Rickettsia asembonensis Characterization by Multilocus Sequence Typing of Complete Genes, Peru.

Science.gov (United States)

Loyola, Steev; Flores-Mendoza, Carmen; Torre, Armando; Kocher, Claudine; Melendrez, Melanie; Luce-Fedrow, Alison; Maina, Alice N; Richards, Allen L; Leguia, Mariana

2018-05-01

While studying rickettsial infections in Peru, we detected Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions (17kDa, gltA, ompA, ompB, and sca4) and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.

A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species

NARCIS (Netherlands)

Boonsilp, Siriphan; Thaipadungpanit, Janjira; Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

2013-01-01

The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. We modified the existing scheme by replacing one of the

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii.

Science.gov (United States)

Tanigawa, Kana; Watanabe, Koichi

2011-03-01

Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing.

Science.gov (United States)

Sharma, Anshul; Kaur, Jasmine; Lee, Sulhee; Park, Young-Seo

2018-06-01

In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence typing (MLST) of seven housekeeping genes (atpA, groEL, gyrB, pheS, pyrG, rpoA, and uvrC). The fragment sizes of the seven amplified housekeeping genes ranged in length from 366 to 1414 bp. Sequence analysis indicated 27 different sequence types (STs) with 25 of them being represented by a single strain indicating high genetic diversity, whereas the remaining 2 were characterized by five strains each. In total, 220 polymorphic nucleotide sites were detected among seven housekeeping genes. The phylogenetic analysis based on the STs of the seven loci indicated that the 35 strains belonged to two major groups, A (28 strains) and B (7 strains). Split decomposition analysis showed that intraspecies recombination played a role in generating diversity among strains. The minimum spanning tree showed that the evolution of the STs was not correlated with food source. This study signifies that the multilocus sequence typing is a valuable tool to access the genetic diversity among L. mesenteroides strains from South Korea and can be used further to monitor the evolutionary changes.

[Standard algorithm of molecular typing of Yersinia pestis strains].

Science.gov (United States)

Eroshenko, G A; Odinokov, G N; Kukleva, L M; Pavlova, A I; Krasnov, Ia M; Shavina, N Iu; Guseva, N P; Vinogradova, N A; Kutyrev, V V

2012-01-01

Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

Population structure of Lactobacillus helveticus isolates from naturally fermented dairy products based on multilocus sequence typing.

Science.gov (United States)

Sun, Zhihong; Liu, Wenjun; Song, Yuqin; Xu, Haiyan; Yu, Jie; Bilige, Menghe; Zhang, Heping; Chen, Yongfu

2015-05-01

Lactobacillus helveticus is an economically important lactic acid bacterium used in industrial dairy fermentation. In the present study, the population structure of 245 isolates of L. helveticus from different naturally fermented dairy products in China and Mongolia were investigated using an multilocus sequence typing scheme with 11 housekeeping genes. A total of 108 sequence types were detected, which formed 8 clonal complexes and 27 singletons. Results from Structure, SplitsTree, and ClonalFrame software analyses demonstrated the presence of 3 subpopulations in the L. helveticus isolates used in our study, namely koumiss, kurut-tarag, and panmictic lineages. Most L. helveticus isolates from particular ecological origins had specific population structures. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Genetic Relationships among Reptilian and Mammalian Campylobacter fetus Strains Determined by Multilocus Sequence Typing

NARCIS (Netherlands)

Dingle, K.E.; Blaser, M.J.; Tu, Z.C.; Pruckler, J.; Fitzgerald, C.; Bergen, van M.A.P.; Lawson, A.J.; Owen, R.J.; Wagenaar, J.A.

2010-01-01

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared similar to 90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two

Genotyping of B. licheniformis based on a novel multi-locus sequence typing (MLST scheme

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Madslien Elisabeth H

2012-10-01

Full Text Available Abstract Background Bacillus licheniformis has for many years been used in the industrial production of enzymes, antibiotics and detergents. However, as a producer of dormant heat-resistant endospores B. licheniformis might contaminate semi-preserved foods. The aim of this study was to establish a robust and novel genotyping scheme for B. licheniformis in order to reveal the evolutionary history of 53 strains of this species. Furthermore, the genotyping scheme was also investigated for its use to detect food-contaminating strains. Results A multi-locus sequence typing (MLST scheme, based on the sequence of six house-keeping genes (adk, ccpA, recF, rpoB, spo0A and sucC of 53 B. licheniformis strains from different sources was established. The result of the MLST analysis supported previous findings of two different subgroups (lineages within this species, named “A” and “B” Statistical analysis of the MLST data indicated a higher rate of recombination within group “A”. Food isolates were widely dispersed in the MLST tree and could not be distinguished from the other strains. However, the food contaminating strain B. licheniformis NVH1032, represented by a unique sequence type (ST8, was distantly related to all other strains. Conclusions In this study, a novel and robust genotyping scheme for B. licheniformis was established, separating the species into two subgroups. This scheme could be used for further studies of evolution and population genetics in B. licheniformis.

Multi-locus variable-number tandem repeat profiling of Salmonella enterica serovar Typhi isolates from blood cultures and gallbladder specimens from Makassar, South-Sulawesi, Indonesia.

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Mochammad Hatta

Full Text Available Multi-locus variable-number tandem repeat analysis differentiated 297 Salmonella enterica serovar Typhi blood culture isolates from Makassar in 76 genotypes and a single unique S. Typhi genotype was isolated from the cholecystectomy specimens of four patients with cholelithiasis. The high diversity in S. Typhi genotypes circulating in Makassar indicates that the number of carriers could be very large, which may complicate disease prevention and control.

Multilocus sequence typing of IncN plasmids

DEFF Research Database (Denmark)

García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

2011-01-01

that spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated......OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid...... in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying blaVIM-1 from Klebsiella pneumoniae and Escherichia coli, isolated during a 5year period in Greece were assigned to ST10, suggesting...

Multilocus sequence typing scheme for the Mycobacterium abscessus complex.

Science.gov (United States)

Macheras, Edouard; Konjek, Julie; Roux, Anne-Laure; Thiberge, Jean-Michel; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby E; Bodmer, Thomas; Jarlier, Vincent; Cambau, Emmanuelle; Brisse, Sylvain; Caro, Valérie; Rastogi, Nalin; Gaillard, Jean-Louis; Heym, Beate

2014-01-01

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Development of a multilocus sequence typing scheme for Ureaplasma.

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Zhang, J; Kong, Y; Feng, Y; Huang, J; Song, T; Ruan, Z; Song, J; Jiang, Y; Yu, Y; Xie, X

2014-04-01

Ureaplasma is a commensal of the human urogenital tract but is always associated with invasive diseases such as non-gonococcal urethritis and infertility adverse pregnancy outcomes. To better understand the molecular epidemiology and population structure of Ureaplasma, a multilocus sequence typing (MLST) scheme based on four housekeeping genes (ftsH, rpL22, valS, thrS) was developed and validated using 283 isolates, including 14 serovars of reference strains and 269 strains obtained from clinical patients. A total of 99 sequence types (STs) were revealed: the 14 type strains of the Ureaplasma serovars were assigned to 12 STs, and 87 novel and special STs appeared among the clinical isolates. ST1 and ST22 were the predominant STs, which contained 68 and 70 isolates, respectively. Two clonal lineages (CC1 and CC2) were shown by eBURST analysis, and linkage disequilibrium was revealed through a standardized index of association (I A (S)). The neighbor-joining tree results of 14 Ureaplasma serovars showed two genetically significantly distant clusters, which was highly congruent with the species taxonomy of ureaplasmas [Ureaplasma parvum (UPA) and Ureaplasma urealyticum (UUR)]. Analysis of the biotypes of 269 clinical isolates revealed that all the isolates of CC1 were UPA and those of CC2 were UUR. Additionally, CC2 was found more often in symptomatic patients with vaginitis, tubal obstruction, and cervicitis. In conclusion, this MLST scheme is adequate for investigations of molecular epidemiology and population structure with highly discriminating capacity.

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

Science.gov (United States)

Chawjiraphan, Wireeya; Sonthayanon, Piengchan; Chanket, Phanita; Benjathummarak, Surachet; Kerdsin, Anusak; Kalambhaheti, Thareerat

2016-11-01

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

Use of multilocus sequence typing for the investigation of colonisation by Candida albicans in intensive care unit patients.

Science.gov (United States)

Cliff, P R; Sandoe, J A T; Heritage, J; Barton, R C

2008-05-01

A prospective study was performed to determine the prevalence of candidal colonisation on the general intensive care unit at a large teaching hospital. Colonisation with Candida spp. was found to be common, occurring in 79% of patients on the unit. C. albicans was the commonest species, colonising 64% of patients, followed by C. glabrata (18%) and C. parapsilosis (14%). Most of the members of staff tested carried Candida spp. at some point, although carriage appeared to be transient. C. parapsilosis was the most commonly isolated species from staff hands, whereas C. albicans was the most commonly isolated species from the mouth. The molecular epidemiology of C. albicans was investigated using Ca3 typing and multilocus sequence typing (MLST). MLST proved to be a reproducible typing method and a useful tool for the investigation of the molecular epidemiology of C. albicans. The results of the molecular typing provided evidence for the presence of an endemic strain on the unit, which was isolated repeatedly from patients and staff. This finding suggests horizontal transmission of C. albicans on the unit though it may also reflect the relative frequency of C. albicans strain types colonising patients on admission. This study has important implications for the epidemiology of systemic candidal infections.

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis.

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Jiufeng Sun

Full Text Available Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb] was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection

Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis

Science.gov (United States)

Sun, Jiufeng; Huang, Yan; Huang, Huaiqiu; Liang, Pei; Wang, Xiaoyun; Mao, Qiang; Men, Jingtao; Chen, Wenjun; Deng, Chuanhuan; Zhou, Chenhui; Lv, Xiaoli; Zhou, Juanjuan; Zhang, Fan; Li, Ran; Tian, Yanli; Lei, Huali; Liang, Chi; Hu, Xuchu; Xu, Jin; Li, Xuerong; XinbingYu

2013-01-01

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the

Multilocus sequence typing of commensal and enteropathogenic Escherichia coli from domestic and wild lagomorphs in Italy

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Giorgia Dotto

2015-12-01

Full Text Available The aim of the study was to determine the multilocus sequence types of Escherichia coli from diseased farm rabbits and apparently healthy wild lagomorphs, and the genetic relatedness among them. Fifty-five enteropathogenic E. coli from reared rabbits and 32 from wild rabbits and hares were characterised by multilocus sequence typing (MLST according to the Michigan State University EcMLST scheme. Isolates were differentiated into 37 sequence types (STs, which were grouped into 8 clonal complexes (CCs. The most common ST was ST140 (CC31, followed by ST238 and ST119 (CC17. MLST analysis revealed 22 novel STs. Phylogenetic analyses showed a heterogeneous distribution of STs into 3 clusters of genetically related strains. The genetic relationship among STs of different origin and the detection of new, as well as previously described STs as human pathogens, indicate a widespread distribution and adaptability of particular lineages to different hosts. These findings highlight the need for further research to improve the knowledge about E. coli populations colonising the gut of lagomorphs and their zoonotic potential.

Typing Chlamydia trachomatis: from egg yolk to nanotechnology

DEFF Research Database (Denmark)

Pedersen, Lisbeth Nørum; Herrmann, Bjørn; Møller, Jens Kjølseth

2009-01-01

A historical review is provided of the various methods used for half a century to differentiate and type Chlamydia trachomatis strains. Typing of C. trachomatis is an important tool for revealing transmission patterns in sexual networks, and enabling association with clinical manifestations......, insufficient epidemiological resolution is achieved by characterization of both MOMP and omp1. This calls for new high-resolution genotyping methods applying for example a multilocus variable number tandem repeat assay (MLVA) or multilocus sequence typing (MLST). The futuristic nanotechnology already seems...

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

Science.gov (United States)

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

2015-12-01

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

Comparison of a newly developed binary typing with ribotyping and multilocus sequence typing methods for Clostridium difficile.

Science.gov (United States)

Li, Zhirong; Liu, Xiaolei; Zhao, Jianhong; Xu, Kaiyue; Tian, Tiantian; Yang, Jing; Qiang, Cuixin; Shi, Dongyan; Wei, Honglian; Sun, Suju; Cui, Qingqing; Li, Ruxin; Niu, Yanan; Huang, Bixing

2018-04-01

Clostridium difficile is the causative pathogen for antibiotic-related nosocomial diarrhea. For epidemiological study and identification of virulent clones, a new binary typing method was developed for C. difficile in this study. The usefulness of this newly developed optimized 10-loci binary typing method was compared with two widely used methods ribotyping and multilocus sequence typing (MLST) in 189 C. difficile samples. The binary typing, ribotyping and MLST typed the samples into 53 binary types (BTs), 26 ribotypes (RTs), and 33 MLST sequence types (STs), respectively. The typing ability of the binary method was better than that of either ribotyping or MLST expressed in Simpson Index (SI) at 0.937, 0.892 and 0.859, respectively. The ease of testing, portability and cost-effectiveness of the new binary typing would make it a useful typing alternative for outbreak investigations within healthcare facilities and epidemiological research. Copyright © 2018 Elsevier B.V. All rights reserved.

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

DEFF Research Database (Denmark)

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

2005-01-01

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

Comparison of multilocus sequence typing, RAPD, and MALDI-TOF mass spectrometry for typing of β-lactam-resistant Klebsiella pneumoniae strains.

Science.gov (United States)

Sachse, Svea; Bresan, Stephanie; Erhard, Marcel; Edel, Birgit; Pfister, Wolfgang; Saupe, Angela; Rödel, Jürgen

2014-12-01

Extended spectrum of β-lactam (ESBL) resistance of Klebsiella pneumoniae has become an increasing problem in hospital infections. Typing of isolates is important to establish the intrahospital surveillance of resistant clones. In this study, the discriminatory potential of randomly amplified polymorphic DNA and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were compared with multilocus sequence typing (MLST) by using 17 β-lactam-resistant K. pneumoniae isolates of different genotypes. MLST alleles were distributed in 8 sequence types (STs). Among ESBL strains of the same ST, the presence of different β-lactamase genes was common. RAPD band patterns also revealed 8 types that corresponded to MLST-defined genotypes in 15 out of 17 cases. MALDI-TOF analysis could differentiate 5 clusters of strains. The results of this work show that RAPD may be usable as a rapid screening method for the intrahospital surveillance of K. pneumoniae, allowing a discrimination of clonally related strains. MALDI-TOF-based typing was not strongly corresponding to genotyping and warrants further investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

Multilocus Sequence Typing for Interpreting Blood Isolates of Staphylococcus epidermidis

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Prannda Sharma

2014-01-01

Full Text Available Staphylococcus epidermidis is an important cause of nosocomial infection and bacteremia. It is also a common contaminant of blood cultures and, as a result, there is frequently uncertainty as to its diagnostic significance when recovered in the clinical laboratory. One molecular strategy that might be of value in clarifying the interpretation of S. epidermidis identified in blood culture is multilocus sequence typing. Here, we examined 100 isolates of this species (50 blood isolates representing true bacteremia, 25 likely contaminant isolates, and 25 skin isolates and the ability of sequence typing to differentiate them. Three machine learning algorithms (classification regression tree, support vector machine, and nearest neighbor were employed. Genetic variability was substantial between isolates, with 44 sequence types found in 100 isolates. Sequence types 2 and 5 were most commonly identified. However, among the classification algorithms we employed, none were effective, with CART and SVM both yielding only 73% diagnostic accuracy and nearest neighbor analysis yielding only 53% accuracy. Our data mirror previous studies examining the presence or absence of pathogenic genes in that the overlap between truly significant organisms and contaminants appears to prevent the use of MLST in the clarification of blood cultures recovering S. epidermidis.

[Multilocus sequence-typing for characterization of Moscow strains of Haemophilus influenzae type b].

Science.gov (United States)

Platonov, A E; Mironov, K O; Iatsyshina, S B; Koroleva, I S; Platonova, O V; Gushchin, A E; Shipulin, G A

2003-01-01

Haemophilius influenzae, type b (Hib) bacteria, were genotyped by multilocus sequence typing (MLST) using 5 loci (adk, fucK, mdh, pgi, recA). 42 Moscow Hib strains (including 38 isolates form cerebrospinal fluid of children, who had purulent meningitis in 1999-2001, and 4 strains isolated from healthy carriers of Hib), as well as 2 strains from Yekaterinburg were studied. In MLST a strain is characterized, by alleles and their combinations (an allele profile) referred to also as sequence-type (ST). 9 Sts were identified within the Russian Hib bacteria: ST-1 was found in 25 strains (57%), ST-12 was found in 8 strains (18%), ST-11 was found in 4 strains (9%) and ST-15 was found in 2 strains (4.5%); all other STs strains (13, 14, 16, 17, 51) were found in isolated cases (2.3%). A comparison of allelic profiles and of nucleotide sequences showed that 93% of Russian isolates, i.e. strain with ST-1, 11, 12, 13, 15 and 17, belong to one and the same clonal complex. 2 isolates from Norway and Sweden from among 7 foreign Hib strains studied up to now can be described as belonging to the same clonal complex; 5 Hib strains were different from the Russian ones.

An Extended Multilocus Sequence Typing (MLST Scheme for Rapid Direct Typing of Leptospira from Clinical Samples.

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Sabrina Weiss

2016-09-01

Full Text Available Rapid typing of Leptospira is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST data direct from patient specimens while minimising costs for subsequent sequencing.An existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK and the Lao Peoples Democratic Republic (Lao PDR. Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine PCR positive for pathogenic Leptospira spp. at least one allele was amplified in 22 samples (50% and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%. No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25% and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was L. interrogans (10/14 and 7/8 from UK and Lao PDR, respectively. All other species were detected in samples from only one country (Lao PDR: L. borgpetersenii [1/8]; UK: L. kirschneri [1/14], L. santarosai [1/14], L. weilii [2/14].Typing information of pathogenic Leptospira spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of Leptospira prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.

Associations between dru Types and SCCmec Cassettes

DEFF Research Database (Denmark)

Bartels, Mette D; Boye, Kit; Oliveira, Duarte C

2013-01-01

SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru......Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct...... repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring...

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

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

2015-08-01

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

A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia.

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Bleidorn, Christoph; Gerth, Michael

2018-01-01

Wolbachia (Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation of Wolbachia strains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST in Wolbachia strain typing has not been assessed objectively. Here, we evaluate the properties of Wolbachia MLST markers and compare it to 252 other single copy loci present in the genome of most Wolbachia strains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of a Wolbachia strain very well. We argue that whole genome typing approaches should be used for Wolbachia typing in the future. Alternatively, if few loci approaches are necessary, we provide a characterisation of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections.

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Laëtitia Fabre

Full Text Available Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.

Molecular Epidemiologic Analysis of Enterococcus faecalis Isolates in Cuba by Multilocus Sequence Typing

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Kobayashi, Nobumichi; Nagashima, Shigeo

2009-01-01

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6′)-aph(2″), aph(3′), ant(6), ant(3″)(9), aph(2″)-Id, aph(2″)-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6′)-Ie-aph(2″)-Ia, aph(3′), ant(6), ant(3″)(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits. PMID:19857135

Population genetic and evolution analysis of controversial genus Edwardsiella by multilocus sequence typing.

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Buján, Noemí; Balboa, Sabela; L Romalde, Jesús; E Toranzo, Alicia; Magariños, Beatriz

2018-05-08

At present, the genus Edwardsiella compiles five species: E. tarda, E. hoshinae, E. ictaluri, E. piscicida and E. anguillarum. Some species of this genus such us E. ictaluri and E. piscicida are important pathogens of numerous fish species. With the description of the two latter species, the phylogeny of Edwardsiella became more complicated. With the aim to clarify the relationships among all species in the genus, a multilocus sequence typing (MLST) approach was developed and applied to characterize 56 isolates and 6 reference strains belonging to the five Edwardsiella species. Moreover, several analyses based on the MLST scheme were performed to investigate the evolution within the genus, as well as the influence of recombination and mutation in the speciation. Edwardsiella isolates presented a high genetic variability reflected in the fourteen sequence types (ST) represented by a single isolates out of eighteen total ST. Mutation events were considerably more frequent than recombination, although both approximately equal influenced the genetic diversification. However, the speciation among species occurred mostly by recombination. Edwardsiella genus displays a non-clonal population structure with some degree of geographical isolation followed by a population expansion of E. piscicida. A database from this study was created and hosted on pubmlst.org (http://pubmlst.org/edwardsiella/). Copyright © 2018 Elsevier Inc. All rights reserved.

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile.

