily selfish genomic-parasites, ITm transposons contribute to the evolution of their hosts because they generate variation and contribute protein domains and regulatory regions. Here we review the molecular mechanism of ITm transposition and its regulation. We focus mostly on the mariner elements, which are understood in the greatest detail owing to in vitro reconstitution and structural analysis. Nevertheless, the most important characteristics are probably shared across the grouping. Members of the ITm family are mobilized by a cut-and-paste mechanism and integrate at 5'-TA dinucleotide target sites. The elements encode a single transposase protein with an N-terminal DNA-binding domain and a C-terminal catalytic domain. The phosphoryl-transferase reactions during the DNA-strand breaking and joining reactions are performed by the two metal-ion mechanism. The metal ions are coordinated by three or four acidic amino acid residues located within an RNase H-like structural fold. Although all of the strand breaking and joining events at a given transposon end are performed by a single molecule of transposase, the reaction is coordinated by close communication between transpososome components. During transpososome assembly, transposase dimers compete for free transposon ends. This helps to protect the host by dampening an otherwise exponential increase in the rate of transposition as the copy number increases.

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota

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Gudeta, Dereje D.; Bortolaia, Valeria; Pollini, Simona

2016-01-01

as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59......% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli...

Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

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Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

2014-09-01

Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

Sequence-function-stability relationships in proteins from datasets of functionally annotated variants: The case of TEM beta-lactamases

NARCIS (Netherlands)

Abriata, L.A.; Salverda, M.L.M.; Tomatis, P.E.

2012-01-01

A dataset of TEM lactamase variants with different substrate and inhibition profiles was compiled and analyzed. Trends show that loops are the main evolvable regions in these enzymes, gradually accumulating mutations to generate increasingly complex functions. Notably, many mutations present in

Molecular Typing of Enterobacteriaceae from Pig Holdings in North-Western Germany Reveals Extended- Spectrum and AmpC β-Lactamases Producing but no Carbapenem Resistant Ones

NARCIS (Netherlands)

Garcia-Cobos, Silvia; Koeck, Robin; Mellmann, Alexander; Frenzel, Julia; Friedrich, Alexander W.; Rossen, John W. A.

2015-01-01

The increase of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in humans and in food-producing animals is of public health concern. The latter could contribute to spreading of these bacteria or their resistance genes to humans. Several studies have reported the isolation of

High prevalence of extensively drug-resistant and metallo beta-lactamase-producing clinical Acinetobacter baumannii in Iran.

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Maspi, Hossein; Mahmoodzadeh Hosseini, Hamideh; Amin, Mohsen; Imani Fooladi, Abbas Ali

2016-09-01

Acinetobacter species particularly Acinetobacter baumannii (A. baumannii) have been widely reported as broad-spectrum antibiotic resistant pathogens. Expression of various types of metallo beta-lactamases (MBL), classified as Ambler class B, has been associated with carbapenem resistance. Here, we attempted to assess the frequency of extensively drug-resistant (XDR) and MBL-producing A. baumannii among clinical isolates. 86 clinical A. baumannii strains were collected from 2014 to 2015 and their susceptibility to meropenem (10 μg), imipenem (10 μg), azteronem (30 μg), pipracillin (100 μg) tazobactam (110 μg), tobramycin (10 μg), fosfomycin (200 μg), rifampicin (5 μg), colistin (10 μg), tigecycline (15 μg), sulbactam/ampicillin (10 μg + 10 μg) and polymixin B (300 U) was evaluated using disk diffusion method. The MBL-producing isolates were screened using combined disc diffusion method. Furthermore, the presence of blaVIM, blaIMP, blaSPM, blaGIM, blaSIM and blaNDM was detected by PCR. 34.9% of isolates were recovered from bronchoalveolar lavage (BAL). 81 (94.2%) and 62 (71.2%) isolates were multidrug resistance (MDR) and XDR, respectively. 44 (51.2%) and 65 (75.6%) isolates were MBL-producing strains with resistance to imipenem and meropenem, respectively. 2 (2.3%), 13 (15.1%), 2 (2.3%), 4 (4.7%) and 2 (2.3%) isolates carried blaVIM, blaIMP, blaSPM, blaGIM and blaSIM genes, respectively. Our data showed that the rate of XDR and MBL A. baumannii is on the rise. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Active miniature inverted-repeat transposable elements transposon in plants: a review].

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Hu, Bingjie; Zhou, Mingbing

2018-02-25

Miniature inverted-repeat transposable elements transposon is a special transposon that could transpose by "cut-paste" mechanism, which is one of characteristics of DNA transposons. Otherwise, the copy number of MITEs is very high, which is one of characteristics of RNA transposons. Many MITE families have been reported, but little about active MITEs. We summarize recent advances in studying active MITEs. Most the MITEs belong to the Tourist-like family, such as mPing, mGing, PhTourist1, Tmi1 and PhTst-3. Additionally, DTstu1 and MITE-39 belong to Stowaway-like family, and AhMITEs1 belongs to Mutator-like family. Moreover, we summarize the structure (terminal inverse repeats and target site duplications), copy number, evolution pattern and transposition characteristics of these active MITEs, to provide the foundation for the identification of other active MITEs and subsequent research on MITE transposition and amplification mechanism.

Successful elimination of extended-spectrum beta-lactamase (ESBL)-producing nosocomial bacteria at a neonatal intensive care unit.

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Szél, Borbála; Reiger, Zsolt; Urbán, Edit; Lázár, Andrea; Mader, Krisztina; Damjanova, Ivelina; Nagy, Kamilla; Tálosi, Gyula

2017-06-01

Extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria are highly dangerous to neonates. At our Neonatal Intensive Care Unit (NICU), the presence of these bacteria became so threatening in 2011 that immediate intervention was required. This study was conducted during a nearly two-year period consisting of three phases: retrospective (9 months), educational (3 months) and prospective (9 months). Based on retrospective data analysis, a complex management plan was devised involving the introduction of the INSURE protocol, changes to the antibiotic regimen, microbiological screening at short intervals, progressive feeding, a safer bathing protocol, staff hand hygiene training and continuous monitoring of the number of newly infected and newly colonized patients. During these intervals, a total of 355 patients were monitored. Both ESBL-producing Enterobacter cloaceae and Klebsiella pneumoniae were found (in both patients and environmental samples). In the prospective period a significant reduction could be seen in the average number of both colonized (26/167 patients; P=0.029) and infected (3/167 patients; P=0.033) patients compared to data from the retrospective period regarding colonized (72/188 patients) and infected (9/188 patients) patients. There was a decrease in the average number of patient-days (from 343.72 to 292.44 days per months), though this difference is not significant (P=0.058). During the prospective period, indirect hand hygiene compliance showed a significant increase (from the previous 26.02 to 33.6 hand hygiene procedures per patient per hospital day, Pinfections were rolled back successfully in a multi-step effort that required an interdisciplinary approach.

In vitro Effectiveness of Commercial Bacteriophage Cocktails on Diverse Extended-Spectrum Beta-Lactamase Producing Escherichia coli Strains.

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Gundogdu, Aycan; Bolkvadze, Darajen; Kilic, Huseyin

2016-01-01

The objective of this study is to determine the in vitro susceptibility of Georgian bacteriophage cocktails on multidrug resistant (MDR) extended-spectrum beta-lactamase producing Escherichia coli (ESBL-EC) isolated from patients' blood and urine cultures. A total of 615 E. coli isolates were included in this study. Phene Plate (PhP)-typing and phylogenetic grouping were used for the typing. Antimicrobial resistance profiles and ESBL production of all isolates were confirmed according to Clinical and Laboratory Standards Institute (CLSI) criteria. The activities of four bacteriophage cocktails (Enko-phage, SES-bacteriophage, Pyo-bacteriophage, and Intesti-bacteriophage) were determined against 142 ESBL-EC using in vitro spot tests. According to this, Enko-phage were active against 87.3% of the tested strains while that ratio was 81.7% for Intesti-bacteriophage, 81.7% for Pyo-bacteriophage, and 59.2% for SES-bacteriophage cocktails. Based on the contingency tests, the phage cocktails were observed to be statistically significantly ( p < 0.001) more effective on ESBL-EC strains belonging to phylogenetic groups D and B2. The employed phage cocktails were found to be affective against all tested resistant types. These results are promising especially for the infections that are caused by MDR pathogens that are difficult to treat. As this is a preliminary step to the potential clinical trials to be designed for the country, in vitro confirmation of their success on a MDR ESBL-EC collection should be accepted as an initial action, which is encouraging to consider clinical trials of phage therapy especially in countries which are not introduce phage therapy.

Comparison between phenotypic and PCR for detection of OXA-23 type and metallo-beta-lactamases producer Acinetobacter spp.

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Azimi, Leila

2013-11-01

Full Text Available [english] Background: Resistance to carbapenems is developing around the world and can cause many problems for treatment of patients. Production of metallo-beta-lactamase (MBL is one of the main mechanism for this type of resistance. So, detection of MBL-producer microorganisms can prevent the spread of this type of resistance.Materials and methods: In this study 94 spp. were investigated. Resistance to imipenem was conducted after purification and identification. Combination disc (CD and Double Disc Synergy Test (DDST were performed for phenotypic detection of MBL and the molecular PCR method was done for vim-1, vim-2, imp-1 and OXA-23 genes.Results: According to TSI, SIM and oxidation-fermentation (OF test and PCR assay 93 and one strain were identified. 85% of them were resistant to imipenem. 34% of them have a positive combination disc test (CD while Double Disc Synergy Test (DDST was negative for all of them. The vim-1, vim-2 and imp-1 genes were not detected in PCR molecular method, however in 74% of strains with positive results in combination disc, were positive for the OXA-23 gene after PCR test. This study shows that the blaOXA-23 resistance determinant may become an emerging therapeutic problem.Discussion: According to the results, it seems that combination disc does not have enough specificity for detection of MBL-producer and using Double Disc Synergy Test (DDST can be more convenient.

Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

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Ruiz, Lorena; Motherway, Mary O'Connell; Lanigan, Noreen; van Sinderen, Douwe

2013-01-01

Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

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Lorena Ruiz

Full Text Available Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

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Clouthier, Christopher M.; Morin, Sébastien; Gobeil, Sophie M. C.; Doucet, Nicolas; Blanchet, Jonathan; Nguyen, Elisabeth; Gagné, Stéphane M.; Pelletier, Joelle N.

2012-01-01

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions. PMID:23284969

Validation of Minim typing for fast and accurate discrimination of extended-spectrum, beta-lactamase-producing Klebsiella pneumoniae isolates in tertiary care hospital.

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Brhelova, Eva; Kocmanova, Iva; Racil, Zdenek; Hanslianova, Marketa; Antonova, Mariya; Mayer, Jiri; Lengerova, Martina

2016-09-01

Minim typing is derived from the multi-locus sequence typing (MLST). It targets the same genes, but sequencing is replaced by high resolution melt analysis. Typing can be performed by analysing six loci (6MelT), four loci (4MelT) or using data from four loci plus sequencing the tonB gene (HybridMelT). The aim of this study was to evaluate Minim typing to discriminate extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KLPN) isolates at our hospital. In total, 380 isolates were analyzed. The obtained alleles were assigned according to both the 6MelT and 4MelT typing scheme. In 97 isolates, the tonB gene was sequenced to enable HybridMelT typing. We found that the presented method is suitable to quickly monitor isolates of ESBL-KLPN; results are obtained in less than 2 hours and at a lower cost than MLST. We identified a local ESBL-KLPN outbreak and a comparison of colonizing and invasive isolates revealed a long term colonization of patients with the same strain. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficacy of a once-a-week screening programme to control extended-spectrum beta-lactamase-producing bacteria in a neonatal intensive care unit.

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Rybczynska, Helena; Melander, Eva; Johansson, Hugo; Lundberg, Fredrik

2014-06-01

Extended-spectrum beta-lactamase (ESBL)-producing bacteria are an escalating problem threatening health. Devastating consequences can result in neonatal intensive care units (NICU) due to these bacteria. The aim of this study was to investigate the efficacy of once-a-week screening (July 2010 to September 2012) versus screening on demand (April 2008 to June 2010). The investigation was an open retrospective descriptive study comparing 2 unpaired groups, the first exposed to screening on demand and the second to screening once a week. All other infection control measures were unchanged. Both groups were cared for in the NICU of Skåne University Hospital. Parameters compared were the proportion of cultured neonates, prevalence, time before detection, number of secondary cases, and clinical infections due to ESBL-producing bacteria. The proportion of cultured neonates increased from 28% to 49% (p control the epidemiology of unwanted pathogens among newborn infants. It provides the opportunity for early intervention, thereby avoiding secondary cases and infections. Premature neonates in particular benefit from this approach. The prevalence of ESBL of 1.77% is low from an international perspective. ESBL appear to be introduced onto the ward by mothers colonized with ESBL.

Induction of rat liver tumor using the Sleeping Beauty transposon and electroporation.

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Park, June-Shine; Kim, Bae-Hwan; Park, Sung Goo; Jung, Sun Young; Lee, Do Hee; Son, Woo-Chan

2013-05-10

The Sleeping Beauty (SB) transposon system has been receiving much attention as a gene transfer method of choice since it allows permanent gene expression after insertion into the host chromosome. However, low transposition frequency in higher eukaryotes limits its use in commonly-used mammalian species. Researchers have therefore attempted to modify gene delivery and expression to overcome this limitation. In mouse liver, tumor induction using SB introduced by the hydrodynamic method has been successfully accomplished. Liver tumor in rat models using SB could also be of great use; however, dose of DNA, injection volume, rate of injection and achieving back pressure limit the use of the hydrodynamics-based gene delivery. In the present study, we combined the electroporation, a relatively simple and easy gene delivery method, with the SB transposon system and as a result successfully induced tumor in rat liver by directly injecting the c-Myc, HRAS and shp53 genes. The tumor phenotype was determined as a sarcomatoid carcinoma. To our knowledge, this is the first demonstration of induction of tumor in the rat liver using the electroporation-enhanced SB transposon system. Copyright © 2013 Elsevier Inc. All rights reserved.

Genetic characterization of antibiotic resistance in enteropathogenic Escherichia coli carrying extended-spectrum beta-lactamases recovered from diarrhoeic rabbits.

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Poeta, P; Radhouani, H; Gonçalves, A; Figueiredo, N; Carvalho, C; Rodrigues, J; Igrejas, G

2010-05-01

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended-spectrum beta-lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K-:H2) was observed among 17 EPEC strains, the O92:K-serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K-serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim-sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla(TEM) gene was demonstrated in the majority of ampicillin-resistant isolates. The aac(3)-II or aac(3)-IV genes were detected in the four gentamicin-resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin-resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline-resistant isolates. A total of nine EPEC isolates showed the phenotype SXT-resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E

Epigenetics and Evolution: Transposons and the Stochastic Epigenetic Modification Model

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Sergio Branciamore

2015-04-01

Full Text Available In addition to genetic variation, epigenetic variation and transposons can greatly affect the evolutionary fitnesses landscape and gene expression. Previously we proposed a mathematical treatment of a general epigenetic variation model that we called Stochastic Epigenetic Modification (SEM model. In this study we follow up with a special case, the Transposon Silencing Model (TSM, with, once again, emphasis on quantitative treatment. We have investigated the evolutionary effects of epigenetic changes due to transposon (T insertions; in particular, we have considered a typical gene locus A and postulated that (i the expression level of gene A depends on the epigenetic state (active or inactive of a cis- located transposon element T, (ii stochastic variability in the epigenetic silencing of T occurs only in a short window of opportunity during development, (iii the epigenetic state is then stable during further development, and (iv the epigenetic memory is fully reset at each generation. We develop the model using two complementary approaches: a standard analytical population genetics framework (di usion equations and Monte-Carlo simulations. Both approaches led to similar estimates for the probability of fixation and time of fixation of locus TA with initial frequency P in a randomly mating diploid population of effective size Ne. We have ascertained the e ect that ρ, the probability of transposon Modification during the developmental window, has on the population (species. One of our principal conclusions is that as ρ increases, the pattern of fixation of the combined TA locus goes from "neutral" to "dominant" to "over-dominant". We observe that, under realistic values of ρ, epigenetic Modifications can provide an e cient mechanism for more rapid fixation of transposons and cis-located gene alleles. The results obtained suggest that epigenetic silencing, even if strictly transient (being reset at each generation, can still have signi cant

Antibiotics as selectors and accelerators of diversity in the mechanisms of resistance: From the resistome to genetic plasticity in the beta-lactamases world

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Juan- Carlos eGalán

2013-02-01

Full Text Available Antibiotics and antibiotic resistance determinants, natural molecules closely related to bacterial physiology and consistent with an ancient origin, are not only present in antibiotic-producing bacteria. Throughput sequencing technologies have revealed an unexpected reservoir of antibiotic resistance in the environment. These data suggest that co-evolution between antibiotic and antibiotic resistance genes has occurred since the beginning of time. This evolutionary race has probably been slow because of highly regulated processes and low antibiotic concentrations. Therefore to understand this global problem, a new variable must be introduced, that the antibiotic resistance is a natural event, inherent to life. However, the industrial production of natural and synthetic antibiotics has dramatically accelerated this race, selecting some of the many resistance genes present in nature and contributing to their diversification. One of the best models available to understand the biological impact of selection and diversification are -lactamases. They constitute the most widespread mechanism of resistance, at least among pathogenic bacteria, with more than 1000 enzymes identified in the literature. In the last years, there has been growing concern about the description, spread and diversification of -lactamases with carbapenemase activity and AmpC-type in plasmids. Phylogenies of these enzymes help the understanding of the evolutionary forces driving their selection. Moreover, understanding the adaptive potential of -lactamases contribute to exploration the evolutionary antagonists trajectories through the design of more efficient synthetic molecules. In this review, we attempt to analyse the antibiotic resistance problem from new perspectives. From intrinsic and environmental resistomes to the adaptive potential of resistance genes and the driving forces involved in their diversification, in order to provide a global perspective of the

Emergence of quinolone resistance among extended-spectrum beta-lactamase-producing Enterobacteriaceae in the Central African Republic: genetic characterization

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Frank Thierry

2011-08-01

Full Text Available Abstract Background Cross-resistance to quinolones and beta-lactams is frequent in Enterobacteriaceae, due to the wide use of these antibiotics clinically and in the food industry. Prescription of one of these categories of antibiotic may consequently select for bacteria resistant to both categories. Genetic mechanisms of resistance may be secondary to a chromosomal mutation located in quinolone resistance determining region of DNA gyrase or topoisomerase IV or to a plasmid acquisition. The insertion sequence ISCR1 is often associated with qnr and may favour its dissemination in Gram-negative bacteria. The aim of this study was to determine the genetic mechanism of quinolone resistance among extended-spectrum beta-lactamase-producing Enterobacteriaceae strains in the Central African Republic. Findings Among seventeen ESBL-producing Enterobacteriaceae isolated from urine, pus or stool between January 2003 and October 2005 in the Central African Republic, nine were resistant to ciprofloxacin (seven from community patients and two from hospitalized patients. The ESBL were previously characterized as CTX-M-15 and SHV-12. Susceptibility to nalidixic acid, norfloxacin and ciprofloxacin, and the minimal inhibitory concentrations of these drugs were determined by disc diffusion and agar dilution methods, respectively. The presence of plasmid-borne ISCR1-qnrA region was determined by PCR and amplicons, if any, were sent for sequencing. Quinolone resistance determining region of DNA gyrase gyrA gene was amplified by PCR and then sequenced for mutation characterization. We found that all CTX-M-producing strains were resistant to the tested quinolones. All the isolates had the same nucleotide mutation at codon 83 of gyrA. Two Escherichia coli strains with the highest MICs were shown to harbour an ISCR1-qnrA1 sequence. This genetic association might favour dissemination of resistance to quinolone and perhaps other antibiotics among Enterobacteriaceae

Isolation and identification of extended-spectrum beta-lactamase (ESBL-producing Escherichia coli from brolier in Erbil, Iraq

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M.N. Al-Sharook

2017-06-01

Full Text Available Extended-spectrum beta-lactamase-producing Escherichia coli isolated from slaughtered broilers in retail market that sell live chickens in Erbil city, Iraq. Forty-one cloacal fecal samples from broiler caecum were investigated from January to April 2016. ESBLs strains were isolated using MacConkey agar supplemented with cefotaxime 1 mg/l and the isolates were identified phynotypically by biochemical tests, TBX agar and VITEK-2 compact system. A total of 34 Escherichia coli and 4 Proteus mirabilis were analysed for determination of ESBL/AmpC by disc diffusion test using antimicrobial 68DC MAST® ESβL discs group including cefpodoxime, cefpodoxime + ESBL inhibitor, cefpodoxime + AmpC inhibitor and cefpodoxime + ESBL inhibitor + AmpC inhibitor and 67DC MAST® ESβL discs group including cefpodoxime, cefpodoxime + clavulanate, ceftazidime, ceftazidime + clavulanate, cefotaxime and cefotaxime + clavulanate. The phenotypic results showed that in group 68DC discs 23.7% E. coli were resistant to cefpodoxime and in group 67DC discs 73.7% of E. coli and 7.9% of P. mirabilis were resistance to one or more of the cefpodoxime, ceftazidime and ceftazidime. Final results revealed that 78.0% of samples were ESBLs/ AmpC positive. This study is the first examination to determine phenorypically E. coli producing ESBLs/AmpC in broiler chickens in Iraq. Conclusion, the healthy broiler can be a major source of ESBLs/AmpC and the possibility that transmitted to humans through the food chain, direct contact and the surrounding environment raises the concerns about public health and safety of poultry meat and the negative consequences of drug therapy that causes the spread of antibiotic resistance.

Synthesis of silver nanoparticles using the Streptomyces coelicolor klmp33 pigment: An antimicrobial agent against extended-spectrum beta-lactamase (ESBL) producing Escherichia coli

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Manikprabhu, Deene; Lingappa, K.

2014-01-01

The increasing emergence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) occurred mainly due to continuous persistent exposure to antibiotics causing high morbidity and mortality so studies in controlling this infection are required. In the present investigation, we developed a synthesis for silver nanoparticles employing a pigment produced by Streptomyces coelicolor klmp33, and assessed the antimicrobial activity of these nanoparticles against ESBL producing E. coli. The ESBL producing E. coli were isolated from urine samples collected from the Gulbarga region in India. As can been seen from our studies, the silver nanoparticles having irregular shapes and size of 28–50 nm showed remarkable antimicrobial activity and moreover the synthesis time is just 20 min and thus the same can be used for formulating pharmaceutical remedies. - Highlights: • Silver nanoparticle synthesis by photo-irradiation method in just 20 min • Isolation of ESBL producing E. coli from urine samples from the Gulbarga region. • Antimicrobial activity of silver nanoparticles against ESBL producing E. coli • The minimum inhibitory concentration of silver nanoparticles against ESBL producing E. coli was 40 μL

Synthesis of silver nanoparticles using the Streptomyces coelicolor klmp33 pigment: An antimicrobial agent against extended-spectrum beta-lactamase (ESBL) producing Escherichia coli

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Manikprabhu, Deene; Lingappa, K., E-mail: lingappak123@gmail.com

2014-12-01

The increasing emergence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) occurred mainly due to continuous persistent exposure to antibiotics causing high morbidity and mortality so studies in controlling this infection are required. In the present investigation, we developed a synthesis for silver nanoparticles employing a pigment produced by Streptomyces coelicolor klmp33, and assessed the antimicrobial activity of these nanoparticles against ESBL producing E. coli. The ESBL producing E. coli were isolated from urine samples collected from the Gulbarga region in India. As can been seen from our studies, the silver nanoparticles having irregular shapes and size of 28–50 nm showed remarkable antimicrobial activity and moreover the synthesis time is just 20 min and thus the same can be used for formulating pharmaceutical remedies. - Highlights: • Silver nanoparticle synthesis by photo-irradiation method in just 20 min • Isolation of ESBL producing E. coli from urine samples from the Gulbarga region. • Antimicrobial activity of silver nanoparticles against ESBL producing E. coli • The minimum inhibitory concentration of silver nanoparticles against ESBL producing E. coli was 40 μL.

The profile of antibiotics resistance and integrons of extended-spectrum beta-lactamase producing thermotolerant coliforms isolated from the Yangtze River basin in Chongqing

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Chen Hao [Department of Environmental Hygiene, School of Military Preventive Medicine, Third Military Medical University, 30 Gaotanyan Street, Chongqing 400038 (China); Shu Weiqun, E-mail: west2003@sohu.co [Department of Environmental Hygiene, School of Military Preventive Medicine, Third Military Medical University, 30 Gaotanyan Street, Chongqing 400038 (China); Chang Xiaosong; Chen Jian; Guo Yebin; Tan Yao [Department of Environmental Hygiene, School of Military Preventive Medicine, Third Military Medical University, 30 Gaotanyan Street, Chongqing 400038 (China)

2010-07-15

The spreading of extended-spectrum {beta}-lactamases (ESBL)-producing thermotolerant coliforms (TC) in the water environment is a threat to human health but little is known about ESBL-producing TCs in the Yangtze River. We received 319 ESBL-producing stains obtained from the Chongqing basin and we investigated antibiotic susceptibility, bla gene types and the presence of integrons and gene cassettes. 16.8% of TC isolates were ESBL-producing bacteria and bla{sub TEM+CTx-M} was the predominant ESBL type. 65.2% of isolates contained class 1 integrons, but only 3 carried intI 2. Gene cassettes were amplified and sequenced. aadA, drfA, cmlA, sat1, aar3 and two ORF cassettes were found. In conclusion, Yangtze River is heavily polluted by ESBL-producing TC bacteria and the combined bla gene type could enhance antibiotic resistance. Class 1 integrons were widespread in ESBL-producing isolates and play an important role in multi-drug resistance. Characterization of gene cassettes could reveal the dissemination of antibiotic resistance genes. - Yangtze River is heavily polluted by ESBL-producing TC bacteria and Class 1 integrons play an important role in multi-drug resistance.

THE NEW METALL-BETA-LACTAMASE’S INHIBITOR EFFICACY IN A MODEL SYSTEM IN VITRO

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A. G. Afinogenova

2016-01-01

Full Text Available The Enterobacteriaceae antibiotics resistance depends on a combination of several mechanisms, such as the beta-lactamases overproduction, the microbial cell reduction outer membrane permeability (usually associated with loss of protein porin, the presence of efflux systems. Particularly noteworthy are the metallo-beta-lactamases (MBL whose presence causes resistance of gram-negative microorganisms to all beta-lactam antibiotics (in some cases except aztreonam. Currently there are no MBL inhibitors permitted for use in the clinic. The effective inhibitors search for carbapenem-resistant bacteria’ MBL authorized for use in the clinic and reinforcing effects of carbapenems, served as the basis for the present study. The work was carried out in three stages: 1 creating a model system using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, to evaluate the increasing of minimal inhibitory concentrations (MIC of carbapenems against previously sensitive Gram-negative microorganisms strains in vitro; 2 evaluation of MBL promising inhibitors in the presence of the same standard enzyme reagent; 3 evaluation of the ability of the identified inhibitors increase the carbapenems effects against clinical isolates of Gram-negative microorganisms producing MBL, in terms of the their MIC and fractional inhibitory concentration index (FIC index. The checkerboard array was modified to evaluate the combined use of carbapenems and potential MBL inhibitor — a drug from the group of bisphosphonates — etidronic acid. Using a standard enzyme reagent metallo-beta-lactamase P. aeruginosa recombinant expressed in E. coli, we created a model system that allows to assess the prospects of new inhibitors MBL gram-negative microorganisms. A dose-dependent effect of increasing the meropenem level MIC from reagent MBL quantity in a model system against previously antibiotic sensitive reference strains of microorganisms was

In vitro selection of resistance in haemophilus influenzae by 4 quinolones and 5 beta-lactams.

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Clark, Catherine; Kosowska, Klaudia; Bozdogan, Bülent; Credito, Kim; Dewasse, Bonifacio; McGhee, Pamela; Jacobs, Michael R; Appelbaum, Peter C

2004-05-01

We tested abilities of ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, amoxicillin, amoxicillin/clavulanate, cefixime, cefpodoxime, and cefdinir to select resistant mutants in 5 beta-lactamase positive and 5 beta-lactamase negative Haemophilus influenzae strains by single and multistep methodology. In multistep tests, amoxicillin, amoxicillin/clavulanate and cefpodoxime exposure did not cause >4-fold minimum inhibitory concentration (MIC) increase after 50 days. One mutant selected by cefdinir had one amino acid substitution (Gly490Glu) in PBP3 and became resistant to cefdinir. Cefixime exposure caused 8-fold MIC-increase in 1 strain with TEM but the mutant remained cefixime susceptible and had no alteration in PBP3 or TEM. Among 10 strains tested, ciprofloxacin, moxifloxacin, gatifloxacin, levofloxacin caused >4-fold MIC increase in 6, 6, 5, and 2 strain, respectively. Despite the increases in quinolone MICs, none of the mutants became resistant to quinolones by established criteria. Quinolone selected mutants had quindone resistance-determining region (QRDR) alterations in GyrA, GyrB, ParC, ParE. Four quinolone mutants had no QRDR alterations. Among beta-lactams cefdinir and cefixime selected one mutant each with higher MICs however amoxicillin, amoxicillin/clavulanate, and cefpodoxime exposure did not select resistant mutants.

Himar1 Transposon for Efficient Random Mutagenesis in Aggregatibacter actinomycetemcomitans

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Qinfeng Ding

2017-09-01

Full Text Available Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism’s genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown. In this study, we found that Himar1 transposon mutagenesis occurs at a high frequency (×10-4, and can be universally applied to wild-type A. actinomycetemcomitans strains of serotypes a, b, and c. The Himar1 transposon inserts were stably inherited in A. actinomycetemcomitans transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of A. actinomycetemcomitans was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the Himar1 mutagenesis system as a novel genetic tool for functional genomic analysis in A. actinomycetemcomitans.

Yersinia enterocolitica and Photorhabdus asymbiotica β-lactamases BlaA are exported by the twin-arginine translocation pathway.

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Schriefer, Eva-Maria; Hoffmann-Thoms, Stephanie; Schmid, Franz X; Schmid, Annika; Heesemann, Jürgen

2013-01-01

In general, β-lactamases of medically important Gram-negative bacteria are Sec-dependently translocated into the periplasm. In contrast, β-lactamases of Mycobacteria spp. (BlaC, BlaS) and the Gram-negative environmental bacteria Stenotrophomonas maltophilia (L2) and Xanthomonas campestris (Bla(XCC-1)) have been reported to be secreted by the twin-arginine translocation (Tat) system. Yersinia enterocolitica carries 2 distinct β-lactamase genes (blaA and blaB) encoding BlaA(Ye) and the AmpC-like β-lactamase BlaB, respectively. By using the software PRED-TAT for prediction and discrimination of Sec from Tat signal peptides, we identified a functional Tat signal sequence for Yersinia BlaA(Ye). The Tat-dependent translocation of BlaA(Ye) could be clearly demonstrated by using a Y. enterocolitica tatC-mutant and cell fractionation. Moreover, we could demonstrate a unique unusual temperature-dependent activity profile of BlaA(Ye) ranging from 15 to 60 °C and a high 'melting temperature' (T(M)=44.3°) in comparison to the related Sec-dependent β-lactamase TEM-1 (20-50°C, T(M)=34.9 °C). Strikingly, the blaA gene of Y. enterocolitica is present in diverse environmental Yersinia spp. and a blaA homolog gene could be identified in the closely related Photorhabdus asymbiotica (BlaA(Pa); 69% identity to BlaA(Ye)). For BlaA(Pa) of P. asymbiotica, we could also demonstrate Tat-dependent secretion. These results suggest that Yersinia BlaA-related β-lactamases may be the prototype of a large Tat-dependent β-lactamase family, which originated from environmental bacteria. Copyright © 2012 Elsevier GmbH. All rights reserved.

The prevalence of extended-spectrum beta-lactamases and CTX-M-1 producing Escherichia coli in urine samples collected at Tabriz city Hospitals

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Soltan Dallal MM

2011-08-01

Full Text Available "nBackground: Numerous use of Beta Lactame in treatment of bacterial infections resulted in increments of drug resistance of such bacteria. One of difficulties in treatment of hospital infections is Extended Spectrum Beta Lactamase (ESBL among isolated clinical strains of E.coli. Since some of ESBL strains shows double reaction in drug sensitivity test at in vitro and in vivo condition, therefore it makes difficulties in selection of right treatment. In the last years, CTX-M enzymes have become the most prevalent ESBLs in worldwide. The prevalence of ESBL types largely remains unknown in many parts of the Iran. This study was made to find the prevalence of ESBL-producing E.coli and molecular detection of CTX-M-1 in Tabriz."n "nMethods: In the present study, 400 urine samples collected between November 2009 and April 2010, from Tabriz Hospitals were studied. Out of the 400 samples, 188 E.coli isolates were detected by standard biochemical tests. Susceptibility to antimicrobial agents was tested to 10 antibiotics by the disk agar diffusion (DAD method. ESBL production was screened by phenotypic test that included both separate and combined disk agar diffusion techniques. The screened isolates were investigated by PCR assay to detect CTX-M-1 gene."n "nResults: From the total 188 E.coli isolates, 82 (43.6% were shown to produce ESBLs by phenotypic test. During the PCR method on the 82 isolates, 69 (84.1% were confirmed as CTX-M-1 producing group."n "nConclusion: The present study showed that CTX-M-producing isolates were increasing among E.coli strains and indicated the need for adequate susceptibility tests in laboratories for choosing the appropriate antibiotics for treatment.

Epidemiology and molecular characterization of extended-spectrum beta-lactamase-producing Enterobacter spp., Pantoea agglomerans, and Serratia marcescens isolates from a Bulgarian hospital.

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Markovska, Rumyana Donkova; Stoeva, Temenuga Jekova; Bojkova, Kalina Dineva; Mitov, Ivan Gergov

2014-04-01

Forty-two extended-spectrum beta-lactamase (ESBL)-producing isolates of Enterobacter aerogenes, Enterobacter cloacae, Pantoea agglomerans, and Serratia marcescens, collected consecutively during the period January-November 2011 from the University Hospital in Varna, Bulgaria, were studied to characterize their ESBLs by isoelectric focusing, group-specific PCR, and sequencing. The epidemiological relationship was evaluated by random amplified polymorphic DNA analysis (RAPD). Transferability of ESBL genes was determined by conjugation experiments. Plasmid analysis was done by replicon typing and PstI fingerprinting. The overall rate of ESBL production was 20%. The most widespread enzyme was CTX-M-3, found in 64%. It was dominant in E. aerogenes (100%) and S. marcescens (83%). SHV-12, CTX-M-3, and CTX-M-15 were found among E. cloacae isolates in 50%, 35%, and 45%, respectively. Three main CTX-M-3-producing epidemic clones of E. aerogenes and S. marcescens have been detected. Among E. cloacae isolates, six different RAPD profiles were discerned. The plasmids harboring blaCTX-M-3 belonged to IncL/M type and demonstrated similar PstI fingerprinting profiles. IncFII plasmids were detected in two CTX-M-15-producing E. cloacae isolates. Our results demonstrate wide intrahospital dissemination of clonal E. aerogenes and S. marcescens isolates, carrying IncL/M conjugative plasmids.

Mariner transposons are sailing in the genome of the blood-sucking bug Rhodnius prolixus.

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Filée, Jonathan; Rouault, Jacques-Deric; Harry, Myriam; Hua-Van, Aurélie

2015-12-15

The Triatomine bug Rhodnius prolixus is a vector of Trypanosoma cruzi, which causes the Chagas disease in Latin America. R. prolixus can also transfer transposable elements horizontally across a wide range of species. We have taken advantage of the availability of the 700 Mbp complete genome sequence of R. prolixus to study the dynamics of invasion and persistence of transposable elements in this species. Using both library-based and de novo methods of transposon detection, we found less than 6 % of transposable elements in the R. prolixus genome, a relatively low percentage compared to other insect genomes with a similar genome size. DNA transposons are surprisingly abundant and elements belonging to the mariner family are by far the most preponderant components of the mobile part of this genome with 11,015 mariner transposons that could be clustered in 89 groups (75 % of the mobilome). Our analysis allowed the detection of a new mariner clade in the R. prolixus genome, that we called nosferatis. We demonstrated that a large diversity of mariner elements invaded the genome and expanded successfully over time via three main processes. (i) several families experienced recent and massive expansion, for example an explosive burst of a single mariner family led to the generation of more than 8000 copies. These recent expansion events explain the unusual prevalence of mariner transposons in the R. prolixus genome. Other families expanded via older bursts of transposition demonstrating the long lasting permissibility of mariner transposons in the R. prolixus genome. (ii) Many non-autonomous families generated by internal deletions were also identified. Interestingly, two non autonomous families were generated by atypical recombinations (5' part replacement with 3' part). (iii) at least 10 cases of horizontal transfers were found, supporting the idea that host/vector relationships played a pivotal role in the transmission and subsequent persistence of transposable

Molecular detection of TEM broad spectrum β-lactamase in clinical ...

African Journals Online (AJOL)

Resistance to β-lactam antibiotics, along with clinical isolates, frequently results to production of β- lactamase enzymes. In recent years, the production of extended spectrum β-lactamases (ESBLs) among clinical isolates, especially Escherichia coli has greatly increased. On the other hand, β lactamase genes have several ...

Genetic markers associated with resistance to beta-lactam and quinolone antimicrobials in non-typhoidal Salmonella isolates from humans and animals in central Ethiopia

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Tadesse Eguale

2017-01-01

Full Text Available Abstract Background Beta-lactam and quinolone antimicrobials are commonly used for treatment of infections caused by non-typhoidal Salmonella (NTS and other pathogens. Resistance to these classes of antimicrobials has increased significantly in the recent years. However, little is known on the genetic basis of resistance to these drugs in Salmonella isolates from Ethiopia. Methods Salmonella isolates with reduced susceptibility to beta-lactams (n = 43 were tested for genes encoding for beta-lactamase enzymes, and those resistant to quinolones (n = 29 for mutations in the quinolone resistance determining region (QRDR as well as plasmid mediated quinolone resistance (PMQR genes using PCR and sequencing. Results Beta-lactamase genes (bla were detected in 34 (79.1% of the isolates. The dominant bla gene was blaTEM, recovered from 33 (76.7% of the isolates, majority being TEM-1 (24, 72.7% followed by TEM-57, (10, 30.3%. The blaOXA-10 and blaCTX-M-15 were detected only in a single S. Concord human isolate. Double substitutions in gyrA (Ser83-Phe + Asp87-Gly as well as parC (Thr57-Ser + Ser80-Ile subunits of the quinolone resistance determining region (QRDR were detected in all S. Kentucky isolates with high level resistance to both nalidixic acid and ciprofloxacin. Single amino acid substitutions, Ser83-Phe (n = 4 and Ser83-Tyr (n = 1 were also detected in the gyrA gene. An isolate of S. Miami susceptible to nalidixic acid but intermediately resistant to ciprofloxacin had Thr57-Ser and an additional novel mutation (Tyr83-Phe in the parC gene. Plasmid mediated quinolone resistance (PMQR genes investigated were not detected in any of the isolates. In some isolates with decreased susceptibility to ciprofloxacin and/or nalidixic acid, no mutations in QRDR or PMQR genes were detected. Over half of the quinolone resistant isolates in the current study 17 (58.6% were also resistant to at least one of the beta-lactam antimicrobials

The expanding universe of transposon technologies for gene and cell engineering

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Ivics Zoltán

2010-12-01

Full Text Available Abstract Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (shRNA expression cassette, a mutagenic gene trap or a therapeutic gene construct cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB, discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.

The expanding universe of transposon technologies for gene and cell engineering.

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Ivics, Zoltán; Izsvák, Zsuzsanna

2010-12-07

Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons) can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (sh)RNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB), discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.

Faecal carriage of extended-spectrum b-lactamase-producing and AMpC b-lactamase-producing bacteria among Danish army recruits

DEFF Research Database (Denmark)

Hammerum, A.M; Lester, C.H; Jakobsen, L

2011-01-01

During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum b-lactamase (ESBL)- producing and AmpC b-lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one Amp...

Rapid optical determination of β-lactamase and antibiotic activity

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2014-01-01

Background The absence of rapid tests evaluating antibiotic susceptibility results in the empirical prescription of antibiotics. This can lead to treatment failures due to escalating antibiotic resistance, and also furthers the emergence of drug-resistant bacteria. This study reports a rapid optical method to detect β-lactamase and thereby assess activity of β-lactam antibiotics, which could provide an approach for targeted prescription of antibiotics. The methodology is centred on a fluorescence quenching based probe (β-LEAF – β-Lactamase Enzyme Activated Fluorophore) that mimics the structure of β-lactam antibiotics. Results The β-LEAF assay was performed for rapid determination of β-lactamase production and activity of β-lactam antibiotic (cefazolin) on a panel of Staphylococcus aureus ATCC strains and clinical isolates. Four of the clinical isolates were determined to be lactamase producers, with the capacity to inactivate cefazolin, out of the twenty-five isolates tested. These results were compared against gold standard methods, nitrocefin disk test for β-lactamase detection and disk diffusion for antibiotic susceptibility, showing results to be largely consistent. Furthermore, in the sub-set of β-lactamase producers, it was demonstrated and validated that multiple antibiotics (cefazolin, cefoxitin, cefepime) could be assessed simultaneously to predict the antibiotic that would be most active for a given bacterial isolate. Conclusions The study establishes the rapid β-LEAF assay for β-lactamase detection and prediction of antibiotic activity using S. aureus clinical isolates. Although the focus in the current study is β-lactamase-based resistance, the overall approach represents a broad diagnostic platform. In the long-term, these studies form the basis for the development of assays utilizing a broader variety of targets, pathogens and drugs. PMID:24708478

A novel thermometric biosensor for fast surveillance of β-lactamase activity in milk.

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Zhou, Shuang; Zhao, Yunfeng; Mecklenburg, Michael; Yang, Dajin; Xie, Bin

2013-11-15

Regulatory restrictions on antibiotic residues in dairy products have resulted in the illegal addition of β-lactamase to lower antibiotic levels in milk in China. Here we demonstrate a fast, sensitive and convenient method based on enzyme thermistor (ET) for the surveillance of β-lactamase in milk. A fixed amount of penicillin G, which is a specific substrate of β-lactamase, was incubated with the milk sample, and an aliquot of the mixture was directly injected into the ET system to give a temperature change corresponding to the remained penicillin G. The amount of β-lactamase present in sample was deduced by the penicillin G consumed during incubation. This method was successfully applied to quantify β-lactamase in milk with the linear range of 1.1-20 UmL(-1) and the detection limit of 1.1 UmL(-1). The recoveries ranged from 93% to 105%, with relative standard deviations (RSDs) below 8%. The stability of the column equipped in ET was also studied, and only 5% decrease of activity was observed after 60 days of use. Compared with the conventional culture-based assay, the advantages of high throughput, timesaving and accurate quantification have made this method an ideal alternative for routine use. Copyright © 2013 Elsevier B.V. All rights reserved.

A plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteria

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Rita eMonson

2015-12-01

Full Text Available Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

How different is the proteome of the extended spectrum β-lactamase producing Escherichia coli strains from seagulls of the Berlengas natural reserve of Portugal?

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Monteiro, R; Hébraud, M; Chafsey, I; Poeta, P; Igrejas, G

2016-08-11

β-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Escherichia coli strains 5A, 10A, 12A and 23B isolated from Seagulls feces, are cefotaxime-resistant strains that produces extended-spectrum beta-lactamases. Bacterial resistance to these antibiotics occurs predominantly through structural modification on the penicillin-binding proteins and enzymatic inactivation by extended-spectrum β-lactamases. Using classical proteomic techniques (2D-GE) coupled to mass spectrometry and bioinformatics extended analysis, in this study, we report several significant differences in cytoplasmic proteins expression when the strains were submitted to antibiotic stress and when the resistant strains were compared with a non-resistant strain. A total of 79 differentially expressed spots were collected for protein identification. Significant level of expression was found in antibiotic resistant proteins like β-lactamase CTX-M-1 and TEM and also in proteins related with oxidative stress. This approach might help us understand which pathways form barriers for antibiotics, another possible new pathways involved in antibiotic resistance to devise appropriate strategies for their control already recognized by the World Health Organization and the European Commission. This study highlights the protein differences when a resistant strain is under antibiotic pressure and how different can be a sensible and resistant strain at the protein level. This survey might help us to understand the specifics barriers for antibiotics and which pathways are involved in its resistance crosswise the wildlife. Copyright © 2016 Elsevier B.V. All rights reserved.

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

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Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A

2001-11-01

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

Genome-scale metabolic network validation of Shewanella oneidensis using transposon insertion frequency analysis.

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Hong Yang

2014-09-01

Full Text Available Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA. TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1 previous genome-wide direct gene-essentiality assignments; and, 2 flux balance analysis (FBA predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.

Molecular determination of extended spectrum b-lactamases antibiotics resistance genes in E.coli isolated from diarrhea in cattle

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Ghassan Khudhair Ismaeel

2017-07-01

Full Text Available None response to the treatment by an antibiotic called antibiotics resistance result from some genes called resistance genes .This mechanism is widespread in most of the bacteria, like E.coli . All of the extended resistance genes called (ESBIS is a typical example for study of some genes that resistance beta-lactam antibiotic is subject of this research. Fifty feces sample were collected from cattle suffering from diarrhea in alqaissiyah city were cultured on selective media for E.coli , then DNA was extracted from all E.coli isolates for antibiotic resistance gene detection by PCR ; The results of this study revealed the prevalence of B-lactamase gene four B-lactamases genes in E.coli blaAmpc gene were (91.4%, the blactx-m gene were (80%, blaTem were (62.8% and finally and blaSHV gene were (22% among isolates E.coli ; blaAMPC gene has high prevalence than others genes while blaSHV was a lower percentage than other genes

Molecular characterization of extended-spectrum beta-lactamases (ESBLs) produced by clinical isolates of Acinetobacter baumannii in Saudi Arabia.

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Alyamani, Essam J; Khiyami, Mohamed A; Booq, Rayan Y; Alnafjan, Basel M; Altammami, Musaad A; Bahwerth, Fayez S

2015-08-20

Acinetobacter baumannii is a common opportunistic pathogen that causes major nosocomial infections in hospitals. In this study, we hypothesized a high prevalence of A. baumanni ESBL (extended-spectrum beta-lactamase) among all collected isolates. A. baumannii isolates (n = 107) from ICU (Intensive care unit) of local hospitals in Makkah were phenotypically and genotypically characterized. The identity and antibiotic susceptibility of A. baumannii strains were determined using the Vitek-2 system. The identified ESBL producers were further analyzed by PCR and sequencing followed by MLST typing. bla TEM , bla SHV , and the bla CTX-M-group genes 1, 2, 8, 9, and 25 were investigated. Furthermore, bla OXA51-like and bla OXA23-like genes were also examined in the carbapenem-resistant A. baumannii isolates. Our data indicated a high prevalence of A. baumannii ESBL producers among the collected strains. Of the 107 A. baumannii isolates, 94 % were found to be resistant to cefepime and ceftazidime, and aztreonam using the Vitek 2 system. The genes detected encoded TEM, OXA-51-like and OXA-23-like enzymes, and CTX-M-group proteins 1, 2, 8, 9, and 25. MLST typing identified eight sequence type (ST) groups. The most dominant STs were ST195 and ST557 and all of them belong to worldwide clonal complex (CC) 2. This study has shown that there is a high prevalence of antimicrobial resistance in A. baumannii. The diversity of STs may suggest that new ESBL strains are constantly emerging. The molecular diversity of the ESBL genes in A. baumannii may have contributed to the increased antimicrobial resistance among all isolates.

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in local and imported poultry meat in Ghana.

Science.gov (United States)

Eibach, Daniel; Dekker, Denise; Gyau Boahen, Kennedy; Wiafe Akenten, Charity; Sarpong, Nimako; Belmar Campos, Cristina; Berneking, Laura; Aepfelbacher, Martin; Krumkamp, Ralf; Owusu-Dabo, Ellis; May, Jürgen

2018-04-01

Antibiotic use in animal husbandry has raised concerns on the spread of resistant bacteria. Currently animal products are traded globally with unprecedented ease, which has been challenging the control of antimicrobial resistance. This study aims to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae from imported and locally produced poultry products sold in Ghana. Local and imported chicken meat was collected from 94 stores and markets throughout Kumasi (Ghana) and cultured on selective ESBL screening agar. Phenotypic ESBL-producing E. coli and K. pneumoniae isolates were confirmed by combined disc test and further characterized by antibiotic susceptibility testing, amplification of the bla CTX-M , bla TEM and bla SHV genes as well as multilocus sequence typing (MLST) and linked to the country of origin. Out of 200 meat samples, 71 (36%) samples revealed 81 ESBL-producing isolates (46 E. coli and 35 K. pneumoniae), with 44% (30/68) of local poultry and 31% (41/132) of imported products being contaminated. Most ESBL-producing isolates harboured the bla CTX-M-15 gene (61/81, 75%) and the dominant Sequence Types (ST) were ST2570 (7/35, 20%) among K. pneumoniae and ST10 (5/46, 11%) among E. coli. High numbers of ESBL-producing bacteria, particularly on local but also imported poultry meat, represent a potential source for human colonization and infection as well as spread within the community. Surveillance along the poultry production-food-consumer chain would be a valuable tool to identify sources of emerging multidrug resistant pathogens in Ghana. Copyright © 2018 Elsevier B.V. All rights reserved.

The effects of lower than conventional doses of oral nadolol on relative beta 1/beta 2-adrenoceptor blockade.

Science.gov (United States)

Wheeldon, N M; McDevitt, D G; Lipworth, B J

1994-08-01

1. The aim of the present study was to evaluate the relative beta 1/beta 2 antagonist selectivity of the beta-adrenoceptor blocker nadolol, in lower than conventional clinical doses. 2. Eight normal volunteers received single oral doses of either placebo (PL), nadolol 5 mg (N5), 20 mg (N20) or 80 mg (N80) in a single-blind, randomised crossover design. beta 1-adrenoceptor antagonism was assessed by attenuation of exercise tachycardia, and beta 2-adrenoceptor blockade by effects on salbutamol-induced chronotropic, hypokalaemic and finger tremor responses. The relative percentage attenuation of beta 2 and beta 1-mediated responses was calculated and expressed as beta 2:beta 1 selectivity ratios. 3. Nadolol produced dose-related reductions in exercise tachycardia in keeping with increasing beta 1-adrenoceptor blockade; mean % reduction (95% CI) compared with placebo: N5 10.7 (6.6 to 14.8), N20 21.4 (17.3 to 25.4), N80 38.9 (34.8 to 42.9). However, even the lowest dose of nadolol (5 mg) produced almost complete blunting of beta 2-mediated effects and significantly increase exercise hyperkalaemia; peak exercise hyperkalaemia (mmol l-1) (means and 95% CI): PL 4.88 (4.68 to 5.07), N5 5.36 (5.17 to 5.55), N20 5.48 (5.28 to 5.67), N80 5.42 (5.22 to 5.61). beta 2:beta 1 selectivity ratios significantly increased as the dose of nadolol was reduced. 4. These data suggest that whereas in the clinical dose range nadolol behaves as a non-selective beta-adrenoceptor antagonist, as the dose is reduced this drug demonstrates an increasing degree of selectivity for the beta 2-adrenoceptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal colonization or infection with extended-spectrum beta-lactamase-producing Enterobacteriaceae in Africa: A systematic review and meta-analysis.

Science.gov (United States)

Bulabula, Andre N H; Dramowski, Angela; Mehtar, Shaheen

2017-11-01

To summarize published studies on the prevalence of and risk factors for maternal bacterial colonization and/or infection with extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in pregnant and/or post-partum women in Africa. A systematic review was conducted using the PubMed, Scopus, and Google Scholar databases. Bibliographies of included eligible studies were manually searched to identify additional relevant articles. No language restriction was applied. The timeframe of the search included all records from electronic database inception to July 15, 2017. A random-effects meta-analysis was performed to summarize the prevalence and the 95% confidence intervals (CI) of ESBL-E colonization or infection in pregnant or post-partum women in Africa. The meta-analysis was conducted using STATA IC 13.1 software and the metaprop function/plugin. Ten studies (seven on pregnant women and three on post-partum women) were included, documenting a 17% prevalence of maternal colonization with ESBL-E in Africa (95% CI 10-23%). The prevalence of ESBL-E in community isolates exceeded that in isolates from the hospital setting (22% vs. 14%). The most frequently reported ESBL-encoding gene was CTX-M (cefotaxime hydrolyzing capabilities). Data on risk factors for maternal ESBL-E colonization and infection are very limited. The prevalence of colonization and/or infection with ESBL-E in pregnant and post-partum women in Africa exceeds that reported from high- and middle-income settings, representing a risk for subsequent neonatal colonization and/or infection with ESBL-E. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

A novel chimeric peptide with antimicrobial activity.

Science.gov (United States)

Alaybeyoglu, Begum; Akbulut, Berna Sariyar; Ozkirimli, Elif

2015-04-01

Beta-lactamase-mediated bacterial drug resistance exacerbates the prognosis of infectious diseases, which are sometimes treated with co-administration of beta-lactam type antibiotics and beta-lactamase inhibitors. Antimicrobial peptides are promising broad-spectrum alternatives to conventional antibiotics in this era of evolving bacterial resistance. Peptides based on the Ala46-Tyr51 beta-hairpin loop of beta-lactamase inhibitory protein (BLIP) have been previously shown to inhibit beta-lactamase. Here, our goal was to modify this peptide for improved beta-lactamase inhibition and cellular uptake. Motivated by the cell-penetrating pVEC sequence, which includes a hydrophobic stretch at its N-terminus, our approach involved the addition of LLIIL residues to the inhibitory peptide N-terminus to facilitate uptake. Activity measurements of the peptide based on the 45-53 loop of BLIP for enhanced inhibition verified that the peptide was a competitive beta-lactamase inhibitor with a K(i) value of 58 μM. Incubation of beta-lactam-resistant cells with peptide decreased the number of viable cells, while it had no effect on beta-lactamase-free cells, indicating that this peptide had antimicrobial activity via beta-lactamase inhibition. To elucidate the molecular mechanism by which this peptide moves across the membrane, steered molecular dynamics simulations were carried out. We propose that addition of hydrophobic residues to the N-terminus of the peptide affords a promising strategy in the design of novel antimicrobial peptides not only against beta-lactamase but also for other intracellular targets. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan.

Science.gov (United States)

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.

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

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Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

Transcriptional Alterations of Virulence-Associated Genes in Extended Spectrum Beta-Lactamase (ESBL-Producing Uropathogenic Escherichia coli during Morphologic Transitions Induced by Ineffective Antibiotics

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Isak Demirel

2017-06-01

Full Text Available It is known that an ineffective antibiotic treatment can induce morphological shifts in uropathogenic Escherichia coli (UPEC but the virulence properties during these shifts remain to be studied. The present study examines changes in global gene expression patterns and in virulence factor-associated genes in an extended spectrum beta-lactamase (ESBL-producing UPEC (ESBL019 during the morphologic transitions induced by an ineffective antibiotic and in the presence of human primary bladder epithelial cells. Microarray results showed that the different morphological states of ESBL019 had significant transcriptional alterations of a large number of genes (Transition; 7%, Filamentation; 32%, and Reverted 19% of the entities on the array. All three morphological states of ESBL019 were associated with a decreased energy metabolism, altered iron acquisition systems and altered adhesion expression. In addition, genes associated with LPS synthesis and bacterial motility was also altered in all the morphological states. Furthermore, the transition state induced a significantly higher release of TNF-α from bladder epithelial cells compared to all other morphologies, while the reverted state was unable to induce TNF-α release. Our findings show that the morphological shifts induced by ineffective antibiotics are associated with significant transcriptional virulence alterations in ESBL-producing UPEC, which may affect survival and persistence in the urinary tract.

Characterization and Diversity of Novel PIF/Harbinger DNA Transposons in Brassica Genomes

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Nouroz, F.; Noreen, S.; Harrison, H.

2016-01-01

Among DNA transposons, PIF/Harbinger is most recently identified superfamily characterized by 3 bp target site duplications (TSDs), flanked by 14-45 bp terminal inverted repeats (TIRs) and displaying DDD or DDE domain displaying transposase. Their autonomous elements contain two open reading frames, ORF1 and ORF2 encoding superfamily specific transposase and DNA-binding domain. Harbinger DNA transposons are recently identified in few plants. In present study, computational and molecular approaches were used for the identification of 8 Harbinger transposons, of which only 2 were complete with putative trans posase, while rest 6 lack transposase and are considered as defective or non-autonomous elements. They ranged in size from 0.5-4 kb with 3 bp TSDs, 15-42 bp TIRs and internal AT richregions. The PCR amplification of Brassica Harbinger transposase revealed diversity and ancient nature of these elements. The amplification polymorphism of some non-autonomous Harbingers showed species specific distribution. Phylogenetic analyses of transposase clustered them into two clades (monocot and dicot) and five sub-clades. The Brassica, Arabidopsis and Malustransposase clustered into genera specific sub-clades; although a lot of homology in transposase was observed. The multiple sequence alignment of Brassica and related transposase showed homology in five conserved blocks. The DD/Sub 35/E triad and sequences showed similarity to already known Pong-like or Arabidopsis ATIS12 Harbinger transposase in contrast to other transposase having DD/Sub 47/E or DD/Sub 48/E motifs. The present study will be helpful in the characterization of Harbingers, their structural diversity in related genera and Harbinger based molecular markers for varietal/lines identifications. (author)

Simultaneous identification of multiple β-lactamases in Acinetobacter baumannii in relation to carbapenem and ceftazidime resistance, using liquid chromatography-tandem mass spectrometry

NARCIS (Netherlands)

Trip, H.; Mende, K.; Majchrzykiewicz-Koehorst, J.A.; Sedee, N.J.A.; Hulst, A.G.; Jansen, H.J.; Murray, C.K.; Paauw, A.

2015-01-01

Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect β-lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of β-lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance

Prevalence of extended-spectrum beta-lactamase-producing bacteria in food

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Tham J

2012-10-01

Full Text Available Johan Tham,1 Mats Walder,2 Eva Melander,2,3 Inga Odenholt11Infectious Diseases Unit, Department of Clinical Sciences, 2Medical Microbiology, Department of Laboratory Medicine, Lund University, Malmö, Sweden; 3Department of Infection Control, Laboratory Medicine, Skåne County, SwedenAbstract: Extended-spectrum β-lactamase (ESBL-producing Enterobacteriaceae with Cefotaximase–München (CTX-M enzymes are rapidly increasing worldwide and pose a threat to health care. ESBLs with CTX-M enzymes have been isolated from animals and different food products, but it is unknown if food imported from the Mediterranean area may be a possible reservoir of these bacteria. During 2007–2008, swab samples from food across different retail outlets (mostly food from the Mediterranean countries and Swedish chicken were collected. Escherichia coli strains from Swedish meat and E. coli isolates from unspecified food from a Swedish food testing laboratory were also examined. In 349 of the 419 swab samples, growth of Enterobacteriaceae was found. In most of the samples, there was also growth of Gram-negative environmental bacteria. Air dry-cured products contained significantly less Enterobacteriaceae isolates compared to lettuces; however, none of the examined Enterobacteriaceae harbored ESBLs. This study did not support the theory that imported food from the Mediterranean area or Swedish domestic food might constitute an important vehicle for the dissemination of ESBL-producing Enterobacteriaceae; however, a spread from food to humans may have occurred after 2008.Keywords: ESBL, antibiotic resistance, zoonosis, food, Enterobacteriaceae

Towards controlled mutagenesis with transposons Ac and Tam3

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Haring, M; Veken, J; Windrich, R; Kneppers, T; Rommens, C; Nijkamp, H J.J.; Hille, J [Department of Genetics, Free University, Amsterdam (Netherlands)

1990-01-01

Full text: The discovery of mobile genetic elements in plants has permitted the use of these transposons for insertional mutagenesis. This applies so far only to Zea mays and Antirrhinum majus, because other plant transposable elements have not been characterised so thoroughly at the genetic and the molecular level. To establish whether transposons (Ac from maize and Tam3 from Antirrhinum) remain mobile in heterologous hosts, either in somatic tissue or after meiosis, a phenotypic assay system for transposition was developed. The separation of the two transposition functions will allow controlled mutagenesis of plant genes. Our results indicate that both transposable elements remain active in heterologous hosts. (author)

Transposon-mediated BAC transgenesis in zebrafish and mice

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Sumiyama Kenta

2009-10-01

Full Text Available Abstract Background Bacterial artificial chromosomes (BACs are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications. Results We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations. Conclusion The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

A passage from India: Association between air traffic and reported cases of New Delhi Metallo-beta-lactamase 1 from 2007 to 2012.