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Bletz, Stefan; Janezic, Sandra; Harmsen, Dag; Rupnik, Maja; Mellmann, Alexander

2018-06-01

Clostridium difficile , recently renamed Clostridioides difficile , is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping ( n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange. Copyright © 2018 American Society for Microbiology.

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

DEFF Research Database (Denmark)

Henri, Clémentine; Félix, Benjamin; Guillier, Laurent

2016-01-01

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

Multilocus microsatellite typing (MLMT of strains from Turkey and Cyprus reveals a novel monophyletic L. donovani sensu lato group.

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Evi Gouzelou

Full Text Available BACKGROUND: New foci of human CL caused by strains of the Leishmania donovani (L. donovani complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE using the Montpellier (MON system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. METHODOLOGY/PRINCIPAL FINDINGS: The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains and from a VL patient in the south-west (Kuşadasi; EP59 strain. These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains and MON-308 (EP59. A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. CONCLUSION: The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding

Multilocus Sequence Analysis for Typing Leptospira interrogans and Leptospira kirschneri▿ †

OpenAIRE

Leon, Albertine; Pronost, Stéphane; Fortier, Guillaume; Andre-Fontaine, Geneviève; Leclercq, Roland

2009-01-01

Fifty-three strains belonging to the pathogenic species Leptospira interrogans and Leptospira kirschneri were analyzed by multilocus sequence analysis. The species formed two distinct branches. In the L. interrogans branch, the phylogenetic tree clustered the strains into three subgroups. Genogroups and serogroups were superimposed but not strictly.

Multilocus Sequence Analysis for Typing Leptospira interrogans and Leptospira kirschneri▿ †

Science.gov (United States)

Leon, Albertine; Pronost, Stéphane; Fortier, Guillaume; Andre-Fontaine, Geneviève; Leclercq, Roland

2010-01-01

Fifty-three strains belonging to the pathogenic species Leptospira interrogans and Leptospira kirschneri were analyzed by multilocus sequence analysis. The species formed two distinct branches. In the L. interrogans branch, the phylogenetic tree clustered the strains into three subgroups. Genogroups and serogroups were superimposed but not strictly. PMID:19955271

Evaluation of 13 short tandem repeated loci for use in personal identification applications

Energy Technology Data Exchange (ETDEWEB)

Hammond, H.A.; Caskey, C.T. (Baylor College of Medicine, Houston, TX (United States)); Jin, L.; Zhong, Y.; Chakraborty, R. (Univ. of Texas Graduate School of Biomedical Sciences, Houston, TX (United States))

1994-07-01

Personal identification by using DNA typing methodologies has been an issue in the popular and scientific press for several years. The authors present a PCR-based DNA-typing method using 13 unlinked short tandem repeat (STR) loci. Validation of the loci and methodology has been performed to meet standards set by the forensic community and the accrediting organization for parentage testing. Extensive statistical analysis has addressed the issues surrounding the presentation of [open quotes]match[close quotes] statistics. The authors have found STR loci to provide a rapid, sensitive, and reliable method of DNA typing for parentage testing, forensic identification, and medical diagnostics. Valid statistical analysis is generally simpler than similar analysis of RFLP-VNTR results and provides powerful statistical evidence of the low frequency of random multilocus genotype matching. 54 refs., 4 figs., 6 tabs.

Multilocus sequence typing reveals two evolutionary lineages of Acidovorax avenae subsp. citrulli.

Science.gov (United States)

Feng, Jianjun; Schuenzel, Erin L; Li, Jianqiang; Schaad, Norman W

2009-08-01

Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, has caused considerable damage to the watermelon and melon industry in China and the United States. Understanding the emergence and spread of this pathogen is important for controlling the disease. To build a fingerprinting database for reliable identification and tracking of strains of A. avenae subsp. citrulli, a multilocus sequence typing (MLST) scheme was developed using seven conserved loci. The study included 8 original strains from the 1978 description of A. avenae subsp. citrulli, 51 from China, and 34 from worldwide collections. Two major clonal complexes (CCs), CC1 and CC2, were identified within A. avenae subsp. citrulli; 48 strains typed as CC1 and 45 as CC2. All eight original 1978 strains isolated from watermelon and melon grouped in CC1. CC2 strains were predominant in the worldwide collection and all but five were isolated from watermelon. In China, a major seed producer for melon and watermelon, the predominant strains were CC1 and were found nearly equally on melon and watermelon.

Short read sequence typing (SRST: multi-locus sequence types from short reads

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Inouye Michael

2012-07-01

Full Text Available Abstract Background Multi-locus sequence typing (MLST has become the gold standard for population analyses of bacterial pathogens. This method focuses on the sequences of a small number of loci (usually seven to divide the population and is simple, robust and facilitates comparison of results between laboratories and over time. Over the last decade, researchers and population health specialists have invested substantial effort in building up public MLST databases for nearly 100 different bacterial species, and these databases contain a wealth of important information linked to MLST sequence types such as time and place of isolation, host or niche, serotype and even clinical or drug resistance profiles. Recent advances in sequencing technology mean it is increasingly feasible to perform bacterial population analysis at the whole genome level. This offers massive gains in resolving power and genetic profiling compared to MLST, and will eventually replace MLST for bacterial typing and population analysis. However given the wealth of data currently available in MLST databases, it is crucial to maintain backwards compatibility with MLST schemes so that new genome analyses can be understood in their proper historical context. Results We present a software tool, SRST, for quick and accurate retrieval of sequence types from short read sets, using inputs easily downloaded from public databases. SRST uses read mapping and an allele assignment score incorporating sequence coverage and variability, to determine the most likely allele at each MLST locus. Analysis of over 3,500 loci in more than 500 publicly accessible Illumina read sets showed SRST to be highly accurate at allele assignment. SRST output is compatible with common analysis tools such as eBURST, Clonal Frame or PhyloViz, allowing easy comparison between novel genome data and MLST data. Alignment, fastq and pileup files can also be generated for novel alleles. Conclusions SRST is a novel

Use of multi-locus sequencing typing as identification method for the food-borne pathogen Listeria monocytogenes: a review

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Sonia Lamon

2015-01-01

Full Text Available Listeria monocytogenes is an ubiquitous, intracellular pathogen which has been implicated within the past decade as the causative organism in several outbreaks of foodborne diseases. In this review, a new approach to molecular typing primarily designed for global epidemiology has been described: multi-locus sequencing typing (MLST. This approach is novel, in that it uses data that allow the unambiguous characterization of bacterial strains via the Internet. Our aim is to present the currently available selection of references on L. monocytogenes MLST detection methods and to discuss its use as gold standard to L. monocytogenes subtyping method.

High-resolution typing of Chlamydia trachomatis: epidemiological and clinical uses.

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de Vries, Henry J C; Schim van der Loeff, Maarten F; Bruisten, Sylvia M

2015-02-01

A state-of-the-art overview of molecular Chlamydia trachomatis typing methods that are used for routine diagnostics and scientific studies. Molecular epidemiology uses high-resolution typing techniques such as multilocus sequence typing, multilocus variable number of tandem repeats analysis, and whole-genome sequencing to identify strains based on their DNA sequence. These data can be used for cluster, network and phylogenetic analyses, and are used to unveil transmission networks, risk groups, and evolutionary pathways. High-resolution typing of C. trachomatis strains is applied to monitor treatment efficacy and re-infections, and to study the recent emergence of lymphogranuloma venereum (LGV) amongst men who have sex with men in high-income countries. Chlamydia strain typing has clinical relevance in disease management, as LGV needs longer treatment than non-LGV C. trachomatis. It has also led to the discovery of a new variant Chlamydia strain in Sweden, which was not detected by some commercial C. trachomatis diagnostic platforms. After a brief history and comparison of the various Chlamydia typing methods, the applications of the current techniques are described and future endeavors to extend scientific understanding are formulated. High-resolution typing will likely help to further unravel the pathophysiological mechanisms behind the wide clinical spectrum of chlamydial disease.

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

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Tellapragada, Chaitanya; Kamthan, Aayushi; Shaw, Tushar; Ke, Vandana; Kumar, Subodh; Bhat, Vinod; Mukhopadhyay, Chiranjay

2016-01-01

There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST) is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate) obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7%) had novel allelic profiles that were not reported previously. Sequence type (ST) 1368 (n = 15, 46.8%) with allelic profile (1, 4, 6, 4, 1, 1, 3) was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST) between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

Unravelling the Molecular Epidemiology and Genetic Diversity among Burkholderia pseudomallei Isolates from South India Using Multi-Locus Sequence Typing.

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Chaitanya Tellapragada

Full Text Available There is a slow but steady rise in the case detection rates of melioidosis from various parts of the Indian sub-continent in the past two decades. However, the epidemiology of the disease in India and the surrounding South Asian countries remains far from well elucidated. Multi-locus sequence typing (MLST is a useful epidemiological tool to study the genetic relatedness of bacterial isolates both with-in and across the countries. With this background, we studied the molecular epidemiology of 32 Burkholderia pseudomallei isolates (31 clinical and 1 soil isolate obtained during 2006-2015 from various parts of south India using multi-locus sequencing typing and analysis. Of the 32 isolates included in the analysis, 30 (93.7% had novel allelic profiles that were not reported previously. Sequence type (ST 1368 (n = 15, 46.8% with allelic profile (1, 4, 6, 4, 1, 1, 3 was the most common genotype observed. We did not observe a genotypic association of STs with geographical location, type of infection and year of isolation in the present study. Measure of genetic differentiation (FST between Indian and the rest of world isolates was 0.14413. Occurrence of the same ST across three adjacent states of south India suggest the dispersion of B.pseudomallei across the south western coastal part of India with limited geographical clustering. However, majority of the STs reported from the present study remained as "outliers" on the eBURST "Population snapshot", suggesting the genetic diversity of Indian isolates from the Australasian and Southeast Asian isolates.

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

Science.gov (United States)

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

2014-01-01

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

Relationships between functional genes in Lactobacillus delbrueckii ssp. bulgaricus isolates and phenotypic characteristics associated with fermentation time and flavor production in yogurt elucidated using multilocus sequence typing.

Science.gov (United States)

Liu, Wenjun; Yu, Jie; Sun, Zhihong; Song, Yuqin; Wang, Xueni; Wang, Hongmei; Wuren, Tuoya; Zha, Musu; Menghe, Bilige; Heping, Zhang

2016-01-01

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is well known for its worldwide application in yogurt production. Flavor production and acid producing are considered as the most important characteristics for starter culture screening. To our knowledge this is the first study applying functional gene sequence multilocus sequence typing technology to predict the fermentation and flavor-producing characteristics of yogurt-producing bacteria. In the present study, phenotypic characteristics of 35 L. bulgaricus strains were quantified during the fermentation of milk to yogurt and during its subsequent storage; these included fermentation time, acidification rate, pH, titratable acidity, and flavor characteristics (acetaldehyde concentration). Furthermore, multilocus sequence typing analysis of 7 functional genes associated with fermentation time, acid production, and flavor formation was done to elucidate the phylogeny and genetic evolution of the same L. bulgaricus isolates. The results showed that strains significantly differed in fermentation time, acidification rate, and acetaldehyde production. Combining functional gene sequence analysis with phenotypic characteristics demonstrated that groups of strains established using genotype data were consistent with groups identified based on their phenotypic traits. This study has established an efficient and rapid molecular genotyping method to identify strains with good fermentation traits; this has the potential to replace time-consuming conventional methods based on direct measurement of phenotypic traits. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Multilocus sequence typing of Xylella fastidiosa causing Pierce's disease and oleander leaf scorch in the United States.

Science.gov (United States)

Yuan, Xiaoli; Morano, Lisa; Bromley, Robin; Spring-Pearson, Senanu; Stouthamer, Richard; Nunney, Leonard

2010-06-01

Using a modified multilocus sequence typing (MLST) scheme for the bacterial plant pathogen Xylella fastidiosa based on the same seven housekeeping genes employed in a previously published MLST, we studied the genetic diversity of two subspecies, X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi, which cause Pierce's disease and oleander leaf scorch, respectively. Typing of 85 U.S. isolates (plus one from northern Mexico) of X. fastidiosa subsp. fastidiosa from 15 different plant hosts and 21 isolates of X. fastidiosa subsp. sandyi from 4 different hosts in California and Texas supported their subspecific status. Analysis using the MLST genes plus one cell-surface gene showed no significant genetic differentiation based on geography or host plant within either subspecies. Two cases of homologous recombination (with X. fastidiosa subsp. multiplex, the third U.S. subspecies) were detected in X. fastidiosa subsp. fastidiosa. Excluding recombination, MLST site polymorphism in X. fastidiosa subsp. fastidiosa (0.048%) and X. fastidiosa subsp. sandyi (0.000%) was substantially lower than in X. fastidiosa subsp. multiplex (0.240%), consistent with the hypothesis that X. fastidiosa subspp. fastidiosa and sandyi were introduced into the United States (probably just prior to 1880 and 1980, respectively). Using whole-genome analysis, we showed that MLST is more effective at genetic discrimination at the specific and subspecific level than other typing methods applied to X. fastidiosa. Moreover, MLST is the only technique effective in detecting recombination.

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

Science.gov (United States)

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

2014-08-01

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

Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria monocytogenes

OpenAIRE

Chen, Yi; Gonzalez-Escalona, Narjol; Hammack, Thomas S.; Allard, Marc W.; Strain, Errol A.; Brown, Eric W.

2016-01-01

ABSTRACT Many listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes. We have developed and evaluated core genome MLST (cgMLST) schemes and applied them to isolates from multiple clonal groups, including those associated with 39 listeriosis outbreaks. The cgMLST...

REPEATABILITY OF THE FRENCH HIGHER VEGETATION TYPES ACCORDING

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H. BRISSE

1998-04-01

Full Text Available Higher vegetation types are generally determined by successive approximations and defined by a common consent. Instead, they might be statistically determined and repeated, according to a numerical method called ‘socio-ecology’. This method deals only with floristical data, but gives them an ecological meaning by a previous calibration of the relations between plants, computed as ecological indices. It is applied to a pair of two homologous samples, each having 2.000 relevés and coming from the 60.000 relevés stored in the French data bank ‘Sophy’. Each sample covers the main ecological gradients of the bank, it defines a hierarchy of vegetation types and it explains half the peculiarity of a type with only 10 to 30 discriminant plants, out of the 5.000 plants observed in the relevés. Results : 1 The discriminant plants may characterize the vegetation types, including the higher ones, in a coherent and readable form. 2 In the two independent classifications, having different structures, the same vegetation types are repeated. They are the reciprocal nearest types, in the socio-ecological space. Though the two classifications have no one relevé in common, the repeated types have nearly the same discriminant plants. 3 At the highest level, two clear-cut main types show the difference between light and shadow. The same herbaceous discriminant plants, for a type, and the ligneous or sciaphilous ones, for the other, have similar fidelities and constancies in the two classifications. 4 Such a numerical agreement, instead of common consent, appears again in the sub-types, which remind the classical ones, but which are repeatable.

Development of a multilocus-based approach for sponge (phylum Porifera) identification: refinement and limitations.

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Yang, Qi; Franco, Christopher M M; Sorokin, Shirley J; Zhang, Wei

2017-02-02

For sponges (phylum Porifera), there is no reliable molecular protocol available for species identification. To address this gap, we developed a multilocus-based Sponge Identification Protocol (SIP) validated by a sample of 37 sponge species belonging to 10 orders from South Australia. The universal barcode COI mtDNA, 28S rRNA gene (D3-D5), and the nuclear ITS1-5.8S-ITS2 region were evaluated for their suitability and capacity for sponge identification. The highest Bit Score was applied to infer the identity. The reliability of SIP was validated by phylogenetic analysis. The 28S rRNA gene and COI mtDNA performed better than the ITS region in classifying sponges at various taxonomic levels. A major limitation is that the databases are not well populated and possess low diversity, making it difficult to conduct the molecular identification protocol. The identification is also impacted by the accuracy of the morphological classification of the sponges whose sequences have been submitted to the database. Re-examination of the morphological identification further demonstrated and improved the reliability of sponge identification by SIP. Integrated with morphological identification, the multilocus-based SIP offers an improved protocol for more reliable and effective sponge identification, by coupling the accuracy of different DNA markers.

Multiple-locus variable-number tandem repeat analysis of Neisseria meningitidis yields groupings similar to those obtained by multilocus sequence typing.

NARCIS (Netherlands)

Schouls, Leo M; Ende, Arie van der; Damen, Marjolein; Pol, Ingrid van de

2006-01-01

We identified many variable-number tandem repeat (VNTR) loci in the genomes of Neisseria meningitidis serogroups A, B, and C and utilized a number of these loci to develop a multiple-locus variable-number tandem repeat analysis (MLVA). Eighty-five N. meningitidis serogroup B and C isolates obtained

Multilocus Sequence Typing of Pathogenic Candida albicans Isolates Collected from a Teaching Hospital in Shanghai, China: A Molecular Epidemiology Study

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Li, Li; Zhang, Qiangqiang; Zhu, Junhao; Gao, Qian; Chen, Min; Zhu, Min

2015-01-01

Molecular typing of Candida albicans is important for studying the population structure and epidemiology of this opportunistic yeast, such as population dynamics, nosocomial infections, multiple infections and microevolution. The genetic diversity of C. albicans has been rarely studied in China. In the present study, multilocus sequence typing (MLST) was used to characterize the genetic diversity and population structure of 62 C. albicans isolates collected from 40 patients from Huashan Hospital in Shanghai, China. A total of 50 diploid sequence types (DSTs) were identified in the 62 C. albicans isolates, with 41 newly identified DSTs. Based on cluster analysis, the 62 isolates were classified into nine existing clades and two new clades (namely clades New 1 and New 2). The majority of the isolates were clustered into three clades, clade 6 (37.5%), clade 1 (15.0%) and clade 17 (15.0%). Isolates of clade New 2 were specifically identified in East Asia. We identified three cases of potential nosocomial transmission based on association analysis between patients’ clinical data and the genotypes of corresponding isolates. Finally, by analyzing the genotypes of serial isolates we further demonstrated that the microevolution of C. albicans was due to loss of heterozygosity. Our study represents the first molecular typing of C. albicans in eastern China, and we confirmed that MLST is a useful tool for studying the epidemiology and evolution of C. albicans. PMID:25919124

An overview of various typing methods for clinical epidemiology of the emerging pathogen Stenotrophomonas maltophilia.

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Gherardi, Giovanni; Creti, Roberta; Pompilio, Arianna; Di Bonaventura, Giovanni

2015-03-01

Typing of bacterial isolates has been used for decades to study local outbreaks as well as in national and international surveillances for monitoring newly emerging resistant clones. Despite being recognized as a nosocomial pathogen, the precise modes of transmission of Stenotrophomonas maltophilia in health care settings are unknown. Due to the high genetic diversity observed among S. maltophilia clinical isolates, the typing results might be better interpreted if also environmental strains were included. This could help to identify preventative measures to be designed and implemented for decreasing the possibility of outbreaks and nosocomial infections. In this review, we attempt to provide an overview on the most common typing methods used for clinical epidemiology of S. maltophilia strains, such as PCR-based fingerprinting analyses, pulsed-field gel electrophoresis, multilocus variable number tandem repeat analysis, and multilocus sequence type. Application of the proteomic-based mass spectrometry by matrix-assisted laser desorption ionization-time of flight is also described. Improvements of typing methods already in use have to be achieved to facilitate S. maltophilia infection control at any level. In the near future, when novel Web-based platforms for rapid data processing and analysis will be available, whole genome sequencing technologies will likely become a highly powerful tool for outbreak investigations and surveillance studies in routine clinical practices. Copyright © 2015 Elsevier Inc. All rights reserved.

Novel Polymorphic Multilocus Microsatellite Markers to Distinguish Candida tropicalis Isolates.

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Xin Fan

Full Text Available Candida tropicalis is an important pathogen. Here we developed and evaluated a polymorphic multilocus microsatellite scheme employing novel genetic markers for genotyping of C. tropicalis. Using 10 isolates from 10 unique (separate patients to screen over 4000 tandem repeats from the C. tropicalis genome (strain MYA-3404, six new candidate microsatellite loci (ctm1, ctm3, ctm8, ctm18, ctm24 and ctm26 were selected according to amplification success, observed polymorphisms and stability of flanking regions by preliminary testing. Two known microsatellite loci CT14 and URA3 were also studied. The 6-locus scheme was then tested against a set of 82 different isolates from 32 patients. Microsatellite genotypes of isolates from the same patient (two to five isolates per patient were identical. The six loci produced eight to 17 allele types and identified 11 to 24 genotypes amongst 32 patients' isolates, achieving a discriminatory power (DP of 0.76 to 0.97 (versus 0.78 for both CT14 and URA3 loci, respectively. Testing of a combination of only three loci, ctm1, ctm3 and ctm24, also achieved maximum typing efficiency (DP = 0.99, 29 genotypes. The microsatellite typing scheme had good correlation compared with pulsed-field gel electrophoresis, although was slightly less discriminatory. The new six-locus microsatellite typing scheme is a potentially valuable tool for genotyping and investigating microevolution of C. tropicalis.