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MacFadden, Derek R; Bogoch, Isaac I; Brownstein, John S; Daneman, Nick; Fisman, David; German, Matthew; Khan, Kamran

2015-01-01

Highly transmissible genes encoding resistance to carbapenems have demonstrated global spread. The New Delhi Metallo-beta-lactamase 1 gene is hypothesized to have originated in India, with subsequent dissemination by colonized or infected travelers. We conducted an ecological study evaluating the association between the cumulative air traffic departing India between 2007 and 2012 and published cases of NDM-1. Receiver operator characteristic curves were generated as well as multivariate logistic regression models. 193 countries with complete flight and World Bank data were included in the analysis. Receiver operator characteristic curves (ROC) of the dichotomous outcome of a published case of NDM-1 were generated, yielding an unadjusted area under the curve (AUC) of 0.88 and adjusted AUC of 0.85. The unadjusted odds ratio of having a reported case of NDM-1, for every percentage increase in cumulative air traffic departing India, was 2.3 (95% CI 1.4 to 3.7) and adjusted was 2.0 (95% CI 1.2 to 3.4). We demonstrate that flows of international travelers departing India by air is associated with published NDM-1 cases, globally. Countries with high passenger flight traffic from India with no reported cases of NDM-1 may be at increased risk of having unreported transmission of NDM-1. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses

NARCIS (Netherlands)

Pacholewicz, Ewa; Liakopoulos, Apostolos; Swart, Arno; Gortemaker, Betty; Dierikx, Cindy; Havelaar, Arie|info:eu-repo/dai/nl/072306122; Schmitt, Heike|info:eu-repo/dai/nl/304831042

2015-01-01

Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the

Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses

NARCIS (Netherlands)

Pacholewicz, Ewa; Liakopoulos, Apostolos; Swart, Arno; Gortemaker, Betty; Dierikx, Cindy; Havelaar, Arie; Schmitt, Heike

2015-01-01

Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the

Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize

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Susan Gabay-Laughnan

2018-01-01

Full Text Available Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds, Enhancer/Suppressor-mutator (En/Spm, and Mutator (Mu transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.

Epidemiology of Extended-Spectrum beta-Lactamase-Producing E-coli and Vancomycin-Resistant Enterococci in the Northern Dutch-German Cross-Border Region

NARCIS (Netherlands)

Zhou, Xuewei; Garcia-Cobos, Silvia; Ruijs, Gijs J. H. M.; Kampinga, Greetje A.; Arends, Jan P.; Borst, Dirk M.; Moller, Lieke V.; Holman, Nicole D.; Schuurs, Theo A.; van Coppenraet, Lesla E. Bruijnesteijn; Weel, Jan F.; van Zeijl, Jan H.; Koeck, Robin; Rossen, John W. A.; Friedrich, Alexander W.

2017-01-01

Objectives: To reveal the prevalence and epidemiology of extended-spectrum β-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- and carbapenemase (CP) producing Enterobacteriaceae and vancomycin-resistant enterococci (VRE) across the Northern Dutch-German border region. Methods: A point-prevalence study

Antimicrobial susceptibility and molecular epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae from intensive care units at Hamad Medical Corporation, Qatar.

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Sid Ahmed, Mazen A; Bansal, Devendra; Acharya, Anushree; Elmi, Asha A; Hamid, Jemal M; Sid Ahmed, Abuelhassan M; Chandra, Prem; Ibrahim, Emad; Sultan, Ali A; Doiphode, Sanjay; Bilal, Naser Eldin; Deshmukh, Anand

2016-01-01

The emergence of extended-spectrum beta-lactamase (ESBL)-producing isolates has important clinical and therapeutic implications. High prevalence of ESBL-producing Enterobacteriaceae has been reported in the literature for clinical samples from a variety of infection sites. The present study was undertaken to evaluate the prevalence of ESBL-producing Enterobacteriaceae, and to perform molecular characterization and antimicrobial susceptibility testing of clinical isolates from patients admitted to the intensive care units at Hamad Medical Corporation, Doha, Qatar, from November 2012 to October 2013. A total of 629 Enterobacteriaceae isolates were included in the study. Identification and susceptibility testing was performed using Phoenix (Becton Dickinson) and the ESBL producers were confirmed by double-disk potentiation as recommended by the Clinical and Laboratory Standards Institute. Molecular analysis of the ESBL producers was performed by polymerase chain reaction. In total, 109 isolates (17.3 %) were confirmed as ESBL producers and all were sensitive to meropenem in routine susceptibility assays. Most of the ESBL producers (99.1 %) were resistant to amoxicillin/clavulanic acid and ceftriaxone and 93.6 % were resistant to cefepime. Among the ESBL-producing genes, bla CTX-M (66.1 %) was the most prevalent, followed by bla SHV (53.2 %) and bla TEM (40.4 %). These findings show the high prevalence of ESBL-producing Enterobacteriaceae within the intensive care units at Hamad Medical Corporation, Qatar, and emphasize the need for judicious use of antibiotics and the implementation of strict infection control measures.

Identification and evolutionary dynamics of cacta DNA transposons in brassica

International Nuclear Information System (INIS)

Nouroz, F.; Noreen, S.; Harrison, J.S.H.

2017-01-01

Transposable elements are the major drivers of genome evolution and plasticity. Due to their transposition mode, they are classified into two major classes as Retrotransposons and DNA transposons. The En/Spm or CACTA elements are diverse group of DNA transposons proliferating in plant genomes. Various bioinformatics and molecular approaches were used for identification and distribution of CACTA transposons in Brassica genome. A combination of dot plot analysis and BLASTN searches yielded 35 autonomous and 7 non-autonomous CACTA elements in Brassica. The elements ranged in sizes from 1.2 kb non-autonomous elements to 11kb autonomous elements, terminated by 3 bp Target Site Duplication (TSD) and ~15 bp conserved Terminal Inverted Repeat (TIR) motifs (5'-CACTACAAGAAAACA-3'), with heterogeneous internal regions. The transposase (TNP) was identified from autonomous CACTA elements, while other protein domains from Brassica and other plants CACTA revealed similar organizations with minor differences. Both transposases (TNPD, TNPA) are present in most CACTA, while a few CACTA harboured an additional ATHILA ORF1-like domain. The PCR analysis amplified the CACTA transposases from 40 Brassica accessions (A, B, and C-genome) suggesting their distribution among various Brassica crops. A detailed characterization and evolutionary analysis of the identified CACTA elements allowed some to be placed in genome-specific groups, while most of them (Brassica-Arabidopsis elements) have followed the same evolutionary line. The distribution of CACTA in Brassica concluded that 3 bp TSDs generating CACTA transposons contributed significantly to genome size and evolution of Brassica genome. (author)

A putative autonomous 20.5 kb-CACTA transposon insertion in an F3'H allele identifies a new CACTA transposon subfamily in Glycine max

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Vodkin Lila

2008-12-01

Full Text Available Abstract Background The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1 from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008. Results We identified a 20.5 kb insertion in a soybean flavonoid 3'-hydroxylase (F3'H gene representing the t* allele (stable gray trichome color whose origin traces to a single mutable chimeric plant displaying both tawny and gray trichomes. This 20.5 kb insertion has the molecular structure of a putative autonomous transposon of the CACTA family, designated Tgmt*. It encodes a large gene that was expressed in two sister isolines (T* and tm of the stable gray line (t* from which Tgmt* was isolated. RT-PCR derived cDNAs uncovered the structure of a large precursor mRNA as well as alternatively spliced transcripts reminiscent of the TNPA-mRNA generated by the En-1 element of maize but without sequence similarity to the maize TNPA. The larger mRNA encodes a transposase with a tnp2 and TNP1-transposase family domains. Because the two soybean lines expressing Tgmt* were derived from the same mutable chimeric plant that created the stable gray trichome t* allele line from which the element was isolated, Tgmt* has the potential to be an autonomous element that was rapidly inactivated in the stable gray trichome t* line. Comparison of Tgmt* to previously described Tgm elements demonstrated that two subtypes of CACTA transposon families exist in soybean based on divergence of their characteristic subterminal repeated motifs and their transposases. In addition, we report the sequence and annotation of a BAC clone containing the F3'H gene (T locus which was interrupted by the novel Tgmt* element

Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.

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Maura McGrail

2011-04-01

Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

Reversible Activation of Halophilic β-lactamase from Methanol-Induced Inactive Form: Contrast to Irreversible Inactivation of Non-Halophilic Counterpart.

Science.gov (United States)

Tokunaga, Hiroko; Maeda, Junpei; Arakawa, Tsutomu; Tokunaga, Masao

2017-06-01

Effects of a water-miscible organic solvent, methanol, on the structure and activity of halophilic β-lactamase derived from Chromohalobacter sp.560 (HaBla), were investigated by means of circular dichroism (CD) measurement and enzymatic activity determination. Beta-lactamase activity was enhanced about 1.2-fold in the presence of 10-20% methanol. CD measurement of HaBla revealed different structures depending on the methanol concentration: native-like active form (Form I) in 10-20% methanol and methanol-induced inactive form at higher concentration (Form II in 40-60% and Form III in 75-80% methanol). Incubation of HaBla with 40% methanol led to the complete loss of activity within ~80 min accompanied by the formation of Form II, whose activity was recovered promptly up to ~80% of full activity upon dilution of the methanol concentration to 10%. In addition, when the protein concentration was sufficiently high (e.g., 0.7 mg/ml), HaBla activity of Form III in 75% methanol could be recovered in the same way (with slightly slower recovery rate), upon dilution of the methanol concentration. In contrast, non-halophilic β-lactamase from Escherichia coli K12 strain MG1655 (EcBla) was irreversibly denatured in the presence of 40% methanol. HaBla showed remarkable ability to renature from the methanol-induced inactive states.

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

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Yokoo, Masako [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Fujita, Ryosuke [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021 (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213 (Japan); Yoshimizu, Mamoru; Kasai, Hisae [Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611 (Japan); Asano, Shin-ichiro [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Bando, Hisanori, E-mail: hban@abs.agr.hokudai.ac.jp [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan)

2013-09-13

Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

International Nuclear Information System (INIS)

Yokoo, Masako; Fujita, Ryosuke; Nakajima, Yumiko; Yoshimizu, Mamoru; Kasai, Hisae; Asano, Shin-ichiro; Bando, Hisanori

2013-01-01

Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells

Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

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Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

2016-04-07

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. Copyright © 2016 Vrljicak et al.

Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

Science.gov (United States)

Hickman, Alison B; Ewis, Hosam E; Li, Xianghong; Knapp, Joshua A; Laver, Thomas; Doss, Anna-Louise; Tolun, Gökhan; Steven, Alasdair C; Grishaev, Alexander; Bax, Ad; Atkinson, Peter W; Craig, Nancy L; Dyda, Fred

2014-07-17

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA. Copyright © 2014 Elsevier Inc. All rights reserved.

The transposon Galileo generates natural chromosomal inversions in Drosophila by ectopic recombination.

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Delprat, Alejandra; Negre, Bàrbara; Puig, Marta; Ruiz, Alfredo

2009-11-18

Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii. To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z(3). In the non inverted chromosome, the 2z(3) distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric. Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.

Occurrence and detection of AmpC β-lactamases among ...

African Journals Online (AJOL)

Aim of the study: We aimed to evaluate the presence of AmpC β-lactamase among Enterobacteriaceae isolates separated from patients with nosocomial infections, and to detect the genetic basis for AmpC production in these strains. Subjects and methods: 50 AmpC β-lactamase Enterobacteriaceae were analyzed for the ...

Variations in the Produce-Associated Microbiota and the Occurrence Frequency of Extended-Spectrum Beta-Lactamase Gram-Negative Bacteria Result in Different Level of Ingestion Risks

KAUST Repository

Bokhari, Osama

2016-04-01

A monitoring effort that spanned across one and a half years was conducted to examine three types of produce-associated microbiota. Produce type was determined to be the predominant factor affecting the microbial communities. Other significant factors that resulted in differences in the microbial populations were the origin and sampling date. Specifically, produce-associated microbiota among lettuce and tomatoes clustered based on the sampling period. Through molecular and cultivation-based approaches, sporadic presence of extended spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae and Acinetobacter baumannii was detected on lettuce and cucumbers during certain periods of sampling. Quantitative microbial risk assessment denoted varying levels of ingestion risks associated with different types of produce. In particular, the risks arising from ESBL-positive K. pneumoniae in the lettuce were higher than the acceptable annual risk of 10-4. Commonly used approaches to clean and wash the produce were insufficient in removing majority of the produce-associated microbiota. More invasive cleaning approaches or thorough cooking of the produce would be required to mitigate the associated risks. Most of the current reports of ESBL-positive bacterial isolates were identified in nosocomial environment. However, the carriage of such drug-resistant bacteria in food that is consumed daily

Antimicrobial Resistance Pattern and Their Beta-Lactamase Encoding Genes among Pseudomonas aeruginosa Strains Isolated from Cancer Patients

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Mai M. Zafer

2014-01-01

Full Text Available This study was designed to investigate the prevalence of metallo-β-lactamases (MBL and extended-spectrum β-lactamases (ESBL in P. aeruginosa isolates collected from two different hospitals in Cairo, Egypt. Antibiotic susceptibility testing and phenotypic screening for ESBLs and MBLs were performed on 122 P. aeruginosa isolates collected in the period from January 2011 to March 2012. MICs were determined. ESBLs and MBLs genes were sought by PCR. The resistant rate to imipenem was 39.34%. The resistance rates for P. aeruginosa to cefuroxime, cefoperazone, ceftazidime, aztreonam, and piperacillin/tazobactam were 87.7%, 80.3%, 60.6%, 45.1%, and 25.4%, respectively. Out of 122 P. aeruginosa, 27% and 7.4% were MBL and ESBL, respectively. The prevalence of blaVIM-2, blaOXA-10-, blaVEB-1, blaNDM-, and blaIMP-1-like genes were found in 58.3%, 41.7%, 10.4%, 4.2%, and 2.1%, respectively. GIM-, SPM-, SIM-, and OXA-2-like genes were not detected in this study. OXA-10-like gene was concomitant with VIM-2 and/or VEB. Twelve isolates harbored both OXA-10 and VIM-2; two isolates carried both OXA-10 and VEB. Only one strain contained OXA-10, VIM-2, and VEB. In conclusion, blaVIM-2- and blaOXA-10-like genes were the most prevalent genes in P. aeruginosa in Egypt. To our knowledge, this is the first report of blaVIM-2, blaIMP-1, blaNDM, and blaOXA-10 in P. aeruginosa in Egypt.

Extended-spectrum beta-lactamases in Klebsiella spp and Escherichia coli obtained in a Brazilian teaching hospital: detection, prevalence and molecular typing beta-lactamases de espectro ampliado em Klebsiella spp e em Escherichia coli obtidas em um hospital escola brasileiro: detecção, prevalência e tipagem molecular

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Ana Lúcia Peixoto de Freitas

2003-12-01

Full Text Available His study was performed to compare the methods of detection and to estimate the prevalence of extended-spectrum beta-lactamases (ESBL among Klebsiella spp and E.coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K.pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clínicas de Porto Alegre, using Kirby-Bauer zone diameter (KB, double-disk diffusion (DD, breakpoint for ceftazidime (MIC CAZ, increased zone diameter with clavulanate (CAZ/CAC and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC. Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E.coli, contrasting with all the other methods (p Este estudo foi desenvolvido para comparar métodos de detecção e para estimar a prevalência de Klebsiella spp e E.coli produtoras de beta-lactamases de espetro ampliado (ESBL em um Hospital Universitário no sul do Brasil. A correlação genética, determinada através de método molecular de tipagem, entre as amostras de K. pneumoniae também foi determinada. A produção de ESBL foi investigada em 95 amostras de Klebsiella spp e E.coli obtidas de pacientes no Hospital de Clínicas de Porto Alegre usando-se: medida do diâmetro a zona de inibição (KB, dupla-difusão de disco (DD, valores de concentração inibitória mínima da ceftazidima (MIC CAZ, aumento do diâmetro da zona de inibição com adição de clavulanato (CAZ/CAC e a relação entre o MIC da ceftazidima/MIC ceftazidima com clavulanato (MIC CAZ/CAC. A tipagem molecular foi realizada utilizando-se o método de macrorestrição de DNA e eletroforese em campo pulsado (PFGE. O método KB apresentou as maiores taxas de produção de ESBL (> 70% para Klebsiella e

TCUP: A novel hAT transposon active in maize tissue culture

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Alan eSmith

2012-01-01

Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

Transposon display supports transpositional activity of P elements in ...

Indian Academy of Sciences (India)

. Abstract. Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances ...

Distribution and classification of Serine β-lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes deposited in Public Databases

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Adriana Fróes

2016-11-01

Full Text Available β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South -SZ- and North Zone -NZ, presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data, exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST against conserved domain database profiles (CDD, Pfam, and COG followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%, while Class D is predominant in NZ (55%. CfxA (65% and ACC (47% types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%, CfxA (28%, ACC (21%, CEPA (20% and FOX (19%. Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of

Distribution and Classification of Serine β-Lactamases in Brazilian Hospital Sewage and Other Environmental Metagenomes Deposited in Public Databases.

Science.gov (United States)

Fróes, Adriana M; da Mota, Fábio F; Cuadrat, Rafael R C; Dávila, Alberto M R

2016-01-01

β-lactam is the most used antibiotic class in the clinical area and it acts on blocking the bacteria cell wall synthesis, causing cell death. However, some bacteria have evolved resistance to these antibiotics mainly due the production of enzymes known as β-lactamases. Hospital sewage is an important source of dispersion of multidrug-resistant bacteria in rivers and oceans. In this work, we used next-generation DNA sequencing to explore the diversity and dissemination of serine β-lactamases in two hospital sewage from Rio de Janeiro, Brazil (South Zone, SZ and North Zone, NZ), presenting different profiles, and to compare them with public environmental data available. Also, we propose a Hidden-Markov-Model approach to screen potential serine β-lactamases genes (in public environments samples and generated hospital sewage data), exploring its evolutionary relationships. Due to the high variability in β-lactamases, we used a position-specific scoring matrix search method (RPS-BLAST) against conserved domain database profiles (CDD, Pfam, and COG) followed by visual inspection to detect conserved motifs, to increase the reliability of the results and remove possible false positives. We were able to identify novel β-lactamases from Brazilian hospital sewage and to estimate relative abundance of its types. The highest relative abundance found in SZ was the Class A (50%), while Class D is predominant in NZ (55%). CfxA (65%) and ACC (47%) types were the most abundant genes detected in SZ, while in NZ the most frequent were OXA-10 (32%), CfxA (28%), ACC (21%), CEPA (20%), and FOX (19%). Phylogenetic analysis revealed β-lactamases from Brazilian hospital sewage grouped in the same clade and close to sequences belonging to Firmicutes and Bacteroidetes groups, but distant from potential β-lactamases screened from public environmental data, that grouped closer to β-lactamases of Proteobacteria. Our results demonstrated that HMM-based approach identified homologs of

Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH

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Burgis Timothy A

2009-07-01

Full Text Available Abstract Background In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. Results We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH, that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. Conclusion We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

Science.gov (United States)

Chen, Allie Y; Thomas, Pei W; Stewart, Alesha C; Bergstrom, Alexander; Cheng, Zishuo; Miller, Callie; Bethel, Christopher R; Marshall, Steven H; Credille, Cy V; Riley, Christopher L; Page, Richard C; Bonomo, Robert A; Crowder, Michael W; Tierney, David L; Fast, Walter; Cohen, Seth M

2017-09-14

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC 50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1 H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Free water surface constructed wetlands limit the dissemination of extended-spectrum beta-lactamase producing Escherichia coli in the natural environment.

Science.gov (United States)

Vivant, Anne-Laure; Boutin, Catherine; Prost-Boucle, Stéphanie; Papias, Sandrine; Hartmann, Alain; Depret, Géraldine; Ziebal, Christine; Le Roux, Sophie; Pourcher, Anne-Marie

2016-11-01

The fates of Escherichia coli and extended-spectrum beta-lactamase-producing E. coli (ESBL E. coli) were studied over a period of one year in a free water surface constructed wetland (FWS CW) with a succession of open water zones and vegetation ponds (Typha or Phragmites), that received the effluent from a wastewater treatment plant. ESBL E. coli were detected and isolated from all sampling areas of the FWS CW throughout the study period. They represented 1‰ of the total E. coli population regardless of the origin of samples. Two main factors affected the log removal of E. coli and of ESBL E. coli: the season and the presence of vegetation. Between the inlet and the outlet of the FWS CW, the log removal of E. coli ranged from 1.5 in the warmer season (summer and fall) to 3.0 in the colder season (winter and spring). The concentrations of E. coli decreased significantly in the vegetated areas during the colder season, but increased in the warmer season, suggesting an effect of the plant growth stage on the survival of E. coli. Among the 369 ESBL E. coli isolates collected during our study, 84% harbored the CTX-M-ESBL type and 55.3% carried bla genes on plasmid DNA. Furthermore, 93% of the ESBL E. coli isolates were multidrug resistant but the proportion of resistant strains did not change significantly along the FWS CW. ESBL E. coli were characterized by MLST analysis using the 7 genes based Achtman Scheme. ESBL E. coli isolated from water, sediments, roots and feces of myocastors collected in the FWS CW and in the recipient river were genotypically related, suggesting persistence and circulation of the ESBL producing E. coli throughout the FWS CW and in the receiving river. Overall, these observations show that FWS CW could be an efficient treatment for ESBL E. coli disinfection of wastewater and could limit their dissemination in the aquatic environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Prevalence and risk factors for extended-spectrum beta-lactamase or AmpC-producing Escherichia coli in organic dairy herds in the Netherlands

NARCIS (Netherlands)

Santman - Berends, Inge; Gonggrijp, M A; Heuvelink, A E; Velthuis, A; Lam, T J G M; van Schaik, Gerdien; Hage, J. J.

2017-01-01

Extended-spectrum β-lactamase and AmpC-producing Escherichia coli (ESBL/AmpC) are an emerging problem and are hypothesized to be associated with antimicrobial use (AMU), and more specifically with the use of third- and fourth-generation cephalosporins. Whether ESBL/AmpC also occur in organic dairy

Pharmacodynamics of Imipenem in Combination with β-Lactamase Inhibitor MK7655 in a Murine Thigh Model

Science.gov (United States)

Mavridou, Eleftheria; Melchers, Ria J. B.; van Mil, Anita C. H. A. M.; Mangin, E.; Motyl, Mary R.

2014-01-01

MK7655 is a newly developed beta-lactamase inhibitor of class A and class C carbapenemases. Pharmacokinetics (PK) of imipenem-cilastatin (IMP/C) and MK7655 were determined for intraperitoneal doses of 4 mg/kg to 128 mg/kg of body weight. MIC and pharmacodynamics (PD) studies of MK7655 were performed against several beta-lactamase producing Pseudomonas aeruginosa and Klebsiella pneumoniae strains to determine its effect in vitro and in vivo. Neutropenic mice were infected in each thigh 2 h before treatment with an inoculum of approximately 5 × 106 CFU. They were treated with IMP/C alone (every 2 hours [q2h], various doses) or in combination with MK7655 in either a dose fractionation study or q2h for 24 h and sacrificed for CFU determinations. IMP/MK7655 decreased MICs regarding IMP MIC. The PK profiles of IMP/C and MK7655 were linear over the dosing range studied and comparable with volumes of distribution (V) of 0.434 and 0.544 liter/kg and half-lives (t1/2) of 0.24 and 0.25 h, respectively. Protein binding of MK7655 was 20%. A sigmoidal maximum effect (Emax) model was fit to the PK/PD index responses. The effect of the inhibitor was not related to the maximum concentration of drug in serum (Cmax)/MIC, and model fits for T>MIC and area under the concentration-time curve (AUC)/MIC were comparable (R2 of 0.7 and 0.75), but there appeared to be no significant relationship of effect with dose frequency. Escalating doses of MK7655 and IMP/C showed that the AUC of MK7655 required for a static effect was dependent on the dose of IMP/C and the MIC of the strain, with a mean area under the concentration-time curve for the free, unbound fraction of the drug (fAUC) of 26.0 mg · h/liter. MK7655 shows significant activity in vivo and results in efficacy of IMP/C in otherwise resistant strains. The exposure-response relationships found can serve as a basis for establishing dosing regimens in humans. PMID:25403667

Detection of β-lactamase encoding genes in feces, soil and water from a Brazilian pig farm.

Science.gov (United States)

Furlan, João Pedro Rueda; Stehling, Eliana Guedes

2018-01-10

β-lactam antibiotics are widely used for the treatment of different types of infections worldwide and the resistance to these antibiotics has grown sharply, which is of great concern. Resistance to β-lactams in gram-negative bacteria is mainly due to the production of β-lactamases, which are classified according to their functional activities. The aim of this study was to verify the presence of β-lactamases encoding genes in feces, soil, and water from a Brazilian pig farm. Different β-lactamases encoding genes were found, including bla CTX-M-Gp1 , bla CTX-M-Gp9 , bla SHV , bla OXA-1-like , bla GES , and bla VEB . The bla SHV and bla CTX-M-Gp1 genes have been detected in all types of samples, indicating the spread of β-lactam resistant bacteria among farm pigs and the environment around them. These results indicate that β-lactamase encoding genes belonging to the cloxacillinase, ESBL, and carbapenemase and they have high potential to spread in different sources, due to the fact that genes are closely related to mobile genetic elements, especially plasmids.

Comprehensive Evaluation of the MBT STAR-BL Module for Simultaneous Bacterial Identification and β-Lactamase-Mediated Resistance Detection in Gram-Negative Rods from Cultured Isolates and Positive Blood Cultures

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Annie W. T. Lee

2018-02-01

Full Text Available Objective: This study evaluated the capability of a MALDI Biotyper system equipped with the newly introduced MBT STAR-BL module to simultaneously perform species identification and β-lactamase-mediated resistance detection in bacteremia -causing bacteria isolated from cultured isolates and patient-derived blood cultures (BCs.Methods: Two hundred retrospective cultured isolates and 153 prospective BCs containing Gram-negative rods (GNR were collected and subjected to direct bacterial identification, followed by the measurement of β-lactamase activities against ampicillin, piperacillin, cefotaxime, ceftazidime, and meropenem using the MBT STAR-BL module. The results and turnaround times were compared with those of routine microbiological processing. All strains were also characterized by beta-lactamase PCR and sequencing.Results: Using the saponin-based extraction method, MALDI-TOF MS correctly identified bacteria in 116/134 (86.6% monomicrobial BCs. The detection sensitivities for β-lactamase activities against ampicillin, piperacillin, third-generation cephalosporin and meropenem were 91.3, 100, 97.9, and 100% for cultured isolates, and 80.4, 100, 68.8, and 40% for monomicrobial BCs (n = 134 respectively. The overall specificities ranged from 91.5 to 100%. Furthermore, the MBT STAR-BL and conventional drug susceptibility test results were concordant in 14/19 (73.7% polymicrobial cultures. Reducing the logRQ cut-off value from 0.4 to 0.2 increased the direct detection sensitivities for β-lactamase activities against ampicillin, cefotaxime and meropenem in BCs to 85.7, 87.5, and 100% respectively. The MBT STAR-BL test enabled the reporting of β-lactamase-producing GNR at 14.16 and 47.64 h before the interim and final reports of routine BCs processing, respectively, were available.Conclusion: The MALDI Biotyper system equipped with the MBT STAR-BL module enables the simultaneous rapid identification of bacterial species and β-lactamase

Comprehensive Evaluation of the MBT STAR-BL Module for Simultaneous Bacterial Identification and β-Lactamase-Mediated Resistance Detection in Gram-Negative Rods from Cultured Isolates and Positive Blood Cultures.

Science.gov (United States)

Lee, Annie W T; Lam, Johnson K S; Lam, Ricky K W; Ng, Wan H; Lee, Ella N L; Lee, Vicky T Y; Sze, Po P; Rajwani, Rahim; Fung, Kitty S C; To, Wing K; Lee, Rodney A; Tsang, Dominic N C; Siu, Gilman K H

2018-01-01

Objective: This study evaluated the capability of a MALDI Biotyper system equipped with the newly introduced MBT STAR-BL module to simultaneously perform species identification and β-lactamase-mediated resistance detection in bacteremia -causing bacteria isolated from cultured isolates and patient-derived blood cultures (BCs). Methods: Two hundred retrospective cultured isolates and 153 prospective BCs containing Gram-negative rods (GNR) were collected and subjected to direct bacterial identification, followed by the measurement of β-lactamase activities against ampicillin, piperacillin, cefotaxime, ceftazidime, and meropenem using the MBT STAR-BL module. The results and turnaround times were compared with those of routine microbiological processing. All strains were also characterized by beta-lactamase PCR and sequencing. Results: Using the saponin-based extraction method, MALDI-TOF MS correctly identified bacteria in 116/134 (86.6%) monomicrobial BCs. The detection sensitivities for β-lactamase activities against ampicillin, piperacillin, third-generation cephalosporin and meropenem were 91.3, 100, 97.9, and 100% for cultured isolates, and 80.4, 100, 68.8, and 40% for monomicrobial BCs ( n = 134) respectively. The overall specificities ranged from 91.5 to 100%. Furthermore, the MBT STAR-BL and conventional drug susceptibility test results were concordant in 14/19 (73.7%) polymicrobial cultures. Reducing the logRQ cut-off value from 0.4 to 0.2 increased the direct detection sensitivities for β-lactamase activities against ampicillin, cefotaxime and meropenem in BCs to 85.7, 87.5, and 100% respectively. The MBT STAR-BL test enabled the reporting of β-lactamase-producing GNR at 14.16 and 47.64 h before the interim and final reports of routine BCs processing, respectively, were available. Conclusion: The MALDI Biotyper system equipped with the MBT STAR-BL module enables the simultaneous rapid identification of bacterial species and β-lactamase

Induction of transposon TN1 translocation under the action of different mutagens

International Nuclear Information System (INIS)

Kubanejshvili, M.G.; Smirnov, S.P.; Tarasov, V.A.

1983-01-01

Migration of ampicillin transposon Tn1 under normal conditions in Escherichia coli cells proceeds with low frequency (10 -4 transpositions for cell). The low transposition frequency is conditioned by the transposition repression, realized by the gene-repressor in transposon structure and, probably, by other regulating genes of the bacterium-host. E. coli cell treatment by physical and chemical mutagens resulted in induction of translocation of ampicillin transposon Tn1 from plasmid RP4 into other replicons. Mitomycin C and ultraviolet radiation produced stronger inducing effect as compared to nitroso-guanidine (NG). The effect of the given mutagens on transposition Tn1 correlated with their activating capacity with respect to inducible SOS-functions of E coli. The mutation of rec A didn't influence on spontaneous Tn1 transposition, but blocked completely the induction of transposition process under mutagen effect. The relationship of inducible transposition with SOS-functions in E. coli cells, controlled by recA and lexA genes, as well as the possible role of the process in genetic microorganism variability are discussed in the paper

Prevalence and Antibiotic Susceptibility Patterns of Extended-Spectrum ß-Lactamase and Metallo-ß-Lactamase-Producing Uropathogenic Escherichia coli Isolates.

Science.gov (United States)

Ghadiri, Hamed; Vaez, Hamid; Razavi-Azarkhiavi, Kamal; Rezaee, Ramin; Haji-Noormohammadi, Mehdi; Rahimi, Ali Asghar; Vaez, Vahid; Kalantar, Enayatollah

2014-01-01

Healthcare professionals worldwide have expressed concern over infections by extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL)-producing bacteria. We evaluated the prevalence of ESBL- and MBL-producing Escherichia coli (E. coli) isolated from community-acquired urinary tract infections (UTIs) and their antibiotic-resistance profiles at 3 private laboratories in Tehran, Iran. E. coli isolates were mostly susceptible to meropenem (90.4%) and imipenem (90.0%), followed by amikacin (89.0%) and gentamicin (84.7%). Moreover, we detected that, of the E. coli isolates, 67 (22.3%) were ESBL producers and 21 (7.0%) of E. coli isolates were MBL positive via the imipenem-ethylenediaminetetraacetic acid (EDTA) combined disc test. This report is the first, to our knowledge, on the prevalence of MBL-producing uropathogenic E. coli (UPEC) strains in Iran. The antibiotic resistance of E. coli isolates revealed that 122 (40.7%) were multidrug resistant. The high number of antibiotic-resistant and ß-lactamase-producing UPEC strains necessitates further attention and consideration, particularly MBL-producing strains. Copyright© by the American Society for Clinical Pathology (ASCP).

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population

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Baier John

2007-05-01

Full Text Available Abstract Background Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations. Results Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill. Conclusion We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.

Extended-Spectrum beta (β)-Lactamases and Antibiogram in Enterobacteriaceae from Clinical and Drinking Water Sources from Bahir Dar City, Ethiopia.

Science.gov (United States)

Abera, Bayeh; Kibret, Mulugeta; Mulu, Wondemagegn

2016-01-01

The spread of Extended-Spectrum beta (β)-Lactamases (ESBL)-producing Enterobacteriaceae has become a serious global problem. ESBL-producing Enterobacteriaceae vary based on differences in antibiotic use, nature of patients and hospital settings. This study was aimed at determining ESBL and antibiogram in Enterobacteriaceae isolates from clinical and drinking water sources in Bahir Dar City, Northwest Ethiopia. Enterobacteriaceae species were isolated from clinical materials and tap water using standard culturing procedures from September 2013 to March 2015. ESBL-producing-Enterobacteriaceae were detected using double-disk method by E-test Cefotaxim/cefotaxim+ clavulanic acid and Ceftazidime/ceftazidime+ clavulanic acid (BioMerieux SA, France) on Mueller Hinton agar (Oxoid, UK). Overall, 274 Enterobacteriaceae were isolated. Of these, 210 (44%) were from patients and 64 (17.1%) were from drinking water. The median age of the patients was 28 years. Urinary tract infection and blood stream infection accounted for 60% and 21.9% of Enterobacteriaceae isolates, respectively. Klebsiella pneumoniae was isolated from 9 (75%) of neonatal sepsis. The overall prevalence of ESBL-producing Enterobacteriaceae in clinical and drinking water samples were 57.6% and 9.4%, respectively. The predominant ESBL-producers were K. pneumoniae 34 (69.4%) and Escherichia coli 71 (58.2%). Statistically significant associations were noted between ESBL-producing and non- producing Enterobacteriaceae with regard to age of patients, infected body sites and patient settings (P = 0.001). ESBL-producing Enterobacteriaceae showed higher levels of resistance against chloramphenicol, ciprofloxacin and cotrimoxazole than non-ESBL producers (P = 0.001). ESBL-producing Enterobacteriaceae coupled with high levels of other antimicrobials become a major concern for treatment of patients with invasive infections such as blood stream infections, neonatal sepsis and urinary tract infections. ESBL

Frequency of Extended-Spectrum Beta-lactamases (ESBLs) in strains of Klebsiella and E. coli isolated from patients hospitalized in Yazd.

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Zandi, Hengameh; Tabatabaei, Seyed Mostafa; Ehsani, Fatemeh; Zarch, Mojtaba Babaei; Doosthosseini, Samira

2017-02-01

Frequency of extended-spectrum beta-lactamases (ESBLs) and its variants may vary in different geographical areas, as reports indicate their spread in some certain communities. The aim of this study was to determine the frequency of ESBLs in strains of Klebsiella and E. coli , isolated from patients hospitalized in teaching hospitals of Yazd. This cross-sectional study was carried out on samples including E. coli and Klebsiella strains collected from laboratories of Shahid Sadoughi and Shahid Rahnemoun hospitals in Yazd, Iran in the period of 2011-2012. The colonies which were positive in lactose Eosin methylene-blue (EMB) medium were identified by biochemical methods, and 270 strains of Klebsiella and E. coli were isolated. Collected data and information were analyzed using Fisher's exact test and descriptive statistics such as mean in SPSS software, version 15, at a significant level of 0.05. In this study, 270 samples were examined, including 152 samples of E. coli (56.3%) and 118 samples of Klebsiella pneumonia (43.7%). Among the 152 samples of E. coli , 45 strains (30%) were producers of ESBLs. In addition, among the 118 samples of Klebsiella pneumonia , 44 strains (37.3%) were producers of ESBLs. E. coli strains showed the most resistance to Cefotaxime (100%), Ceftazidime (97.7%), and Cefepime (75.5%) respectively and Klebsiella strains showed the most resistance to Cefotaxime (100%), Ceftazidime (100%) and Cefepime (79.5%), respectively. Frequency of ESBLs in Klebsiella strains was higher than E. coli strains. No significant relationship was found between frequency of ESBLs and age or gender. In addition, E. coli strains showed the highest sensitivity to Imipenem, Amoxicillin/clavulanate, and Ciprofloxacin, while the highest antibiotic sensitivity of Klebsiella strains was shown to be to Piperacillin, Imipenem, and Amoxicillin/clavulanate.

Structural Variabilities in β-Lactamase (blaA of Different Biovars of Yersinia enterocolitica: Implications for β-Lactam Antibiotic and β-Lactamase Inhibitor Susceptibilities.

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Neelja Singhal

Full Text Available Yersiniosis caused by Yersinia enterocolitica has been reported from all continents. The bacterial species is divided into more than fifty serovars and six biovars viz. 1A, 1B, 2, 3, 4 and 5 which differ in geographical distribution, ecological niches and pathogenicity. Most Y.enterocolitica strains harbor chromosomal genes for two β-lactamases, blaA an Ambler class A penicillinase and blaB an Ambler class C inducible cephalosporinase. In the present study, susceptibility to b-lactam antibiotics and β-lactamase inhibitor was studied for Y. enterocolitica strains of biovars 1A, 1B, 2 and 4. We observed that β-lactamases were expressed differentially among strains of different biovars. To understand the molecular mechanisms underlying such differential expression, the sequences of genes and promoters of blaA were compared. Also, the variants of blaA present in different biovars were modeled and docked with amoxicillin and clavulanic acid. The mRNA secondary structures of blaA variants were also predicted in-silico. Our findings indicated that neither variations in the promoter regions, nor the secondary structures of mRNA contributed to higher/lower expression of blaA in different biovars. Analysis of H-bonding residues of blaA variants with amoxicillin and clavulanic acid revealed that if amino acid residues of a β-lactamase interacting with amoxicillin and the clavulanic acid were similar, clavulanic acid was effective in engaging the enzyme, accounting for a significant reduction in MIC of amoxicillin-clavulanate. This finding might aid in designing better β-lactamase inhibitors with improved efficiencies in future.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

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Viviane Figueira Marques

Full Text Available Abstract Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

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Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

2016-07-01

The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

Design, Synthesis, and Crystal Structures of 6-Alkylidene-2 -Substituted Penicillanic Acid Sulfones as Potent Inhibitors of Acinetobacter baumannii OXA-24 Carbapenemase

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Bou, G.; Santillana, E; Sheri, A; Beceiro, A; Sampson, J; Kalp, M; Bethel, C; Distler, A; Drawz, S; et. al.

2010-01-01

Class D {beta}-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial {beta}-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel {beta}-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2{prime}-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important {beta}-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 {beta}-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC{sub 50} values against OXA-24 and two OXA-24 {beta}-lactamase variants ranged from 10 {+-} 1 (4 vs WT) to 338 {+-} 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K{sub i} (500 {+-} 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k{sub inact}/K{sub i} = 0.21 {+-} 0.02 {micro}M{sup -1}s{sup -1}). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 {angstrom}) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2{prime}-substituted penicillin sulfones are effective mechanism-based inactivators of class D {beta}-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D {beta}-lactamases is proposed.

Anti-bacterial Efficacy of Bacteriocin Produced by Marine Bacillus subtilis Against Clinically Important Extended Spectrum Beta-Lactamase Strains and Methicillin-Resistant Staphylococcus aureus

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Suresh Mickymaray

2018-02-01

Full Text Available Objective: To investigate the anti-bacterial efficacy of bacteriocin produced by Bacillus subtilis SM01 (GenBank accession no: KY612347, a Gram-positive marine bacterium, against Extended Spectrum Beta-Lactamase (ESBL producing Gram-negative pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli, and Gram-positive pathogen Methicillin-Resistant Staphylococcus aureus (MRSA. Methods: A marine bacterium was isolated from mangrove sediment from the Red Sea coast of Jeddah, Kingdom of Saudi Arabia, and identified based on its morphological, biochemical, and molecular characteristics. The bacteriocin production using this isolate was carried out in brain heart infusion broth (BHIB medium. The Anti-bacterial activity of bacteriocin was evaluated against selected ESBL strains and MRSA by the well agar method. The effects of incubation time, pH, and temperature on the Anti-bacterial activity were studied. Results: The bacteriocin Bac-SM01 produced by B. subtilis SM01 demonstrated broad-spectrum Anti-bacterial activity against both Gram-negative and -positive bacteria. The present study is the first report that the bacteriocin Bac-SM01 inhibits the growth of ESBL producing Gram-negative strains A. baumannii, P. aeruginosa, and E. coli, and a Gram-positive MRSA strain. The optimum incubation time, pH, and temperature for the Anti-bacterial activity of Bac-SM01 was 24 h, 7, and 37°C respectively. Conclusion: The overall investigation can conclude that the bacteriocin Bac-SM01 from the marine isolate Bacillus subtilis SM01 could be used as an alternative Anti-bacterial agent in pharmaceutical products.

Case of Meningitis in a Neonate Caused by an Extended-Spectrum-Beta-Lactamase-Producing Strain of Hypervirulent Klebsiella pneumoniae

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Khalit S. Khaertynov

2017-08-01

Full Text Available Klebsiella pneumoniae is one of the most important infectious agents among neonates. This pathogen has a potential to develop an increased antimicrobial resistance and virulence. The classic non-virulent strain of K. pneumoniae, producing an extended-spectrum beta-lactamases (ESBL, is associated with nosocomial infection mainly in preterm neonates. Hypervirulent K. pneumoniae strains are associated with invasive infection among previously healthy ambulatory patients, and most of them exhibit antimicrobial susceptibility. During the last few years, several cases of diseases caused by hypervirulent K. pneumoniae producing ESBL have been registered in different geographical regions of the world. However, reports of such cases in neonates are rare. Here, we reported that this pathogen can cause pyogenic meningitis in full-term neonate with poor prognosis. A previously healthy, full-term, 12-day-old neonate was admitted to the infectious diseases hospital with suspected meningitis. The clinical symptoms included loss of appetite, irritability, fever, seizures, and a bulging anterior fontanelle. The analysis of the cerebrospinal fluid confirmed the diagnosis of meningitis. Blood and cerebrospinal fluid cultures were positive for K. pneumoniae, producing ESBL. K. pneumoniae isolates were resistant to aminopenicillins, 3rd generation cephalosporins but were sensitive to imipenem and meropenem. The “string test” was positive. The study of the virulence factors of K. pneumoniae by PCR revealed the presence of the rmpA gene. A combination of K. pneumoniae virulence and drug resistance complicated by cerebral oedema led to the death of the neonate. We concluded that both the risk of developing severe forms of infection and the outcome of the disease due to K. pneumonia are associated with the phenotypic features of the pathogen such as its antibiotic susceptibility and virulence factors. Emergence of the ESBL-producing strain of hypervirulent K

Geographical evolution of the CTX-M ß-lactamase – an update

African Journals Online (AJOL)

The CTX-M- type extended spectrum -lactamases (ESBLs) that preferentially hydrolyze cefotaxime are emerging globally and comprise of more than 50 enzymes. The emergence of novel CTX-M - lactamases in several countries is noted as opposed to the transfer of established CTX-M genes from one country to another, ...

Detection of AmpC β lactamases in gram-negative bacteria

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Gunjan Gupta

2014-01-01

Full Text Available Amp C β-lactamases are clinically important cephalosporinases encoded on the chromosomes of many Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor/β-lactam combinations. The increase in antibiotic resistance among Gram-negative bacteria is a notable example of how bacteria can procure, maintain and express new genetic information that can confer resistance to one or several antibiotics. Detection of organisms producing these enzymes can be difficult, because their presence does not always produce a resistant phenotype on conventional disc diffusion or automated susceptibility testing methods. These enzymes are often associated with potentially fatal laboratory reports of false susceptibility to β-lactams phenotypically. With the world-wide increase in the occurrence, types and rate of dissemination of these enzymes, their early detection is critical. AmpC β-lactamases show tremendous variation in geographic distribution. Thus, their accurate detection and characterization are important from epidemiological, clinical, laboratory, and infection control point of view. This document describes the methods for detection for AmpC β-lactamases, which can be adopted by routine diagnostic laboratories.

Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers.

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Tängdén, Thomas; Cars, Otto; Melhus, Asa; Löwdin, Elisabeth

2010-09-01

Foreign travel has been suggested to be a risk factor for the acquisition of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. To our knowledge, this has not previously been demonstrated in a prospective study. Healthy volunteers traveling outside Northern Europe were enrolled. Rectal swabs and data on potential travel-associated risk factors were collected before and after traveling. A total of 105 volunteers were enrolled. Four of them did not complete the study, and one participant carried ESBL-producing Escherichia coli before travel. Twenty-four of 100 participants with negative pretravel samples were colonized with ESBL-producing Escherichia coli after the trip. All strains produced CTX-M enzymes, mostly CTX-M-15, and some coproduced TEM or SHV enzymes. Coresistance to several antibiotic subclasses was common. Travel to India was associated with the highest risk for the acquisition of ESBLs (88%; n = 7). Gastroenteritis during the trip was an additional risk factor (P = 0.003). Five of 21 volunteers who completed the follow-up after 6 months had persistent colonization with ESBLs. This is the first prospective study demonstrating that international travel is a major risk factor for colonization with ESBL-producing Enterobacteriaceae. Considering the high acquisition rate of 24%, it is obvious that global efforts are needed to meet the emergence and spread of CTX-M enzymes and other antimicrobial resistances.

Determinação da produção de metalo-b-lactamases em amostras de Pseudomonas aeruginosa isoladas em João Pessoa, Paraíba

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Santos Filho Lauro; Santos Isabele Beserra; Assis Alexandro Mangueira Lima de; Xavier Danilo Elias

2002-01-01

Bactérias produtoras de metalo-beta-lactamases (MBLs) são em grande parte resistentes aos betalactâmicos de largo espectro, incluindo oximino-aminotiazol cefalosporinas e também aos carbapenens. O objetivo deste trabalho foi detectar cepas de Pseudomonas aeruginosa resistentes ao imipenem e à ceftazidima, assim como identificar aquelas produtoras de MBLs. Foram estudadas 198 linhagens não-repetitivas isoladas de diversas amostras clínicas, hospitalares e comunitárias, identificadas bioquimica...

Clinical and economic outcomes associated with community-acquired intra-abdominal infections caused by extended spectrum beta-lactamase (ESBL) producing bacteria in China.

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Hu, Bijie; Ye, Huifeng; Xu, Yingchun; Ni, Yuxing; Hu, Yunjian; Yu, Yunsong; Huang, Zhenfei; Ma, Larry

2010-06-01

To compare clinical and economic outcomes in patients with community-acquired intra-abdominal infection (IAI) due to extended spectrum beta-lactamase (ESBL) producing (ESBL-positive) bacteria versus non-ESBL-producing (ESBL-negative) bacteria in China. This was a retrospective chart review study of patients hospitalized with community-acquired IAI due to ESBL-positive or ESBL-negative infections caused by Escherichia coli or Klebsiella spp. Data were collected from six hospitals in China that participated in the Study for Monitoring Antibiotic Resistance Trends (SMART) during 2006-2007. Outcomes included clinical response at discharge and following first-line antibiotic, number of antibiotic agents and classes, duration of hospitalization, and overall hospitalization and intravenous antibiotic costs. Of the 85 patients included in the study, 32 (37.6%) had ESBL-positive and 53 (62.4%) had ESBL-negative infections; E. coli was responsible for 77.6% of infections. Infection resolved at discharge in 30 (93.8%) ESBL-positive and 48 (90.6%) ESBL-negative patients (P = NS). Fewer ESBL-positive patients achieved complete response following first-line antibiotics (56.3% versus 83.0%; P = 0.01). ESBL-positive patients required longer antibiotic treatment, more antibiotics, longer hospitalization (24.3 versus 14.6 days; 1.67-fold ratio; P = 0.001), and incurred higher hospitalization costs ( yen24,604 vs. yen13,788; $3604 vs. $2020; 1.78-fold ratio; P < 0.001). Patients with ESBL-positive infection had similar resolution rates at discharge compared to those with ESBL-negative infection, despite poorer first-line antibiotic response. However, ESBL-positive infection led to significantly greater hospitalization cost and intravenous antibiotic cost, and longer hospital stay.

Risk factors associated with the community-acquired colonization of extended-spectrum beta-lactamase (ESBL) positive Escherichia Coli. an exploratory case-control study.

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Leistner, Rasmus; Meyer, Elisabeth; Gastmeier, Petra; Pfeifer, Yvonne; Eller, Christoph; Dem, Petra; Schwab, Frank

2013-01-01

The number of extended-spectrum beta-lactamase (ESBL) positive (+) Escherichia coli is increasing worldwide. In contrast with many other multidrug-resistant bacteria, it is suspected that they predominantly spread within the community. The objective of this study was to assess factors associated with community-acquired colonization of ESBL (+) E. coli. We performed a matched case-control study at the Charité University Hospital Berlin between May 2011 and January 2012. Cases were defined as patients colonized with community-acquired ESBL (+) E. coli identified language most commonly spoken at home (mother tongue). An additional rectal swab was obtained together with the questionnaire to verify colonization status. Genotypes of ESBL (+) E. coli strains were determined by PCR and sequencing. Risk factors associated with ESBL (+) E. coli colonization were analyzed by a multivariable conditional logistic regression analysis. We analyzed 85 cases and 170 controls, respectively. In the multivariable analysis, speaking an Asian language most commonly at home (OR = 13.4, CI 95% 3.3-53.8; p<0.001) and frequently eating pork (≥ 3 meals per week) showed to be independently associated with ESBL colonization (OR = 3.5, CI 95% 1.8-6.6; p<0.001). The most common ESBL genotypes were CTX-M-1 with 44% (n = 37), CTX-M-15 with 28% (n = 24) and CTX-M-14 with 13% (n = 11). An Asian mother tongue and frequently consuming certain types of meat like pork can be independently associated with the colonization of ESBL-positive bacteria. We found neither frequent consumption of poultry nor previous use of antibiotics to be associated with ESBL colonization.

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota.

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Gudeta, Dereje D; Bortolaia, Valeria; Pollini, Simona; Docquier, Jean-Denis; Rossolini, Gian M; Amos, Gregory C A; Wellington, Elizabeth M H; Guardabassi, Luca

2016-01-01

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural ( n = 6) and grassland ( n = 4) soil into Escherichia coli . The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria , two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes . All MBLs detected in soil microbiota were functional when expressed in E. coli , resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant ß-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota.

Extended-spectrum beta-lactamase-producing bacteria are not detected in supragingival plaque samples from human fecal carriers of ESBL-producing Enterobacteriaceae

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Arne Søraas

2014-08-01

Full Text Available Background: The prevalence of infections caused by Cefotaximase-Munich (CTX-M-type extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E has rapidly increased during the past 15 years. Enterobacteriaceae are commonly found in the gastrointestinal tract and long-term intestinal carriage is considered important for the spread of ESBL and as a source of clinical infections. Oral biofilm such as supragingival plaque is known to contain numerous antibiotic resistance determinants and may also represent a poorly investigated site for ESBL carriage and further spread. Objective: To investigate possible carriage of ESBL-producing bacteria in supragingival plaque of known fecal carriers of these bacteria. Design: We screened for the presence of aerobic and anaerobic ESBL-producing bacteria and blaCTX-M in supragingival plaque samples from healthy human adults with culture-verified fecal carriage of CTX-M-producing Escherichia coli. The presence or absence of Enterobacteriaceae and ESBL-producing bacteria in plaque samples was evaluated using culture-based methods and consensus CTX-M PCR. Results: Oral samples were obtained from 17 participants with known previous carriage of ESBL-producing E. coli. No ESBL-producing bacteria or ESBL genes were detected using culture-based and molecular methods. One colony of Rahnella aquatilis harboring the class A ESBL gene bla RAHN-1/2 was identified in an oral sample from one of the participants. Conclusion: This pilot study supports the notion that the presence of CTX-M-producing bacteria is uncommon in oral plaque of healthy human adult fecal carriers. Due to the limited number of persons tested, a low prevalence of oral ESBL-carriage in healthy adults or carriage in selected groups of patients cannot be excluded. To our knowledge, this is the first description of an R. aquatilis with the RAHN-1/2 gene in the oral cavity.

Chromosome-Encoded Broad-Spectrum Ambler Class A β-Lactamase RUB-1 from Serratia rubidaea.

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Bonnin, Rémy A; Didi, Jennifer; Ergani, Ayla; Lima, Sandra; Naas, Thierry

2017-02-01

Whole-genome sequencing of Serratia rubidaea CIP 103234 T revealed a chromosomally located Ambler class A β-lactamase gene. The gene was cloned, and the β-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter β-lactamases. Analysis by 5' rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ 70 consensus sequence. This work further illustrates the heterogeneity of β-lactamases among Serratia spp. Copyright © 2017 American Society for Microbiology.

Characterization of CIA-1, an Ambler class A extended-spectrum β-lactamase from Chryseobacterium indologenes.

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Matsumoto, Takehisa; Nagata, Mika; Ishimine, Nau; Kawasaki, Kenji; Yamauchi, Kazuyoshi; Hidaka, Eiko; Kasuga, Eriko; Horiuchi, Kazuki; Oana, Kozue; Kawakami, Yoshiyuki; Honda, Takayuki

2012-01-01

An Ambler class A β-lactamase gene, bla(CIA-1), was cloned from the reference strain Chryseobacterium indologenes ATCC 29897 and expressed in Escherichia coli BL21. The bla(CIA-1) gene encodes a novel extended-spectrum β-lactamase (ESBL) that shared 68% and 60% identities with the CGA-1 and CME-1 β-lactamases, respectively. bla(CIA-1)-like genes were detected from clinical isolates. In addition to the metallo-β-lactamase IND of Ambler class B, C. indologenes has a class A ESBL gene, bla(CIA-1), located on the chromosome.

Epidemiology and Burden of Bloodstream Infections Caused by Extended-Spectrum Beta-Lactamase Producing Enterobacteriaceae in a Pediatric Hospital in Senegal.

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Awa Ndir

Full Text Available Severe bacterial infections are not considered as a leading cause of death in young children in sub-Saharan Africa. The worldwide emergence of extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E could change the paradigm, especially in neonates who are at high risk of developing healthcare-associated infections.To evaluate the epidemiology and the burden of ESBL-E bloodstream infections (BSI.A case-case-control study was conducted in patients admitted in a pediatric hospital during two consecutive years. Cases were patients with Enterobacteriaceae BSI and included ESBL-positive (cases 1 and ESBL-negative BSI (cases 2. Controls were patients with no BSI. Multivariate analysis using a stepwise logistic regression was performed to identify risk factors for ESBL acquisition and for fatal outcomes. A multistate model was used to estimate the excess length of hospital stay (LOS attributable to ESBL production while accounting for time of infection. Cox proportional hazards models were performed to assess the independent effect of ESBL-positive and negative BSI on LOS.The incidence rate of ESBL-E BSI was of 1.52 cases/1000 patient-days (95% CI: 1.2-5.6 cases per 1000 patient-days. Multivariate analysis showed that independent risk factors for ESBL-BSI acquisition were related to underlying comorbidities (sickle cell disease OR = 3.1 (95%CI: 2.3-4.9, malnutrition OR = 2.0 (95%CI: 1.7-2.6 and invasive procedures (mechanical ventilation OR = 3.5 (95%CI: 2.7-5.3. Neonates were also identified to be at risk for ESBL-E BSI. Inadequate initial antibiotic therapy was more frequent in ESBL-positive BSI than ESBL-negative BSI (94.2% versus 5.7%, p<0.0001. ESBL-positive BSI was associated with higher case-fatality rate than ESBL-negative BSI (54.8% versus 15.4%, p<0.001. Multistate modelling indicated an excess LOS attributable to ESBL production of 4.3 days. The adjusted end-of-LOS hazard ratio for ESBL-positive BSI was 0.07 (95%CI, 0

PREVALENCE OF EXTENDED SPECTRUM β-LACTAMASES ...

African Journals Online (AJOL)

DR. AMINU

ABSTRACT. Confirmed variants of enterobacteriaceae isolated from 143 patients that attended Murtala. Mohammed Specialist Hospital Kano, were screened for extended spectrum β-lactamases (ESBLs) production using Clinical Laboratory Standards Institute (CLSI) breakpoint. Suspected ESBLs producers were ...

Frequent use of colistin-based drug treatment to eliminate extended-spectrum beta-lactamase-producing Escherichia coli in backyard chicken farms in Thai Binh Province, Vietnam.

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Nakayama, Tatsuya; Jinnai, Michio; Kawahara, Ryuji; Diep, Khong Thi; Thang, Nguyen Nam; Hoa, Tran Thi; Hanh, Le Kieu; Khai, Pham Ngoc; Sumimura, Yoshinori; Yamamoto, Yoshimasa

2017-01-01

Reports of livestock infections with extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E) are increasing. Based on interviews conducted over a 6-month period, we found that veterinarians in the Vietnamese province of Thai Binh prefer to prescribe colistin-based drugs (CBD) in chicken farms. We aimed to clarify whether CBD use selects for strains of colistin-resistant ESBL-E. With the cooperation of seven local households, we detected ESBL-E in chickens' feces after treating chickens with CBD. Phylogenetic groupings and the presence of CTX-M/AmpC genes were determined, and the multi-antibiotic susceptibility of isolates was analyzed. Our results showed that ESBL-E presented in seven chickens' feces from two households. Seventy-two percent of ESBL-E isolates harbored CTX-M9 and the phylogenetic group A; the colistin minimum inhibitory concentration (MIC) of all isolated ESBL-E ranged from 0.064 to 1 μg mL -1 . Moreover, ESBL-E isolates were used to experimentally select for colistin resistance, and the effect of commercial CBD on ESBL-E was investigated. The results showed that an ESBL-E strain with a colistin MIC of 4 μg mL -1 was able to grow in media with CBD. Although CBD treatment was effective, in vitro experiments demonstrated that ESBL-E can easily acquire colistin resistance. Therefore, restrictions on colistin use are necessary to prevent the emergence of colistin-resistant bacteria.

Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

Science.gov (United States)

Ruggiero, Melina; Kerff, Frédéric; Herman, Raphaël; Sapunaric, Frédéric; Galleni, Moreno; Gutkind, Gabriel; Charlier, Paulette; Sauvage, Eric

2014-01-01

PER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior. PMID:25070104

Autoantibodies to IA-2beta improve diabetes risk assessment in high-risk relatives

DEFF Research Database (Denmark)

Achenbach, P; Bonifacio, E; Williams, A J K

2008-01-01

-positive participants (median age 12.1 years; 57% male), 113 developed diabetes (5 year cumulative risk 56%), and 148 were also GADA-positive and IAA-positive (4Ab-positive). IA2betaA were detected in 137 (65%) ICA/IA2A-positive participants and were associated with an increased 5 year diabetes risk (IA2beta......A-positive 65 vs 39% in IA2betaA-negative, p=0.0002). The effect was most marked in 4Ab-positive relatives (72% vs 52%, p=0.003). Metabolic testing further refined risk assessment. Among 101 4Ab-positive relatives with IA2betaA, the 5 year risk was 94% in those with a low FPIR (vs 50% in those with a normal...... FPIR, p4Ab/IA2betaA-positive participants with a low FPIR was 1.5 years. Multivariate analysis confirmed IA2betaA status, antibody number, young...

Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC β-lactamases and/or carbapenemases in Spain.

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Machuca, Jesús; Agüero, Jesús; Miró, Elisenda; Conejo, María Del Carmen; Oteo, Jesús; Bou, Germán; González-López, Juan José; Oliver, Antonio; Navarro, Ferran; Pascual, Álvaro; Martínez-Martínez, Luis

2017-10-01

Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC β-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC β-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC β-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints. Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Imported chicken meat as a potential source of quinolone-resistant Escherichia coli producing extended-spectrum beta-lactamases in the UK.

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Warren, R E; Ensor, V M; O'Neill, P; Butler, V; Taylor, J; Nye, K; Harvey, M; Livermore, D M; Woodford, N; Hawkey, P M

2008-03-01

Escherichia coli producing CTX-M-15 enzyme began to rapidly spread in the UK from around 2003 but other types also occur, notably CTX-M-14. We examined breasts from UK-reared (n = 62) and imported (n = 27) chickens as potential sources of quinolone-resistant E. coli with bla(CTX-M) genes. A further 40 samples for which the country of rearing could not be identified were examined. During 2006, 129 fresh and frozen chicken breast fillets were purchased from retail outlets in the West Midlands. These were cultured for E. coli on CLED agar containing 8 mg/L ciprofloxacin and carrying a 10 microg cefpodoxime disc. Resistant isolates were identified and typed by RAPD fingerprinting; bla(CTX-M) was identified by PCR and genotyped by reverse-line hybridization. The country of rearing was identified from the packaging for 89 of 129 purchased samples. Only one of the 62 UK-reared chicken samples carried E. coli producing a CTX-M-1 enzyme, whereas 10 of 27 samples reared overseas had E. coli with CTX-M enzymes. Specifically, 4/10 Brazilian, 3/4 Brazilian/Polish/French, and 2/2 Dutch samples had E. coli with CTX-M-2 enzymes. Six of 40 samples for which the country of rearing was not known had producers of CTX-M enzymes, 5 of them with CTX-M-14. Quinolone-resistant E. coli with various CTX-M beta-lactamase genes that are common in human infections worldwide were found in imported chicken breasts, indicating a possible source for gut colonization. Samples from Brazil were commonly positive for E. coli with CTX-M-2, the dominant bla(CTX-M) genotype from human infections in South America, which is currently rare in clinical infections in the UK. CTX-M-15, the dominant CTX-M type in human infections in the UK, was not found in chicken isolates, suggesting that the UK-reared chickens are not a reservoir of CTX-M-15.