Genotyping of Indian antigenic, vaccine, and field Brucella spp. using multilocus sequence typing.

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Shome, Rajeswari; Krithiga, Natesan; Shankaranarayana, Padmashree B; Jegadesan, Sankarasubramanian; Udayakumar S, Vishnu; Shome, Bibek Ranjan; Saikia, Girin Kumar; Sharma, Narendra Kumar; Chauhan, Harshad; Chandel, Bharat Singh; Jeyaprakash, Rajendhran; Rahman, Habibur

2016-03-31

Brucellosis is one of the most important zoonotic diseases that affects multiple livestock species and causes great economic losses. The highly conserved genomes of Brucella, with > 90% homology among species, makes it important to study the genetic diversity circulating in the country. A total of 26 Brucella spp. (4 reference strains and 22 field isolates) and 1 B. melitensis draft genome sequence from India (B. melitensis Bm IND1) were included for sequence typing. The field isolates were identified by biochemical tests and confirmed by both conventional and quantitative polymerase chain reaction (qPCR) targeting bcsp 31Brucella genus-specific marker. Brucella speciation and biotyping was done by Bruce ladder, probe qPCR, and AMOS PCRs, respectively, and genotyping was done by multilocus sequence typing (MLST). The MLST typing of 27 Brucella spp. revealed five distinct sequence types (STs); the B. abortus S99 reference strain and 21 B. abortus field isolates belonged to ST1. On the other hand, the vaccine strain B. abortus S19 was genotyped as ST5. Similarly, B. melitensis 16M reference strain and one B. melitensis field isolate were grouped into ST7. Another B. melitensis field isolate belonged to ST8 (draft genome sequence from India), and only B. suis 1330 reference strain was found to be ST14. The sequences revealed genetic similarity of the Indian strains to the global reference and field strains. The study highlights the usefulness of MLST for typing of field isolates and validation of reference strains used for diagnosis and vaccination against brucellosis.

Relationships between emm and multilocus sequence types within a global collection of Streptococcus pyogenes

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McGregor Karen F

2008-04-01

Full Text Available Abstract Background The M type-specific surface protein antigens encoded by the 5' end of emm genes are targets of protective host immunity and attractive vaccine candidates against infection by Streptococcus pyogenes, a global human pathogen. A history of genetic change in emm was evaluated for a worldwide collection of > 500 S. pyogenes isolates that were defined for genetic background by multilocus sequence typing of housekeeping genes. Results Organisms were categorized by genotypes that roughly correspond to throat specialists, skin specialists, and generalists often recovered from infections at either tissue site. Recovery of distant clones sharing the same emm type was ~4-fold higher for skin specialists and generalists, as compared to throat specialists. Importantly, emm type was often a poor marker for clone. Recovery of clones that underwent recombinational replacement with a new emm type was most evident for the throat and skin specialists. The average ratio of nonsynonymous substitutions per nonsynonymous site (Ka and synonymous substitutions per synonymous site (Ks was 4.9, 1.5 and 1.3 for emm types of the throat specialist, skin specialist and generalist groups, respectively. Conclusion Data indicate that the relationships between emm type and genetic background differ among the three host tissue-related groups, and that the selection pressures acting on emm appear to be strongest for the throat specialists. Since positive selection is likely due in part to a protective host immune response, the findings may have important implications for vaccine design and vaccination strategies.

Towards multilocus sequence typing of the Leishmania donovani complex: Resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD)

Czech Academy of Sciences Publication Activity Database

Mauricio, I. L.; Yeo, M.; Baghaei, M.; Doto, D.; Pratlong, F.; Zemanová, Eva; Dedet, J.-P.; Lukeš, Julius; Miles, M. A.

2006-01-01

Roč. 36, č. 7 (2006), s. 757-769 ISSN 0020-7519 Grant - others:European Comission(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Keywords : Leishmania donovani * Leishmania infantum * multilocus sequence typing Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.337, year: 2006

Multilocus sequence typing of Trichomonas vaginalis clinical samples from Amsterdam, the Netherlands.

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van der Veer, C; Himschoot, M; Bruisten, S M

2016-10-13

In this cross-sectional epidemiological study we aimed to identify molecular profiles for Trichomonas vaginalis and to determine how these molecular profiles were related to patient demographic and clinical characteristics. Molecular typing methods previously identified two genetically distinct subpopulations for T. vaginalis; however, few molecular epidemiological studies have been performed. We now increased the sensitivity of a previously described multilocus sequence typing (MLST) tool for T. vaginalis by using nested PCR. This enabled the typing of direct patient samples. From January to December 2014, we collected all T. vaginalis positive samples as detected by routine laboratory testing. Samples from patients either came from general practitioners offices or from the sexually transmitted infections (STI) clinic in Amsterdam. Epidemiological data for the STI clinic patients were retrieved from electronic patient files. The primary outcome was the success rate of genotyping direct T. vaginalis positive samples. The secondary outcome was the relation between T. vaginalis genotypes and risk factors for STI. All 7 MLST loci were successfully typed for 71/87 clinical samples. The 71 typed samples came from 69 patients, the majority of whom were women (n=62; 90%) and half (n=34; 49%) were STI clinic patients. Samples segregated into a two population structure for T. vaginalis representing genotypes I and II. Genotype I was most common (n=40; 59.7%). STI clinic patients infected with genotype II reported more sexual partners in the preceding 6 months than patients infected with genotype I (p=0.028). No other associations for gender, age, ethnicity, urogenital discharge or co-occurring STIs with T. vaginalis genotype were found. MLST with nested PCR is a sensitive typing method that allows typing of direct (uncultured) patient material. Genotype II is possibly more prevalent in high-risk sexual networks. Published by the BMJ Publishing Group Limited. For

A multi-locus phylogenetic evaluation of Diaporthe (Phomopsis)

NARCIS (Netherlands)

Udayanga, D.; Liu, X.; Crous, P.W.; McKenzie, E.H.C.; Chukeatirote, E.; Hyde, K.D.

2012-01-01

The genus Diaporthe (Phomopsis) includes important plant pathogenic fungi with wide host ranges and geographic distributions. In the present study, phylogenetic species recognition in Diaporthe is re-evaluated using a multi-locus phylogeny based on a combined data matrix of rDNA ITS, and partial

An Improved MLVF Method and Its Comparison with Traditional MLVF, spa Typing, MLST/SCCmec and PFGE for the Typing of Methicillin-Resistant Staphylococcus aureus

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Du, Xue-Fei; Xiao, Meng; Liang, Hong-Yan; Sun, Zhe; Jiang, Yue-Hong; Chen, Guo-Yu; Meng, Xiao-Yu; Zou, Gui-Ling; Zhang, Li; Liu, Ya-Li; Zhang, Hui; Sun, Hong-Li; Jiang, Xiao-Feng; Xu, Ying-Chun

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important nosocomial pathogen, causing considerable morbidity and mortality. During the last 20 years, a variety of genotyping methods have been introduced for screening the prevalence of MRSA. In this study, we developed and evaluated an improved approach capillary gel electrophoresis based multilocus variable-number tandem-repeat fingerprinting (CGE/MLVF) for rapid MRSA typing. A total of 42 well-characterized strains and 116 non-repetitive clinical MRSA isolates collected from six hospitals in northeast China between 2009 and 2010 were tested. The results obtained by CGE/MLVF against clinical isolates were compared with traditional MLVF, spa typing, Multilocus sequence typing/staphylococcal cassette chromosome mec (MLST/SCCmec) and pulse field gel electrophoresis (PFGE). The discriminatory power estimated by Simpson’s index of diversity was 0.855 (28 types), 0.855 (28 patterns), 0.623 (11 types), 0.517 (8 types) and 0.854 (28 patterns) for CGE/MLVF, traditional MLVF, spa typing, MLST/SCCmec and PFGE, respectively. All methods tested showed a satisfied concordance in clonal complex level calculated by adjusted Rand’s coefficient. CGE/MLVF showed better reproducibility and accuracy than traditional MLVF and PFGE methods. In addition, the CGE/MLVF has potential to produce portable results. In conclusion, CGE/MLVF is a rapid and easy to use MRSA typing method with lower cost, good reproducibility and high discriminatory power for monitoring the outbreak and clonal spread of MRSA isolates. PMID:24406728

Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae.

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Dijkman, R; Feberwee, A; Landman, W J M

2016-08-01

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.

Multilocus resolution of Mugilidae phylogeny (Teleostei: Mugiliformes): Implications for the family's taxonomy.

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Xia, Rong; Durand, Jean-Dominique; Fu, Cuizhang

2016-03-01

The interrelationships among mugilids (Mugiliformes: Mugilidae) remain highly debated. Using a mitochondrial gene-based phylogeny as criterion, a revised classification with 25 genera in the Mugilidae has recently been proposed. However, phylogenetic relationships of major mitochondrial lineages remain unresolved and to gain a general acceptance the classification requires confirmation based on multilocus evidence and diagnostic morphological characters. Here, we construct a species-tree using twelve nuclear and three mitochondrial loci and infer the evolution of 71 morphological characters. Our multilocus phylogeny does not agree with previous morphology-based hypotheses for the relationships within Mugilidae, confirms the revised classification with 25 genera and further resolves their phylogenetic relationships. Using the well-resolved multilocus phylogeny as the criterion, we reclassify Mugilidae genera into three new subfamilies (Myxinae, Rhinomugilinae, and Cheloninae) and one new, recombined, subfamily (Mugilinae). The Rhinomugilinae subfamily is further divided into four tribes. The revised classification of Mugilidae is supported by morpho-anatomical synapomorphies or a combination of characters. These characters are used to erect a key to the subfamilies and genera. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of Campylobacter jejuni

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Nash John HE

2008-08-01

Full Text Available Abstract Background Multi-Locus Sequence Typing (MLST has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of Campylobacter jejuni strains using MLST and Comparative Genomic Hybridization (CGH on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci. Results Our results indicate that, in general, there is significant concordance between strain relationships established by MLST and those based on shared gene content as established by CGH. While MLST has significant predictive power with respect to overall genome similarity of isolates, we also found evidence for significant differences in genomic content among strains that would otherwise appear to be highly related based on their MLST profiles. Conclusion The extensive genomic mosaicism between closely related strains has important implications in the context of establishing strain to strain relationships because it suggests that the exact gene content of strains, and by extension their phenotype, is less likely to be "predicted" based on a small number of typing loci. This in turn suggests that a greater emphasis should be placed on analyzing genes of clinical interest as we forge ahead with the next generation of molecular typing methods.

The Comparison of Streptococcus agalactiae Isolated from Fish and Bovine using Multilocus Sequence Typing

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ANGELA MARIANA LUSIASTUTI

2013-12-01

Full Text Available Multilocus sequence typing (MLST has greater utility for determining the recent ancestral lineage and the relatedness of individual strains. Group B streptococci (GBS is one of the major causes of subclinical mastitis of dairy cattle in several countries. GBS also sporadically causes epizootic infections in fish. The aim of this study was to compare the evolutionary lineage of fish and bovine isolates in relation to the S. agalactiae global population as a whole by comparing the MLST profiles. Twenty S. agalactiae isolates were obtained from dairy cattle and fish. PCR products were amplified with seven different oligonucleotide primer pairs designed from the NEM316 GBS genome sequence. Clone complexes demonstrated that bovine and fish isolates were separate populations. These findings lead us to conclude that fish S. agalactiae is not a zoonotic agent for bovine. MLST could help clarify the emergence of pathogenic clones and to decide whether the host acts as a reservoir for another pathogenic lineage.

Human Campylobacteriosis in Luxembourg, 2010?2013: A Case-Control Study Combined with Multilocus Sequence Typing for Source Attribution and Risk Factor Analysis

OpenAIRE

Mossong, Jo?l; Mughini-Gras, Lapo; Penny, Christian; Devaux, Anthony; Olinger, Christophe; Losch, Serge; Cauchie, Henry-Michel; van Pelt, Wilfrid; Ragimbeau, Catherine

2016-01-01

Campylobacteriosis has increased markedly in Luxembourg during recent years. We sought to determine which Campylobacter genotypes infect humans, where they may originate from, and how they may infect humans. Multilocus sequence typing was performed on 1153 Campylobacter jejuni and 136 C. coli human strains to be attributed to three putative animal reservoirs (poultry, ruminants, pigs) and to environmental water using the asymmetric island model. A nationwide case-control study (2010?2013) for...

Insights into the emergent bacterial pathogen Cronobacter spp., generated by multilocus sequence typing and analysis

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Susan eJoseph

2012-11-01

Full Text Available Cronobacter spp. (previously known as Enterobacter sakazakii is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognised route of infection being the consumption of contaminated infant formula. As a recently recognised bacterial pathogen of considerable importance and regulatory control, appropriate detection and identification schemes are required. The application of multilocus sequence typing (MLST and analysis (MLSA of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB and ppsA (concatenated length 3036 base pairs has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognised genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health.

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

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Blanchard, Adam M; Jolley, Keith A; Maiden, Martin C J; Coffey, Tracey J; Maboni, Grazieli; Staley, Ceri E; Bollard, Nicola J; Warry, Andrew; Emes, Richard D; Davies, Peers L; Tötemeyer, Sabine

2018-01-01

Dichelobacter nodosus ( D. nodosus ) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China

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Mingjun Sun

2017-12-01

Full Text Available Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2 was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2 and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS-single-nucleotide polymorphism (SNP-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

The Leishmania donovani complex: Genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH and FH), new targets for multilocus sequence typing

Czech Academy of Sciences Publication Activity Database

Zemanová, Eva; Jirků, Milan; Mauricio, I. L.; Horák, Aleš; Miles, M. A.; Lukeš, Julius

2007-01-01

Roč. 37, č. 2 (2007), s. 149-160 ISSN 0020-7519 R&D Projects: GA MŠk 2B06129 Grant - others:EU(EU) QLK2-CT-2001-01810 Institutional research plan: CEZ:AV0Z60220518 Source of funding: R - rámcový projekt EK Keywords : Leishmania donovani complex * zymodeme * multilocus sequence typing * Leishmania * phylogenetic network Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.392, year: 2007

A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions.

Science.gov (United States)

Ohshima, Chihiro; Takahashi, Hajime; Iwakawa, Ai; Kuda, Takashi; Kimura, Bon

2017-07-17

Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future. Copyright © 2017. Published by Elsevier B.V.

Multilocus genotyping of Giardia duodenalis in captive non-human primates in Sichuan and Guizhou provinces, Southwestern China.

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Zhijun Zhong

Full Text Available Giardia duodenalis is a common human and animal pathogen. It has been increasingly reported in wild and captive non-human primates (NHPs in recent years. However, multilocus genotyping information for G. duodenalis infecting NHPs in southwestern China is limited. In the present study, the prevalence and multilocus genotypes (MLGs of G. duodenalis in captive NHPs in southwestern China were determined. We examined 207 fecal samples from NHPs in Sichuan and Guizhou provinces, and 16 specimens were positive for G. duodenalis. The overall infection rate was 7.7%, and only assemblage B was identified. G. duodenalis was detect positive in northern white-cheeked gibbon (14/36, 38.9%, crab-eating macaque (1/60, 1.7% and rhesus macaques (1/101, 0.9%. Multilocus sequence typing based on beta-giardin (bg, triose phosphate isomerase (tpi and glutamate dehydrogenase (gdh revealed nine different assemblage B MLGs (five known genotypes and four novel genotypes. Based on a phylogenetic analysis, one potentially zoonotic genotype of MLG SW7 was identified in a northern white-cheeked gibbon. A high degree of genetic diversity within assemblage B was observed in captive northern white-cheeked gibbons in Southwestern China, including a potentially zoonotic genotype, MLG SW7. To the best of our knowledge, this is the first report using a MLGs approach to identify G. duodenalis in captive NHPs in Southwestern China.

Determining Clostridium difficile intra-taxa diversity by mining multilocus sequence typing databases.

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Muñoz, Marina; Ríos-Chaparro, Dora Inés; Patarroyo, Manuel Alfonso; Ramírez, Juan David

2017-03-14

Multilocus sequence typing (MLST) is a highly discriminatory typing strategy; it is reproducible and scalable. There is a MLST scheme for Clostridium difficile (CD), a gram positive bacillus causing different pathologies of the gastrointestinal tract. This work was aimed at describing the frequency of sequence types (STs) and Clades (C) reported and evalute the intra-taxa diversity in the CD MLST database (CD-MLST-db) using an MLSA approach. Analysis of 1778 available isolates showed that clade 1 (C1) was the most frequent worldwide (57.7%), followed by C2 (29.1%). Regarding sequence types (STs), it was found that ST-1, belonging to C2, was the most frequent. The isolates analysed came from 17 countries, mostly from the United Kingdom (UK) (1541 STs, 87.0%). The diversity of the seven housekeeping genes in the MLST scheme was evaluated, and alleles from the profiles (STs), for identifying CD population structure. It was found that adk and atpA are conserved genes allowing a limited amount of clusters to be discriminated; however, different genes such as drx, glyA and particularly sodA showed high diversity indexes and grouped CD populations in many clusters, suggesting that these genes' contribution to CD typing should be revised. It was identified that CD STs reported to date have a mostly clonal population structure with foreseen events of recombination; however, one group of STs was not assigned to a clade being highly different containing at least nine well-supported clusters, suggesting a greater amount of clades for CD. This study shows the usefulness of CD-MLST-db as a tool for studying CD distribution and population structure, identifying the need for reviewing the usefulness of sodA as housekeeping gene within the MLST scheme and suggesting the existence of a greater amount of CD clades. The study also shows the plausible exchange of genetic material between STs, contributing towards intra-taxa genetic diversity.

Rank-based characterization of pollen assemblages collected by honey bees using a multi-locus metabarcoding approach.

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Richardson, Rodney T; Lin, Chia-Hua; Quijia, Juan O; Riusech, Natalia S; Goodell, Karen; Johnson, Reed M

2015-11-01

Difficulties inherent in microscopic pollen identification have resulted in limited implementation for large-scale studies. Metabarcoding, a relatively novel approach, could make pollen analysis less onerous; however, improved understanding of the quantitative capacity of various plant metabarcode regions and primer sets is needed to ensure that such applications are accurate and precise. We applied metabarcoding, targeting the ITS2, matK, and rbcL loci, to characterize six samples of pollen collected by honey bees, Apis mellifera. Additionally, samples were analyzed by light microscopy. We found significant rank-based associations between the relative abundance of pollen types within our samples as inferred by the two methods. Our findings suggest metabarcoding data from plastid loci, as opposed to the ribosomal locus, are more reliable for quantitative characterization of pollen assemblages. Furthermore, multilocus metabarcoding of pollen may be more reliable than single-locus analyses, underscoring the need for discovering novel barcodes and barcode combinations optimized for molecular palynology.

Multilocus Sequence Typing of the Clinical Isolates of Salmonella Enterica Serovar Typhimurium in Tehran Hospitals

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Reza Ranjbar

2017-09-01

Full Text Available Background: Salmonella enterica serovar Typhimurium is one of the most important serovars of Salmonella enterica and is associated with human salmonellosis worldwide. Many epidemiological studies have focused on the characteristics of Salmonella Typhimurium in many countries as well as in Asia. This study was conducted to investigate the genetic characteristics of Salmonella Typhimurium using multilocus sequence typing (MLST. Methods: Clinical samples (urine, blood, and stool were collected from patients, who were admitted to 2 hospitals in Tehran between April and September, 2015. Salmonella Typhimurium strains were identified by conventional standard biochemical and serological testing. The antibiotic susceptibility patterns of the Salmonella Typhimurium isolates against 16 antibiotics was determined using the disk diffusion assay. The clonal relationship between the strains of Salmonella Typhimurium was analyzed using MLST. Results: Among the 68 Salmonella isolates, 31% (n=21 were Salmonella Typhimurium. Of the total 21 Salmonella Typhimurium isolates, 76% (n=16 were multidrug-resistant and showed resistance to 3 or more antibiotic families. The Salmonella Typhimurium isolates were assigned to 2 sequence types: ST19 and ST328. ST19 was more common (86%. Both sequence types were further assigned to 1 eBURST group. Conclusion: This is the first study of its kind in Iran to determine the sequence types of the clinical isolates of Salmonella Typhimurium in Tehran hospitals using MLST. ST19 was detected as the major sequence type of Salmonella Typhimurium.

A novel multi-locus sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional dairy products in China and Mongolia.

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Dan, Tong; Liu, Wenjun; Sun, Zhihong; Lv, Qiang; Xu, Haiyan; Song, Yuqin; Zhang, Heping

2014-06-09

Economically, Leuconostoc lactis is one of the most important species in the genus Leuconostoc. It plays an important role in the food industry including the production of dextrans and bacteriocins. Currently, traditional molecular typing approaches for characterisation of this species at the isolate level are either unavailable or are not sufficiently reliable for practical use. Multilocus sequence typing (MLST) is a robust and reliable method for characterising bacterial and fungal species at the molecular level. In this study, a novel MLST protocol was developed for 50 L. lactis isolates from Mongolia and China. Sequences from eight targeted genes (groEL, carB, recA, pheS, murC, pyrG, rpoB and uvrC) were obtained. Sequence analysis indicated 20 different sequence types (STs), with 13 of them being represented by a single isolate. Phylogenetic analysis based on the sequences of eight MLST loci indicated that the isolates belonged to two major groups, A (34 isolates) and B (16 isolates). Linkage disequilibrium analyses indicated that recombination occurred at a low frequency in L. lactis, indicating a clonal population structure. Split-decomposition analysis indicated that intraspecies recombination played a role in generating genotypic diversity amongst isolates. Our results indicated that MLST is a valuable tool for typing L. lactis isolates that can be used for further monitoring of evolutionary changes and population genetics.

Multilocus Sequence Typing of Historical Burkholderia pseudomallei Isolates Collected in Southeast Asia from 1964 to 1967 Provides Insight into the Epidemiology of Melioidosis

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McCombie, Roberta L.; Finkelstein, Richard A.; Woods, Donald E.

2006-01-01

A collection of 207 historically relevant Burkholderia pseudomallei isolates was analyzed by multilocus sequence typing (MLST). The strain collection contains environmental isolates obtained from a geographical distribution survey of B. pseudomallei isolates in Thailand (1964 to 1967), as well as stock cultures and colony variants from the U.S. Army Medical Research Unit (Malaysia), the Walter Reed Army Institute for Research, and the Pasteur Institute (Vietnam). The 207 isolates of the colle...

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

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Guinard, Jérémy; Latreille, Anne; Guérin, Fabien; Poussier, Stéphane

2016-01-01

ABSTRACT Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations. IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the

Multilocus sequence typing as a replacement for serotyping in Salmonella enterica.

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Mark Achtman

Full Text Available Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs. The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs. Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

Rank-based characterization of pollen assemblages collected by honey bees using a multi-locus metabarcoding approach1

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Richardson, Rodney T.; Lin, Chia-Hua; Quijia, Juan O.; Riusech, Natalia S.; Goodell, Karen; Johnson, Reed M.

2015-01-01

Premise of the study: Difficulties inherent in microscopic pollen identification have resulted in limited implementation for large-scale studies. Metabarcoding, a relatively novel approach, could make pollen analysis less onerous; however, improved understanding of the quantitative capacity of various plant metabarcode regions and primer sets is needed to ensure that such applications are accurate and precise. Methods and Results: We applied metabarcoding, targeting the ITS2, matK, and rbcL loci, to characterize six samples of pollen collected by honey bees, Apis mellifera. Additionally, samples were analyzed by light microscopy. We found significant rank-based associations between the relative abundance of pollen types within our samples as inferred by the two methods. Conclusions: Our findings suggest metabarcoding data from plastid loci, as opposed to the ribosomal locus, are more reliable for quantitative characterization of pollen assemblages. Furthermore, multilocus metabarcoding of pollen may be more reliable than single-locus analyses, underscoring the need for discovering novel barcodes and barcode combinations optimized for molecular palynology. PMID:26649264

Evaluation of two multi-locus sequence typing schemes for commensal Escherichia coli from dairy cattle in Washington State.

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Ahmed, Sara; Besser, Thomas E; Call, Douglas R; Weissman, Scott J; Jones, Lisa P; Davis, Margaret A

2016-05-01

Multi-locus sequence typing (MLST) is a useful system for phylogenetic and epidemiological studies of multidrug-resistant Escherichiacoli. Most studies utilize a seven-locus MLST, but an alternate two-locus typing method (fumC and fimH; CH typing) has been proposed that may offer a similar degree of discrimination at lower cost. Herein, we compare CH typing to the standard seven-locus method for typing commensal E. coli isolates from dairy cattle. In addition, we evaluated alternative combinations of eight loci to identify combinations that maximize discrimination and congruence with standard seven-locus MLST among commensal E. coli while minimizing the cost. We also compared both methods when used for typing uropathogenic E. coli (UPEC). CH typing was less discriminatory for commensal E. coli than the standard seven-locus method (Simpson's Index of Diversity=0.933 [0.902-0.964] and 0.97 [0.96-0.979], respectively). Combining fimH with housekeeping gene loci improved discriminatory power for commensal E. coli from cattle but resulted in poor congruence with MLST. We found that a four-locus typing method including the housekeeping genes adk, purA, gyrB and recA could be used to minimize cost without sacrificing discriminatory power or congruence with Achtman seven-locus MLST when typing commensal E. coli. Copyright © 2016 Elsevier B.V. All rights reserved.

Major clades of Agaricales: a multilocus phylogenetic overview.

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P. Brandon Matheny; Judd M. Curtis; Valerie Hofstetter; M. Catherine Aime; Jean-Marc Moncalvo; Zai-Wei Ge; Zhu-Liang Yang; Joseph F. Ammirati; Timothy J. Baroni; Neale L. Bougher; Karen W. Lodge Hughes; Richard W. Kerrigan; Michelle T. Seidl; Aanen; Matthew Duur K. DeNitis; Graciela M. Daniele; Dennis E. Desjardin; Bradley R. Kropp; Lorelei L. Norvell; Andrew Parker; Else C. Vellinga; Rytas Vilgalys; David S. Hibbett

2006-01-01

An overview of the phylogeny of the Agaricales is presented based on a multilocus analysis of a six-gene region supermatrix. Bayesian analyses of 5611 nucleotide characters of rpb1, rpb1-intron 2, rpb2 and 18S, 25S, and 5.8S ribosomal RNA genes recovered six major clades, which are recognized informally and labeled the Agaricoid, Tricholomatoid, Marasmioid, Pluteoid,...

Does typing of Chlamydia trachomatis using housekeeping multilocus sequence typing reveal different sexual networks among heterosexuals and men who have sex with men?

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Versteeg, Bart; Bruisten, Sylvia M; van der Ende, Arie; Pannekoek, Yvonne

2016-04-18

Chlamydia trachomatis infections remain the most common bacterial sexually transmitted infection worldwide. To gain more insight into the epidemiology and transmission of C. trachomatis, several schemes of multilocus sequence typing (MLST) have been developed. We investigated the clustering of C. trachomatis strains derived from men who have sex with men (MSM) and heterosexuals using the MLST scheme based on 7 housekeeping genes (MLST-7) adapted for clinical specimens and a high-resolution MLST scheme based on 6 polymorphic genes, including ompA (hr-MLST-6). Specimens from 100 C. trachomatis infected men who have sex with men (MSM) and 100 heterosexual women were randomly selected from previous studies and sequenced. We adapted the MLST-7 scheme to a nested assay to be suitable for direct typing of clinical specimens. All selected specimens were typed using both the adapted MLST-7 scheme and the hr-MLST-6 scheme. Clustering of C. trachomatis strains derived from MSM and heterosexuals was assessed using minimum spanning tree analysis. Sufficient chlamydial DNA was present in 188 of the 200 (94 %) selected samples. Using the adapted MLST-7 scheme, full MLST profiles were obtained for 187 of 188 tested specimens resulting in a high success rate of 99.5 %. Of these 187 specimens, 91 (48.7 %) were from MSM and 96 (51.3 %) from heterosexuals. We detected 21 sequence types (STs) using the adapted MLST-7 and 79 STs using the hr-MLST-6 scheme. Minimum spanning tree analyses was used to examine the clustering of MLST-7 data, which showed no reflection of separate transmission in MSM and heterosexual hosts. Moreover, typing using the hr-MLST-6 scheme identified genetically related clusters within each of clusters that were identified by using the MLST-7 scheme. No distinct transmission of C. trachomatis could be observed in MSM and heterosexuals using the adapted MLST-7 scheme in contrast to using the hr-MLST-6. In addition, we compared clustering of both MLST schemes and

Multilocus DNA fingerprints in gallinaceous birds: general approach and problems.

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Hanotte, O; Bruford, M W; Burke, T

1992-06-01

Multilocus profiles were investigated in five different species of Galliformes (ring-necked pheasant Phasianus colchicus, Indian peafowl Pavo cristatus, Japanese quail Coturnix coturnix japonica, domestic chicken Gallus gallus, and red grouse Lagopus lagopus scoticus) using two human multilocus probes (33.6 and 33.15) in combination with each of four restriction enzymes (AluI, DdeI, HaeIII or HinfI). All the species show a DNA fingerprint-like pattern using at least one restriction enzyme in combination with each multilocus probe. The number of bands detected and the value of the index of similarity for each species differ significantly between the profiles obtained with each multilocus probe. Some enzyme/probe combinations reveal strong cross-hybridization of the multilocus probes with satellite or satellite-like DNA sequences in pheasant, peacock, quail and chicken, which partially or completely prevented scoring of the profile. The choice of restriction enzyme was found to influence the number of bands, the value of the index of similarity and the probability of obtaining an identical fingerprint between unrelated individuals. The Mendelian inheritance and independent segregation of the fragments detected using AluI was investigated in three species (ring-necked pheasant, Indian peafowl and red grouse). Some bands were shown to be tightly linked. An extreme case was encountered in the red grouse, where 12 of the 15 bands scored in one parent represented only two, apparently allelic, haplotypes and so derived from a single locus. However, fingerprint patterns will often be adequate for use in paternity analyses, such as in behavioural studies, despite the occurrence of haplotypic sets of bands. Identical DNA multilocus profiles were sometimes observed between captive-bred siblings in one species. These results emphasize the desirability of determining, in each new species, the optimal experimental conditions as a preliminary to any behavioural or population

Multilocus inference of species trees and DNA barcoding.

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Mallo, Diego; Posada, David

2016-09-05

The unprecedented amount of data resulting from next-generation sequencing has opened a new era in phylogenetic estimation. Although large datasets should, in theory, increase phylogenetic resolution, massive, multilocus datasets have uncovered a great deal of phylogenetic incongruence among different genomic regions, due both to stochastic error and to the action of different evolutionary process such as incomplete lineage sorting, gene duplication and loss and horizontal gene transfer. This incongruence violates one of the fundamental assumptions of the DNA barcoding approach, which assumes that gene history and species history are identical. In this review, we explain some of the most important challenges we will have to face to reconstruct the history of species, and the advantages and disadvantages of different strategies for the phylogenetic analysis of multilocus data. In particular, we describe the evolutionary events that can generate species tree-gene tree discordance, compare the most popular methods for species tree reconstruction, highlight the challenges we need to face when using them and discuss their potential utility in barcoding. Current barcoding methods sacrifice a great amount of statistical power by only considering one locus, and a transition to multilocus barcodes would not only improve current barcoding methods, but also facilitate an eventual transition to species-tree-based barcoding strategies, which could better accommodate scenarios where the barcode gap is too small or inexistent.This article is part of the themed issue 'From DNA barcodes to biomes'. © 2016 The Authors.

The relation between multilocus population genetics and social evolution theory.

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Gardner, Andy; West, Stuart A; Barton, Nicholas H

2007-02-01

Evolution at multiple gene positions is complicated. Direct selection on one gene disturbs the evolutionary dynamics of associated genes. Recent years have seen the development of a multilocus methodology for modeling evolution at arbitrary numbers of gene positions with arbitrary dominance and epistatic relations, mode of inheritance, genetic linkage, and recombination. We show that the approach is conceptually analogous to social evolutionary methodology, which focuses on selection acting on associated individuals. In doing so, we (1) make explicit the links between the multilocus methodology and the foundations of social evolution theory, namely, Price's theorem and Hamilton's rule; (2) relate the multilocus approach to levels-of-selection and neighbor-modulated-fitness approaches in social evolution; (3) highlight the equivalence between genetical hitchhiking and kin selection; (4) demonstrate that the multilocus methodology allows for social evolutionary analyses involving coevolution of multiple traits and genetical associations between nonrelatives, including individuals of different species; (5) show that this methodology helps solve problems of dynamic sufficiency in social evolution theory; (6) form links between invasion criteria in multilocus systems and Hamilton's rule of kin selection; (7) illustrate the generality and exactness of Hamilton's rule, which has previously been described as an approximate, heuristic result.

Multilocus Sequence Typing and Staphylococcal Protein A Typing Revealed Novel and Diverse Clones of Methicillin-Resistant Staphylococcus aureus in Seafood and the Aquatic Environment.

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Murugadas, V; Toms, C Joseph; Reethu, Sara A; Lalitha, K V

2017-03-01

Methicillin-resistant Staphylococcus aureus (MRSA) has been a global health concern since the 1960s, and isolation of this pathogen from food-producing animals has been increasing. However, little information is available on the prevalence of MRSA and its clonal characteristics in seafood and the aquatic environment. In this study, 267 seafood and aquatic environment samples were collected from three districts of Kerala, India. Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) was performed for 65 MRSA strains isolated from 20 seafood and aquatic environment samples. The MRSA clonal profiles were t657-ST772, t002-ST5, t334-ST5, t311-ST5, t121-ST8, t186-ST88, t127-ST1, and two non-spa assignable strains. Whole spa gene sequence analysis along with MLST confirmed one strain as t711-ST6 and another as a novel MRSA clone identified for the first time in seafood and the aquatic environment with a t15669 spa type and a new MLST profile of ST420-256-236-66-82-411-477. The MRSA strains were clustered into five clonal complexes based on the goeBURST algorithm, indicating high diversity among MRSA strains in seafood and the aquatic environment. The novel clone formed a separate clonal complex with matches to three loci. This study recommends large-scale spa typing and MLST of MRSA isolates from seafood and the aquatic environment to determine the prevalence of new MRSA clones. This monitoring process can be useful for tracing local spread of MRSA isolates into the seafood production chain in a defined geographical area.

Complete Deletion of the Fucose Operon in Haemophilus influenzae Is Associated with a Cluster in Multilocus Sequence Analysis-Based Phylogenetic Group II Related to Haemophilus haemolyticus: Implications for Identification and Typing.

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de Gier, Camilla; Kirkham, Lea-Ann S; Nørskov-Lauritsen, Niels

2015-12-01

Nonhemolytic variants of Haemophilus haemolyticus are difficult to differentiate from Haemophilus influenzae despite a wide difference in pathogenic potential. A previous investigation characterized a challenging set of 60 clinical strains using multiple PCRs for marker genes and described strains that could not be unequivocally identified as either species. We have analyzed the same set of strains by multilocus sequence analysis (MLSA) and near-full-length 16S rRNA gene sequencing. MLSA unambiguously allocated all study strains to either of the two species, while identification by 16S rRNA sequence was inconclusive for three strains. Notably, the two methods yielded conflicting identifications for two strains. Most of the "fuzzy species" strains were identified as H. influenzae that had undergone complete deletion of the fucose operon. Such strains, which are untypeable by the H. influenzae multilocus sequence type (MLST) scheme, have sporadically been reported and predominantly belong to a single branch of H. influenzae MLSA phylogenetic group II. We also found evidence of interspecies recombination between H. influenzae and H. haemolyticus within the 16S rRNA genes. Establishing an accurate method for rapid and inexpensive identification of H. influenzae is important for disease surveillance and treatment. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

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Wang, Liyan; Ma, Lina; Liu, Yongan; Gao, Pengcheng; Li, Youquan; Li, Xuerui; Liu, Yongsheng

2016-10-01

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels

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Hall, Miquette; Chattaway, Marie A.; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R.

2014-01-01

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica. PMID:25339391

Development and validation of a single-tube multiple-locus variable number tandem repeat analysis for Klebsiella pneumoniae.

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Antoinette A T P Brink

Full Text Available Genotyping of Klebsiella pneumoniae is indispensable for management of nosocomial infections, monitoring of emerging strains--including extended-spectrum beta-lactamase (ESBL producers-, and general epidemiology. Such objectives require a high-resolution genotyping method with a fixed scheme that allows (1 long-term retrospective and prospective assessment, (2 objective result readout and (3 library storage for database development and exchangeable results. We have developed a multiple-locus variable number tandem repeat analysis (MLVA using a single-tube fluorescently primed multiplex PCR for 8 Variable Number Tandem Repeats (VNTRs and automated fragment size analysis. The type allocation scheme was optimized using 224 K. pneumoniae clinical isolates, which yielded 101 MLVA types. The method was compared to the gold standard multilocus sequence typing (MLST using a subset of these clinical isolates (n = 95 and found to be highly concordant, with at least as high a resolution but with considerably less hands-on time. Our results position this MLVA scheme as an appropriate, high-throughput and relatively low-cost tool for K. pneumoniae epidemiology.

A multi locus variable number of tandem repeat analysis (MLVA scheme for Streptococcus agalactiae genotyping

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Mereghetti Laurent

2011-07-01

Full Text Available Abstract Background Multilocus sequence typing (MLST is currently the reference method for genotyping Streptococcus agalactiae strains, the leading cause of infectious disease in newborns and a major cause of disease in immunocompromised children and adults. We describe here a genotyping method based on multiple locus variable number of tandem repeat (VNTR analysis (MLVA applied to a population of S. agalactiae strains of various origins characterized by MLST and serotyping. Results We studied a collection of 186 strains isolated from humans and cattle and three reference strains (A909, NEM316 and 2603 V/R. Among 34 VNTRs, 6 polymorphic VNTRs loci were selected for use in genotyping of the bacterial population. The MLVA profile consists of a series of allele numbers, corresponding to the number of repeats at each VNTR locus. 98 MLVA genotypes were obtained compared to 51 sequences types generated by MLST. The MLVA scheme generated clusters which corresponded well to the main clonal complexes obtained by MLST. However it provided a higher discriminatory power. The diversity index obtained with MLVA was 0.960 compared to 0.881 with MLST for this population of strains. Conclusions The MLVA scheme proposed here is a rapid, cheap and easy genotyping method generating results suitable for exchange and comparison between different laboratories and for the epidemiologic surveillance of S. agalactiae and analyses of outbreaks.

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

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Skurnik Mikael

2011-02-01

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

A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

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Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

2013-01-01

Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis. PMID:23359622

Development of new multilocus variable number of tandem repeat analysis (MLVA) for Listeria innocua and its application in a food processing plant.

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Takahashi, Hajime; Ohshima, Chihiro; Nakagawa, Miku; Thanatsang, Krittaporn; Phraephaisarn, Chirapiphat; Chaturongkasumrit, Yuphakhun; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

2014-01-01

Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE). The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Development of new multilocus variable number of tandem repeat analysis (MLVA for Listeria innocua and its application in a food processing plant.

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Hajime Takahashi

Full Text Available Listeria innocua is an important hygiene indicator bacterium in food industries because it behaves similar to Listeria monocytogenes, which is pathogenic to humans. PFGE is often used to characterize bacterial strains and to track contamination source. However, because PFGE is an expensive, complicated, time-consuming protocol, and poses difficulty in data sharing, development of a new typing method is necessary. MLVA is a technique that identifies bacterial strains on the basis of the number of tandem repeats present in the genome varies depending on the strains. MLVA has gained attention due to its high reproducibility and ease of data sharing. In this study, we developed a MLVA protocol to assess L. innocua and evaluated it by tracking the contamination source of L. innocua in an actual food manufacturing factory by typing the bacterial strains isolated from the factory. Three VNTR regions of the L. innocua genome were chosen for use in the MLVA. The number of repeat units in each VNTR region was calculated based on the results of PCR product analysis using capillary electrophoresis (CE. The calculated number of repetitions was compared with the results of the gene sequence analysis to demonstrate the accuracy of the CE repeat number analysis. The developed technique was evaluated using 60 L. innocua strains isolated from a food factory. These 60 strains were classified into 11 patterns using MLVA. Many of the strains were classified into ST-6, revealing that this MLVA strain type can contaminate each manufacturing process in the factory. The MLVA protocol developed in this study for L. innocua allowed rapid and easy analysis through the use of CE. This technique was found to be very useful in hygiene control in factories because it allowed us to track contamination sources and provided information regarding whether the bacteria were present in the factories.

Evaluation of a Multilocus Sequence Typing (MLST) scheme for Leishmania (Viannia) braziliensis and Leishmania (Viannia) panamensis in Colombia.