Two hAT transposon genes were transferred from Brassicaceae to broomrapes and are actively expressed in some recipients

Science.gov (United States)

Sun, Ting; Renner, Susanne S.; Xu, Yuxing; Qin, Yan; Wu, Jianqiang; Sun, Guiling

2016-01-01

A growing body of evidence is pointing to an important role of horizontal gene transfer (HGT) in the evolution of higher plants. However, reports of HGTs of transposable elements (TEs) in plants are still scarce, and only one case is known of a class II transposon horizontally transferred between grasses. To investigate possible TE transfers in dicots, we performed transcriptome screening in the obligate root parasite Phelipanche aegyptiaca (Orobanchaceae), data-mining in the draft genome assemblies of four other Orobanchaceae, gene cloning, gene annotation in species with genomic information, and a molecular phylogenetic analysis. We discovered that the broomrape genera Phelipanche and Orobanche acquired two related nuclear genes (christened BO transposase genes), a new group of the hAT superfamily of class II transposons, from Asian Sisymbrieae or a closely related tribe of Brassicaceae, by HGT. The collinearity of the flanking genes, lack of a classic border structure, and low expression levels suggest that BO transposase genes cannot transpose in Brassicaceae, whereas they are highly expressed in P. aegyptiaca. PMID:27452947

Frequency of β-lactamase enzyme and antibiogram pattern in bacterial flora isolated from staffs hands

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Shilla Jalalpoor

2011-12-01

Full Text Available Background: β-lactamase is an enzyme that can inactivate β–Lactam family antibiotics. High prevalence of β-lactamase producer bacteria on the staff hands, due to antibiotic resistance and nosocomial infection in hospitalized patients. The objective of this study was to assess the frequency of β-lactamase positive bacteria and antibiogram pattern in bacterial flora isolated from staff hands of the Al-Zahra hospital in Isfahan.Materials and Method: This laboratory research was performed during of 2005-2007 in Al-Zahra hospital in Isfahan. According to statistical formula, we randomly selected 80 samples from staff hands. Staff hand samples collected with finger print method. Bacterial identification was performed with microbiological methods and β–lactamase production was performed with Acidometric method and antibiogram pattern was performed with Kirby Bauer method.Results: According to the acidometric test results of 80 isolated staff hands, 61.85% of strains produce β–lactamase. Staphylococcus spp., Bacillus spp. and Enterobacteriaceae were the most important producers respectively (70.83%, 64.72% and 50%. According to antibiogram test results, penicillin and vancomycin had the highest and lowest resistance. Conclusion: High frequency of β–lactamase in bacterial survey represents colonization of bacteria in staff hands; may be due to facility transmission β–lactamase plasmid genes in bacteria. We suggest better hand washing in hospitals and prescription of β–lactame antibiotics was based only on antibiogram results

A trial with IgY chicken antibodies to eradicate faecal carriage of Klebsiella pneumoniae and Escherichia coli producing extended-spectrum beta-lactamases

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Anna-Karin Jonsson

2015-11-01

Full Text Available Background: Extended-spectrum beta-lactamase (ESBL-producing Enterobacteriaceae is an emerging therapeutic challenge, especially in the treatment of urinary tract infections. Following an outbreak of CTX-M-15 Klebsiella pneumoniae in Uppsala, Sweden, an orphan drug trial on IgY chicken antibodies was undertaken in an attempt to eradicate faecal carriage of ESBL-producing K. pneumoniae and Escherichia coli. Methods: Hens were immunised with epitopes from freeze-dried, whole-cell bacteria (ESBL-producing K. pneumoniae and E. coli and recombinant proteins of two K. pneumoniae fimbriae subunits (fimH and mrkD. The egg yolks were processed according to good manufacturing practice and the product was stored at−20°C until used. Using an internal database from the outbreak and the regular laboratory database, faecal carriers were identified and recruited from May 2005 to December 2013. The participants were randomised in a placebo-controlled 1:1 manner. Results: From 749 eligible patients, 327 (44% had deceased, and only 91 (12% were recruited and signed the informed consent. In the initial screening performed using the polymerase chain reaction, 24 participants were ESBL positive and subsequently randomised and treated with either the study drug or a placebo. The study was powered for 124 participants. Because of a very high dropout rate, the study was prematurely terminated. From the outbreak cohort (n=247, only eight patients were screened, and only one was positive with the outbreak strain in faeces. Conclusions: The present study design, using IgY chicken antibodies for the eradication of ESBL-producing K. pneumonia and E. coli, was ineffective in reaching its goal due to high mortality and other factors resulting in a low inclusion rate. Spontaneous eradication of ESBL-producing bacteria was frequently observed in recruited participants, which is consistent with previous reports.

A trial with IgY chicken antibodies to eradicate faecal carriage of Klebsiella pneumoniae and Escherichia coli producing extended-spectrum beta-lactamases

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Jonsson, Anna-Karin; Larsson, Anders; Tängdén, Thomas; Melhus, Åsa; Lannergård, Anders

2015-01-01

Background Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is an emerging therapeutic challenge, especially in the treatment of urinary tract infections. Following an outbreak of CTX-M-15 Klebsiella pneumoniae in Uppsala, Sweden, an orphan drug trial on IgY chicken antibodies was undertaken in an attempt to eradicate faecal carriage of ESBL-producing K. pneumoniae and Escherichia coli. Methods Hens were immunised with epitopes from freeze-dried, whole-cell bacteria (ESBL-producing K. pneumoniae and E. coli) and recombinant proteins of two K. pneumoniae fimbriae subunits (fimH and mrkD). The egg yolks were processed according to good manufacturing practice and the product was stored at−20°C until used. Using an internal database from the outbreak and the regular laboratory database, faecal carriers were identified and recruited from May 2005 to December 2013. The participants were randomised in a placebo-controlled 1:1 manner. Results From 749 eligible patients, 327 (44%) had deceased, and only 91 (12%) were recruited and signed the informed consent. In the initial screening performed using the polymerase chain reaction, 24 participants were ESBL positive and subsequently randomised and treated with either the study drug or a placebo. The study was powered for 124 participants. Because of a very high dropout rate, the study was prematurely terminated. From the outbreak cohort (n=247), only eight patients were screened, and only one was positive with the outbreak strain in faeces. Conclusions The present study design, using IgY chicken antibodies for the eradication of ESBL-producing K. pneumonia and E. coli, was ineffective in reaching its goal due to high mortality and other factors resulting in a low inclusion rate. Spontaneous eradication of ESBL-producing bacteria was frequently observed in recruited participants, which is consistent with previous reports. PMID:26560861

Extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae: critical tools for antibiotic resistance pattern.

Science.gov (United States)

Padmini, Nagarajan; Ajilda, Antony Alex Kennedy; Sivakumar, Natesan; Selvakumar, Gopal

2017-06-01

Drug resistance is a phenomenon where by an organism becomes fully or partially resistant to drugs or antibiotics being used against it. Antibiotic resistance poses an exacting intimidation for people with underlying medical immune conditions or weakened immune systems. Infections caused by the enzyme extended spectrum β-lactamase (ESBL) producing multi drug resistance (MDR) Enterobacteriaceae especially Escherichia coli and Klebsiella pneumoniae are resistant to a broad range of beta lactams, including third generation cephalosporins. Among all the pathogens, these two MDR E. coli and K. pneumoniae have emerged as one of the world's greatest health threats in past two decades. The nosocomial infections caused by these ESBL producing MDR E. coli and K. pneumoniae complicated the therapy and limit treatment options. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Escherichia coli producing CMY-2 β-lactamase in bovine mastitis milk.

Science.gov (United States)

Endimiani, Andrea; Bertschy, Isabelle; Perreten, Vincent

2012-01-01

An Escherichia coli isolate producing the CMY-2 β-lactamase was found in the milk of a cow with recurrent subclinical mastitis. The isolate was resistant to the antibiotics commonly used for intramammary mastitis treatment, such as penicillins, cephalosporins, β-lactam/β-lactamase inhibitor combinations, aminoglycosides, tetracyclines, and sulfonamides. This is the first report of a plasmid-mediated AmpC-producing Enterobacteriaceae in bovine milk.

Evolution of Metallo-β-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution

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María-Rocío Meini

2014-07-01

Full Text Available The production of β-lactamase enzymes is one of the most distributed resistance mechanisms towards β-lactam antibiotics. Metallo-β-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-β-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-β-lactamases.The production of β-lactamase enzymes is one of the most distributed resistance mechanisms towards β-lactam antibiotics. Metallo-β-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-β-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict

Facile construction of a random protein domain insertion library using an engineered transposon.

Science.gov (United States)

Shah, Vandan; Pierre, Brennal; Kim, Jin Ryoun

2013-01-15

Insertional fusion between multiple protein domains represents a novel means of creating integrated functionalities. Currently, there is no robust guideline for selection of insertion sites ensuring the desired functional outcome of insertional fusion. Therefore, construction and testing of random domain insertion libraries, in which a host protein domain is randomly inserted into a guest protein domain, significantly benefit extensive exploration of sequence spaces for insertion sites. Short peptide residues are usually introduced between protein domains to alleviate structural conflicts, and the interdomain linker residues may affect the functional outcome of protein insertion complexes. Unfortunately, optimal control of interdomain linker residues is not always available in conventional methods used to construct random domain insertion libraries. Moreover, most conventional methods employ blunt-end rather than sticky-end ligation between host and guest DNA fragments, thus lowering library construction efficiency. Here, we report the facile construction of random domain insertion libraries using an engineered transposon. We show that random domain insertion with optimal control of interdomain linker residues was possible with our engineered transposon-based method. In addition, our method employs sticky-end rather than blunt-end ligation between host and guest DNA fragments, thus allowing for facile construction of relatively large sized libraries. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Science.gov (United States)

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.

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

Science.gov (United States)

Maglennon, Gareth A; Cook, Beth S; Deeney, Alannah S; Bossé, Janine T; Peters, Sarah E; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N

2013-12-21

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

Extended spectrum beta-lactamase-producing Escherichia coli forms filaments as an initial response to cefotaxime treatment

DEFF Research Database (Denmark)

Kjeldsen, Thea S. B.; Sommer, Morten Otto Alexander; Olsen, John E.

2015-01-01

Background: beta-lactams target the peptidoglycan layer in the bacterial cell wall and most beta-lactam antibiotics cause filamentation in susceptible Gram-negative bacteria at low concentrations. The objective was to determine the initial morphological response of cephalosporin resistant CTX-M-1......-producing E. coli to cefotaxime and to determine whether the response depended on the growth phase of the bacterium and the concentration of antibiotic. Results: Two antibiotic resistant strains carrying bla(CTX-M-1) on the chromosome and on an IncI1 plasmid and three sensitive strains were used...... to cefotaxime. The filament formation was restricted to early growth phases and the time the cells grew as filaments was antibiotic concentration dependent. This indicates that antibiotic resistant E. coli undergo the same morphological changes as sensitive bacteria in the presence of beta-lactam antibiotic...

Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation

Science.gov (United States)

2012-01-01

Background In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet. Results Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries. Conclusions Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype. PMID:22452797

Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

DEFF Research Database (Denmark)

Staunstrup, Nicklas H; Moldt, Brian; Mátés, Lajos

2009-01-01

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral...... Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase......-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans...

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

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Tabara, H; Sarkissian, M; Kelly, W G; Fleenor, J; Grishok, A; Timmons, L; Fire, A; Mello, C C

1999-10-15

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Evaluation of restoration of sensitivities of resistant staphylococcus aureus isolates by using cefuroxime and clavulanic acid in combination

International Nuclear Information System (INIS)

Jalil, A.; Din, I.; Khan, S.U.

2008-01-01

The present study was planned to observe the activity of cefuroxime, a second generation cephalosporin after combining it with a beta-lactamase inhibitor calvulanic acid. The study was conducted to evaluate the restoration or increase in sensitivity of beta-lactamase producing isolates of Staphylococcus aureus. Staphylococcus aureus were identified by standard procedures. For beta-lactamase detection chromogenic Nitrocefin impregnated sticks were used. The sensitivity of the bacteria to the antibiotic disks was measured by disk diffusion method using standard zone diameter criteria given by National Committee of Clinical Laboratory Standards. The disks of cefuroxime with clavulanic acid had developed larger zones of inhibition. The activity of cefuroxime against Staphylococcus areus was significantly increased by clavulanic acid. Clavulanic acid if used in combination with cefuroxime, can improve the antimicrobial activity of cefuroxime against beta - lactamase producing Staphylococcus aureus. (author)

Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster.

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Venken, Koen J T; Bellen, Hugo J

2014-06-15

The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters. Copyright © 2014 Elsevier Inc. All rights reserved.

Rim 2/Hipa CACTA transposon display ; A new genetic marker technique in Oryza species

Directory of Open Access Journals (Sweden)

Lee Ju

2005-03-01

Full Text Available Abstract Background Transposons constitute the major fractions of repetitive sequences in eukaryotes, and have been crucial in the shaping of current genomes. Transposons are generally divided into two classes according to the mechanism underlying their transposition: RNA intermediate class 1 and DNA intermediate class 2. CACTA is a class 2 transposon superfamily, which is found exclusively in plants. As some transposons, including the CACTA superfamily, are highly abundant in plant species, and their nucleotide sequences are highly conserved within a family, they can be utilized as genetic markers, using a slightly modified version of the conventional AFLP protocol. Rim2 /Hipa is a CACTA transposon family having 16 bp consensus TIR sequences to be present in high copy numbers in rice genome. This research was carried out in order to develop a Rim2/Hipa CACTA-AFLP or Rim2/Hipa CACTA-TD (transposon display, hereafter Rim2/Hipa-TD protocol for the study of genetic markers in map construction and the study of genetic diversity in rice. Results Rim2/Hipa-TD generated ample polymorphic profiles among the different rice accessions, and the amplification profiles were highly reproducible between different thermocyclers and Taq polymerases. These amplification profiles allowed for clear distinction between two different ecotypes, Japonica and Indica, of Oryza sativa. In the analysis of RIL populations, the Rim2/Hipa-TD markers were found to be segregated largely in a dominant manner, although in a few cases, non-parental bands were observed in the segregating populations. Upon linkage analysis, the Rim2/Hipa-TD markers were found to be distributed in the regions proximal to the centromeres of the chromosomes. The distribution of the Rim2/Hipa CACTA elements was surveyed in 15 different Oryza species via Rim2/Hipa-TD. While Rim2/Hipa-TD yielded ample amplification profiles between 100 to 700 bp in the AA diploid Oryza species, other species having BB, CC

Lack of Correlation Between β-Lactamase Production and Susceptibility to Cefamandole or Cefoxitin Among Spontaneous Mutants of Enterobacteriaceae

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Ott, John L.; Turner, J. R.; Mahoney, David F.

1979-01-01

A large number of cultures of gram-negative bacteria were examined for their susceptibility to various concentrations of cefamandole, cefoxitin, carbenicillin, and nalidixic acid. Heterogeneity of susceptibility was demonstrated in individual cultures to all of these antibiotics. Resistant clones isolated from cefamandole or cefoxitin plates were examined for β-lactamase production. Approximately 13% of 262 resistant clones acquired the ability to produce a β-lactamase. Examination of the substrate profile of the β-lactamases from some of these clones revealed no change in the specific activity of these enzymes for cefamandole, cephaloridine, or compound 87/312 as compared with their parental enzymes. This study clearly shows that some resistant clones do not produce β-lactamases, whereas some susceptible strains produced significant amounts of these enzymes. We conclude from these findings that little correlation exists between β-lactamase production and decreased susceptibility to cefamandole or cefoxitin. The results suggest the possibility that characteristics other than β-lactamase production may be responsible for resistance in Enterobacteriaceae. PMID:311615

The BDGP gene disruption project: Single transposon insertions associated with 40 percent of Drosophila genes

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Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson; Evans-Holm, Martha; Hiesinger, P. Robin; Schulze, Karen L.; Rubin, Gerald M.; Hoskins, Roger A.; Spradling, Allan C.

2004-01-13

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

A large inversion in the linear chromosome of Streptomyces griseus caused by replicative transposition of a new Tn3 family transposon.

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Murata, M; Uchida, T; Yang, Y; Lezhava, A; Kinashi, H

2011-04-01

We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.

High Prevalence of Escherichia coli-Producing CTX-M-15 Extended-Spectrum Beta-Lactamases in Poultry and Human Clinical Isolates in Romania.

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Maciuca, Iuliana E; Williams, Nicola J; Tuchilus, Cristina; Dorneanu, Olivia; Guguianu, Eleonora; Carp-Carare, Catalin; Rimbu, Cristina; Timofte, Dorina

2015-12-01

Use of antibiotics in food animals may contribute to development and spread of resistant organisms, particularly so in some countries. The aim of this study was two-fold; first, to establish the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in chicken production in a region within Romania. Second, to study the relatedness of ESBL-producing E. coli isolates recovered from broilers, abattoir workers where the chickens were slaughtered and from the human clinical specimens from two regional hospitals. The results indicated a very high (69%) rate of carriage of ESBL and AmpC-producing E. coli in chickens with 36% CTX-M producers. Sequencing showed that chickens in Romania have the highest worldwide prevalence (53%) of blaCTX-M-15 reported in poultry E. coli isolates. The majority (53%) of the extended-spectrum cephalosporin-resistant E. coli carried plasmid-mediated blaampC genes, mostly blaCMY-2 type, one of the highest prevalences reported in Europe. The predominant CTX-M type found in the human clinical E. coli isolates was blaCTX-M-15 and most isolates coharbored blaOXA-1, blaTEM, and aac(6')-ib-cr. The majority (60%) of the human clinical isolates belonged to the pandemic virulent clone B2-ST131. The clonal relationship between broiler and the human CTX-M-producing E. coli isolates was assessed by macrorestriction pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), which indicated strain diversity with no common STs found between human and poultry isolates. Moreover, IncI1 was the most prevalent replicon found in broiler ESBL-producing E. coli isolates and also in transconjugants, indicating that plasmids and not clonal spread may play a role in the transfer of blaCTX-M genes. This study identifies a high prevalence of ESBL-producing E. coli from broiler chickens in Romania with a high occurrence incidence of blaCTX-M-15, which reflects the main ESBL type found in human E. coli infections in this

Efficacy of non-carbapenem antibiotics for pediatric patients with first febrile urinary tract infection due to extended-spectrum beta-lactamase-producing Escherichia coli.

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Abe, Yoshifusa; Inan-Erdogan, Işil; Fukuchi, Kunihiko; Wakabayashi, Hitomi; Ogawa, Yasuha; Hibino, Satoshi; Sakurai, Shunsuke; Matsuhashi, Kazuhiko; Watanabe, Yoshitaka; Hashimoto, Kaori; Ugajin, Kazuhisa; Itabashi, Kazuo

2017-08-01

Although carbapenem is the recommended for urinary tract infection (UTI) caused by extended-spectrum beta-lactamase (ESBL)-producing organisms, non-carbapenems have been reported to be effective for adult patients with UTI caused by ESBL-producing organisms. The purpose of this study was to evaluate the efficacy of non-carbapenems for pediatric patients with UTI due to ESBL-producing Escherichia coli (E. coli) based on the microbiologic and clinical outcomes. Fifteen children, who were treated for first febrile UTI caused by ESBL-producing E. coli were enrolled in this study. Antimicrobial susceptibilities and ESBL production were determined according to the Clinical and Laboratory Standards Institute guidelines. To detect CTX-M genes, polymerase chain reaction was performed with specific primers for bla CTX-M detection. Of the 15 enrolled patients, 10 (66.7%) were boys and 5 (33.3%) were girls, with a median age of four months. VUR was detected in six patients (40%). For detection of bla CTX-M by PCR, CTX-M-3, CTX-M-8, CTX-M-14, and CTX-M-15 were detected in five, one, eight, and one patient, respectively. Overall, 14 of the 15 isolates (93.3%) were susceptible for fosfomycin (FOM), and all isolates were susceptible for cefmetazole (CMZ), flomoxef (FMOX), and imipenem/cilastatin (IPM/CS). Of the 15 patients, 12 (80%) clinically improved without the use of carbapenems. In conclusion, even if isolates of ESBL-producing E. coli are multidrug resistant based on MIC assessment, clinical susceptibility to non-carbapenems, such as CMZ, FMOX, and FOM, is possible. Accordingly, carbapenems may not be required all the time for treatment of pediatric UTI in clinical practice. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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

Double beta decays and related subjects for particle and nuclear physics

International Nuclear Information System (INIS)

Ejiri, Hiroyasu

1991-01-01

Present status and some perspectives in 1990's are briefly given on double beta decays and related subjects. Subjects discussed are as follows I) Double beta decays without neutrinos, which require lepton number non-conservations and finite neutrino mass. II) Double beta decays followed by two neutrinos. III) Double weak processes with strangeness change ΔS = 2, leading to the H particle with 6 quarks of ss uu dd. IV) Charge non-conservation and electron decays. These are very rare nuclear processes studied by Ultra RAre-process NUclear Spectroscopy (URANUS). It is shown that URANUS is an important detector frontier of non-accelerator nuclear physics in 1990's. (orig.)

Distribution of Extended-Spectrum β-Lactamases, AmpC β-Lactamases, and Carbapenemases among Enterobacteriaceae Isolates Causing Intra-Abdominal Infections in the Asia-Pacific Region: Results of the Study for Monitoring Antimicrobial Resistance Trends (SMART)

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Sheng, Wang-Huei; Badal, Robert E.

2013-01-01

The increasing trend of β-lactam resistance among Enterobacteriaceae is a worldwide threat. Enterobacteriaceae isolates causing intra-abdominal infections (IAI) from the Study for Monitoring Antimicrobial Resistance Trends (SMART) collected in 2008 and 2009 from the Asia-Pacific region were investigated. Detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases was performed by multiplex PCR. A total of 699 Enterobacteriaceae isolates with positive genotypic results, included Escherichia coli (n = 443), Klebsiella pneumoniae (n = 187), Enterobacter cloacae (n = 45), Klebsiella oxytoca (n = 9), Citrobacter freundii (n = 5), Proteus mirabilis (n = 3), Enterobacter aerogenes (n = 2), Morganella morganii (n = 2), and one each of Enterobacter asburiae, Proteus vulgaris, and Providencia rettgeri were analyzed. Nearly 20% of these β-lactamase-producing Enterobacteriaceae isolates were from community-associated IAI. CTX-M (588 isolates, including 428 [72.8%] with CTX-M-15) was the most common ESBL, followed by SHV (n = 59) and TEM (n = 4). CMY (n = 110, including 102 [92.7%] with CMY-2) was the most common AmpC β-lactamase, followed by DHA (n = 46) and ACT/MIR (n = 40). NDM (n = 65, including 62 [95.4%] with NDM-1) was the most common carbapenemase, followed by IMP (n = 7) and OXA (n = 7). Isolates from hospital-associated IAI had more complicated β-lactamase combinations than isolates from the community. Carbapenemases were all exclusively detected in Enterobacteriaceae isolates from India, except that IMP β-lactamases were also detected in Philippines and Australia. CTX-M β-lactamases were the predominant ESBLs produced by Enterobacteriaceae causing IAI in the Asia-Pacific region. Emergence of CTX-M-15-, CMY-2-, and NDM-1-producing Enterobacteriaceae isolates is of major concern and highlights the need for further surveillance in this area. PMID:23587958

Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae

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Kim, Hyun-Soo [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Sciences; Kim, Na-Rae [Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Yang, Jungwoo [Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center; Choi, Wonja [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Sciences; Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center

2011-08-15

Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1-5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. (orig.)

Trapping and Characterization of a Reaction Intermediate in Carbapenem Hydrolysis by B. cereus Metallo-β-lactamase

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Tioni, Mariana F.; Llarrull, Leticia I.; Poeylaut-Palena, Andrés A.; Martí, Marcelo A.; Saggu, Miguel; Periyannan, Gopal R.; Mata, Ernesto G.; Bennett, Brian; Murgida, Daniel H.; Vila, Alejandro J.

2009-01-01

Metallo-β-lactamases hydrolyze most β-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze-quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)-BcII with imipenem. These studies show that Co(II)-BcII is able to hydrolyze imipenem both in the mono- and dinuclear forms. In contrast to the situation met for penicillin, the species that accumulates during turnover is an enzyme-intermediate adduct in which the β-lactam bond has already been cleaved. This intermediate is a metal-bound anionic species, with a novel resonant structure, that is stabilized by the metal ion at the DCH or Zn2 site. This species has been characterized based on its spectroscopic features. This represents a novel, previously unforeseen intermediate, that is related to the chemical nature of carbapenems, as confirmed by the finding of a similar intermediate for meropenem. Since carbapenems are the only substrates cleaved by B1, B2 and B3 lactamases, the identification of this intermediate could be exploited as a first step towards the design of transition state based inhibitors for all three classes of metallo-β-lactamases. PMID:18980308

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase.

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Ruggiero, Melina; Papp-Wallace, Krisztina M; Taracila, Magdalena A; Mojica, Maria F; Bethel, Christopher R; Rudin, Susan D; Zeiser, Elise T; Gutkind, Gabriel; Bonomo, Robert A; Power, Pablo

2017-06-01

PER β-lactamases are an emerging family of extended-spectrum β-lactamases (ESBL) found in Gram-negative bacteria. PER β-lactamases are unique among class A enzymes as they possess an inverted omega (Ω) loop and extended B3 β-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-β-lactam β-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 / K = 2 × 10 3 ± 0.1 × 10 3 M -1 s -1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D β-lactamases (i.e., k 2 / K range of ≈10 3 M -1 s -1 ) and not class A β-lactamases (i.e., k 2 / K range, 10 4 to 10 5 M -1 s -1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 ± 3 atomic mass units [amu] → 31,604 ± 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the β-lactamase and that the sulfate of AVI formed interactions with the β-lactam carboxylate binding site of the PER-2 β-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 β-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation ( k 2 / K ) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 β-lactamase, we tested different β-lactam-AVI combinations. By lowering MICs to ≤2 mg/liter, the ceftaroline-AVI combination could represent a favorable

Salmonella Heidelberg: Genetic profile of its antimicrobial resistance related to extended spectrum β-lactamases (ESBLs).

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Giuriatti, Jéssica; Stefani, Lenita Moura; Brisola, Maiara Cristina; Crecencio, Regiane Boaretto; Bitner, Dinael Simão; Faria, Gláucia Amorim

2017-08-01

The objective of this study was to evaluate the phenotypic and genotypic profile of antimicrobial susceptibility and the possible involvement of extended spectrum beta-lactamases (ESBLs) in the resistance profile of Salmonella Heidelberg (SH) isolated from chicken meat. We used 18 SH isolates from chicken meat produced in 2013 in the state of Paraná, Southern Brazil. The isolates were submitted to disk-diffusion tests and from these results it was possible to determine the number of isolates considered multiresistant and the index of multiple antimicrobial resistance (IRMA) against ten antimicrobials routinely used in human and veterinary medicine. It was considered multidrug resistant the isolate that showed resistance to three or more classes of antibiotics. Another test performed was the disc-approximation in order to investigate interposed zones of inhibition, indicative of ESBLs production. In the isolates that presented multidrug resistance (18/18), a search of resistance genes involved in the production of ESBLs was performed using PCR: blaCMY-2, blaSHV-1, blaTEM-1, blaCTX-M2, blaOXA-1, blaPSE-1 and AmpC. The overall antimicrobial resistance was 80.55%. The highest levels of resistance were observed for nalidixic acid and ceftiofur (100%). The most commonly resistance pattern found (42.1%) was A (penicillin-cephalosporin-quinolone-tetracycline). The results were negative for ghost zone formation, indicative of ESBLs. However, PCR technique was able to detect resistance genes via ESBLs where the blaTEM-1 gene showed the highest amplification (83.33%), and the second most prevalent genes were blaCMY-2 (38.88%) and AmpC gene (38.88%). The blaOXA-1 and blaPSE-1 genes were not detected. These results are certainly of concern since SH is becoming more prevalent in the South of Brazil and able to cause severe disease in immune compromised individuals, showing high antimicrobial resistance to those drugs routinely used in the treatment and control of human and

Horizontol dissemination of TEM- and SHV-typr beta-lactamase genes-carrying resistance plasmids amongst clonical isolates of Enterobacteriaceae Disseminação horizontal de plasmídios de resistência contendo genes de beta-lactamase dos tipos TEM e SHV entre isolados clínicos de Enterobacteriaceae

Directory of Open Access Journals (Sweden)

Osman Birol Ozgumus

2008-12-01

Full Text Available The extended-spectrum β-lactamase (ESBL-producing bacteria have been isolated at increasing frequency worldwide. Expression of ESBL is often associated with multidrug resistance and dissemination by resistance plasmids. During a two-month period in 2000, 133 clinical isolates of enterobacterial strains were randomly collected from outpatients and inpatients at a university hospital in Turkey. The ESBL producing strains were determined by double-disk synergy (DDS testing. Twenty ESBL producing strains (15% including Escherichia coli (n = 9, Klebsiella pneumoniae (n = 7, Klebsiella oxytoca (n = 2 and Enterobacter aerogenes (n = 2 were detected and further analyzed for their resistance transfer features, plasmid profile and nature of the resistance genes. Plasmid transfer assays were performed using broth mating techniques. TEM- and SHV- genes were analyzed by polymerase chain reaction (PCR and hybridization using specific probes. EcoRI restriction enzyme analyses of R plasmids were used in the detection of epidemic plasmids. Fourteen plasmid profiles (A, B1, B2, C1, and C2 to L were obtained with EcoRI restriction enzyme analysis. Most of these plasmids were detected to carry both TEM- and SHV-derived genes by PCR, and confirmed by localizing each gene by hybridization assay. Epidemiological evidence indicated that there was an apparent horizontal dissemination of conjugative R plasmids among multidrug-resistant enterobacterial genera and species in this hospitalO isolamento de bactérias produtoras de beta-lactamases de espectro expandido (ESBL está aumentando no mundo todo. Freqüentemente, a expressão de ESBL está associada com resistência a múltiplas drogas e disseminação por plasmídios de resistência. Durante um período de dois meses em 2000, 133 isolados clínicos de cepas de enterobactérias foram obtidos aleatoriamente de pacientes internos e externos de um hospital universitário na Turquia. As cepas produtoras de ESBL foram

Horizontal transfers of Mariner transposons between mammals and insects.