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Herrera, Giovanny; Hernández, Carolina; Ayala, Martha S; Flórez, Carolina; Teherán, Aníbal A; Ramírez, Juan David

2017-05-12

Leishmaniases are parasitic vector-borne diseases affecting more than 12 million people in 98 countries. In Colombia, leishmaniasis is widespread and the most common clinical manifestation is cutaneous, mainly caused by L. panamensis and L. braziliensis. Currently, the genetic diversity of these species in Colombia is unknown. To address this, we applied molecular techniques for their characterization, using multilocus sequence typing (MLST) to explore the genetic variability and phylodynamics of the disease. Seven previously described genetic markers were selected highlighting the implementation of a mitochondrial marker. Markers were applied to 163 samples from isolates obtained between 1980 and 2001. The identification of the samples showed an excellent correlation with typing tests previously applied (MLEE, monoclonal antibodies). Isolates of L. braziliensis showed greater genetic diversity than L. panamensis, and a greater number of diploid sequence types (DSTs). In addition, the geographical distribution of DSTs for each species were obtained through georeferencing maps. To our knowldge, this study represents the first description of the genetic variability of L. panamensis in Colombia and South America, and is the first to propose a scheme of MLST for epidemiological surveillance of leishmaniasis in the country.

Jammer Type Estimation in LTE with a Smart Jammer Repeated Game

KAUST Repository

Aziz, Farhan

2017-02-22

LTE/LTE-Advanced networks are known to be vulnerable to denial-of-service (DOS) and loss-of-service attacks from smart jammers. The interaction between the network and the smart jammer has been modeled as an infinite-horizon general-sum (non-zero-sum) Bayesian game with asymmetric information, with the network being the uninformed player. Although significant work has been done on optimal strategy computation and control of information revelation of the informed player in repeated asymmetric information games, it has been limited to zero-sum games with perfect monitoring. Recent progress on the strategy computation of the uninformed player is also limited to zero-sum games with perfect monitoring and is focused on expected payoff formulations. Since the proposed formulation is a general-sum game with imperfect monitoring, existing formulations cannot be leveraged for estimating true state of nature (the jammer type). Hence, a threat-based mechanism is proposed for the uninformed player (the network) to estimate the informed player’s type (jammer type). The proposed mechanism helps the network resolve uncertainty about the state of nature (jammer type) so that it can compute a repeated-game strategy conditioned on its estimate. The proposed algorithm does not rely on the commonly assumed “full monitoring” premise, and uses a combination of threat-based mechanism and non-parametric estimation to estimate the jammer type. In addition, it does not require any explicit feedback from the network users nor does it rely on a specific distribution (e.g., Gaussian) of test statistic. It is shown that the proposed algorithm’s estimation performance is quite robust under realistic modeling and observational constraints despite all the aforementioned challenges.

Evaluation of tandem repeats for MLVA typing of Streptococcus uberis isolated from bovine mastitis

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Lamoureux Jérémy

2006-11-01

Full Text Available Abstract Background Streptococcus uberis is a common cause of bovine mastitis and recommended control measures, based on improved milking practice, teat dipping and antibiotic treatment at drying-off, are poorly efficient against this environmental pathogen. A simple and efficient typing method would be helpful in identifying S.uberis sources, virulent strains and cow to cow transmission. The potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats for S. uberis mastitis isolates genotyping was investigated. Results The genomic sequence of Streptococcus uberis (strain 0104J was analyzed for potential variable number tandem repeats (VNTRs. Twenty-five tandem repeats were identified and amplified by PCR with DNA samples from 24 S. uberis strains. A set of seven TRs were found to be polymorphic and used for MLVA typing of 88 S. uberis isolates. A total of 82 MLVA types were obtained with 22 types among 26 strains isolated from the milk of mastitic cows belonging to our experimental herd, and 61 types for 62 epidemiologically unrelated strains, i.e. collected in different herds and areas. Conclusion The MLVA method can be applied to S. uberis genotyping and constitutes an interesting complement to existing typing methods. This method, which is easy to perform, low cost and can be used in routine, could facilitate investigations of the epidemiology of S. uberis mastitis in dairy cows.

Expression of Sme efflux pumps and multilocus sequence typing in clinical isolates of Stenotrophomonas maltophilia.

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Cho, Hye Hyun; Sung, Ji Youn; Kwon, Kye Chul; Koo, Sun Hoe

2012-01-01

Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia. In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays. The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed. The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.

Multi-locus sequence typing of Bartonella henselae isolates from three continents reveals hypervirulent and feline-associated clones.

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Mardjan Arvand

Full Text Available Bartonella henselae is a zoonotic pathogen and the causative agent of cat scratch disease and a variety of other disease manifestations in humans. Previous investigations have suggested that a limited subset of B. henselae isolates may be associated with human disease. In the present study, 182 human and feline B. henselae isolates from Europe, North America and Australia were analysed by multi-locus sequence typing (MLST to detect any associations between sequence type (ST, host species and geographical distribution of the isolates. A total of 14 sequence types were detected, but over 66% (16/24 of the isolates recovered from human disease corresponded to a single genotype, ST1, and this type was detected in all three continents. In contrast, 27.2% (43/158 of the feline isolates corresponded to ST7, but this ST was not recovered from humans and was restricted to Europe. The difference in host association of STs 1 (human and 7 (feline was statistically significant (P< or =0.001. eBURST analysis assigned the 14 STs to three clonal lineages, which contained two or more STs, and a singleton comprising ST7. These groups were broadly consistent with a neighbour-joining tree, although splits decomposition analysis was indicative of a history of recombination. These data indicate that B. henselae lineages differ in their virulence properties for humans and contribute to a better understanding of the population structure of B. henselae.

Molecular Epidemiologic Typing Systems of Bacterial Pathogens: Current Issues and Perpectives

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Struelens Marc J

1998-01-01

Full Text Available The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s ? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of molecular typing methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission and divergent, epidemiologically-unrelated isolates (arising from independent sources of infection. Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in development for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with molecular typing systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.

Serotypes, antibiotic susceptibilities, and multi-locus sequence type profiles of Streptococcus agalactiae isolates circulating in Beijing, China.

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Wang, Ping; Tong, Jing-jing; Ma, Xiu-hua; Song, Feng-li; Fan, Ling; Guo, Cui-mei; Shi, Wei; Yu, Sang-jie; Yao, Kai-hu; Yang, Yong-hong

2015-01-01

To investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of Streptococcus agalactiae (S. agalactiae) in Beijing to provide references for the prevention and treatment of S. agalactiae infections. All isolates were identified using the CAMP test and the latex-agglutination assay and serotyped using a Strep-B-Latex kit, after which they were assessed for antibiotic susceptibility, macrolide-resistance genes, and MLST profiles. In total, 56 S. agalactiae isolates were identified in 863 pregnant women (6.5%). Serotypes Ia, Ib, II, III, and V were identified, among which types III (32.1%), Ia (17.9%), Ib (16.1%), and V (14.3%) were the predominant serotypes. All isolates were susceptible to penicillin and ceftriaxone. The nonsusceptiblity rates measured for erythromycin, clarithromycin, azithromycin, telithromycin, clindamycin, tetracycline, and levofloxacin were 85.7%, 92.9%, 98.2%, 30.4%, 73.2%, 91%, and 39.3%, respectively. We identified 14 sequence types (STs) for the 56 isolates, among which ST19 (30.4%) was predominant. The rate of fluoroquinolone resistance was higher in serotype III than in the other serotypes. Among the 44 erythromycin-resistant isolates, 32 (72.7%) carried ermB. S. agalactiae isolates of the serotypes Ia, Ib, III, and V are common in Beijing. Among the S. agalactiae isolates, the macrolide and clindamycin resistance rates are extremely high. Most of the erythromycin-resistant isolates carry ermB.

Multilocus Sequence Typing and Virulence Profiles in Uropathogenic Escherichia coli Isolated from Cats in the United States.

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Xiaoqiang Liu

Full Text Available The population structure, virulence, and antimicrobial resistance of uropathogenic E. coli (UPEC from cats are rarely characterized. The aim of this study was to compare and characterize the UPEC isolated from cats in four geographic regions of USA in terms of their multilocus sequence typing (MLST, virulence profiles, clinical signs, antimicrobial resistance and phylogenetic grouping. The results showed that a total of 74 E. coli isolates were typed to 40 sequence types with 10 being novel. The most frequent phylogenetic group was B2 (n = 57. The most frequent sequence types were ST73 (n = 12 and ST83 (n = 6, ST73 was represented by four multidrug resistant (MDR and eight non-multidrug resistant (SDR isolates, and ST83 were significantly more likely to exhibit no drug resistant (NDR isolates carrying the highest number of virulence genes. Additionally, MDR isolates were more diverse, and followed by SDR and NDR isolates in regards to the distribution of the STs. afa/draBC was the most prevalent among the 29 virulence-associated genes. Linking virulence profile and antimicrobial resistance, the majority of virulence-associated genes tested were more prevalent in NDR isolates, and followed by SDR and MDR isolates. Twenty (50% MLST types in this study have previously been associated with human isolates, suggesting that these STs are potentially zoonotic. Our data enhanced the understanding of E. coli population structure and virulence association from cats. The diverse and various combinations of virulence-associated genes implied that the infection control may be challenging.

Y-Chromosome short tandem repeat, typing technology, locus ...

African Journals Online (AJOL)

Aghomotsegin

2015-07-08

Jul 8, 2015 ... Y-Chromosome short tandem repeat, typing technology, locus information and allele frequency in different population: A review. Muhanned Abdulhasan Kareem1, Ameera Omran Hussein2 and Imad Hadi Hameed2*. 1Babylon University, Centre of Environmental Research, Hilla City, Iraq. 2Department of ...

Zinc Resistance within Swine-Associated Methicillin-Resistant Staphylococcus aureus Isolates in the United States Is Associated with Multilocus Sequence Type Lineage.

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Hau, Samantha J; Frana, Timothy; Sun, Jisun; Davies, Peter R; Nicholson, Tracy L

2017-08-01

Zinc resistance in livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence type 398 (ST398) is primarily mediated by the czrC gene colocated with the mecA gene, encoding methicillin resistance, within the type V staphylococcal cassette chromosome mec (SCC mec ) element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of zinc in feed as an antidiarrheal agent has the potential to contribute to the emergence and spread of methicillin-resistant S. aureus (MRSA) in swine, through increased selection pressure to maintain the SCC mec element in isolates obtained from pigs. In this study, we report the prevalence of the czrC gene and phenotypic zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and U.S. swine-associated LA-MRSA ST398 isolates. We demonstrated that the prevalence of zinc resistance in U.S. swine-associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact and swine-associated LA-MRSA ST398 isolates, as well as prevalences from previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively, our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the U.S. swine population. Additionally, our data indicate that zinc resistance is associated with the multilocus sequence type lineage, suggesting a potential link between the genetic lineage and the carriage of resistance determinants. IMPORTANCE Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST5 in the U.S. swine population. Additionally, our data indicate

Core Genome Multilocus Sequence Typing Scheme for Stable, Comparative Analyses of Campylobacter jejuni and C. coli Human Disease Isolates.

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Cody, Alison J; Bray, James E; Jolley, Keith A; McCarthy, Noel D; Maiden, Martin C J

2017-07-01

Human campylobacteriosis, caused by Campylobacter jejuni and C. coli , remains a leading cause of bacterial gastroenteritis in many countries, but the epidemiology of campylobacteriosis outbreaks remains poorly defined, largely due to limitations in the resolution and comparability of isolate characterization methods. Whole-genome sequencing (WGS) data enable the improvement of sequence-based typing approaches, such as multilocus sequence typing (MLST), by substantially increasing the number of loci examined. A core genome MLST (cgMLST) scheme defines a comprehensive set of those loci present in most members of a bacterial group, balancing very high resolution with comparability across the diversity of the group. Here we propose a set of 1,343 loci as a human campylobacteriosis cgMLST scheme (v1.0), the allelic profiles of which can be assigned to core genome sequence types. The 1,343 loci chosen were a subset of the 1,643 loci identified in the reannotation of the genome sequence of C. jejuni isolate NCTC 11168, chosen as being present in >95% of draft genomes of 2,472 representative United Kingdom campylobacteriosis isolates, comprising 2,207 (89.3%) C. jejuni isolates and 265 (10.7%) C. coli isolates. Validation of the cgMLST scheme was undertaken with 1,478 further high-quality draft genomes, containing 150 or fewer contiguous sequences, from disease isolate collections: 99.5% of these isolates contained ≥95% of the 1,343 cgMLST loci. In addition to the rapid and effective high-resolution analysis of large numbers of diverse isolates, the cgMLST scheme enabled the efficient identification of very closely related isolates from a well-defined single-source campylobacteriosis outbreak. Copyright © 2017 Cody et al.

A fast multilocus test with adaptive SNP selection for large-scale genetic-association studies

KAUST Repository

Zhang, Han

2013-09-11

As increasing evidence suggests that multiple correlated genetic variants could jointly influence the outcome, a multilocus test that aggregates association evidence across multiple genetic markers in a considered gene or a genomic region may be more powerful than a single-marker test for detecting susceptibility loci. We propose a multilocus test, AdaJoint, which adopts a variable selection procedure to identify a subset of genetic markers that jointly show the strongest association signal, and defines the test statistic based on the selected genetic markers. The P-value from the AdaJoint test is evaluated by a computationally efficient algorithm that effectively adjusts for multiple-comparison, and is hundreds of times faster than the standard permutation method. Simulation studies demonstrate that AdaJoint has the most robust performance among several commonly used multilocus tests. We perform multilocus analysis of over 26,000 genes/regions on two genome-wide association studies of pancreatic cancer. Compared with its competitors, AdaJoint identifies a much stronger association between the gene CLPTM1L and pancreatic cancer risk (6.0 × 10(-8)), with the signal optimally captured by two correlated single-nucleotide polymorphisms (SNPs). Finally, we show AdaJoint as a powerful tool for mapping cis-regulating methylation quantitative trait loci on normal breast tissues, and find many CpG sites whose methylation levels are jointly regulated by multiple SNPs nearby.

Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing

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Pruckler James M

2008-11-01

Full Text Available Abstract Background Bacillus cereus is most commonly associated with foodborne illness (diarrheal and emetic but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST schemes have recently been developed to genotype B. cereus and analysis has suggested a clonal or weakly clonal population structure for B. cereus and its close relatives B. anthracis and B. thuringiensis. In this study we used MLST to determine if B. cereus isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI illness were clonal or formed clonal complexes. Results A retrospective analysis of 55 clinical B. cereus isolates submitted to the Centers for Disease Control and Prevention between 1954 and 2004 was conducted. Clinical isolates from severe infections (n = 27, gastrointestinal (GI illness (n = 18, and associated isolates from food (n = 10 were selected for analysis using MLST. The 55 isolates were diverse and comprised 38 sequence types (ST in two distinct clades. Of the 27 isolates associated with serious illness, 13 clustered in clade 1 while 14 were in clade 2. Isolates associated with GI illness were also found throughout clades 1 and 2, while no isolates in this study belonged to clade 3. All the isolates from this study belonging to the clade 1/cereus III lineage were associated with severe disease while isolates belonging to clade1/cereus II contained isolates primarily associated with severe disease and emetic illness. Only three STs were observed more than once for epidemiologically distinct isolates. Conclusion STs of clinical B. cereus isolates were phylogenetically diverse and distributed among two of three previously described clades. Greater numbers of strains will need to be analyzed to confirm if specific lineages or clonal complexes are more likely to contain clinical isolates or be associated with specific illness, similar to B. anthracis and

Multilocus sequence typing (MLST methods for the emerging Campylobacter species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus and C. curvus

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William G Miller

2012-04-01

Full Text Available Multilocus sequence typing (MLST systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g. C. jejuni, C. coli, C. lari and C. fetus to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g. C. jejuni or veterinary (e.g. C. fetus relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal or human clinical samples. We describe herein MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt. Multiple food animal and human clinical C. hyointestinalis (n=48, C. lanienae (n=34 and C. sputorum (n=24 isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types (STs were identified using all four MLST methods. Similar to Campylobacter MLST methods described previously, these novel MLST methods identified mixed isolates containing two or more strains of the same species. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in speciating and differentiating strains of multiple, emerging Campylobacter species.

Optimization of analytical parameters for inferring relationships among Escherichia coli isolates from repetitive-element PCR by maximizing correspondence with multilocus sequence typing data.

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Goldberg, Tony L; Gillespie, Thomas R; Singer, Randall S

2006-09-01

Repetitive-element PCR (rep-PCR) is a method for genotyping bacteria based on the selective amplification of repetitive genetic elements dispersed throughout bacterial chromosomes. The method has great potential for large-scale epidemiological studies because of its speed and simplicity; however, objective guidelines for inferring relationships among bacterial isolates from rep-PCR data are lacking. We used multilocus sequence typing (MLST) as a "gold standard" to optimize the analytical parameters for inferring relationships among Escherichia coli isolates from rep-PCR data. We chose 12 isolates from a large database to represent a wide range of pairwise genetic distances, based on the initial evaluation of their rep-PCR fingerprints. We conducted MLST with these same isolates and systematically varied the analytical parameters to maximize the correspondence between the relationships inferred from rep-PCR and those inferred from MLST. Methods that compared the shapes of densitometric profiles ("curve-based" methods) yielded consistently higher correspondence values between data types than did methods that calculated indices of similarity based on shared and different bands (maximum correspondences of 84.5% and 80.3%, respectively). Curve-based methods were also markedly more robust in accommodating variations in user-specified analytical parameter values than were "band-sharing coefficient" methods, and they enhanced the reproducibility of rep-PCR. Phylogenetic analyses of rep-PCR data yielded trees with high topological correspondence to trees based on MLST and high statistical support for major clades. These results indicate that rep-PCR yields accurate information for inferring relationships among E. coli isolates and that accuracy can be enhanced with the use of analytical methods that consider the shapes of densitometric profiles.

Multi-locus sequence typing provides epidemiological insights for diseased sharks infected with fungi belonging to the Fusarium solani species complex.

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Desoubeaux, Guillaume; Debourgogne, Anne; Wiederhold, Nathan P; Zaffino, Marie; Sutton, Deanna; Burns, Rachel E; Frasca, Salvatore; Hyatt, Michael W; Cray, Carolyn

2018-07-01

Fusarium spp. are saprobic moulds that are responsible for severe opportunistic infections in humans and animals. However, we need epidemiological tools to reliably trace the circulation of such fungal strains within medical or veterinary facilities, to recognize environmental contaminations that might lead to infection and to improve our understanding of factors responsible for the onset of outbreaks. In this study, we used molecular genotyping to investigate clustered cases of Fusarium solani species complex (FSSC) infection that occurred in eight Sphyrnidae sharks under managed care at a public aquarium. Genetic relationships between fungal strains were determined by multi-locus sequence typing (MLST) analysis based on DNA sequencing at five loci, followed by comparison with sequences of 50 epidemiologically unrelated FSSC strains. Our genotyping approach revealed that F. keratoplasticum and F. solani haplotype 9x were most commonly isolated. In one case, the infection proved to be with another Hypocrealian rare opportunistic pathogen Metarhizium robertsii. Twice, sharks proved to be infected with FSSC strains with the same MLST sequence type, supporting the hypothesis the hypothesis that common environmental populations of fungi existed for these sharks and would suggest the longtime persistence of the two clonal strains within the environment, perhaps in holding pools and life support systems of the aquarium. This study highlights how molecular tools like MLST can be used to investigate outbreaks of microbiological disease. This work reinforces the need for regular controls of water quality to reduce microbiological contamination due to waterborne microorganisms.

Distribution and factors associated with Salmonella enterica genotypes in a diverse population of humans and animals in Qatar using multi-locus sequence typing (MLST).

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Chang, Yu C; Scaria, Joy; Ibraham, Mariamma; Doiphode, Sanjay; Chang, Yung-Fu; Sultan, Ali; Mohammed, Hussni O

2016-01-01

Salmonella enterica is one of the most commonly reported causes of bacterial foodborne illness around the world. Understanding the sources of this pathogen and the associated factors that exacerbate its risk to humans will help in developing risk mitigation strategies. The genetic relatedness among Salmonella isolates recovered from human gastroenteritis cases and food animals in Qatar were investigated in the hope of shedding light on these sources, their possible transmission routes, and any associated factors. A repeat cross-sectional study was conducted in which the samples and associated data were collected from both populations (gastroenteritis cases and animals). Salmonella isolates were initially analyzed using multi-locus sequence typing (MLST) to investigate the genetic diversity and clonality. The relatedness among the isolates was assessed using the minimum spanning tree (MST). Twenty-seven different sequence types (STs) were identified in this study; among them, seven were novel, including ST1695, ST1696, ST1697, ST1698, ST1699, ST1702, and ST1703. The pattern of overall ST distribution was diverse; in particular, it was revealed that ST11 and ST19 were the most common sequence types, presenting 29.5% and 11.5% within the whole population. In addition, 20 eBurst Groups (eBGs) were identified in our data, which indicates that ST11 and ST19 belonged to eBG4 and eBG1, respectively. In addition, the potential association between the putative risk factors and eBGs were evaluated. There was no significant clustering of these eBGs by season; however, a significant association was identified in terms of nationality in that Qataris were six times more likely to present with eBG1 compared to non-Qataris. In the MST analysis, four major clusters were presented, namely, ST11, ST19, ST16, and ST31. The linkages between the clusters alluded to a possible transmission route. The results of the study have provided insight into the ST distributions of S. enterica and

Genome-scale multilocus microsatellite typing of Trypanosoma cruzi discrete typing unit I reveals phylogeographic structure and specific genotypes linked to human infection.