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Oliveira, Sarah G; Bao, Weidong; Martins, Cesar; Jurka, Jerzy

2012-09-26

Active transposable elements (TEs) can be passed between genomes of different species by horizontal transfer (HT). This may help them to avoid vertical extinction due to elimination by natural selection or silencing. HT is relatively frequent within eukaryotic taxa, but rare between distant species. Closely related Mariner-type DNA transposon families, collectively named as Mariner-1_Tbel families, are present in the genomes of two ants and two mammalian genomes. Consensus sequences of the four families show pairwise identities greater than 95%. In addition, mammalian Mariner1_BT family shows a close evolutionary relationship with some insect Mariner families. Mammalian Mariner1_BT type sequences are present only in species from three groups including ruminants, tooth whales (Odontoceti), and New World leaf-nosed bats (Phyllostomidae). Horizontal transfer accounts for the presence of Mariner_Tbel and Mariner1_BT families in mammals. Mariner_Tbel family was introduced into hedgehog and tree shrew genomes approximately 100 to 69 million years ago (MYA). Most likely, these TE families were transferred from insects to mammals, but details of the transfer remain unknown.

Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

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Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

2004-06-01

In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

Interactions of "bora-penicilloates" with serine β-lactamases and DD-peptidases.

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Dzhekieva, Liudmila; Adediran, S A; Pratt, R F

2014-10-21

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

[Analysis of resistant genes of beta-lactam antibiotics from Pseudomonas aeruginosa in pediatric patients].

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Dong, Fang; Xu, Xi-wei; Song, Wen-qi; Lü, Ping; Yang, Yong-hong; Shen, Xu-zhuang

2008-11-18

To analyze the antibiotic resistance of the Pseudomonas aeruginosa (PA) isolated from pediatric patients and the resistant genes of beta-lactam antibiotics thereof. 146 PA strains were isolated from pediatric patients. Agar dilution method recommended by the Clinical and Laboratory Standards Institute was used to examine the minimum inhibitory concentrations (MICs) of 12 antimicrobial agents, including the penicillins, third and fourth genet ration cephalosporins, carbapenemase, Aztreonam, beta-lactamase inhibitors, quinolones, and aminoglycosides. PCR was used to detect the expression of the genes TEM, SHV, OXA, PER, GES, CTX-M, IMP, VIM, DHA, MIR, FOX, and oprD2. The multi-drug resistance rates against different antibiotic were high among the 146 PA strains. The rates of imipenem and meropenem resistance were 41.1% and 35.6% respectively. Among the 146 PA strains, 46 (31.5%) were positive for the MBL genotype; 38 (82.6%) carried the blaIMP gene, 8 (17.4%) carried the blaVIM gene, and 114 (78.1%) were oprD2 negative. The genes TEM, SHV, OXA, CTX-M, PER, VEB, GES, FOX, MIR, and DHA were not found in all strains. Many PA isolated from pediatric patients carry the genes IMP or VIM and losses oprD2 gene related to the expression of the outer membrane porin OprD2. The loss of the gene oprD2 is essential mechanism of beta-lactam antibiotics resistance in PA.

Association of high mortality with extended-spectrum β-lactamase (ESBL) positive cultures in community acquired infections.

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Ray, Sumit; Anand, Dimple; Purwar, Sankalp; Samanta, Arijit; Upadhye, Kaustubh V; Gupta, Prasoon; Dhar, Debashis

2018-04-01

Infections due to multidrug resistant organisms have become a serious health concern worldwide. The present study was conducted to investigate the spectrum of microbial resistance pattern in the community and their effects on mortality. A retrospective review and analysis of prospectively collected data was done of all patients admitted with diagnosis of sepsis in two tertiary care ICU's for a period of two years. Demographics, culture positivity, microbial spectrum, resistance pattern and outcome data were collected. Out of 5309 patients enrolled; 3822 had suspected clinical infection on admission with 1452 patients growing positive microbial cultures. Among these, 201 bacterial strains were isolated from patients who had community acquired infections. 73% were Gram negative bacilli, commonest being E. coli (63%). 63.4% E. coli and 60.7% Klebsiella isolates were ESBL producers. The mortality in ESBL positive infections was significantly higher as compared to ESBL negative infections (Odds ratio 2.756). Moreover, ESBL positive patients empirically treated with Beta Lactams+Beta Lactamase inhibitors (BL+BLI) had significantly higher mortality as compared to patients treated with carbapenems. More data from multiple centres need to be gathered to formulate appropriate antibiotic policy for critically ill patients admitted from the community. Copyright © 2017 Elsevier Inc. All rights reserved.

Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of beta-Lactamase-Producing Escherichia coli

DEFF Research Database (Denmark)

Baker, Kristin R.; Sigurdardottir, Helga Høeg; Jana, Bimal

2017-01-01

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for “helper” drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid...... was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dap...... for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness...

Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae

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N O Yilmaz

2013-01-01

Full Text Available Background: Detecting plasmid-mediated AmpC (pAmpC β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82 and Escherichia coli (n: 191 were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX screening test, modified three dimensional test (M3DT, and multiplex polymerase chain reaction (PCR. Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.

Novel Aminoglycoside Resistance Transposons and Transposon-Derived Circular Forms Detected in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

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Dwibedi, Chinmay Kumar; Sjöström, Karin; Edquist, Petra; Wai, Sun Nyunt; Uhlin, Bernt Eric

2016-01-01

Acinetobacter baumannii has emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates of A. baumannii collected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n = 16) and CC25 (n = 7). Resistance to carbapenems was related to blaOXA-23 (20 isolates), blaOXA-24/40-like (6 isolates), blaOXA-467 (1 isolate), and ISAba1-blaOXA-69 (1 isolate). Ceftazidime resistance was associated with blaPER-7 in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylase armA gene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020 and TnaphA6. Importantly, a number of circular forms related to the IS26 or ISAba125 composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes. PMID:26824943

Lactam hydrolysis catalyzed by mononuclear metallo-ß-bactamases

DEFF Research Database (Denmark)

Olsen, Lars; Antony, J; Ryde, U

2003-01-01

Two central steps in the hydrolysis of lactam antibiotics catalyzed by mononuclear metallo-beta-lactamases, formation of the tetrahedral intermediate and its breakdown by proton transfer, are studied for model systems using the density functional B3LYP method. Metallo-beta-lactamases have two metal...

Presence of ESBL/AmpC-producing Escherichia coli in the broiler production pyramid: a descriptive study.

NARCIS (Netherlands)

Dierikx, C.M.; Goot, van der J.A.; Smith, H.E.; Kant, A.; Mevius, D.J.

2013-01-01

Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the

Impact of empirical treatment in extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. bacteremia. A multicentric cohort study

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Peralta Galo

2012-10-01

Full Text Available Abstract Background The objective of this study is to analyze the factors that are associated with the adequacy of empirical antibiotic therapy and its impact in mortality in a large cohort of patients with extended-spectrum β-lactamase (ESBL - producing Escherichia coli and Klebsiella spp. bacteremia. Methods Cases of ESBL producing Enterobacteriaceae (ESBL-E bacteremia collected from 2003 through 2008 in 19 hospitals in Spain. Statistical analysis was performed using multivariate logistic regression. Results We analyzed 387 cases ESBL-E bloodstream infections. The main sources of bacteremia were urinary tract (55.3%, biliary tract (12.7%, intra-abdominal (8.8% and unknown origin (9.6%. Among all the 387 episodes, E. coli was isolated from blood cultures in 343 and in 45.71% the ESBL-E was multidrug resistant. Empirical antibiotic treatment was adequate in 48.8% of the cases and the in hospital mortality was 20.9%. In a multivariate analysis adequacy was a risk factor for death [adjusted OR (95% CI: 0.39 (0.31-0.97; P = 0.04], but not in patients without severe sepsis or shock. The class of antibiotic used empirically was not associated with prognosis in adequately treated patients. Conclusion ESBL-E bacteremia has a relatively high mortality that is partly related with a low adequacy of empirical antibiotic treatment. In selected subgroups the relevance of the adequacy of empirical therapy is limited.

An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries

DEFF Research Database (Denmark)

Westh, Henrik Torkil; Zinn, Christina Scheel; Rosdahl, Vibeke Thamdrup

2004-01-01

of therapeutical subgroups of antimicrobials varied significantly between hospitals. A positive correlation was found between S. aureus resistance to methicillin (MRSA) and consumption of beta-lactam combinations, between resistance to quinolones and consumption of beta-lactam combinations and carbapenems....... Consumption of beta-lactamase-sensitive antibiotics (penicillin) was positively correlated to consumption of beta-lactamase-resistant penicillins and negatively correlated to consumption of carbapenems, quinolones, and glycopeptides, whereas consumption of cephalosporins was positively correlated...

Caracterización de Klebsiella pneumoniae productora de la beta-lactamasa SHV-5, en una unidad de cuidados intensivos Characterization of SHV-5 beta-lactamase-producing Klebsiella pneumoniae in an intensive care unit

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Verónica Andrade

2004-12-01

chain reaction (RAPD-PCR and serotyping, beta-Lactamase isoelectric focusing (IEF, and nucleotide sequencing of PCR products. RESULTS: Serotype 61 was predominant and correlated with genomic fingerprints of RAPD and PFGE in 11 of 15 isolates. One SHV-5-producer predominant clone with a high case-fatality rate was identified. CONCLUSIONS: Molecular biology techniques are useful tools to characterize the K. pneumoniae clone isolated from patients and health care workers, suggesting potential cross-transmission. These data call for strengthening control programs to prevent dissemination of nosocomial infections in the studied hospital.

Risk factors associated with the community-acquired colonization of extended-spectrum beta-lactamase (ESBL positive Escherichia Coli. an exploratory case-control study.

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Rasmus Leistner

Full Text Available BACKGROUND: The number of extended-spectrum beta-lactamase (ESBL positive (+ Escherichia coli is increasing worldwide. In contrast with many other multidrug-resistant bacteria, it is suspected that they predominantly spread within the community. The objective of this study was to assess factors associated with community-acquired colonization of ESBL (+ E. coli. METHODS: We performed a matched case-control study at the Charité University Hospital Berlin between May 2011 and January 2012. Cases were defined as patients colonized with community-acquired ESBL (+ E. coli identified <72 h after hospital admission. Controls were patients that carried no ESBL-positive bacteria but an ESBL-negative E.coli identified <72 h after hospital admission. Two controls per case were chosen from potential controls according to admission date. Case and control patients completed a questionnaire assessing nutritional habits, travel habits, household situation and language most commonly spoken at home (mother tongue. An additional rectal swab was obtained together with the questionnaire to verify colonization status. Genotypes of ESBL (+ E. coli strains were determined by PCR and sequencing. Risk factors associated with ESBL (+ E. coli colonization were analyzed by a multivariable conditional logistic regression analysis. RESULTS: We analyzed 85 cases and 170 controls, respectively. In the multivariable analysis, speaking an Asian language most commonly at home (OR = 13.4, CI 95% 3.3-53.8; p<0.001 and frequently eating pork (≥ 3 meals per week showed to be independently associated with ESBL colonization (OR = 3.5, CI 95% 1.8-6.6; p<0.001. The most common ESBL genotypes were CTX-M-1 with 44% (n = 37, CTX-M-15 with 28% (n = 24 and CTX-M-14 with 13% (n = 11. CONCLUSION: An Asian mother tongue and frequently consuming certain types of meat like pork can be independently associated with the colonization of ESBL-positive bacteria. We found neither frequent consumption

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase

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Wong Kwok-Yin

2011-03-01

Full Text Available Abstract Background β-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for β-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood. Results Here we report the crystal structure of a Class A β-lactamase PenP from Bacillus licheniformis 749/C with fluorescein conjugated at residue 166 after E166C mutation, both in apo form (PenP-E166Cf and in covalent complex form with cefotaxime (PenP-E166Cf-cefotaxime, to illustrate its biosensing mechanism. In the apo structure the fluorescein molecule partially occupies the antibiotic binding site and is highly dynamic. In the PenP-E166Cf-cefatoxime complex structure the binding and subsequent acylation of cefotaxime to PenP displaces fluorescein from its original location to avoid steric clash. Such displacement causes the well-folded Ω-loop to become fully flexible and the conjugated fluorescein molecule to relocate to a more solvent exposed environment, hence enhancing its fluorescence emission. Furthermore, the fully flexible Ω-loop enables the narrow-spectrum PenP enzyme to bind cefotaxime in a mode that resembles the extended-spectrum β-lactamase. Conclusions Our structural studies indicate the biosensing mechanism of a fluorescein-labelled β-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of β-lactamase-based biosensor to detect the wide spectrum of β-lactam antibiotics. The observation of increased Ω-loop flexibility upon conjugation of fluorophore may have the potential to serve as a screening tool for novel β-lactamase inhibitors that target the Ω-loop and not the active site.

Occurrence and Variety of β-Lactamase Genes among Aeromonas spp. Isolated from Urban Wastewater Treatment Plant

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Marta Piotrowska

2017-05-01

Full Text Available Members of the genus Aeromonas that commonly occur in various aquatic ecosystems are taken into account as vectors spreading antibiotic resistance genes (ARGs in the environment. In our study strains of Aeromonas spp. (n = 104 not susceptible to ampicillin were isolated from municipal sewage of different levels of purification – raw sewage, activated sludge and treated wastewater. The crucial step of the study was the identification of β-lactamase resistance genes. The identified genes encode β-lactamases from 14 families – blaTEM, blaOXA, blaSHV, blaCTX-M, blaMOX, blaACC, blaFOX, blaGES, blaPER, blaV EB, blaKPC, cphA, imiH, and cepH. There were no significant differences in number of identified ARGs between isolation points. BlaOXA, blaFOX variants and, characteristic for Aeromonas genus, metallo-β-lactamase cphA-related genes were the most commonly identified types of β-lactam resistance determinants. Moreover, we found four extended-spectrum β-lactamases (blaSHV -11, blaCTX-M-27, blaCTX-M-98, and blaPER-4 – and seven AmpC (blaACC, blaFOX-2-like, blaFOX-3, blaFOX-4-like, blaFOX-9, blaFOX-10-like, and blaFOX-13-like types and variants of genes that had never been found among Aeromonas spp. before. Five of the β-lactamases families (blaTEM, blaOXA, blaFOX, blaV EB, and cphA were identified in all three isolation sites, which supports the hypothesis that wastewater treatment plants (WWTPs are hot spots of ARGs dissemination. The obtained ARGs sequences share high identity with previously described β-lactamases, but new variants of those genes have to be considered as well. Characterization of antibiotic susceptibility was performed using disk the diffusion method with 12 different antibiotics according to CLSI guidelines. Over 60% of the strains are unsusceptible to cefepime and chloramphenicol and the majority of the strains have a multidrug resistance phenotype (68%. Finally, analysis of plasmid profiles among the resistant strains

High β-Lactamase Levels Change the Pharmacodynamics of β-Lactam Antibiotics in Pseudomonas aeruginosa Biofilms

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Ciofu, Oana; Yang, Liang; Wu, Hong; Song, Zhijun; Oliver, Antonio; Høiby, Niels

2013-01-01

Resistance to β-lactam antibiotics is a frequent problem in Pseudomonas aeruginosa lung infection of cystic fibrosis (CF) patients. This resistance is mainly due to the hyperproduction of chromosomally encoded β-lactamase and biofilm formation. The purpose of this study was to investigate the role of β-lactamase in the pharmacokinetics (PK) and pharmacodynamics (PD) of ceftazidime and imipenem on P. aeruginosa biofilms. P. aeruginosa PAO1 and its corresponding β-lactamase-overproducing mutant, PAΔDDh2Dh3, were used in this study. Biofilms of these two strains in flow chambers, microtiter plates, and on alginate beads were treated with different concentrations of ceftazidime and imipenem. The kinetics of antibiotics on the biofilms was investigated in vitro by time-kill methods. Time-dependent killing of ceftazidime was observed in PAO1 biofilms, but concentration-dependent killing activity of ceftazidime was observed for β-lactamase-overproducing biofilms of P. aeruginosa in all three models. Ceftazidime showed time-dependent killing on planktonic PAO1 and PAΔDDh2Dh3. This difference is probably due to the special distribution and accumulation in the biofilm matrix of β-lactamase, which can hydrolyze the β-lactam antibiotics. The PK/PD indices of the AUC/MBIC and Cmax/MBIC (AUC is the area under concentration-time curve, MBIC is the minimal biofilm-inhibitory concentration, and Cmax is the maximum concentration of drug in serum) are probably the best parameters to describe the effect of ceftazidime in β-lactamase-overproducing P. aeruginosa biofilms. Meanwhile, imipenem showed time-dependent killing on both PAO1 and PAΔDDh2Dh3 biofilms. An inoculum effect of β-lactams was found for both planktonic and biofilm P. aeruginosa cells. The inoculum effect of ceftazidime for the β-lactamase-overproducing mutant PAΔDDh2Dh3 biofilms was more obvious than for PAO1 biofilms, with a requirement of higher antibiotic concentration and a longer period of treatment

Epidemiology of extended-spectrum β-lactamase, AmpC, and carbapenemase production in Proteus mirabilis.

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Datta, Priya; Gupta, Varsha; Arora, Shilpa; Garg, Shivani; Chander, Jagdish

2014-01-01

Proteus mirabilis strains that produce extended-spectrum β-lactamase (ESBL), AmpC β-lactamase, and carbapenemase pose potential threats to patient care because most clinical diagnostic laboratories may not attempt to detect these three major groups of enzymes. Therefore, the objective of this study was to ascertain if P. mirabilis isolates collected from our heathcare facility possess various mechanisms of resistance to β-lactams (i.e., ESBL, AmpC, and carbapenemases) and to additionally arrive at conclusions regarding concurrent testing for these three mechanism of drug resistance in order to reduce cost and time in routine diagnostic testing. Between January 2011 and June 2011, 60 consecutive non-repeated strains of P. mirabilis were evaluated for production of ESBLs, AmpC β-lactamases, and carbapenemases. Of these, 36 isolates were found to be ESBL producers, and 7 (12%) were positive for production of AmpC β-lactamases and ESBLs. Therefore, 19.4% of ESBL-producing Proteus strains coproduced AmpC enzymes. The modified Hodge test confirmed carbapenemase production in only 1 isolate (1.7%), which was also ESBL- and AmpC-positive. The clinical impact of additional AmpC expression in ESBL-producing P. mirabilis results in a newly acquired resistance to β-lactamase inhibitors. In addition, to save time and costs, we recommend the use of cefepime/cefepime-clavulanate or boronic acid for the ESBL detection but in only those strains that were positive for ESBL by screening and negative by confirmatory tests.

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

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Kim, Jaewon; Lee, Jihun; Brych, Stephen R; Logan, Timothy M; Blaber, Michael

2005-02-01

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

[The outpatient use of beta lactam antibiotics in Montenegro before the introduction of new reform strategy on drug market].

Science.gov (United States)

Duborija-Kovacević, Natasa

2006-01-01

The study represents the first investigation of outpatient use of beta lactam antibiotics in Montenegro carried out in accordance with internationally approved methodology (DDD/ATC). The objective of our study was to establish both the scope and overall use of beta lactam antibiotics, and to assess their compatibility with current pharmacotherapeutic guidelines and their use in developed countries. The retrospective pharmaco-epidemiological study comprised a 100%-sample of beta lactams that were used in the period prior to introduction of new reform strategy on drug market. Beta lactam antibiotics (J01C, J01D) were the most frequently applied anti-infectives for systemic use (ATC group J) in 2000 (11.3 DDD/1000 inh./day, 61%). Penicillins (J01C) were the most utilized (8.0 DDD/1000 inh./day, 71%). Cephalosporin derivatives (cephalexin and cefaclor) accounted for the remaining 29% (3.3 DDD/1000 inh./day). Aminopenicillins were prevailing among penicillins (85%). Beta lactamase sensitive penicillins were in the second place and approximately accounted for 14%. The results of our study showed that the use of beta lactam antibacterials could be estimated as partially satisfactory. There is a need to make additional efforts with a view of further rationalization.

blaCTX-M-I group extended spectrum beta lactamase-producing Salmonella typhi from hospitalized patients in Lagos, Nigeria

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Akinyemi KO

2015-05-01

Full Text Available Kabiru O Akinyemi,1 Bamidele A Iwalokun,2 Olajide O Alafe,1 Sulaiman A Mudashiru,1 Christopher Fakorede,11Department of Microbiology, Lagos State University, Ojo, Lagos, Nigeria; 2Biochemistry and Nutrition Division, Nigerian Institute of Medical Research, Yaba, Lagos, NigeriaPurpose: The global spread of blaCTX-M-I extended-spectrum beta-lactamase (ESBL-producing Salmonella spp. remains a major threat to treatment and control. Evidence of emergence and spread of this marker are lacking in Nigeria. This study investigated blaCTX-M-I ESBL production among Salmonella isolates from hospitalized patients.Methods: Patients (158 total made up of two groups were evaluated. Group A was composed of 135 patients with persistent pyrexia and group B was composed of 23 gastroenteritis patients and their stool samples. Samples were cultured, and isolates were identified and were subjected to antibiotic susceptibility testing by standard methods. Isolates were further screened for ESBL production, blaCTX-M-I genes and transferability by double disk synergy test, plasmid extraction, polymerase chain reaction, and conjugation experiment.Results: Thirty-five (25.9% Salmonella isolates were identified from group A, of which 74.3% were S. typhi, 22.9% were S. paratyphi and two (5.7% were invasive non-typhoidal S. enteritidis. Nine Plasmodium falciparum infections were recorded, four of which were identified as co-infections with typhoidal Salmonella. Only two (8.7% S. enteritidis samples were obtained from group B (P>0.05. A total of 24 isolates were ESBL-positive, eliciting resistance to five to seven antibiotics, and were multiple-drug resistant. ESBL production due to the blaCTX-M-I gene cluster was detected in eleven (45.8% Salmonella isolates. Nine (81.8% of the eleven blaCTX-M-I ESBL producers were S. typhi and two (18.2% isolates were S. enteritidis. Four of nine S. typhi blaCTX-M-I ESBL-producing strains harbored 23 kb self-transmissible plasmid that was co

Epidemiology of infections caused by multiresistant gram-negatives: ESBLs, MBLs, panresistant strains.

Science.gov (United States)

Rossolini, Gian Maria; Mantengoli, Elisabetta; Docquier, Jean-Denis; Musmanno, Rosa Anna; Coratza, Grazietta

2007-07-01

Microbial drug resistance is a growing problem of global magnitude. In gram-negative pathogens, the most important resistance problems are encountered in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter, with increasing trends observed for all major anti-gram-negative agents (beta-lactams, fluoroquinolones and aminoglycosides). A matter of major concern is the emergence of new beta-lactamases capable of degrading the expanded-spectrum cephalosporins and/or carbapenems, such as the extended-spectrum beta-lactamases (ESBLs) and the carbapenemases. These beta-lactamase genes are often associated with resistance determinants to non-beta-lactam agents (e.g. aminoglycosides and fluoroquinolones), and strains producing ESBLs or carbapenemases often exhibit complex multidrug resistant phenotypes and sometimes are panresistant. The problem is worsened by the dearth of new agents active on multidrug-resistant Gram-negatives in the pipeline. The importance to develop better strategies to control resistance is underscored.

Horizontal transfers of Mariner transposons between mammals and insects

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Oliveira Sarah G

2012-09-01

Full Text Available Abstract Background Active transposable elements (TEs can be passed between genomes of different species by horizontal transfer (HT. This may help them to avoid vertical extinction due to elimination by natural selection or silencing. HT is relatively frequent within eukaryotic taxa, but rare between distant species. Findings Closely related Mariner-type DNA transposon families, collectively named as Mariner-1_Tbel families, are present in the genomes of two ants and two mammalian genomes. Consensus sequences of the four families show pairwise identities greater than 95%. In addition, mammalian Mariner1_BT family shows a close evolutionary relationship with some insect Mariner families. Mammalian Mariner1_BT type sequences are present only in species from three groups including ruminants, tooth whales (Odontoceti, and New World leaf-nosed bats (Phyllostomidae. Conclusions Horizontal transfer accounts for the presence of Mariner_Tbel and Mariner1_BT families in mammals. Mariner_Tbel family was introduced into hedgehog and tree shrew genomes approximately 100 to 69 million years ago (MYA. Most likely, these TE families were transferred from insects to mammals, but details of the transfer remain unknown.

Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

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Hackett Perry B

2006-06-01

Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

Extended Spectrum β-Lactamase (ESBL) in Klebsiella Pneumoniae ...

African Journals Online (AJOL)

Pseudomonas aeruginosa, Streptococcus Pneumoniae and Serratia spp. Two of the children died in spite of early use of appropriate antibiotics as determined by antibiotic susceptibility testing. Phenotypic and molecualr investigation showed extended-spectrum β-lactamase (ESBL) producing K. pneumoniae to be ...

Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

DEFF Research Database (Denmark)

Kues, Wilfried A.; Herrmann, Doris; Barg-Kues, Brigitte

2013-01-01

the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (i......PS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives......The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing...

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

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Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

1989-01-01

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291

Genetic and biochemical characterization of HMB-1, a novel subclass B1 metallo-β-lactamase found in a Pseudomonas aeruginosa clinical isolate.

Science.gov (United States)

Pfennigwerth, Niels; Lange, Felix; Belmar Campos, Cristina; Hentschke, Moritz; Gatermann, Sören G; Kaase, Martin

2017-04-01

To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate. The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat . P. aeruginosa NRZ-03096 was resistant to all tested β-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to β-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem. The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa . © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Occurrence and sensitivity profile of extended spectrum beta-lactamase-producing Enterobacteriaceae at a tertiary hospital in Southern Brazil

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Cristina Letícia Rugini

2015-12-01

Full Text Available Abstract: INTRODUCTION: Nosocomial infections are closely associated with antimicrobial drug resistance. One of the most important mechanisms of resistance to β-lactam antibiotics is the production of extended spectrum β-lactamases (ESBLs. The objective of the present study was to evaluate the prevalence and antimicrobial susceptibility profile of ESBL-producing strains and to assess the evolution of antimicrobial drug resistance between 2007 and 2013 at the Hospital São Vicente de Paulo, Passo Fundo, State of Rio Grande do Sul, Brazil. METHODS: We conducted a descriptive, observational, cross-sectional study. Bacterial culture was performed from January to December 2013. The antimicrobial susceptibility profile of these cultures was determined using the disk diffusion method. Phenotypic screening for ESBL production was performed using the disk approximation method. RESULTS : We analyzed a total of 19,112 cultures, 11.5% of which were positive for Enterobacteriaceae. Of these, 30.3% of the isolates were positive for ESBL production, and the most prevalent species was Klebsiella sp. (37.5%. Over 95% of these isolates showed reduced susceptibility to all cephalosporins, aztreonam, and amoxicillin/clavulanic acid. The isolates also showed high sensitivity to the following antimicrobials: amikacin, meropenem, and piperacillin/tazobactam. Overall, the resistance rates among ESBL-producing Enterobacteriaceae decreased from 2007 to 2013. CONCLUSIONS : In our hospital, the increased sensitivity to certain antimicrobial agents seems to be directly related to the implementation of improvements in the methods to prevent and control nosocomial infections in addition to the natural development of other resistance mechanisms.

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and DD-peptidases.

Science.gov (United States)

Pelto, Ryan B; Pratt, R F

2012-09-28

The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

Efflux pump, the masked side of beta-lactam resistance in Klebsiella pneumoniae clinical isolates.

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Jean-Marie Pages

Full Text Available BACKGROUND: Beta-lactamase production and porin decrease are the well-recognized mechanisms of acquired beta-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX and susceptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this beta-lactam phenotype was the aim of this study. METHODOLOGY/FINDINGS: MICs of 9 beta-lactams, including cloxacillin (CLX, and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI, then with both CLX (subinhibitory concentrations and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other beta-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also 16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other beta-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of beta-lactams. CONCLUSION: This is the first study demonstrating that efflux mechanism plays a key role in the beta-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in beta-lactam resistance is specially underestimated in clinical isolates.

Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

International Nuclear Information System (INIS)

Chen, Li; Schmidt, Emmett V; Stuart, Lynda; Ohsumi, Toshiro K; Burgess, Shawn; Varshney, Gaurav K; Dastur, Anahita; Borowsky, Mark; Benes, Cyril; Lacy-Hulbert, Adam

2013-01-01

The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens

Dissemination and genetic support of broad-spectrum beta-lactam-resistant Escherichia coli strain isolated from two Tunisian hospitals during 2004-2012.

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Ayari, Khaoula; Bourouis, Amel; Chihi, Hela; Mahrouki, Sihem; Naas, Thierry; Belhadj, Omrane

2017-06-01

The dissemination of extended-spectrum β-lactamase (ESBL)-producing bacteria presented a great concern worldwide. Gram-negative organisms such as Escherichia coli and Klebsiella pneumoniae are the most frequently isolated pathogens responsible for nosocomial infections. The aim of this study was to investigate and to follow the emergence of resistance and the characterization of Extended-Spectrum Beta-Lactamases (ESBL) among broad-spectrum beta-lactam- Escherichia coli clinical isolates recovered from the military hospital and Habib Thameur hospital in Tunisia. A total of 113 E.coli isolates obtained during the period 2004 through 2012 showed a significant degree of multi-resistance. Among these strains, the double-disk synergy test confirmed the ESBL phenotype in 46 isolates. These included 32(70%) strains from Hospital A and 14(30%) from Hospital B. The ESBL was identified as CTX-M-15. The ESBL resistance was transferred by a 60 kb plasmid CTXM-15-producing isolates were unrelated according to the PFGE analysis and characterization of the regions surrounding the blaCTX-M-15 showed the ISEcp1 elements located in the upstream region of the bla gene and 20 of them truncated by IS26. ESBL producing E. coli strains are a serious threat in the community in Tunisia and we should take into consideration any possible spread of such epidemiological resistance.