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Martin S Llewellyn

2009-05-01

Full Text Available Trypanosoma cruzi is the most important parasitic infection in Latin America and is also genetically highly diverse, with at least six discrete typing units (DTUs reported: Tc I, IIa, IIb, IIc, IId, and IIe. However, the current six-genotype classification is likely to be a poor reflection of the total genetic diversity present in this undeniably ancient parasite. To determine whether epidemiologically important information is "hidden" at the sub-DTU level, we developed a 48-marker panel of polymorphic microsatellite loci to investigate population structure among 135 samples from across the geographic distribution of TcI. This DTU is the major cause of resurgent human disease in northern South America but also occurs in silvatic triatomine vectors and mammalian reservoir hosts throughout the continent. Based on a total dataset of 12,329 alleles, we demonstrate that silvatic TcI populations are extraordinarily genetically diverse, show spatial structuring on a continental scale, and have undergone recent biogeographic expansion into the southern United States of America. Conversely, the majority of human strains sampled are restricted to two distinct groups characterised by a considerable reduction in genetic diversity with respect to isolates from silvatic sources. In Venezuela, most human isolates showed little identity with known local silvatic strains, despite frequent invasion of the domestic setting by infected adult vectors. Multilocus linkage indices indicate predominantly clonal parasite propagation among all populations. However, excess homozygosity among silvatic strains and raised heterozygosity among domestic populations suggest that some level of genetic recombination cannot be ruled out. The epidemiological significance of these findings is discussed.

Microsatellite instability at a tetranucleotide repeat in type I endometrial carcinoma

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Choi Ho

2008-12-01

Full Text Available Abstract Background Microsatellite instability (MSI at tri- or tetranucleotide repeat markers (elevated microsatellite alterations at selected tetranucleotide repeat, EMAST has been recently described. But, the underlying genetic mechanism of EMAST is unclear. This study was to investigate the prevalence of EMAST, in type I endometrial carcinoma, and to determine the correlation between the MSI status and mismatch repair genes (MMR or p53. Methods We examined the 3 mono-, 3 di-, and 6 tetranucleotide repeat markers by PCR in 39 cases of type I endometrial carcinoma and performed the immunohistochemistry of hMSH2, hMLH1, and p53 protein. Results More than two MSI at mono- and dinucleotide repeat markers was noted in 8 cases (MSI-H, 20.5%. MSI, at a tetranucleotide repeat, was detected in 15 cases (EMAST, 38.5%. In remaining 16 cases, any MSI was not observed. (MSS, 42.1%, MSI status was not associated with FIGO stage, grade or depth of invasion. The absence of expression of either one of both hMSH2 or hMLH1 was noted in seven (87.5% of eight MSI-H tumors, one (6.3% of 16 MSS tumors, and five (33.3% of 15 EMAST tumors. (p = 0.010 The expression of p53 protein was found in one (12.5% of eight MSI-H tumors, five (31.3% of 16 MSS tumors, and seven of 15 EMAST tumors. (p = 0.247 Conclusion Our results showed that about 38.5% of type I endometrial carcinomas exhibited EMAST, and that EMAST was rarely associated with alteration of hMSH2 or hMLH1.

Internalin profiling and multilocus sequence typing suggest four Listeria innocua subgroups with different evolutionary distances from Listeria monocytogenes.

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Chen, Jianshun; Chen, Qiaomiao; Jiang, Lingli; Cheng, Changyong; Bai, Fan; Wang, Jun; Mo, Fan; Fang, Weihuan

2010-03-31

Ecological, biochemical and genetic resemblance as well as clear differences of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as a model to examine evolution of pathogenicity. This study was attempted to examine the population structure of L. innocua and the microevolution in the L. innocua-L. monocytogenes clade via profiling of 37 internalin genes and multilocus sequence typing based on the sequences of 9 unlinked genes gyrB, sigB, dapE, hisJ, ribC, purM, gap, tuf and betL. L. innocua was genetically monophyletic compared to L. monocytogenes, and comprised four subgroups. Subgroups A and B correlated with internalin types 1 and 3 (except the strain 0063 belonging to subgroup C) and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. Subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed higher recombination rates than those of subgroup B, including the relative frequency of occurrence of recombination versus mutation (rho/theta) and the relative effect of recombination versus point mutation (r/m). Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. The phylogram based on the analysis with correction for recombination revealed that the time to the most recent common ancestor (TMRCA) of L. innocua subgroups A and B were similar. Additionally, subgroup D, which correlated with internalin type 5, branched off from the other three subgroups. All L. innocua strains lacked seventeen virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ and two subgroup B strains bearing bsh) and were nonpathogenic to mice. L. innocua represents a young species descending from L. monocytogenes and comprises four subgroups: two major subgroups A and B

Internalin profiling and multilocus sequence typing suggest four Listeria innocua subgroups with different evolutionary distances from Listeria monocytogenes

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2010-01-01

Background Ecological, biochemical and genetic resemblance as well as clear differences of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as a model to examine evolution of pathogenicity. This study was attempted to examine the population structure of L. innocua and the microevolution in the L. innocua-L. monocytogenes clade via profiling of 37 internalin genes and multilocus sequence typing based on the sequences of 9 unlinked genes gyrB, sigB, dapE, hisJ, ribC, purM, gap, tuf and betL. Results L. innocua was genetically monophyletic compared to L. monocytogenes, and comprised four subgroups. Subgroups A and B correlated with internalin types 1 and 3 (except the strain 0063 belonging to subgroup C) and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. Subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed higher recombination rates than those of subgroup B, including the relative frequency of occurrence of recombination versus mutation (ρ/θ) and the relative effect of recombination versus point mutation (r/m). Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. The phylogram based on the analysis with correction for recombination revealed that the time to the most recent common ancestor (TMRCA) of L. innocua subgroups A and B were similar. Additionally, subgroup D, which correlated with internalin type 5, branched off from the other three subgroups. All L. innocua strains lacked seventeen virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ and two subgroup B strains bearing bsh) and were nonpathogenic to mice. Conclusions L. innocua represents a young species descending from L. monocytogenes and comprises four subgroups: two

Correlation between Ureaplasma subgroup 2 and genitourinary tract disease outcomes revealed by an expanded multilocus sequence typing (eMLST) scheme.

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Zhang, Jun; Kong, Yingying; Ruan, Zhi; Huang, Jun; Song, Tiejun; Song, Jingjuan; Jiang, Yan; Yu, Yunsong; Xie, Xinyou

2014-01-01

The multilocus sequence typing (MLST) scheme of Ureaplasma based on four housekeeping genes (ftsH, rpL22, valS, and thrS) was described in our previous study; here we introduced an expanded MLST (eMLST) scheme with improved discriminatory power, which was developed by adding two putative virulence genes (ureG and mba-np1) to the original MLST scheme. To evaluate the discriminatory power of eMLST, a total of 14 reference strains of Ureaplasma serovars and 269 clinical strains (134 isolated from symptomatic patients and 135 obtained from asymptomatic persons) were investigated. Our study confirmed that all 14 serotype strains could successfully be differentiated into 14 eMLST STs (eSTs), while some of them could not even be differentiated by the MLST, and a total of 136 eSTs were identified among the clinical isolates we investigated. In addition, phylogenetic analysis indicated that two genetically significantly distant clusters (cluster I and II) were revealed and most clinical isolates were located in cluster I. These findings were in accordance with and further support for the concept of two well-known genetic lineages (Ureaplasma parvum and Ureaplasma urealyticum) in our previous study. Interestingly, although both clusters were associated with clinical manifestation, the sub-group 2 of cluster II had pronounced and adverse effect on patients and might be a potential risk factor for clinical outcomes. In conclusion, the eMLST scheme offers investigators a highly discriminative typing tool that is capable for precise epidemiological investigations and clinical relevance of Ureaplasma.

Correlation between Ureaplasma subgroup 2 and genitourinary tract disease outcomes revealed by an expanded multilocus sequence typing (eMLST scheme.

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Jun Zhang

Full Text Available The multilocus sequence typing (MLST scheme of Ureaplasma based on four housekeeping genes (ftsH, rpL22, valS, and thrS was described in our previous study; here we introduced an expanded MLST (eMLST scheme with improved discriminatory power, which was developed by adding two putative virulence genes (ureG and mba-np1 to the original MLST scheme. To evaluate the discriminatory power of eMLST, a total of 14 reference strains of Ureaplasma serovars and 269 clinical strains (134 isolated from symptomatic patients and 135 obtained from asymptomatic persons were investigated. Our study confirmed that all 14 serotype strains could successfully be differentiated into 14 eMLST STs (eSTs, while some of them could not even be differentiated by the MLST, and a total of 136 eSTs were identified among the clinical isolates we investigated. In addition, phylogenetic analysis indicated that two genetically significantly distant clusters (cluster I and II were revealed and most clinical isolates were located in cluster I. These findings were in accordance with and further support for the concept of two well-known genetic lineages (Ureaplasma parvum and Ureaplasma urealyticum in our previous study. Interestingly, although both clusters were associated with clinical manifestation, the sub-group 2 of cluster II had pronounced and adverse effect on patients and might be a potential risk factor for clinical outcomes. In conclusion, the eMLST scheme offers investigators a highly discriminative typing tool that is capable for precise epidemiological investigations and clinical relevance of Ureaplasma.

Multilocus sequence typing of Xylella fastidiosa isolated from olive affected by “olive quick decline syndrome” in Italy

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Toufic ELBEAINO

2015-01-01

Full Text Available The recent finding of Xylella fastidiosa (Xf in olive trees in southern Italy, the scanty molecular information on this bacterium and its association with the olive quick decline syndrome (OQDS prompted the necessity to isolate and acquire more genetic data on the type of strain present in that region. For the first time, the bacterium was isolated from infected olive on culture media. Genetic information were obtained through genomic comparison with other subspecies or strains. The sequences of thirteen genes from its genome, comprising seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT usually used in multilocus sequence typing (MLST systems, and six genes involved in different biochemical functions (RNA Pol sigma-70 factor, hypothetical protein HL, 16S rRNA, rfbD, nuoN, and pilU, were analyzed. The sequences of the biochemical function genes were explored  individually to study the genetic structure of this bacterium, while the MLST genes were linked together into one concatameric sequence (4161 bp long to increase the resolution of the phylogenetic analysis when compared with Xf strains previously reported. Sequence analyses of single genes showed that the Xf olive strain is distinct from the four previously defined taxons (Xf subsp. fastidiosa, Xf subsp. multiplex, Xf subsp. sandyi and Xf subsp. pauca with a dissimilarity rate that reached 4%. In particular, Xf from olive shared the greatest identity with the strain “9a5c” (subsp. pauca, but was nevertheless distinct from it. Similarly, the MLST based on concatameric sequences confirmed the genetic variance of Xf from olive by generating a novel sequence type profile (ST53. Phylogenetic tree analyses showed that Xf from olive clustered in one clade close to subspecies pauca (strains “9a5c” and “CVC0018”, but was nevertheless distinct from them. These results indicate molecular divergence of this olive bacterium with all other strains yet reported.

Taxonomic evaluation of putative Streptomyces scabiei strains held in the ARS (NRRL) Culture Collection using multi-locus sequence analysis

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Multi-locus sequence analysis has been demonstrated to be a useful tool for identification of Streptomyces species and was previously applied to phylogenetically differentiate the type strains of species pathogenic on potatoes (Solanum tuberosum L.). The ARS Culture Collection (NRRL) contains 43 str...

Multilocus sequence typing of Lactococcus lactis from naturally fermented milk foods in ethnic minority areas of China.

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Xu, Haiyan; Sun, Zhihong; Liu, Wenjun; Yu, Jie; Song, Yuqin; Lv, Qiang; Zhang, Jiachao; Shao, Yuyu; Menghe, Bilige; Zhang, Heping

2014-05-01

To determine the genetic diversity and phylogenetic relationships among Lactococcus lactis isolates, 197 strains isolated from naturally homemade yogurt in 9 ethnic minority areas of 6 provinces of China were subjected to multilocus sequence typing (MLST). The MLST analysis was performed using internal fragment sequences of 12 housekeeping genes (carB, clpX, dnaA, groEL, murC, murE, pepN, pepX, pyrG, recA, rpoB, and pheS). Six (dnaA) to 8 (murC) different alleles were detected for these genes, which ranged from 33.62 (clpX) to 41.95% (recA) GC (guanine-cytosine) content. The nucleotide diversity (π) ranged from 0.00362 (murE) to 0.08439 (carB). Despite this limited allelic diversity, the allele combinations of each strain revealed 72 different sequence types, which denoted significant genotypic diversity. The dN/dS ratios (where dS is the number of synonymous substitutions per synonymous site, and dN is the number of nonsynonymous substitutions per nonsynonymous site) were lower than 1, suggesting potential negative selection for these genes. The standardized index of association of the alleles IA(S)=0.3038 supported the clonality of Lc. lactis, but the presence of network structure revealed by the split decomposition analysis of the concatenated sequence was strong evidence for intraspecies recombination. Therefore, this suggests that recombination contributed to the evolution of Lc. lactis. A minimum spanning tree analysis of the 197 isolates identified 14 clonal complexes and 23 singletons. Phylogenetic trees were constructed based on the sequence types, using the minimum evolution algorithm, and on the concatenated sequence (6,192 bp), using the unweighted pair-group method with arithmetic mean, and these trees indicated that the evolution of our Lc. lactis population was correlated with geographic origin. Taken together, our results demonstrated that MLST could provide a better understanding of Lc. lactis genome evolution, as well as useful information for

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

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

2013-04-01

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

Multiple-locus variant-repeat assay (MLVA) is a useful tool for molecular epidemiologic analysis of Streptococcus agalactiae strains causing bovine mastitis.

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Radtke, Andreas; Bruheim, Torkjel; Afset, Jan Egil; Bergh, Kåre

2012-06-15

Group B streptococci (GBS) were considered a major cause of mastitis in cattle until preventive measures succeeded in controlling the disease in the 1970s and 1980s. During the last 5-6 years an increasing number of cases have been observed in some Scandinavian countries. A total of 187 GBS isolates from mastitis cases were collected from 119 animals in 34 Norwegian farms in the period from April 2007 to November 2010. 133 (71%) of the isolates were from farms with automated milking systems. The strains underwent typing of capsular polysaccharides (CPS) and surface proteins, and were analyzed by multi-locus variable repeat assay (MLVA) to investigate the epidemiological relationship of strains within and between farms. The GBS strains were differentiated into 12 types by CPS and surface protein analysis, with CPS types V (54%) and IV (34%) predominating. MLVA was superior to CPS and protein typing for strain differentiation, resolving the 187 strains into 37 types. In 29 of 34 farms all GBS strains had identical MLVA profiles specific for each farm. However, in one farm represented with 48 isolates, four MLVA variants with differences in one repeat locus were observed during the almost 3-year long collection period. Similar variations were observed at four other farms. This might reflect the stability of repeat loci under in vivo conditions. Farms with automated milking systems were overrepresented in this material. In conclusion, the five-loci MLVA allowed rapid high-resolution genotyping of the bovine GBS strains within and between farms. Copyright © 2012 Elsevier B.V. All rights reserved.

Internalin profiling and multilocus sequence typing suggest four Listeria innocua subgroups with different evolutionary distances from Listeria monocytogenes

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Wang Jun

2010-03-01

Full Text Available Abstract Background Ecological, biochemical and genetic resemblance as well as clear differences of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as a model to examine evolution of pathogenicity. This study was attempted to examine the population structure of L. innocua and the microevolution in the L. innocua-L. monocytogenes clade via profiling of 37 internalin genes and multilocus sequence typing based on the sequences of 9 unlinked genes gyrB, sigB, dapE, hisJ, ribC, purM, gap, tuf and betL. Results L. innocua was genetically monophyletic compared to L. monocytogenes, and comprised four subgroups. Subgroups A and B correlated with internalin types 1 and 3 (except the strain 0063 belonging to subgroup C and internalin types 2 and 4 respectively. The majority of L. innocua strains belonged to these two subgroups. Subgroup A harbored a whole set of L. monocytogenes-L. innocua common and L. innocua-specific internalin genes, and displayed higher recombination rates than those of subgroup B, including the relative frequency of occurrence of recombination versus mutation (ρ/θ and the relative effect of recombination versus point mutation (r/m. Subgroup A also exhibited a significantly smaller exterior/interior branch length ratio than expected under the coalescent model, suggesting a recent expansion of its population size. The phylogram based on the analysis with correction for recombination revealed that the time to the most recent common ancestor (TMRCA of L. innocua subgroups A and B were similar. Additionally, subgroup D, which correlated with internalin type 5, branched off from the other three subgroups. All L. innocua strains lacked seventeen virulence genes found in L. monocytogenes (except for the subgroup D strain L43 harboring inlJ and two subgroup B strains bearing bsh and were nonpathogenic to mice. Conclusions L. innocua represents a young species descending from L. monocytogenes and

Single-Locus versus Multilocus Patterns of Local Adaptation to Climate in Eastern White Pine (Pinus strobus, Pinaceae.

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Om P Rajora

Full Text Available Natural plant populations are often adapted to their local climate and environmental conditions, and populations of forest trees offer some of the best examples of this pattern. However, little empirical work has focused on the relative contribution of single-locus versus multilocus effects to the genetic architecture of local adaptation in plants/forest trees. Here, we employ eastern white pine (Pinus strobus to test the hypothesis that it is the inter-genic effects that primarily drive climate-induced local adaptation. The genetic structure of 29 range-wide natural populations of eastern white pine was determined in relation to local climatic factors using both a reference set of SSR markers, and SNPs located in candidate genes putatively involved in adaptive response to climate. Comparisons were made between marker sets using standard single-locus outlier analysis, single-locus and multilocus environment association analyses and a novel implementation of Population Graphs. Magnitudes of population structure were similar between the two marker sets. Outlier loci consistent with diversifying selection were rare for both SNPs and SSRs. However, genetic distances based on the multilocus among population covariances (cGD were significantly more correlated to climate, even after correcting for spatial effects, for SNPs as compared to SSRs. Coalescent simulations confirmed that the differences in mutation rates between SSRs and SNPs did not affect the topologies of the Population Graphs, and hence values of cGD and their correlations with associated climate variables. We conclude that the multilocus covariances among populations primarily reflect adaptation to local climate and environment in eastern white pine. This result highlights the complexity of the genetic architecture of adaptive traits, as well as the need to consider multilocus effects in studies of local adaptation.

Prevalence of blaZ gene types and the cefazolin inoculum effect among methicillin-susceptible Staphylococcus aureus blood isolates and their association with multilocus sequence types and clinical outcome.

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Chong, Y P; Park, S-J; Kim, E S; Bang, K-M; Kim, M-N; Kim, S-H; Lee, S-O; Choi, S-H; Jeong, J-Y; Woo, J H; Kim, Y S

2015-02-01

Cefazolin treatment failures have been described for bacteraemia caused by methicillin-susceptible Staphylococcus aureus (MSSA) with type A β-lactamase and inoculum effect (InE). We investigated the prevalence of blaZ (β-lactamase) gene types and a cefazolin InE among MSSA blood isolates in South Korea and evaluated their association with specific genotypes. The clinical impact of the cefazolin InE was also evaluated. A total of 220 MSSA isolates were collected from a prospective cohort study of S. aureus bacteraemia. A pronounced InE with cefazolin was defined as a ≥4-fold increase in the minimum inhibitory concentration (MIC) between a standard and high inoculum, resulting in a non-susceptible MIC. Sequencing of blaZ and multilocus sequence typing (MLST) were performed. Clinical outcomes were assessed in 77 patients treated with cefazolin. The blaZ gene was detected in 92 % of the 220 MSSA isolates. Type C β-lactamase was the most common (53 %), followed by type B (20 %) and type A (17 %). Certain genotypes were significantly associated with specific β-lactamase types (notably, ST30 and type A β-lactamase). A pronounced cefazolin InE was observed in 13 % of isolates. Most of these (79 %) expressed type A β-lactamase and ST30 was the predominant (55 %) clone amongst them. Cefazolin treatment failure was not observed in patients infected with strains exhibiting a pronounced InE. These strains had no impact on other clinical outcomes. In conclusion, the prevalence of a pronounced InE with cefazolin could be dependent upon distributions of MSSA genotypes. Cefazolin can likely be used for the treatment of MSSA bacteraemia (except endocarditis), without consideration of an InE.