Relative response of TL and component-resolved OSL to alpha and beta radiations in annealed sedimentary quartz

International Nuclear Information System (INIS)

Polymeris, George S.; Afouxenidis, Dimitrios; Raptis, Spyridoula; Liritzis, Ioannis; Tsirliganis, Nestor C.; Kitis, George

2011-01-01

Knowledge of the relative luminescence response to alpha and beta radiation is very important in TL and OSL dating. In the present study the relative alpha to beta response is studied in a sedimentary quartz sample, previously fired at 900 deg. C for 1 h, in the dose region between 1 and 128 Gy, for both thermoluminescence (TL) and linearly modulated optically stimulated luminescence (LM - OSL). The LM - OSL measurements were performed at room temperature and at 125 deg. C. All OSL signals were deconvolved into their individual components. Comparison of OSL curves after alpha and beta irradiation strongly supports that quartz OSL components follow first order kinetics in both cases. In the case of TL, the relative alpha to beta response is found to be very different for each TL glow-peak, but it does not depend strongly on irradiation dose. In the case of LM - OSL measurements, it is found that the relative behaviour of the alpha to beta response is different for three distinct regions, namely the fast OSL component, the region of medium OSL component originating from the TL glow-peak at 110 deg. C when stimulation takes place at room temperature and finally the region of slow OSL component. Following stimulation at ambient temperature, the relative alpha to beta response of all components was not observed to depend significantly on dose, with the value of ratio being 0.03 and a tendency to decrease with increasing dose. However, in the case of measurements performed at 125 deg. C, the relative response of the fast components is much enhanced, and for the remaining components it increases with increasing dose. Special care must be taken to examine the relative alpha to beta response of the fast component at 125 deg. C which contrasts the relative response of the TL peak at ca. 325 deg. C. The implications for the dating of annealed quartz are also briefly discussed. - Highlights: → Relative alpha to beta response for TL and LM-OSL is studied in annealed

Determination of antimicrobial resistance pattern and Extended-Spectrum Beta Lactamases producing Pseudomonas aeruginosa strains isolated from clinical specimens of Hajar and Kashani Hospitals,Shahrekord 1387

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Mana Shojapour

2011-09-01

Full Text Available Background: Pseudomonas aeruginosa is one of the leading causes of hospital infections in patients hospitalized for a 10 day period or over. It is also considered to be the most important cause of the burn wound infection. Approximately 75% of deaths in burned patients are due to wound infection and the subsequent septicemia. Clinical use of antibiotics has increasingly led to the global distribution of P. aeruginosa isolates with multi-drug resistance. The study was launched to determine the antimicrobial susceptibility pattern and the presence of the extended-spectrum-beta lactamase (ESBL in P.aeruginosa strains isolated from clinical specimens. Methods: Totally, 175 P. aeruginosa strains were isolated from clinical samples and identified by standard methods. The pattern of antimicrobial resistance was then performed on the isolates using Disk Agar Diffusion (DAD according to CLSI Guideline. Primary screening test for ESBL producing strains was performed by ceftazidim antibiotic disk using disk diffusion method. Combined disk method was used to confirm ESBL producing bacteria. Results: The rate of antimicrobial resistance of P.aeruginosa isolates were 64% to ticarcillin, 52.2% to cefepime, 68.6% to ticarcillin/clavolanic acid, 68.6% to ceftazidime, 67.4% to amikacin, 68.6% to gentamicin, 48% to imipenem, 77.7% to ciprofloxacin and 5.1% to polymixcine B. In the primary screening test, 120 isolates of P.aeruginosa strains were resistant to ceftazidime. In the combined disk method, 66 isolates (55% were positive for ESBLs. Conclusion: Polymixcine B was found to be the most effective antimicrobial agent in this study. Bacteria carrying ESBL genes may increase mortality and morbidity. Thus, their accurate diagnosis is of extreme importance to prevent from the treatment failure resulted from improper antibiotic administration.

Clinical patterns, epidemiology and risk factors of community-acquired urinary tract infection caused by extended-spectrum beta-lactamase producers: a prospective hospital case-control study.

Science.gov (United States)

Almomani, Basima A; Hayajneh, Wail A; Ayoub, Abeer M; Ababneh, Mera A; Al Momani, Miral A

2018-05-10

To assess incidence rate, risk factors and susceptibility patterns associated with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli or Klebsiella pneumoniae in community-acquired urinary tract infections (CA-UTIs). A prospective, case-control study was conducted at a tertiary teaching hospital from Jan 2015 to Dec 2016. The results of microbiology cultures were initially screened to include only patients with positive E. coli or K. pneumoniae urine cultures. Afterwards, clinical symptoms were assessed to confirm the UTI. To investigate the risk factors, patients with a positive urine culture for ESBL-producing isolates were assigned as cases, while patients with non-ESBL were assigned as controls. Out of 591 patients included in this study, 57.5% (n = 340) were included in the control group and 42.5% (n = 251) were in the case group. The incidence rate of ESBL-producing isolates was 3.465 cases per 1000-patient hospital admissions. Male gender (OR = 1.856, 95% CI = 1.192-2.889, p = 0.006), pediatrics (OR = 1.676, 95% CI = 1.117-2.517, p = 0.013), patients with comorbidity (OR = 1.542, 95% CI = 1.029-2.312, p = 0.036) and UTI in the previous 12 months (OR = 1.705, 95% CI = 1.106-2.628, p = 0.016) were independently associated with a higher risk of infection. The resistance rate for most commonly prescribed antibiotics was high. Our results suggest that the incidence of ESBL producers among CA-UTIs is high. Male gender, pediatrics, comorbidity and UTI in the previous 12 months were associated with a higher risk for infection. Continuous surveillance and prudent antibiotic use by healthcare professionals are important factors for effective control of ESBL associated infections.

An analysis of the relation between return and beta for portfolios of Turkish equities

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Salvatore J. Terregrossa

2016-12-01

Full Text Available The present study investigates the possible existence of a systematic relation between beta and excess-return for portfolios of Turkish equities. In the process, no systematic relation is found between beta and realized portfolio excess-return, in an unconditional sense. However, the study does find a systematic relation between realized portfolio excess-return and beta, conditioned upon the sign of realized market-portfolio excess-return. Moreover, an even stronger systematic relation is found between realized portfolio excess-return and beta, conditioned not only upon the sign, but also the magnitude of realized market-portfolio excess-return, with the estimation of the security market plane (SMP model. The study has several useful implications for portfolio managers. Firstly, the empirical findings strongly suggest that employment of the SMP model may generate more accurate estimations of expected asset-return, compared with straightforward application of the capital asset pricing model (CAPM. Enhanced accuracy of expected asset-return, in turn, may lead to more accurate appraisals of asset value, resulting in more profitable investment opportunities and decisions. Employment of the SMP model may thus lead to enhanced efficient-portfolio development, by leading to construction of portfolios with greater expected-return, for a given class of quantifiable-risk.

Using PATIMDB to create bacterial transposon insertion mutant libraries.

Science.gov (United States)

Urbach, Jonathan M; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M

2009-04-01

PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software.

Variants of β-lactamase KPC-2 that are resistant to inhibition by avibactam.

Science.gov (United States)

Papp-Wallace, Krisztina M; Winkler, Marisa L; Taracila, Magdalena A; Bonomo, Robert A

2015-07-01

KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R

Detection and molecular characterization of β-lactamase genes in clinical isolates of Gram-negative bacteria in Southern Ecuador

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Diana Yessenia Calva Delgado

2016-11-01

Full Text Available This work performed a phenotypic and genotypic characterization of 79 clinical isolates of Enterobacteriaceae and Pseudomonadaceae collected in hospitals of Southern Ecuadorin 2013. Our results showed a high incidence of β-lactamases and ESBLs with blaTEM and blaCTX-M as the prevalent genes, respectively. By direct sequencing of PCR amplicons, the different β-lactamases and variants of the genes were also distinguished. Our results revealed a predominance of TEM-1 β-lactamase and the presence of different CTX-M variants with a prevalence of CTX-M-15. Two infrequent CTX-M variants in South America were also identified. To the best of our knowledge, this is one of the first studies describing the genetic characteristics of β-lactamases in Ecuador.

Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

DEFF Research Database (Denmark)

Becker, F.; Schnorr, K.; Wilting, R.

2004-01-01

To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...... minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium...... Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation...

Beta-hydroxy-beta-methylbutyrate (HMB) ameliorates age-related deficits in water maze performance, especially in male rats.

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Kougias, Daniel G; Hankosky, Emily R; Gulley, Joshua M; Juraska, Janice M

2017-03-01

Beta-hydroxy-beta-methylbutyrate (HMB) is commonly supplemented to maintain muscle in elderly and clinical populations and has potential as a nootropic. Previously, we have shown that in both male and female rats, long-term HMB supplementation prevents age-related dendritic shrinkage within the medial prefrontal cortex (mPFC) and improves cognitive flexibility and working memory performance that are both age- and sex-specific. In this study, we further explore the cognitive effects by assessing visuospatial learning and memory with the Morris water maze. Female rats were ovariectomized at 11months of age to model human menopause. At 12months of age, male and female rats received relatively short- or long-term (1- or 7-month) dietary HMB (450mg/kg/dose) supplementation twice a day prior to testing. Spatial reference learning and memory was assessed across four days in the water maze with four trials daily and a probe trial on the last day. Consistent with previous work, there were age-related deficits in water maze performance in both sexes. However, these deficits were ameliorated in HMB-treated males during training and in both sexes during probe trial performance. Thus, HMB supplementation prevented the age-related decrement in water maze performance, especially in male rats. Copyright © 2016 Elsevier Inc. All rights reserved.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Science.gov (United States)

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

lactamases genes among0 Escherichia coli from patients with ...

African Journals Online (AJOL)

-lactamases (ESBLs) that mediate resistance to b-lactam drugs among Escherichia coli and other uropathogens have been reported worldwide. However, there is little information on the detection of ESBLs genes in E. coli from patients with ...

The role of outer membrane in Serratia marcescens intrinsic resistance to antibiotics.

Science.gov (United States)

Sánchez, L; Ruiz, N; Leranoz, S; Viñas, M; Puig, M

1997-09-01

Three different porins from Serratia marcescens were described. They were named Omp1, Omp2 and Omp3 and their molecular weights were 42, 40 and 39 kDa respectively. Omp2 and Omp3 showed osmoregulation and thermoregulation in a similar way to OmpC and OmpF of Escherichia coli. Permeability coefficients of the outer membrane of this species were calculated following the Zimmermann and Rosselet method. P values were similar to those obtained in Escherichia coli, which suggests that the chromosomal beta-lactamase would play a major role in the resistance of Serratia marcescens to beta-lactam antibiotics. Both MIC values and permeabilities were modified by salycilates and acetylsalycilate. Synergism between the outer membrane and the beta-lactamase was also evaluated. When bacteria grew in the presence of a beta-lactam in the medium, the beta-lactamase accounted for most of the resistance.

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

Science.gov (United States)

Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

2011-01-01

We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit. PMID:21985007

Biological markers of amyloid beta-related mechanisms in Alzheimer's disease.

LENUS (Irish Health Repository)

Hampel, Harald

2010-06-01

Recent research progress has given detailed knowledge on the molecular pathogenesis of Alzheimer\\'s disease (AD), which has been translated into an intense, ongoing development of disease-modifying treatments. Most new drug candidates are targeted on inhibiting amyloid beta (Abeta) production and aggregation. In drug development, it is important to co-develop biomarkers for Abeta-related mechanisms to enable early diagnosis and patient stratification in clinical trials, and to serve as tools to identify and monitor the biochemical effect of the drug directly in patients. Biomarkers are also requested by regulatory authorities to serve as safety measurements. Molecular aberrations in the AD brain are reflected in the cerebrospinal fluid (CSF). Core CSF biomarkers include Abeta isoforms (Abeta40\\/Abeta42), soluble APP isoforms, Abeta oligomers and beta-site APP-cleaving enzyme 1 (BACE1). This article reviews recent research advances on core candidate CSF and plasma Abeta-related biomarkers, and gives a conceptual review on how to implement biomarkers in clinical trials in AD.

Biological markers of amyloid beta-related mechanisms in Alzheimer's disease.

LENUS (Irish Health Repository)

Hampel, Harald

2012-02-01

Recent research progress has given detailed knowledge on the molecular pathogenesis of Alzheimer\\'s disease (AD), which has been translated into an intense, ongoing development of disease-modifying treatments. Most new drug candidates are targeted on inhibiting amyloid beta (Abeta) production and aggregation. In drug development, it is important to co-develop biomarkers for Abeta-related mechanisms to enable early diagnosis and patient stratification in clinical trials, and to serve as tools to identify and monitor the biochemical effect of the drug directly in patients. Biomarkers are also requested by regulatory authorities to serve as safety measurements. Molecular aberrations in the AD brain are reflected in the cerebrospinal fluid (CSF). Core CSF biomarkers include Abeta isoforms (Abeta40\\/Abeta42), soluble APP isoforms, Abeta oligomers and beta-site APP-cleaving enzyme 1 (BACE1). This article reviews recent research advances on core candidate CSF and plasma Abeta-related biomarkers, and gives a conceptual review on how to implement biomarkers in clinical trials in AD.

Differential beta-band event-related desynchronization during categorical action sequence planning.

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Hame Park

Full Text Available A primate study reported the existence of neurons from the dorso-lateral prefrontal cortex which fired prior to executing categorical action sequences. The authors suggested these activities may represent abstract level information. Here, we aimed to find the neurophysiological representation of planning categorical action sequences at the population level in healthy humans. Previous human studies have shown beta-band event-related desynchronization (ERD during action planning in humans. Some of these studies showed different levels of ERD according to different types of action preparation. Especially, the literature suggests that variations in cognitive factors rather than physical factors (force, direction, etc modulate the level of beta-ERD. We hypothesized that the level of beta-band power will differ according to planning of different categorical sequences. We measured magnetoencephalography (MEG from 22 subjects performing 11 four-sequence actions--each consisting of one or two of three simple actions--in 3 categories; 'Paired (ooxx', 'Alternative (oxox' and 'Repetitive (oooo' ('o' and 'x' each denoting one of three simple actions. Time-frequency representations were calculated for each category during the planning period, and the corresponding beta-power time-courses were compared. We found beta-ERD during the planning period for all subjects, mostly in the contralateral fronto-parietal areas shortly after visual cue onset. Power increase (transient rebound followed ERD in 20 out of 22 subjects. Amplitudes differed among categories in 20 subjects for both ERD and transient rebound. In 18 out of 20 subjects 'Repetitive' category showed the largest ERD and rebound. The current result suggests that beta-ERD in the contralateral frontal/motor/parietal areas during planning is differentiated by the category of action sequences.

The relative importance of relativistic induced interactions in the beta decay of 170Tm

International Nuclear Information System (INIS)

Bogdan, D.; Cristu, M.I.; Holan, S.; Faessler, A.

1982-09-01

The log ft-values, the spectrum shape functions, and the beta-gamma angular correlation coefficients of the 170 Tm beta decay are computed in the framework of relativistic formfactor formalism using asymmetric rotor model wavefunctions. Main vector and axial vector hadron currents being strongly hindered, the relative importance of induced interaction matrix elements is accurately estimated. Good agreement with experiment is obtained for the beta decay observables when the main induced interaction terms were taken into account. The contribution of the pseudoscalar term was found insignificant. (authors)

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

DEFF Research Database (Denmark)

Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

2001-01-01

no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial......beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N...

Environmental pollution with antimicrobial agents from bulk drug manufacturing industries in Hyderabad, South India, is associated with dissemination of extended-spectrum beta-lactamase and carbapenemase-producing pathogens.

Science.gov (United States)

Lübbert, Christoph; Baars, Christian; Dayakar, Anil; Lippmann, Norman; Rodloff, Arne C; Kinzig, Martina; Sörgel, Fritz

2017-08-01

High antibiotic and antifungal concentrations in wastewater from anti-infective drug production may exert selection pressure for multidrug-resistant (MDR) pathogens. We investigated the environmental presence of active pharmaceutical ingredients and their association with MDR Gram-negative bacteria in Hyderabad, South India, a major production area for the global bulk drug market. From Nov 19 to 28, 2016, water samples were collected from the direct environment of bulk drug manufacturing facilities, the vicinity of two sewage treatment plants, the Musi River, and habitats in Hyderabad and nearby villages. Samples were analyzed for 25 anti-infective pharmaceuticals with liquid chromatography-tandem mass spectrometry and for MDR Gram-negative bacteria using chromogenic culture media. In addition, specimens were screened with PCR for bla VIM , bla KPC , bla NDM , bla IMP-1 , and bla OXA-48 resistance genes. All environmental specimens from 28 different sampling sites were contaminated with antimicrobials. High concentrations of moxifloxacin, voriconazole, and fluconazole (up to 694.1, 2500, and 236,950 µg/L, respectively) as well as increased concentrations of eight other antibiotics were found in sewers in the Patancheru-Bollaram industrial area. Corresponding microbiological analyses revealed an extensive presence of extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae and non-fermenters (carrying mainly bla OXA-48 , bla NDM , and bla KPC ) in more than 95% of the samples. Insufficient wastewater management by bulk drug manufacturing facilities leads to unprecedented contamination of water resources with antimicrobial pharmaceuticals, which seems to be associated with the selection and dissemination of carbapenemase-producing pathogens. The development and global spread of antimicrobial resistance present a major challenge for pharmaceutical producers and regulatory agencies.

Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes

NARCIS (Netherlands)

Kieboom, J; Bruinenberg, R; Keizer-Gunnink, [No Value; de Bont, JAM

2001-01-01

Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMOD-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a

Detection and genetic characterization of β-lactamases in Prevotella intermedia and Prevotella nigrescens isolated from oral cavity infections and peritonsillar abscesses.

Science.gov (United States)

Fernández-Canigia, Liliana; Cejas, Daniela; Gutkind, Gabriel; Radice, Marcela

2015-06-01

A prospective analysis on β-lactam resistance mechanisms and β-lactamase prevalence was conducted on Prevotella intermedia and Prevotella nigrescens recovered from patients with chronic periodontitis and peritonsillar abscesses. Both phenotypic and genotypic methods were performed to characterize the β-lactamases, their coding genes and their genetic contexts. Overall, β-lactamase production was observed in 64% (16/25) P. intermedia and 23.8% (5/21) P. nigrescens (p intermedia (8/16) than in P. nigrescens (2/16) recovered from chronic periodontitis, almost all isolates from peritonsillar abscesses were producers (8/9 and 3/3, respectively). cfxA, but not cepA and cblA, was detected in those isolates, which were previously categorized as β-lactamase producers. CfxA producing isolates displayed higher β-lactam MICs than non-producers in both species. The most frequent allele was cfxA2, followed by cfxA3 and a new allelic variant named cfxA6. The analysis of the downstream flanking region in the three cfxA variants revealed the association with mobA of Tn4555, suggesting their localization in a mobilizable element. β-lactam resistance and cfxA carriage prevalence seems to be not only related to the bacterial species but also to the infection site. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcription and somatic transposition of the maize En/Spm transposon system in rice

NARCIS (Netherlands)

Greco, R.; Ouwerkerk, P.B.F.; Taal, A.J.C.; Sallaud, C.; Guiderdoni, E.; Meijer, A.H.; Hoge, J.H.C.; Pereira, A.B.

2004-01-01

Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision

Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL.

Directory of Open Access Journals (Sweden)

Claudia Stein

Full Text Available The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT, amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.

SME-Type Carbapenem-Hydrolyzing Class A β-Lactamases from Geographically Diverse Serratia marcescens Strains

Science.gov (United States)

Queenan, Anne Marie; Torres-Viera, Carlos; Gold, Howard S.; Carmeli, Yehuda; Eliopoulos, George M.; Moellering, Robert C.; Quinn, John P.; Hindler, Janet; Medeiros, Antone A.; Bush, Karen

2000-01-01

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two β-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 β-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U.S. isolates had β-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing β-lactamases first described in London has now been identified in S. marcescens isolates across the United States. PMID:11036019

TcA, the putative transposase of the C. elegans Tc1 transposon, has an N-terminal DNA binding domain.

OpenAIRE

Schukkink, R F; Plasterk, R H

1990-01-01

Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion pr...

Phenotypic methods for detection of various β-lactamases in Gram-negative clinical isolates: Need of the hour

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Neena V Nagdeo

2012-01-01

Full Text Available Background: Many clinical laboratories have problems detecting various β-lactamases. Confusion exists about the importance of these resistance mechanisms, optimal test methods, and appropriate reporting conventions. It is more imperative to use various phenotypic methods for detection of various β-lactamases in routine microbiology laboratory on day-to-day basis to prevent antimicrobial resistance by evidence-based judicious use of antimicrobials. Aims: In view of the multidrug-resistant organisms being reported world over, we planned a cross-sectional prospective analytical study to determine resistance mechanism by various β-lactamases in Gram-negative clinical isolates using various phenotypic methods. Materials and Methods: All nonrepeat, nonenteric clinical isolates of Gram-negative bacilli, resistant to at least two third-generation cephalosporins, were first screened by Novel disc placement method, and isolates showing multiple mechanisms of resistance and reduced zone of inhibition for imipenem were further confirmed for AmpC and metallo β-lactamases. Statistical Analysis: All the data was managed and analyzed in Microsoft Excel. Results: Out of 807 isolates tested, as many as 795 (98.51% revealed the presence of extended-spectrum β-lactamases (ESBLs. Only 10 isolates of Escherichia coli and 2 of Klebsiella pneumoniae did not show production of ESBL. A total of 450 (55.76% isolates produced single enzyme,while 345 (42.75% strains revealed multiple enzyme production simultaneously. Only ESBL production was seen in 315 (39.03% strains, only AmpC in 75 (9.29% and only MBL in 60 (7.44% strains, while ESBL and AmpC together were seen in 219 (27.14% and AmpC plus MBL in 92 (11.40% strains. However, ESBL plus MBL were never observed together. All three enzymes were simultaneously detected in 34 (4.21% strains. Conclusion: This innovative method of disc placement makes it easy, affordable, and reliable method for routine use by basic

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

Science.gov (United States)

Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder

2016-10-01

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

Microbial Fe (III) reduction and hydrogen production by a transposon-mutagenized strain of Pantoea agglomerans BH18

International Nuclear Information System (INIS)

Liu, Hongyan; Wang, Guangce

2015-01-01

Based on the transposon-mutagenized library of Pantoea agglomerans BH18, mutant screens were conducted to obtain the strain with the highest Fe (III) reduction and hydrogen production. Of these transposon-mutagenized mutants, the mutant strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. The PCR amplification and kanamycin resistance selection results indicated that the transposon insertion of the mutant strain TB230 was stable. Hydrogen production of the mutant strain TB230 was (2.21 ± 0.34) mol H 2 /mol glucose, which increased hydrogen production by over 40% compared with that of the wild type strain. The accumulation concentration of Fe (II) in the medium of the mutant strain TB230 with Fe (OH) 3 as the sole electron acceptor was (7.39 ± 0.49) mmol/l, which was approximately 3-fold greater than that of the wild type strain. The mutant strain TB230 showed high Fe (III)-reducing activity and hydrogen production by adopting glucose and pyruvate as the carbon source. In addition, the mutant strain TB230 was capable of Fe (III) reduction and hydrogen production under fresh or marine conditions. This result indicates that the mutant strain with high microbial Fe (III) reduction and hydrogen production is beneficial for the improvement of anaerobic performance. - Highlights: • The mutant strain TB230 was a transposon-mutagenized strain of Pantoea agglomerans BH18. • Strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. • H 2 yield and Fe (III)-reducing activity were 2.21 ± 0.34 and 7.39 ± 0.49 in marine condition. • Strain TB230 was capable of Fe (III) reduction and hydrogen production in fresh or marine condition

Purification, crystallization and preliminary crystallographic analysis of Est-Y29: a novel oligomeric β-lactamase

International Nuclear Information System (INIS)

Kim, SeungBum; Joo, Sangbum; Yoon, Sangyoung; Kim, Sungsoo; Moon, Jongkook; Ryu, Yeonwoo; Kim, Kyeong Kyu; Kim, T. Doohun

2009-01-01

Est-Y29, a novel oligomeric class C β-lactamase from a metagenomic library, was crystallized in space group I4 1 and diffraction data were collected to 1.49 Å resolution. β-Lactam antibiotics such as penicillins and cephalosporins have a four-atom ring as a common element in their structure. The β-lactamases, which catalyze the inactivation of these antibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C β-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized. The recombinant protein was expressed in Escherichia coli with an N-terminal 6×His tag and purified to homogeneity. EstY-29 was crystallized and X-ray intensity data were collected to 1.49 Å resolution using synchrotron radiation

Occurrence and detection of AmpC β-lactamases among ...

African Journals Online (AJOL)

Soha A. El-Hady

2015-04-06

Apr 6, 2015 ... Molecular detection of plasmid mediated AmpC by multiplex PCR was conducted on them. Results: The .... b-lactamases into the external environment. AmpC .... prove satisfactory) and should not display significant homol-.

Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery

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Edith eCoronado

2014-11-01

Full Text Available The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested, which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

TED, an autonomous and rare maize transposon of the mutator superfamily with a high gametophytic excision frequency.

Science.gov (United States)

Li, Yubin; Harris, Linda; Dooner, Hugo K

2013-09-01

Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.

SME-3, a Novel Member of the Serratia marcescens SME Family of Carbapenem-Hydrolyzing β-Lactamases

Science.gov (United States)

Queenan, Anne Marie; Shang, Wenchi; Schreckenberger, Paul; Lolans, Karen; Bush, Karen; Quinn, John

2006-01-01

Imipenem-resistant Serratia marcescens isolates were cultured from a lung transplant patient given multiple antibiotics over several months. The strains expressed SME-3, a β-lactamase of the rare SME carbapenem-hydrolyzing family. SME-3 differed from SME-1 by a single amino acid substitution of tyrosine for histidine at position 105, but the two β-lactamases displayed similar hydrolytic profiles. PMID:17005839

Transposon fingerprinting using low coverage whole genome shotgun sequencing in cacao (Theobroma cacao L.) and related species.

Science.gov (United States)

Sveinsson, Saemundur; Gill, Navdeep; Kane, Nolan C; Cronk, Quentin

2013-07-24

Transposable elements (TEs) and other repetitive elements are a large and dynamically evolving part of eukaryotic genomes, especially in plants where they can account for a significant proportion of genome size. Their dynamic nature gives them the potential for use in identifying and characterizing crop germplasm. However, their repetitive nature makes them challenging to study using conventional methods of molecular biology. Next generation sequencing and new computational tools have greatly facilitated the investigation of TE variation within species and among closely related species. (i) We generated low-coverage Illumina whole genome shotgun sequencing reads for multiple individuals of cacao (Theobroma cacao) and related species. These reads were analysed using both an alignment/mapping approach and a de novo (graph based clustering) approach. (ii) A standard set of ultra-conserved orthologous sequences (UCOS) standardized TE data between samples and provided phylogenetic information on the relatedness of samples. (iii) The mapping approach proved highly effective within the reference species but underestimated TE abundance in interspecific comparisons relative to the de novo methods. (iv) Individual T. cacao accessions have unique patterns of TE abundance indicating that the TE composition of the genome is evolving actively within this species. (v) LTR/Gypsy elements are the most abundant, comprising c.10% of the genome. (vi) Within T. cacao the retroelement families show an order of magnitude greater sequence variability than the DNA transposon families. (vii) Theobroma grandiflorum has a similar TE composition to T. cacao, but the related genus Herrania is rather different, with LTRs making up a lower proportion of the genome, perhaps because of a massive presence (c. 20%) of distinctive low complexity satellite-like repeats in this genome. (i) Short read alignment/mapping to reference TE contigs provides a simple and effective method of investigating

Chromobacterium spp. harbour Ambler class A β-lactamases showing high identity with KPC.

Science.gov (United States)

Gudeta, Dereje Dadi; Bortolaia, Valeria; Jayol, Aurélie; Poirel, Laurent; Nordmann, Patrice; Guardabassi, Luca

2016-06-01

The origin of KPC is unknown. The aim of this study was to detect progenitors of KPC in silico and to functionally verify their β-lactam hydrolysis activity. The sequence of KPC-2 was used to mine the NCBI protein sequence database. The best non-KPC hits were analysed by amino acid (aa) alignment and phylogenetic tree construction. Genes encoding KPC-2 homologues were expressed in Escherichia coli. The carbapenemase activities of the recombinant strains were characterized by the CarbaNP test and UV spectrophotometry and MICs of selected β-lactams were determined. Genes encoding the closest KPC-2 homologues were identified on the chromosome of Chromobacterium piscinae strain ND17 (CRP-1, 76% aa identity), Chromobacterium sp. C-61 (CRS-1, 70% aa identity) and Chromobacterium haemolyticum DSM19808 (CRH-1, 69% aa identity). All three Chromobacterium β-lactamases were phylogenetically more related to KPC than to other Ambler class A β-lactamases. The 27 bp region preceding the start codon of blaCRP-1 displayed high nucleotide identity to the corresponding region upstream from blaKPC (74%). Heterologous expression of blaCRP-1 and to a lesser extent of blaCRH-1 in E. coli significantly increased the MICs of meropenem and most cephalosporins. The CarbaNP test was positive for both recombinant strains, but spectrophotometric analysis confirmed higher carbapenemase activity for CRP-1-producing clones. The recovery of three class A β-lactamases with up to 76% aa identity to KPC from distinct Chromobacterium species is highly indicative of the role played by this genus in the evolution of KPC. © The Author 2016. 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.

Comparison of three different methods to detect the production of β-lactamase enzyme by staphylococci

OpenAIRE

Jagad, Beena P.; Vakanee, Amit

2017-01-01

Production β-lactamase enzyme is the most common and important mode of exhibiting resistance to β-lactam antibiotics. Manifestation of this enzyme is difficult to demonstrate in routine antibiotic sensitivity testing. Sensitivity to penicillin is not sufficient to indicate whether this antibiotic should be used clinically and main factor compromising the clinical efficacy of these drugs is the production of β-lactamase enzyme. Only feasible method for determination of β-lactam resistance is t...

Characterization of Silver Nanomaterials Derived from Marine Streptomyces sp. Al-Dhabi-87 and Its In Vitro Application against Multidrug Resistant and Extended-Spectrum Beta-Lactamase Clinical Pathogens

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Naif Abdullah Al-Dhabi

2018-04-01

Full Text Available A novel antagonistic marine Streptomyces sp. Al-Dhabi-87 that was recovered from the Gulf region of Saudi Arabia was used to synthesize silver nanoparticles (NP from the culture free extract. The produced NP were confirmed by UV-visible spectroscopy (UV-Vis, high-resolution scanning electron microscope (HRSEM, transmission electron microscope (TEM, Fourier-transform infrared spectroscopy (FTIR, Energy Dispersive Spectroscopy (EDAX, and X-ray Powder Diffraction (XRD, and broth micro dilution techniques were employed for the determination of minimum inhibitory concentrations (MIC values. The synthesized NP was authenticated by alterations in color and wavelength scanning. HRSEM and TEM analysis confirmed that the size of the NP ranged from 10 to 17 nm and that it was spherical in shape. In addition, the FTIR spectrum revealed a variation in the band values from 500 to 3300 cm−1 respectively. Rietveld refinement analysis of the XRD data confirmed the size of the NP, which coincided with the results of the TEM analysis. In addition, the Riveted refinement analysis supported the TEM data. The NP documented significant activity against the wound infection microbial strains, such as Enterococcus faecalis, Staphylococcus epidermidis, and Staphylococcus aureus. Gram negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumonia, and Escherichia coli revealed MIC values of 0.039, 0.078, and 0.152 mg/mL, respectively. The promising activity of NP towards extended-spectrum beta-lactamases E.coli, drug resistant Acinetobacter baumannii, and multidrug resistant S. aureus (at 0.018, 0.039, and 0.039 mg/mL, respectively was advantageous. Overall, NP that were obtained from the novel Streptomyces sp. Al-Dhabi-87, with its promising antimicrobial activity towards the drug resistant pathogens, would be useful for healing infectious diseases.

Antibacterial activity of the Antarctic bacterium Janthinobacterium sp. SMN 33.6 against multi-resistant Gram-negative bacteria

Directory of Open Access Journals (Sweden)

Geraldine Asencio

2014-01-01

Conclusions: The ethanolic extract of Janthinobacterium sp. SMN 33.6 possesses antibacterial activity against a chromosomal AmpC beta-lactamase-producing strain of Serratia marcescens, an extended-spectrum beta-lactamase-producing Escherichia coli and also against carbapenemase-producing strains of Acinetobacter baumannii and Pseudomonas aeruginosa. This becomes a potential and interesting biotechnological tool for the control of bacteria with multi-resistance to commonly used antibiotics.

[Antibacterial activity of panipenem against clinical isolates in 2000 and 2001].

Science.gov (United States)

Abe, Tomomi; Fukuoka, Takashi; Sato, Yuki; Ito, Kazuyoshi; Sei, Masami

2002-12-01

As the post-marketing surveillance of panipenem/betamipron (Carbenin), MICs of panipenem (PAPM) against 1355 clinical isolates of 28 species from 15 medical institutions all over Japan from June 2000 to March 2001 were measured using the broth microdilution method approved by the Japanese Society of Chemotherapy and compared with those of parenteral carbapenem antibacterials, imipenem (IPM) and meropenem (MEPM), and parenteral cephem antibacterials, cefozopran, cefepime, and sulbactam/cefoperazone. The activity of PAPM was comparable to that of IPM against almost all species tested. Compared with MEPM, PAPM was more active against Gram-positive bacteria and Bacteroides spp., and less active against Gram-negative bacteria. Compared with the parenteral cephems, PAPM was more active against most of species tested and its MIC ranges were narrower than those of the cephems as were those of other carbapenems. In this surveillance study, the incidence of resistance in various species were as follows: 39.3% for methicillin-resistant Staphylococcus aureus, 47.3% for penicillin-intermediate Streptococcus pneumoniae (PISP), 15.1% for penicillin-resistant S. pneumoniae (PRSP), 0.9% for extended-spectrum beta-lactamase (ESBL) producing Escherichia coli, 3.4% for ESBL producing Klebsiella pneumoniae, 19.2% for beta-lactamase producing Haemophilus influenzae, 24.0% for beta-lactamase-negative ampicillin-resistant (BLNAR) H. influenzae, and 1.0% for metallo-beta-lactamase producing Pseudomonas aeruginosa. Against these resistant strains, carbapenems including PAPM showed generally more potent activity than cephems. It was noted that PAPM showed the most potent activity against PISP and PRSP, which showed high incidence of 62.4% totally, among tested drugs. Metallo-beta-lactamase producing P. aeruginosa exhibited high resistance and BLNAR H. influenzae also exhibited low susceptibility against all tested drugs. But no remarkable change in the activity of PAPM against other species

Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

Science.gov (United States)

Dubois, Emeline; Bischerour, Julien; Marmignon, Antoine; Mathy, Nathalie; Régnier, Vinciane; Bétermier, Mireille

2012-01-01

Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes. PMID:22888464

Randomized controlled trial of piperacillin-tazobactam, cefepime and ertapenem for the treatment of urinary tract infection caused by extended-spectrum beta-lactamase-producing Escherichia coli.

Science.gov (United States)

Seo, Yu Bin; Lee, Jacob; Kim, Young Keun; Lee, Seung Soon; Lee, Jeong-A; Kim, Hyo Youl; Uh, Young; Kim, Han-Sung; Song, Wonkeun

2017-06-07

Due to limited therapeutic options, the spread of extended-spectrum beta-lactamases (ESBLs) have become a major public health concern. We conducted a prospective, randomized, open-label comparison of the therapeutic efficacy of piperacillin-tazobactam (PTZ), cefepime, and ertapenem in febrile nosocomial urinary tract infection with ESBL-producing Escherichia coli (ESBL-EC). This study was conducted at three university hospitals between January 2013 and August 2015. Hospitalized adult patients presenting with fever were screened for healthcare-associated urinary tract infection (HA-UTI). When ESBL-EC was solely detected and susceptible to a randomized antibiotic in vitro, the case was included in the final analysis. Participants were treated for 10-14 days with PTZ, cefepime, or ertapenem. A total of 66 participants were evenly assigned to the PTZ and ertapenem treatment groups. After the recruitment of six participants, assignment to the cefepime treatment group was stopped because of an unexpectedly high treatment failure rate. The baseline characteristics of these participants did not differ from participants in other treatment groups. The clinical and microbiological response to PTZ treatment was estimated to be 94% and was similar to the response to ertapenem treatment. The efficacy of cefepime was 33.3%. In the cefepime group, age, Charlson comorbidity index, genotype, and minimal inhibitory concentration (MIC) did not significantly affect the success of treatment. Similarly, genotype seemed to be irrelevant with respect to clinical outcome in the PTZ group. Expired cases tended to involve septic shock with a high Charlson comorbidity index and high MIC. Results from this study suggest that PTZ is effective in the treatment of urinary tract infection caused by ESBL-EC when the in vitro test indicates susceptibility. In addition, cefepime should not be used as an alternative treatment for urinary tract infection caused by ESBL-EC. The trial was registered with

Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution.

Science.gov (United States)

Lewandowski, Eric M; Lethbridge, Kathryn G; Sanishvili, Ruslan; Skiba, Joanna; Kowalski, Konrad; Chen, Yu

2018-01-01

The β-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A β-lactamases, the β-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A β-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A β-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A β-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE. © 2017 Federation of European Biochemical Societies.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

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Felipe Lira de Sá Cavalcanti

2012-05-01

Full Text Available In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

TED, an Autonomous and Rare Maize Transposon of the Mutator Superfamily with a High Gametophytic Excision Frequency[W

Science.gov (United States)

Li, Yubin; Harris, Linda; Dooner, Hugo K.

2013-01-01

Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor. PMID:24038653

Enterobacteriaceae isolates carrying the New Delhi metallo-β-lactamase gene in Yemen.

Science.gov (United States)

Gharout-Sait, Alima; Alsharapy, Samer-Ahmed; Brasme, Lucien; Touati, Abdelaziz; Kermas, Rachida; Bakour, Sofiane; Guillard, Thomas; de Champs, Christophe

2014-10-01

Ten carbapenem-resistant Enterobacteriaceae (eight Klebsiella pneumoniae isolates and two Enterobacter cloacae) isolates from Yemen were investigated using in vitro antimicrobial susceptibility testing, phenotypic carbapenemase detection, multilocus sequence typing (MLST) and replicon typing. Carbapenemase, extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance determinant genes were identified using PCR and sequencing. All of the 10 carbapenem-resistant Enterobacteriaceae were resistant to β-lactams, tobramycin, ciprofloxacin and cotrimoxazole. Imipenem, doripenem and meropenem MICs ranged from 2 to >32 mg l(-1) and ertapenem MICs ranged from 6 to >32 mg l(-1). All of the K. pneumoniae isolates showed ESBL activity in phenotypic tests. Genes encoding blaNDM were detected in all strains. All K. pneumoniae strains produced CTX-M-15 ESBL and SHV β-lactamases. TEM-1 β-lactamase was detected in seven isolates. Nine isolates were qnr positive including QnrB1, QnrA1 and QnrS1, and six isolates produced AAC-6'-Ib-cr. MLST identified five different sequence types (STs): ST1399, ST147, ST29, ST405 and ST340. Replicon typing showed the presence of IncFII1K plasmids in four transformants. To the best of our knowledge, this is the first report of NDM-1-producing Enterobacteriaceae isolates in Yemen. © 2014 The Authors.

Development of a high-frequency in vivo transposon mutagenesis system for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942.

Science.gov (United States)

Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru

2014-11-01

Synechocystis sp. PCC 6803 (Synechocystis) is the first sequenced photosynthetic organism and has two advantages: natural transformation and light-activated heterotrophic growth. Such characteristics have mainly promoted reverse genetic analysis in this organism, however, to date approximately 50% of genes are still annotated as 'unknown protein' or 'hypothetical protein'. Therefore, forward genetic analysis is required for the identification of significant genes responsible for photosynthesis and other physiological phenomena among the genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly frequent transposition (9×10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of 10 randomly selected mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the 10 mutants exhibited the slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942, with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis of a broad range of cyanobacteria. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Indication of viruses and virus-specific antibodies by ELISA using conjugates based on β-lactamase obtained by genetic engineering

International Nuclear Information System (INIS)

Kharitonenkov, I.G.; Kordym, V.A.; Khristova, M.L.; Leonov, S.V.; Kirillova, V.S.; Chernykh, S.I.

1987-01-01

The method of enzyme-linked immunosorbent assay (ELISA), by means of which antigens and antibodies of different origin can be detected with high sensitivity and specificity, is an immunoenzymatic technique based on the use of conjugates, or macromolecular complexes formed by covalent attachment of enzyme molecules to antigen or antibody molecules. Conjugates based on peroxidase, alkaline phosphatase, and beta-galactosidase are most frequently used to construct immunoenzymatic test systems. The use of these enzymes in ELISA, however, is complicated by the fact that they are often present in free or bound form in the biological material under study, and that their substrates either possess low stability, are difficult to synthesize, or are toxic. In this paper, in order to avoid these shortcomings, the authors develop a method for the biosynthesis of lactamase conjugates which is based on genetic engineering, and demonstrate the viability and stability of these conjugates in radioimmunoenzymatic assay of viruses

Free Energy Contribution Analysis Using Response Kernel Approximation: Insights into the Acylation Reaction of a Beta-Lactamase.

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Asada, Toshio; Ando, Kanta; Bandyopadhyay, Pradipta; Koseki, Shiro

2016-09-08

A widely applicable free energy contribution analysis (FECA) method based on the quantum mechanical/molecular mechanical (QM/MM) approximation using response kernel approaches has been proposed to investigate the influences of environmental residues and/or atoms in the QM region on the free energy profile. This method can evaluate atomic contributions to the free energy along the reaction path including polarization effects on the QM region within a dramatically reduced computational time. The rate-limiting step in the deactivation of the β-lactam antibiotic cefalotin (CLS) by β-lactamase was studied using this method. The experimentally observed activation barrier was successfully reproduced by free energy perturbation calculations along the optimized reaction path that involved activation by the carboxylate moiety in CLS. It was found that the free energy profile in the QM region was slightly higher than the isolated energy and that two residues, Lys67 and Lys315, as well as water molecules deeply influenced the QM atoms associated with the bond alternation reaction in the acyl-enzyme intermediate. These facts suggested that the surrounding residues are favorable for the reactant complex and prevent the intermediate from being too stabilized to proceed to the following deacylation reaction. We have demonstrated that the free energy contribution analysis should be a useful method to investigate enzyme catalysis and to facilitate intelligent molecular design.

Beta-lactamic resistance profiles in Porphyromonas, Prevotella, and Parvimonas species isolated from acute endodontic infections.

Science.gov (United States)

Montagner, Francisco; Jacinto, Rogério Castilho; Correa Signoretti, Fernanda Graziela; Scheffer de Mattos, Vanessa; Grecca, Fabiana Soares; Gomes, Brenda Paula Figueiredo de Almeida

2014-03-01

Susceptibility to beta-lactamic agents has changed among anaerobic isolates from acute endodontic infections. The aim of the present study was to determine the prevalence of the cfxA/cfxA2 gene in Prevotella spp., Porphyromonas spp., and Parviomonas micra strains and show its phenotypic expression. Root canal samples from teeth with acute endodontic infections were collected and Porphyromonas, Prevotella, and Parvimonas micra strains were isolated and microbiologically identified with conventional culture techniques. The susceptibility of the isolates was determined by the minimum inhibitory concentration of benzylpenicillin, amoxicillin, and amoxicillin + clavulanate using the E-test method (AB BIODISK, Solna, Sweden). The presence of the cfxA/cfxA2 gene was determined through primer-specific polymerase chain reaction. The nitrocefin test was used to determine the expression of the lactamase enzyme. Prevotella disiens, Prevotella oralis, Porphyromonas gingivalis, and P. micra strains were susceptible to benzylpenicillin, amoxicillin, and amoxicillin + clavulanate. The cfxA/cfxA2 gene was detected in 2 of 29 isolates (6.9%). Simultaneous detection of the cfxA/cfxA2 gene and lactamase production was observed for 1 Prevotella buccalis strain. The gene was in 1 P. micra strain but was not expressed. Three strains were positive for lactamase production, but the cfxA/cfxA2 gene was not detected through polymerase chain reaction. There is a low prevalence of the cfxA/cfxA2 gene and its expression in Porphyromonas spp., Prevotella spp., and P. micra strains isolated from acute endodontic infections. Genetic and phenotypic screening must be performed simultaneously to best describe additional mechanisms involved in lactamic resistance for strict anaerobes. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission.

Science.gov (United States)

Projahn, Michaela; Daehre, Katrin; Roesler, Uwe; Friese, Anika

2017-01-01

Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n = 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low. ESBL-/pAmpC-producing enterobacteria occur frequently in broiler-fattening farms. Recent studies investigated the prevalence and

Rectal carriage of extended-spectrum beta-lactamase-producing gram-negative bacilli in community settings in Madagascar.

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Perlinot Herindrainy

Full Text Available BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacteria (ESBL-PE emerged at the end of the 1980s, causing nosocomial outbreaks and/or hyperendemic situations in hospitals and long-term care facilities. In recent years, community-acquired infections due to ESBL-PE have spread worldwide, especially across developing countries including Madagascar. OBJECTIVES: This study aimed to determine the prevalence and risk factors of intestinal carriage of ESBL-PE in the community of Antananarivo. METHODS: Non-hospitalized patients were recruited in three health centers in different socio economic settings. Fresh stool collected were immediately plated on Drigalski agar containing 3 mg/liter of ceftriaxone. Gram-negative bacilli species were identified and ESBL production was tested by a double disk diffusion (cefotaxime and ceftazidime +/- clavulanate assay. Characterization of ESBLs were perfomed by PCR and direct sequencing. Molecular epidemiology was analysed by Rep-PCR and ERIC-PCR. RESULTS: 484 patients were screened (sex ratio  =  1.03, median age 28 years. 53 ESBL-PE were isolated from 49 patients (carrier rate 10.1%. The isolates included Escherichia coli (31, Klebsiella pneumoniae (14, Enterobacter cloacae (3, Citrobacter freundii (3, Kluyvera spp. (1 and Pantoae sp. (1. In multivariate analysis, only the socioeconomic status of the head of household was independently associated with ESBL-PE carriage, poverty being the predominant risk factor. CONCLUSIONS: The prevalence of carriage of ESBL in the community of Antananarivo is one of the highest reported worldwide. This alarming spread of resistance genes should be stopped urgently by improving hygiene and streamlining the distribution and consumption of antibiotics.

Oral Gram-negative anaerobic bacilli as a reservoir of β-lactam resistance genes facilitating infections with multiresistant bacteria.

Science.gov (United States)

Dupin, Clarisse; Tamanai-Shacoori, Zohreh; Ehrmann, Elodie; Dupont, Anais; Barloy-Hubler, Frédérique; Bousarghin, Latifa; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne

2015-02-01

Many β-lactamases have been described in various Gram-negative bacilli (Capnocytophaga, Prevotella, Fusobacterium, etc.) of the oral cavity, belonging to class A of the Ambler classification (CepA, CblA, CfxA, CSP-1 and TEM), class B (CfiA) or class D in Fusobacterium nucleatum (FUS-1). The minimum inhibitory concentrations of β-lactams are variable and this variation is often related to the presence of plasmids or other mobile genetic elements (MGEs) that modulate the expression of resistance genes. DNA persistence and bacterial promiscuity in oral biofilms also contribute to genetic transformation and conjugation in this particular microcosm. Overexpression of efflux pumps is facilitated because the encoding genes are located on MGEs, in some multidrug-resistant clinical isolates, similar to conjugative transposons harbouring genes encoding β-lactamases. All these facts lead us to consider the oral cavity as an important reservoir of β-lactam resistance genes and a privileged place for genetic exchange, especially in commensal strictly anaerobic Gram-negative bacilli. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Métodos alternativos para detecção de betalactamase de espectro estendido em Escherichia coli e Klebsiella pneumoniae Alternative methods for the detection of extended-spectrum-beta-lactamase in Escherichia coli and Klebsiella pneumoniae

Directory of Open Access Journals (Sweden)

Alexsander Costa Martins

2011-08-01

Full Text Available INTRODUÇÃO: A resistência a antimicrobianos tem aumentado rapidamente nos últimos anos no Brasil e no mundo e, embora exista uma variedade de mecanismos de resistência, destaca-se a produção de betalactamase de espectro estendido (ESBL como um dos principais. Essas enzimas são mediadas por plasmídios, conferem resistência a vários antimicrobianos betalactâmicos e são inibidas por compostos, como ácido clavulânico, sulbactam e tazobactam. OBJETIVO: O objetivo deste estudo foi comparar metodologias alternativas à técnica padrão preconizada pelo Clinical and Laboratory Standards Institute (CLSI para detecção de ESBL. MATERIAL E MÉTODO: Foram realizados testes com 36 isolados (26 de E. coli e 10 de K. pneumoniae mediante disco combinado (CLSI e técnicas alternativas designadas meio disco (MD e substituição de discos (SD. CONCLUSÃO: As duas metodologias propostas apresentaram resultados satisfatórios com sensibilidade superior a 90% e custo inferior à técnica de referência (disco combinado, podendo ser utilizadas na pesquisa de ESBL.INTRODUCTION: Antimicrobial resistance has increased apace in Brazil and worldwide in the last years, even though there is a great variety of resistance mechanisms and extended spectrum beta-lactamases (ESBL is among the main ones. These enzymes are plasmid mediated, which causes resistance to some beta-lactam antimicrobials and are inhibited by compounds such as clavulanic acid, sulbactam and tazobactam. OBJECTIVE: The objective of this study was to compare alternative methods to the standard ESBL detection technique recommended by the Clinical and Laboratory Standards Institute (CLSI. MATERIAL AND METHOD: Tests with 36 isolates (26 E. coli and 10 K. pneumoniae were performed by means of CLSI disk diffusion method and alternative techniques designated as half disk (HD and disk substitution (SD. CONCLUSION: Both methods yielded satisfactory results with higher sensitivity (90% and lower costs

PLE-wu, a new member of piggyBac transposon family from insect, is active in mammalian cells.

Science.gov (United States)

Wu, Chunxiao; Wang, Shu

2014-10-01

piggyBac, a highly active transposon in insect and mammalian cells, is a very useful tool in genome manipulation. A new piggyBac-like element (PLE), named PLE-wu, was identified from a mutant baculovirus cultured in sf9 insect cells. This new transposon is 2931 bp in length and encodes two active forms of transposase, a 708-amino acid-long transposase and a short 576-residue-long transposase translated from a downstream in-frame initiation codon. PLE-wu has asymmetric terminal structures, containing 6-bp inverted terminal repeats, 32-bp imperfect inverted and direct sub-terminal repeats. Similar to piggyBac, PLE-wu exhibits traceless excision activity in both insect and mammalian cells, restoring the original TTAA target sequence upon excision. It also retains the insertion activity in mammalian cells with a plasmid to chromosome transposition rate about 10-fold higher than random integration. Plasmid rescue assays revealed that the TTAA target sequence was duplicated at the junctions of the insertion site. Deletion of the terminal sequences including the sub-terminal repeats decreased the transposition activity of the 708-residue-long transposase, while the transposition activity of the short form of transposase was not affected. With its low sequence similarity to piggyBac, PLE-wu will contribute to the understanding the mechanism of PLE transposition, as well as design of new transposon systems with higher activity. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Prevalence of AmpC β-lactamase among Gram-negative bacteria ...

African Journals Online (AJOL)

Purpose: Infections caused by AmpC-positive bacteria results in high patient morbidity and mortality making their detection clinically important as they cannot be detected in routine susceptibility testing. This study aim to determine the prevalence of AmpC β-lactamase among Gram negative bacteria recovered from clinical ...

Metabolic effects of beta2-agonists in relation to exercise performance

DEFF Research Database (Denmark)

Kalsen, Anders

2015-01-01

athletes. The present PhD thesis is based on four manuscripts in which the acute effects of beta2-agonists on exercise performance were investigated. The aims were 1) to investigate whether supratherapeutic inhalation of beta2-agonists enhances muscle strength, anaerobic performance and aerobic performance......, 2) to uncover the mechanisms behind potential beta2-adrenergic improvements in anaerobic performance, 3) to investigate whether inhalation of beta2-agonists is ergogenic in elite athletes with or without airway hyperresponsiveness (AHR). Results from the studies of the thesis show...... administration of a certain dose, but a further increase in dose does not seem to elicit a greater performance-enhancing effect. Moreover, the effects of beta2-agonists on performance are unaffected by training status and AHR, but athletes with AHR who regularly use beta2-agonists get a reduced ergogenic...

Combinatorial events of insertion sequences and ICE in Gram-negative bacteria.

Science.gov (United States)

Toleman, Mark A; Walsh, Timothy R

2011-09-01

The emergence of antibiotic and antimicrobial resistance in Gram-negative bacteria is incremental and linked to genetic elements that function in a so-called 'one-ended transposition' manner, including ISEcp1, ISCR elements and Tn3-like transposons. The power of these elements lies in their inability to consistently recognize one of their own terminal sequences, while recognizing more genetically distant surrogate sequences. This has the effect of mobilizing the DNA sequence found adjacent to their initial location. In general, resistance in Gram-negatives is closely linked to a few one-off events. These include the capture of the class 1 integron by a Tn5090-like transposon; the formation of the 3' conserved segment (3'-CS); and the fusion of the ISCR1 element to the 3'-CS. The structures formed by these rare events have been massively amplified and disseminated in Gram-negative bacteria, but hitherto, are rarely found in Gram-positives. Such events dominate current resistance gene acquisition and are instrumental in the construction of large resistance gene islands on chromosomes and plasmids. Similar combinatorial events appear to have occurred between conjugative plasmids and phages constructing hybrid elements called integrative and conjugative elements or conjugative transposons. These elements are beginning to be closely linked to some of the more powerful resistance mechanisms such as the extended spectrum β-lactamases, metallo- and AmpC type β-lactamases. Antibiotic resistance in Gram-negative bacteria is dominated by unusual combinatorial mistakes of Insertion sequences and gene fusions which have been selected and amplified by antibiotic pressure enabling the formation of extended resistance islands. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Tc7, a Tc1-hitch hiking transposon in Caenorhabditis elegans.

OpenAIRE

Rezsohazy, R; van Luenen, H G; Durbin, R M; Plasterk, R H

1997-01-01

We have found a novel transposon in the genome of Caenorhabditis elegans. Tc7 is a 921 bp element, made up of two 345 bp inverted repeats separated by a unique, internal sequence. Tc7 does not contain an open reading frame. The outer 38 bp of the inverted repeat show 36 matches with the outer 38 bp of Tc1. This region of Tc1 contains the Tc1-transposase binding site. Furthermore, Tc7 is flanked by TA dinucleotides, just like Tc1, which presumably correspond to the target duplication generated...

Characteristics of β-lactamases and their genes (blaA and blaB in Yersinia intermedia and Y. frederiksenii

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Sharma Sachin

2007-04-01

Full Text Available Abstract Background The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India. Results The enzymes, Bla-A (a constitutive class A penicillinase and Bla-B (an inducible class C cephalosporinase were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126–1024 mg/L and MIC90 (256–1024 mg/L of β-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87–96% with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77–79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85% with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7 and "Bla-B like" (pI 5.5–7.1 enzymes. Conclusion Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two β-lactamases. Limited heterogeneity

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

OpenAIRE

Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

1989-01-01

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled tra...

Low-beta investment strategies

OpenAIRE

Korn, Olaf; Kuntz, Laura-Chloé

2015-01-01

This paper investigates investment strategies that exploit the low-beta anomaly. Although the notion of buying low-beta stocks and selling high-beta stocks is natural, a choice is necessary with respect to the relative weighting of high-beta stocks and low-beta stocks in the investment portfolio. Our empirical results for US large-cap stocks show that this choice is very important for the risk-return characteristics of the resulting portfolios and their sensitivities to common risk factors. W...

Beta-glucosidase variants and polynucleotides encoding same

Science.gov (United States)

Wogulis, Mark; Harris, Paul; Osborn, David

2017-06-27

The present invention relates to beta-glucosidase variants, e.g. beta-glucosidase variants of a parent Family GH3A beta-glucosidase from Aspergillus fumigatus. The present invention also relates to polynucleotides encoding the beta-glucosidase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the beta-glucosidase variants.

Fast and efficient Drosophila melanogaster gene knock-ins using MiMIC transposons.

Science.gov (United States)

Vilain, Sven; Vanhauwaert, Roeland; Maes, Ine; Schoovaerts, Nils; Zhou, Lujia; Soukup, Sandra; da Cunha, Raquel; Lauwers, Elsa; Fiers, Mark; Verstreken, Patrik

2014-10-08

Modern molecular genetics studies necessitate the manipulation of genes in their endogenous locus, but most of the current methodologies require an inefficient donor-dependent homologous recombination step to locally modify the genome. Here we describe a methodology to efficiently generate Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an attP-site. These restriction enzyme sites allow for double-strand break-mediated removal of unwanted flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes. As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRK(HA) knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the fruit fly genome without the need for inefficient donor-dependent homologous recombination events. Copyright © 2014 Vilain et al.

A Fitness Cost Associated With the Antibiotic Resistance Enzyme SME-1 β-Lactamase

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Marciano, David C.; Karkouti, Omid Y.; Palzkill, Timothy

2007-01-01

The blaTEM-1 β-lactamase gene has become widespread due to the selective pressure of β-lactam use and its stable maintenance on transferable DNA elements. In contrast, blaSME-1 is rarely isolated and is confined to the chromosome of carbapenem-resistant Serratia marcescens strains. Dissemination of blaSME-1 via transfer to a mobile DNA element could hinder the use of carbapenems. In this study, blaSME-1 was determined to impart a fitness cost upon Escherichia coli in multiple genetic contexts and assays. Genetic screens and designed SME-1 mutants were utilized to identify the source of this fitness cost. These experiments established that the SME-1 protein was required for the fitness cost but also that the enzyme activity of SME-1 was not associated with the fitness cost. The genetic screens suggested that the SME-1 signal sequence was involved in the fitness cost. Consistent with these findings, exchange of the SME-1 signal sequence for the TEM-1 signal sequence alleviated the fitness cost while replacing the TEM-1 signal sequence with the SME-1 signal sequence imparted a fitness cost to TEM-1 β-lactamase. Taken together, these results suggest that fitness costs associated with some β-lactamases may limit their dissemination. PMID:17565956

Prevalence of Multiple Drug Resistant Clinical Isolates of Extended-Spectrum Beta-Lactamase Producing Enterobacteriaceae in Southeast Iran

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Shahla Mansouri

2010-06-01

Full Text Available AbstractBackground: Multidrug resistance and production of extendedspectrum β-lactamases (ESBLs by enteric gramnegativerods in hospitals and community continue to beworsened. We aimed to characterize the multidrug resistanceand determine the prevalence of ESBL production by clinicalisolates of Enterobacteriaceae in southeast Iran.Methods: Gram-negative bacteria isolated from clinical samplesof hospital inpatients and outpatients from three hospitalsin southeast Iran were tested for susceptibility to 10commonly used antimicrobials. For 500 isolates whichshowed resistance to ≥3 antibiotics from different classes,minimum inhibitory concentration, and prevalence of ESBLproduction were determined by agar dilution and double discsynergy method respectively. The isolated bacterial specieswere compared in respect of antibacterial resistance, ESBLproduction, patients' gender, hospital ward, and type ofspecimen.Results: The most frequent resistance was to trimethoprim/sulfamethoxazole, amoxicillin, and tetracycline. Imipenemwith 99.8% and ceftizoxime with 83% susceptibility were themost active agents. A total of 53.8% of isolates expressedESBL production. Escherichia coli and Klebsiella pneumoniaewere most common in outpatients, and inpatients samplesrespectively. Higher rate of resistance to most antibacterialagents and ESBL production was found in samples ofinpatients.Conclusion: The present study showed high prevalence ofESBL-producing Enterobacteriaceae especially in the patientsadmitted to hospital. Infection control strategy with continuousresistance surveillance is essential to monitor in vitro susceptibilityto antibacterial agents currently used in clinicalpractice. Determination of the type of involved ESBL enzymesis important for a better antimicrobial control and empiricaltherapy of critically ill patients in hospitals.Iran J Med Sci 2010; 35(2: 101-108.

Phenotypic Tests for the Detection of β-Lactamase-Producing Enterobacteriaceae Isolated from Different Environments.

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de Oliveira, Daniele V; Van Der Sand, Sueli T

2016-07-01

Some bacteria from the Enterobacteriaceae family are showing a significant capability to disseminate β-lactams resistance mechanisms among them, and these same mechanisms can be carried out from the hospital environment to superficial water. The aim of this study was to evaluate different phenotypic methods for the detection β-lactamases production by enterobacteria isolated from the anthropogenic environment: hospital wastewater and from a stream that cross the city of Porto Alegre. The applied tests were the modified Hodge test (MHT) and phenotypic tests with the following inhibitors: carbapenemase-phenylboronic acid (APB), metallo-β-lactamase-EDTA, AmpC β-lactamase-cloxacillin, and the confirmatory test for extended-spectrum β-lactamase (ESBL)-clavulanic acid. For this evaluation, 131 isolates were initially subjected to antibiogram using the following antimicrobials: cefotaxime (30 µg), cefpodoxime (10 μg), ceftazidime (30 µg), ertapenem (10 μg), meropenem (10 μg), and aztreonam (30 μg). After this first screening, 62 isolates showed a profile resistance for at least one antimicrobial. These isolates were subjected to all phenotypic tests. Of those, 40 isolates were positive for at least one phenotypic test. In MHT test, one isolate was positive and five were with inconclusive results. The results achieved with the inhibitors are as follows: APB 25/40 positive strains; EDTA 8/40 positive strains; and with CLOXA 2/40 positive strains. ESBL production was observed for 34/40 strains. This assessment shows a high level of bacteria which can produce enzymes that inactivate β-lactams present in the different environment like the stream waters and from the hospital settings.