Integration of least angle regression with empirical Bayes for multi-locus genome-wide association studies

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Multi-locus genome-wide association studies has become the state-of-the-art procedure to identify quantitative trait loci (QTL) associated with traits simultaneously. However, implementation of multi-locus model is still difficult. In this study, we integrated least angle regression with empirical B...

Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples

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Ningxin Zhang

2018-06-01

Full Text Available The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS modification of the highly discriminatory C. albicans MLST (multilocus sequence typing method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type. Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to

Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria monocytogenes.

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Chen, Yi; Gonzalez-Escalona, Narjol; Hammack, Thomas S; Allard, Marc W; Strain, Errol A; Brown, Eric W

2016-10-15

Many listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes. We have developed and evaluated core genome MLST (cgMLST) schemes and applied them to isolates from multiple clonal groups, including those associated with 39 listeriosis outbreaks. The cgMLST clusters were congruent with MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish among outbreak strains and epidemiologically unrelated strains of the same clonal group, which could not be achieved using classic MLST schemes. The precise selection of cgMLST gene targets may not be critical for the general identification of clonal groups and outbreak strains. cgMLST analyses further identified outbreak strains, including those associated with recent outbreaks linked to contaminated French-style cheese, Hispanic-style cheese, stone fruit, caramel apple, ice cream, and packaged leafy green salad, as belonging to major clonal groups. We further developed lineage-specific cgMLST schemes, which can include accessory genes when core genomes do not possess sufficient diversity, and this provided additional resolution over species-specific cgMLST. Analyses of isolates from different common-source listeriosis outbreaks revealed various degrees of diversity, indicating that the numbers of allelic differences should always be combined with cgMLST clustering and epidemiological evidence to define a listeriosis outbreak. Classic multilocus sequence typing (MLST) schemes targeting internal fragments of 6 to 8 genes that define clonal complexes or epidemic clones have been widely employed to study L. monocytogenes biodiversity and its relation to pathogenicity potential and epidemiology. We demonstrated that core genome MLST

Delineation of the species Haemophilus influenzae by phenotype, multilocus sequence phylogeny, and detection of marker genes

DEFF Research Database (Denmark)

Nørskov-Lauritsen, Niels; Overballe, MD; Kilian, Mogens

2009-01-01

To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic genospec......To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic...... genospecies biotype IV, and the never formally validated species "Haemophilus intermedius". Multilocus sequence phylogeny based on six housekeeping genes separated a cluster encompassing the type and the reference strains of H. influenzae from 31 more distantly related strains. Comparison of 16S rRNA gene...

Multilocus sequence typing and rtxA toxin gene sequencing analysis of Kingella kingae isolates demonstrates genetic diversity and international clones.

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Romain Basmaci

Full Text Available BACKGROUND: Kingella kingae, a normal component of the upper respiratory flora, is being increasingly recognized as an important invasive pathogen in young children. Genetic diversity of this species has not been studied. METHODS: We analyzed 103 strains from different countries and clinical origins by a new multilocus sequence-typing (MLST schema. Putative virulence gene rtxA, encoding an RTX toxin, was also sequenced, and experimental virulence of representative strains was assessed in a juvenile-rat model. RESULTS: Thirty-six sequence-types (ST and nine ST-complexes (STc were detected. The main STc 6, 14 and 23 comprised 23, 17 and 20 strains respectively, and were internationally distributed. rtxA sequencing results were mostly congruent with MLST, and showed horizontal transfer events. Of interest, all members of the distantly related ST-6 (n = 22 and ST-5 (n = 4 harboured a 33 bp duplication or triplication in their rtxA sequence, suggesting that this genetic trait arose through selective advantage. The animal model revealed significant differences in virulence among strains of the species. CONCLUSION: MLST analysis reveals international spread of ST-complexes and will help to decipher acquisition and evolution of virulence traits and diversity of pathogenicity among K. kingae strains, for which an experimental animal model is now available.

High genetic diversity among strains of the unindustrialized lactic acid bacterium Carnobacterium maltaromaticum in dairy products as revealed by multilocus sequence typing.

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Rahman, Abdur; Cailliez-Grimal, Catherine; Bontemps, Cyril; Payot, Sophie; Chaillou, Stéphane; Revol-Junelles, Anne-Marie; Borges, Frédéric

2014-07-01

Dairy products are colonized with three main classes of lactic acid bacteria (LAB): opportunistic bacteria, traditional starters, and industrial starters. Most of the population structure studies were previously performed with LAB species belonging to these three classes and give interesting knowledge about the population structure of LAB at the stage where they are already industrialized. However, these studies give little information about the population structure of LAB prior their use as an industrial starter. Carnobacterium maltaromaticum is a LAB colonizing diverse environments, including dairy products. Since this bacterium was discovered relatively recently, it is not yet commercialized as an industrial starter, which makes C. maltaromaticum an interesting model for the study of unindustrialized LAB population structure in dairy products. A multilocus sequence typing scheme based on an analysis of fragments of the genes dapE, ddlA, glpQ, ilvE, pyc, pyrE, and leuS was applied to a collection of 47 strains, including 28 strains isolated from dairy products. The scheme allowed detecting 36 sequence types with a discriminatory index of 0.98. The whole population was clustered in four deeply branched lineages, in which the dairy strains were spread. Moreover, the dairy strains could exhibit a high diversity within these lineages, leading to an overall dairy population with a diversity level as high as that of the nondairy population. These results are in agreement with the hypothesis according to which the industrialization of LAB leads to a diversity reduction in dairy products. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Multilocus genetics to reconstruct aeromonad evolution

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Roger Frédéric

2012-04-01

Full Text Available Abstract Background Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. Results The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Conclusions Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or “disease specialized” while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been

Human Campylobacteriosis in Luxembourg, 2010-2013: A Case-Control Study Combined with Multilocus Sequence Typing for Source Attribution and Risk Factor Analysis.

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Mossong, Joël; Mughini-Gras, Lapo; Penny, Christian; Devaux, Anthony; Olinger, Christophe; Losch, Serge; Cauchie, Henry-Michel; van Pelt, Wilfrid; Ragimbeau, Catherine

2016-02-10

Campylobacteriosis has increased markedly in Luxembourg during recent years. We sought to determine which Campylobacter genotypes infect humans, where they may originate from, and how they may infect humans. Multilocus sequence typing was performed on 1153 Campylobacter jejuni and 136 C. coli human strains to be attributed to three putative animal reservoirs (poultry, ruminants, pigs) and to environmental water using the asymmetric island model. A nationwide case-control study (2010-2013) for domestic campylobacteriosis was also conducted, including 367 C. jejuni and 48 C. coli cases, and 624 controls. Risk factors were investigated by Campylobacter species, and for strains attributed to different sources using a combined case-control and source attribution analysis. 282 sequence types (STs) were identified: ST-21, ST-48, ST-572, ST-50 and ST-257 were prevailing. Most cases were attributed to poultry (61.2%) and ruminants (33.3%). Consuming chicken outside the home was the dominant risk factor for both Campylobacter species. Newly identified risk factors included contact with garden soil for either species, and consuming beef specifically for C. coli. Poultry-associated campylobacteriosis was linked to poultry consumption in wintertime, and ruminant-associated campylobacteriosis to tap-water provider type. Besides confirming chicken as campylobacteriosis primary source, additional evidence was found for other reservoirs and transmission routes.

Multilocus sequence typing, biochemical and antibiotic resistance characterizations reveal diversity of North American strains of the honey bee pathogen Paenibacillus larvae.

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Krongdang, Sasiprapa; Evans, Jay D; Pettis, Jeffery S; Chantawannakul, Panuwan

2017-01-01

Paenibacillus larvae is a Gram positive bacterium and the causative agent of the most widespread fatal brood disease of honey bees, American foulbrood (AFB). A total of thirty-three independent Paenibacillus larvae isolates from various geographical origins in North America and five reference strains were investigated for genetic diversity using multilocus sequence typing (MLST). This technique is regarded to be a powerful tool for epidemiological studies of pathogenic bacteria and is widely used in genotyping assays. For MLST, seven housekeeping gene loci, ilvD (dihydroxy-acid dyhydrogenase), tri (triosephosphate isomerase), purH (phospharibosyl-aminoimidazolecarboxamide), recF (DNA replication and repair protein), pyrE (orotate phosphoribosyltransferase), sucC (succinyl coenzyme A synthetase β subunit) and glpF (glycerol uptake facilitator protein) were studied and applied for primer designs. Previously, ERIC type DNA fingerprinting was applied to these same isolates and the data showed that almost all represented the ERIC I type, whereas using BOX-PCR gave an indication of more diversity. All isolates were screened for resistance to four antibiotics used by U.S. beekeepers, showing extensive resistance to tetracycline and the first records of resistance to tylosin and lincomycin. Our data highlight the intraspecies relationships of P. larvae and the potential application of MLST methods in enhancing our understanding of epidemiological relationships among bacterial isolates of different origins.

Multilocus sequence typing, biochemical and antibiotic resistance characterizations reveal diversity of North American strains of the honey bee pathogen Paenibacillus larvae.

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Sasiprapa Krongdang

Full Text Available Paenibacillus larvae is a Gram positive bacterium and the causative agent of the most widespread fatal brood disease of honey bees, American foulbrood (AFB. A total of thirty-three independent Paenibacillus larvae isolates from various geographical origins in North America and five reference strains were investigated for genetic diversity using multilocus sequence typing (MLST. This technique is regarded to be a powerful tool for epidemiological studies of pathogenic bacteria and is widely used in genotyping assays. For MLST, seven housekeeping gene loci, ilvD (dihydroxy-acid dyhydrogenase, tri (triosephosphate isomerase, purH (phospharibosyl-aminoimidazolecarboxamide, recF (DNA replication and repair protein, pyrE (orotate phosphoribosyltransferase, sucC (succinyl coenzyme A synthetase β subunit and glpF (glycerol uptake facilitator protein were studied and applied for primer designs. Previously, ERIC type DNA fingerprinting was applied to these same isolates and the data showed that almost all represented the ERIC I type, whereas using BOX-PCR gave an indication of more diversity. All isolates were screened for resistance to four antibiotics used by U.S. beekeepers, showing extensive resistance to tetracycline and the first records of resistance to tylosin and lincomycin. Our data highlight the intraspecies relationships of P. larvae and the potential application of MLST methods in enhancing our understanding of epidemiological relationships among bacterial isolates of different origins.

Repeatability of Cryogenic Multilayer Insulation

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Johnson, W. L.; Vanderlaan, M.; Wood, J. J.; Rhys, N. O.; Guo, W.; Van Sciver, S.; Chato, D. J.

2017-12-01

Due to the variety of requirements across aerospace platforms, and one off projects, the repeatability of cryogenic multilayer insulation (MLI) has never been fully established. The objective of this test program is to provide a more basic understanding of the thermal performance repeatability of MLI systems that are applicable to large scale tanks. There are several different types of repeatability that can be accounted for: these include repeatability between identical blankets, repeatability of installation of the same blanket, and repeatability of a test apparatus. The focus of the work in this report is on the first two types of repeatability. Statistically, repeatability can mean many different things. In simplest form, it refers to the range of performance that a population exhibits and the average of the population. However, as more and more identical components are made (i.e. the population of concern grows), the simple range morphs into a standard deviation from an average performance. Initial repeatability testing on MLI blankets has been completed at Florida State University. Repeatability of five Glenn Research Center (GRC) provided coupons with 25 layers was shown to be +/- 8.4% whereas repeatability of repeatedly installing a single coupon was shown to be +/- 8.0%. A second group of 10 coupons has been fabricated by Yetispace and tested by Florida State University, the repeatability between coupons has been shown to be +/- 15-25%. Based on detailed statistical analysis, the data has been shown to be statistically significant.

Multilocus sequence typing of Streptococcus thermophilus from naturally fermented dairy foods in China and Mongolia.

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Yu, Jie; Sun, Zhihong; Liu, Wenjun; Xi, Xiaoxia; Song, Yuqin; Xu, Haiyan; Lv, Qiang; Bao, Qiuhua; Menghe, Bilige; Sun, Tiansong

2015-10-26

Streptococcus thermophilus is a major dairy starter used for manufacturing of dairy products. In the present study, we developed a multilocus sequence typing (MLST) scheme for this important food bacterium. Sequences of 10 housekeeping genes (carB, clpX, dnaA, murC, murE, pepN, pepX, pyrG, recA, and rpoB) were obtained for 239 S. thermophilus strains, which were isolated from home-made fermented dairy foods in 18 different regions of Mongolia and China. All 10 genes of S. thermophilus were sequenced, aligned, and defined sequence types (STs) using the BioNumerics Software. The nucleotide diversity was calculated by START v2.0. The population structure, phylogenetic relationships and the role of recombination were inferred using ClonalFrame v1.2, SplitsTree 4.0 and Structure v2.3. The 239 S. thermophilus isolates and 18 reference strains could be assigned into 119 different STs, which could be further separated into 16 clonal complexes (CCs) and 38 singletons. Among the 10 loci, a total of 132 polymorphic sites were detected. The standardized index of association (IAS=0.0916), split-decomposition and ρ/θ (relative frequency of occurrence of recombination and mutation) and r/m value (relative impact of recombination and mutation in the diversification) confirms that recombination may have occurred, but it occurred at a low frequency in these 10 loci. Phylogenetic trees indicated that there were five lineages in the S. thermophilus isolates used in our study. MSTree and ClonalFrame tree analyses suggest that the evolution of S. thermophilus isolates have little relationship with geographic locality, but revealed no association with the types of fermented dairy product. Phylogenetic analysis of 36 whole genome strains (18 S. thermophilus, 2 S. vestibularis and 16 S. salivarius strains) indicated that our MLST scheme could clearly separate three closely related species within the salivarius group and is suitable for analyzing the population structure of the

Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

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Denoeud France

2006-02-01

Full Text Available Abstract Background The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA for both typing and species identification. Results Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species. The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1 and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2. Conclusion The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

Typing of Candida isolates from patients with invasive infection and concomitant colonization

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Brillowska-Dabrowska, A.; Bergmann, O.; Jensen, Irene Møller

2010-01-01

We investigated the relationship between colonizing and invasive isolates from patients with candidaemia. Molecular typing was performed using random amplification of polymorphic DNA (RAPD) and multilocus sequence typing (MLST). We found MLST to be sufficient for typing Candida isolates, and that......We investigated the relationship between colonizing and invasive isolates from patients with candidaemia. Molecular typing was performed using random amplification of polymorphic DNA (RAPD) and multilocus sequence typing (MLST). We found MLST to be sufficient for typing Candida isolates...

Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis.

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Račić, Ivana; Spičić, Silvio; Galov, Ana; Duvnjak, Sanja; Zdelar-Tuk, Maja; Vujnović, Anja; Habrun, Boris; Cvetnić, Zeljko

2014-10-10

Although Q fever affects humans and animals in Croatia, we are unaware of genotyping studies of Croatian strains of the causative pathogen Coxiella burnetii, which would greatly assist monitoring and control efforts. Here 3261 human and animal samples were screened for C. burnetii DNA by conventional PCR, and 335 (10.3%) were positive. Of these positive samples, 82 were genotyped at 17 loci using the relatively new method of multi-locus variable number tandem repeat analysis (MLVA). We identified 13 C. burnetii genotypes not previously reported anywhere in the world. Two of these 13 genotypes are typical of the continental part of Croatia and share more similarity with genotypes outside Croatia than with genotypes within the country. The remaining 11 novel genotypes are typical of the coastal part of Croatia and show more similarity to one another than to genotypes outside the country. Our findings shed new light on the phylogeny of C. burnetii strains and may help establish MLVA as a standard technique for Coxiella genotyping. Copyright © 2014 Elsevier B.V. All rights reserved.

Multilocus Sequence Typing Reveals Relevant Genetic Variation and Different Evolutionary Dynamics among Strains of Xanthomonas arboricola pv. juglandis

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Marco Scortichini

2010-11-01

Full Text Available Forty-five Xanthomonas arboricola pv. juglandis (Xaj strains originating from Juglans regia cultivation in different countries were molecularly typed by means of MultiLocus Sequence Typing (MLST, using acnB, gapA, gyrB and rpoD gene fragments. A total of 2.5 kilobases was used to infer the phylogenetic relationship among the strains and possible recombination events. Haplotype diversity, linkage disequilibrium analysis, selection tests, gene flow estimates and codon adaptation index were also assessed. The dendrograms built by maximum likelihood with concatenated nucleotide and amino acid sequences revealed two major and two minor phylotypes. The same haplotype was found in strains originating from different continents, and different haplotypes were found in strains isolated in the same year from the same location. A recombination breakpoint was detected within the rpoD gene fragment. At the pathovar level, the Xaj populations studied here are clonal and under neutral selection. However, four Xaj strains isolated from walnut fruits with apical necrosis are under diversifying selection, suggesting a possible new adaptation. Gene flow estimates do not support the hypothesis of geographic isolation of the strains, even though the genetic diversity between the strains increases as the geographic distance between them increases. A triplet deletion, causing the absence of valine, was found in the rpoD fragment of all 45 Xaj strains when compared with X. axonopodis pv. citri strain 306. The codon adaptation index was high in all four genes studied, indicating a relevant metabolic activity.

Coexistence of 3G repeaters with LTE base stations.

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Yeo, Woon-Young; Lee, Sang-Min; Hwang, Gyung-Ho; Kim, Jae-Hoon

2013-01-01

Repeaters have been an attractive solution for mobile operators to upgrade their wireless networks at low cost and to extend network coverage effectively. Since the first LTE commercial deployment in 2009, many mobile operators have launched LTE networks by upgrading their 3G and legacy networks. Because all 3G frequency bands are shared with the frequency bands for LTE deployment and 3G mobile operators have an enormous number of repeaters, reusing 3G repeaters in LTE networks is definitely a practical and cost-efficient solution. However, 3G repeaters usually do not support spatial multiplexing with multiple antennas, and thus it is difficult to reuse them directly in LTE networks. In order to support spatial multiplexing of LTE, the role of 3G repeaters should be replaced with small LTE base stations or MIMO-capable repeaters. In this paper, a repeater network is proposed to reuse 3G repeaters in LTE deployment while still supporting multilayer transmission of LTE. Interestingly, the proposed network has a higher cluster throughput than an LTE network with MIMO-capable repeaters.

Enrichment of Multilocus Sequence Typing Clade 1 with Oral Candida albicans Isolates in Patients with Untreated Periodontitis

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McManus, Brenda A.; Maguire, Rory; Cashin, Phillipa J.; Claffey, Noel; Flint, Stephen; Abdulrahim, Mohammed H.

2012-01-01

This study investigated the prevalence and cell density of Candida species in periodontal pockets, healthy subgingival sites, and oral rinse samples of patients with untreated periodontitis. Twenty-one periodontitis patients underwent sampling at two periodontitis sites, and 19/21 of these patients underwent sampling at one periodontally healthy site. Both paper point and curette sampling techniques were employed. The periodontitis patients and 50 healthy subjects were also sampled by oral rinse. Candida isolates were recovered on CHROMagar Candida medium, and representative isolates were identified. Candida spp. were recovered from 10/21 (46.7%) periodontitis patients and from 16/50 (32%) healthy subjects. C. albicans predominated in both groups and was recovered from all Candida-positive subjects. Candida-positive periodontitis patients yielded Candida from periodontal pockets with average densities of 3,528 and 3,910 CFU/sample from curette and paper point samples, respectively, and 1,536 CFU/ml from oral rinse samples. The majority (18/19) of the healthy sites sampled from periodontitis patients were Candida negative. The 16 Candida-positive healthy subjects yielded an average of 279 CFU/ml from oral rinse samples. C. albicans isolates were investigated by multilocus sequence typing (MLST) to determine if specific clonal groups were associated with periodontitis. MLST analysis of 31 C. albicans isolates from periodontitis patients yielded 19 sequence types (STs), 13 of which were novel. Eleven STs belonged to MLST clade 1. In contrast, 16 C. albicans isolates from separate healthy subjects belonged to 16 STs, with 4 isolates belonging to clade 1. The distributions of STs between both groups were significantly different (P = 0.04) and indicated an enrichment of C. albicans isolates in periodontal pockets, which warrants a larger study. PMID:22875886

Prevalence of Thermotolerant Campylobacter spp. in Chicken Meat in Croatia and Multilocus Sequence Typing of a Small Subset of Campylobacter jejuni and Campylobacter coli Isolates

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Andrea Humski

2016-01-01

Full Text Available In order to detect thermotolerant Campylobacter spp., 241 samples of fresh chicken meat, at retail in Croatia, were analysed according to a standard method, followed by biochemical test and molecular polymerase chain reaction/restriction enzyme analysis for exact species determination. Campylobacter spp. prevalence was 73.86 %. Campylobacter jejuni and Campylobacter coli were isolated from 53.53 and 15.35 % of the samples, respectively. In 4.98 % of isolates thermotolerant Campylobacter spp. were not determined. The multi locus sequence typing method was used to evaluate genetic diversity of eight Campylobacter jejuni and four Campylobacter coli isolates. To our knowledge, these results of genotyping provided the first data on the presence of sequence types (STs and clonal complexes (CCs of Campylobacter jejuni and C. coli isolates in Croatia. By applying the multilocus sequence typing, a new allele of tkt gene locus was discovered and marked tkt508. The C. jejuni ST 6182 and C. coli ST 6183 genotypes were described for the fi rst time, and all other identified genotypes were clustered in the previously described sequence types and clonal complexes. These findings provide useful information on the prevalence and epidemiology of Campylobacter jejuni and C. coli in Croatia.

Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations.

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Gilmore, Simon; Peakall, Rod; Robertson, James

2003-01-09

Short tandem repeat (STR) markers are the DNA marker of choice in forensic analysis of human DNA. Here we extend the application of STR markers to Cannabis sativa and demonstrate their potential for forensic investigations. Ninety-three individual cannabis plants, representing drug and fibre accessions of widespread origin were profiled with five STR makers. A total of 79 alleles were detected across the five loci. All but four individuals from a single drug-type accession had a unique multilocus genotype. An analysis of molecular variance (AMOVA) revealed significant genetic variation among accessions, with an average of 25% genetic differentiation. By contrast, only 6% genetic difference was detected between drug and fibre crop accessions and it was not possible to unequivocally assign plants as either drug or fibre type. However, our results suggest that drug strains may typically possess lower genetic diversity than fibre strains, which may ultimately provide a means of genetic delineation. Our findings demonstrate the promise of cannabis STR markers to provide information on: (1) agronomic type, (2) the geographical origin of drug seizures, and (3) evidence of conspiracy in production of clonally propagated drug crops.

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

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

2018-03-01

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

Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry.

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Zautner, Andreas Erich; Masanta, Wycliffe Omurwa; Tareen, Abdul Malik; Weig, Michael; Lugert, Raimond; Groß, Uwe; Bader, Oliver

2013-11-07

Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt). Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism. Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.

Epidemiological analysis of Leishmania tropica strains and giemsa-stained smears from Syrian and Turkish leishmaniasis patients using multilocus microsatellite typing (MLMT.

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Mehmet Karakuş

2017-04-01

Full Text Available Turkey is located in an important geographical location, in terms of the epidemiology of vector-borne diseases, linking Asia and Europe. Cutaneous leishmaniasis (CL is one of the endemic diseases in a Turkey and according to the Ministry Health of Turkey, 45% of CL patients originate from Şanlıurfa province located in southeastern Turkey. Herein, the epidemiological status of CL, caused by L. tropica, in Turkey was examined using multilocus microsatellite typing (MLMT of strains obtained from Turkish and Syrian patients. A total of 38 cryopreserved strains and 20 Giemsa-stained smears were included in the present study. MLMT was performed using 12 highly specific microsatellite markers. Delta K (ΔK calculation and Bayesian statistics were used to determine the population structure. Three main populations (POP A, B and C were identified and further examination revealed the presence of three subpopulations for POP B and C. Combined analysis was performed using the data of previously typed L. tropica strains and Mediterranean and Şanlıurfa populations were identified. This finding suggests that the epidemiological status of L. tropica is more complicated than expected when compared to previous studies. A new population, comprised of Syrian L. tropica samples, was reported for the first time in Turkey, and the data presented here will provide new epidemiological information for further studies.

Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

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Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

2017-02-01

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including bla CMY and bla NDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a bla NDM-1 -positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of bla NDM -positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this bla NDM -containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

Outbreak of listeriosis caused by infected beef meat from a meals-on-wheels delivery in Denmark 2009

DEFF Research Database (Denmark)

Smith, B; Larsson, J T; Lisby, M

2011-01-01

An outbreak of listeriosis in Denmark occurred in May 2009. Multilocus variable number of tandem repeats analysis typing, later confirmed by pulsed-field gel electrophoresis typing, showed that isolates from eight patients had identical patterns and were distinguishable from Listeria monocytogene...

Differentiation of Xylella fastidiosa Strains via Multilocus Sequence Analysis of Environmentally Mediated Genes (MLSA-E)

OpenAIRE

Parker, Jennifer K.; Havird, Justin C.; De La Fuente, Leonardo

2012-01-01

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of enviro...

Genotyping analysis of Helicobacter pylori using multiple-locus variable-number tandem-repeats analysis in five regions of China and Japan

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Zhang Jinyong

2011-09-01

Full Text Available Abstract Background H. pylori (Helicobacter pylori is the major causative agent of chronic active gastritis. The population of H. pylori shows a high genomic variability among isolates. And the polymorphism of repeat-units of genomics had participated the important process of evolution. Its long term colonization of the stomach caused different clinical outcomes, which may relate to the high degree of genetic variation of H. pylori. A variety of molecular typing tools have been developed to access genetic relatedness in H. pylori isolates. However, there is still no standard genotyping system of this bacterium. The MLVA (Multi-locus of variable number of tandem repeat analysis method is useful for performing phylogenetic analysis and is widely used in bacteria genotyping; however, there's little application in H. pylori analysis. This article is the first application of the MLVA method to investigate H. pylori from different districts and ethnic groups of China. Results MLVA of 12 VNTR loci with high discrimination power based on 30 candidates were performed on a collection of 202 strains of H. pylori which originated from five regions of China and Japan. Phylogenetic tree was constructed using MLVA profiles. 12 VNTR loci presented with high various polymorphisms, and the results demonstrated very close relationships between genotypes and ethnic groups. Conclusions This study used MLVA methodology providing a new perspective on the ethnic groups and distribution characteristics of H. pylori.

Multilocus sequence analysis of phytopathogenic species of the genus Streptomyces

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The identification and classification of species within the genus Streptomyces is difficult because there are presently 576 validly described species and this number increases every year. The value of the application of multilocus sequence analysis scheme to the systematics of Streptomyces species h...

Systematic characterization of Bacillus Genetic Stock Center Bacillus thuringiensis strains using Multi-Locus Sequence Typing.

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Wang, Kui; Shu, Changlong; Soberón, Mario; Bravo, Alejandra; Zhang, Jie

2018-04-30

The goal of this work was to perform a systematic characterization of Bacillus thuringiensis (Bt) strains from the Bacillus Genetic Stock Center (BGSC) collection using Multi-Locus Sequence Typing (MLST). Different genetic markers of 158 Bacillus thuringiensis (Bt) strains from 73 different serovars stored in the BGSC, that represented 92% of the different Bt serovars of the BGSC were analyzed, the 8% that were not analyzed were not available. In addition, we analyzed 72 Bt strains from 18 serovars available at the pubMLST bcereus database, and Bt strains G03, HBF18 and Bt185, with no H serovars provided by our laboratory. We performed a systematic MLST analysis using seven housekeeping genes (glpF, gmK, ilvD, pta, pur, pycA and tpi) and analyzed correlation of the results of this analysis with strain serovars. The 233 Bt strains analyzed were assigned to 119 STs from which 19 STs were new. Genetic relationships were established by phylogenetic analysis and showed that STs could be grouped in two major Clusters containing 21 sub-groups. We found that a significant number of STs (101 in total) correlated with specific serovars, such as ST13 that corresponded to nine Bt isolates from B. thuringiensis serovar kenyae. However, other serovars showed high genetic variability and correlated with multiple STs; for example, B. thuringiensis serovar morrisoni correlated with 11 different STs. In addition, we found that 16 different STs correlated with multiple serovars (2-4 different serovars); for example, ST12 correlated with B. thuringiensis serovar alesti, dakota, palmanyolensis and sotto/dendrolimus. These data indicated that only partial correspondence between MLST and serotyping can be established. Copyright © 2018 Elsevier Inc. All rights reserved.

Class 1 integrons characterization and multilocus sequence typing of Salmonella spp. from swine production chains in Chiang Mai and Lamphun provinces, Thailand.

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Boonkhot, Phacharaporn; Tadee, Pakpoom; Yamsakul, Panuwat; Pocharoen, Chairoj; Chokesajjawatee, Nipa; Patchanee, Prapas

2015-05-01

Pigs and pork products are well known as an important source of Salmonella, one of the major zoonotic foodborne pathogens. The emergence and spread of antimicrobial resistance is becoming a major public health concern worldwide. Integrons are genetic elements known to have a role in the acquisition and expression of genes conferring antibiotic resistance. This study focuses on the prevalence of class 1 integrons-carrying Salmonella, the genetic diversity of strains of those organisms obtained from swine production chains in Chiang Mai and Lamphun provinces, Thailand, using multilocus sequence typing (MLST) and comparison of genetic diversity of sequence types of Salmonella from this study with pulsotypes identified in previous study. In 175 Salmonella strains, the overall prevalence of class 1 integrons-carrying-Salmonella was 14%. The gene cassettes array pattern "dfrA12-orfF-aadA2" was the most frequently observed. Most of the antimicrobial resistance identified was not associated with related gene cassettes harbored by Salmonella. Six sequence types were generated from 30 randomly selected strains detected by MLST. Salmonella at the human-animal-environment interface was confirmed. Linkages both in the farm to slaughterhouse contamination route and the horizontal transmission of resistance genes were demonstrated. To reduce this problem, the use of antimicrobials in livestock should be controlled by veterinarians. Education and training of food handlers as well as promotion of safe methods of food consumption are important avenues for helping prevent foodborne illness.

Development of a Multilocus Sequence Typing (MLST) scheme for Treponema pallidum subsp. pertenue: Application to yaws in Lihir Island, Papua New Guinea.

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Godornes, Charmie; Giacani, Lorenzo; Barry, Alyssa E; Mitja, Oriol; Lukehart, Sheila A

2017-12-01

Yaws is a neglected tropical disease, caused by Treponema pallidum subsp. pertenue. The disease causes chronic lesions, primarily in young children living in remote villages in tropical climates. As part of a global yaws eradication campaign initiated by the World Health Organization, we sought to develop and evaluate a molecular typing method to distinguish different strains of T. pallidum subsp. pertenue for disease control and epidemiological purposes. Published genome sequences of strains of T. pallidum subsp. pertenue and pallidum were compared to identify polymorphic genetic loci among the strains. DNA from a number of existing historical Treponema isolates, as well as a subset of samples from yaws patients collected in Lihir Island, Papua New Guinea, were analyzed using these targets. From these data, three genes (tp0548, tp0136 and tp0326) were ultimately selected to give a high discriminating capability among the T. pallidum subsp. pertenue samples tested. Intragenic regions of these three target genes were then selected to enhance the discriminating capability of the typing scheme using short readily amplifiable loci. This 3-gene multilocus sequence typing (MLST) method was applied to existing historical human yaws strains, the Fribourg-Blanc simian isolate, and DNA from 194 lesion swabs from yaws patients on Lihir Island, Papua New Guinea. Among all samples tested, fourteen molecular types were identified, seven of which were found in patient samples and seven among historical isolates or DNA. Three types (JG8, TD6, and SE7) were predominant on Lihir Island. This MLST approach allows molecular typing and differentiation of yaws strains. This method could be a useful tool to complement epidemiological studies in regions where T. pallidum subsp. pertenue is prevalent with the overall goals of improving our understanding of yaws transmission dynamics and helping the yaws eradication campaign to succeed.

Development of a Multilocus Sequence Typing (MLST) scheme for Treponema pallidum subsp. pertenue: Application to yaws in Lihir Island, Papua New Guinea

Science.gov (United States)

Godornes, Charmie; Giacani, Lorenzo; Barry, Alyssa E.; Mitja, Oriol

2017-01-01

Background Yaws is a neglected tropical disease, caused by Treponema pallidum subsp. pertenue. The disease causes chronic lesions, primarily in young children living in remote villages in tropical climates. As part of a global yaws eradication campaign initiated by the World Health Organization, we sought to develop and evaluate a molecular typing method to distinguish different strains of T. pallidum subsp. pertenue for disease control and epidemiological purposes. Methods and principal findings Published genome sequences of strains of T. pallidum subsp. pertenue and pallidum were compared to identify polymorphic genetic loci among the strains. DNA from a number of existing historical Treponema isolates, as well as a subset of samples from yaws patients collected in Lihir Island, Papua New Guinea, were analyzed using these targets. From these data, three genes (tp0548, tp0136 and tp0326) were ultimately selected to give a high discriminating capability among the T. pallidum subsp. pertenue samples tested. Intragenic regions of these three target genes were then selected to enhance the discriminating capability of the typing scheme using short readily amplifiable loci. This 3-gene multilocus sequence typing (MLST) method was applied to existing historical human yaws strains, the Fribourg-Blanc simian isolate, and DNA from 194 lesion swabs from yaws patients on Lihir Island, Papua New Guinea. Among all samples tested, fourteen molecular types were identified, seven of which were found in patient samples and seven among historical isolates or DNA. Three types (JG8, TD6, and SE7) were predominant on Lihir Island. Conclusions This MLST approach allows molecular typing and differentiation of yaws strains. This method could be a useful tool to complement epidemiological studies in regions where T. pallidum subsp. pertenue is prevalent with the overall goals of improving our understanding of yaws transmission dynamics and helping the yaws eradication campaign to succeed

Population genetics and new insight into range of CAG repeats of spinocerebellar ataxia type 3 in the Han Chinese population.

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Shi-Rui Gan

Full Text Available Spinocerebellar ataxia type 3 (SCA3, also called Machado-Joseph disease (MJD, is one of the most common SCAs worldwide and caused by a CAG repeat expansion located in ATXN3 gene. Based on the CAG repeat numbers, alleles of ATXN3 can be divided into normal alleles (ANs, intermediate alleles (AIs and expanded alleles (AEs. It was controversial whether the frequency of large normal alleles (large ANs is related to the prevalence of SCA3 or not. And there were huge chaos in the comprehension of the specific numbers of the range of CAG repeats which is fundamental for genetic analysis of SCA3. To illustrate these issues, we made a novel CAG repeat ladder to detect CAG repeats of ATXN3 in 1003 unrelated Chinese normal individuals and studied haplotypes defined by three single nucleotide polymorphisms (SNPs closed to ATXN3. We found that the number of CAG repeats ranged from 13 to 49, among them, 14 was the most common number. Positive skew, the highest frequency of large ANs and 4 AIs which had never been reported before were found. Also, AEs and large ANs shared the same haplotypes defined by the SNPs. Based on these data and other related studies, we presumed that de novo mutations of ATXN3 emerging from large ANs are at least one survival mechanisms of mutational ATXN3 and we can redefine the range of CAG repeats as: ANs≤44, 45 ≤AIs ≤49 and AEs≥50.

Particular Candida albicans strains in the digestive tract of dyspeptic patients, identified by multilocus sequence typing.

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Yan-Bing Gong

Full Text Available BACKGROUND: Candida albicans is a human commensal that is also responsible for chronic gastritis and peptic ulcerous disease. Little is known about the genetic profiles of the C. albicans strains in the digestive tract of dyspeptic patients. The aim of this study was to evaluate the prevalence, diversity, and genetic profiles among C. albicans isolates recovered from natural colonization of the digestive tract in the dyspeptic patients. METHODS AND FINDINGS: Oral swab samples (n = 111 and gastric mucosa samples (n = 102 were obtained from a group of patients who presented dyspeptic symptoms or ulcer complaints. Oral swab samples (n = 162 were also obtained from healthy volunteers. C. albicans isolates were characterized and analyzed by multilocus sequence typing. The prevalence of Candida spp. in the oral samples was not significantly different between the dyspeptic group and the healthy group (36.0%, 40/111 vs. 29.6%, 48/162; P > 0.05. However, there were significant differences between the groups in the distribution of species isolated and the genotypes of the C. albicans isolates. C. albicans was isolated from 97.8% of the Candida-positive subjects in the dyspeptic group, but from only 56.3% in the healthy group (P < 0.001. DST1593 was the dominant C. albicans genotype from the digestive tract of the dyspeptic group (60%, 27/45, but not the healthy group (14.8%, 4/27 (P < 0.001. CONCLUSIONS: Our data suggest a possible link between particular C. albicans strain genotypes and the host microenvironment. Positivity for particular C. albicans genotypes could signify susceptibility to dyspepsia.

Epidemiological characterization of a nosocomial outbreak of extended spectrum β-lactamase Escherichia coli ST-131 confirms the clinical value of core genome multilocus sequence typing.

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Woksepp, Hanna; Ryberg, Anna; Berglind, Linda; Schön, Thomas; Söderman, Jan

2017-12-01

Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST-131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST-131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

Energy Technology Data Exchange (ETDEWEB)

Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

2017-09-01

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

Minim typing--a rapid and low cost MLST based typing tool for Klebsiella pneumoniae.

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Andersson, Patiyan; Tong, Steven Y C; Bell, Jan M; Turnidge, John D; Giffard, Philip M

2012-01-01

Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.

Minim typing--a rapid and low cost MLST based typing tool for Klebsiella pneumoniae.

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Patiyan Andersson

Full Text Available Here we report a single nucleotide polymorphism (SNP based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM analysis of fragments within the multilocus sequence typing (MLST loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D. DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929, and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995. The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit.

Multilocus Sequence Typing Reveals a New Cluster of Closely Related Candida tropicalis Genotypes in Italian Patients With Neurological Disorders.

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Scordino, Fabio; Giuffrè, Letterio; Barberi, Giuseppina; Marino Merlo, Francesca; Orlando, Maria Grazia; Giosa, Domenico; Romeo, Orazio

2018-01-01

Candida tropicalis is a pathogenic yeast that has emerged as an important cause of candidemia especially in elderly patients with hematological malignancies. Infections caused by this species are mainly reported from Latin America and Asian-Pacific countries although recent epidemiological data revealed that C. tropicalis accounts for 6-16.4% of the Candida bloodstream infections (BSIs) in Italy by representing a relevant issue especially for patients receiving long-term hospital care. The aim of this study was to describe the genetic diversity of C. tropicalis isolates contaminating the hands of healthcare workers (HCWs) and hospital environments and/or associated with BSIs occurring in patients with different neurological disorders and without hematological disease. A total of 28 C. tropicalis isolates were genotyped using multilocus sequence typing analysis of six housekeeping ( ICL1, MDR1, SAPT2, SAPT4, XYR1 , and ZWF1 ) genes and data revealed the presence of only eight diploid sequence types (DSTs) of which 6 (75%) were completely new. Four eBURST clonal complexes (CC2, CC10, CC11, and CC33) contained all DSTs found in this study and the CC33 resulted in an exclusive, well-defined, clonal cluster from Italy. In conclusion, C. tropicalis could represent an important cause of BSIs in long-term hospitalized patients with no underlying hematological disease. The findings of this study also suggest a potential horizontal transmission of a specific C. tropicalis clone through hands of HCWs and expand our understanding of the molecular epidemiology of this pathogen whose population structure is still far from being fully elucidated as its complexity increases as different categories of patients and geographic areas are examined.

A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination.

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Ankarklev, Johan; Lebbad, Marianne; Einarsson, Elin; Franzén, Oscar; Ahola, Harri; Troell, Karin; Svärd, Staffan G

2018-06-01

Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia. Copyright © 2018. Published by Elsevier B.V.

Suppression of Repeat-Intensive False Targets Based on Temporal Pulse Diversity

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Gang Lu

2013-01-01

Full Text Available This paper considers the problem of suppressing the repeat-intensive false targets produced by a deception electronic attack (EA system equipped with a Digital Radio Frequency Memory (DRFM device. Different from a conventional repeat jammer, this type of jamming intensively retransmits the intercepted signal stored in a DRFM to the victim radar in a very short time-delay interval relative to a radar pulse wide. A multipeak matched-filtering output is then produced other than the merely expected true target. An electronic protection (EP algorithm based on the space time block code (STBC is proposed to suppress the adverse effects of this jammer. By transmitting a pulse sequence generated from the STBC in succession and the following cancellation process applied upon the received signal, this algorithm performs successfully in a single antenna system provided that the target models are nonfluctuating or slow fluctuating and the pulse repetition frequency (PRF is comparatively high. The performance in white and correlated Gaussian disturbance is evaluated by means of Monte Carlo simulations.

Development of a Hierarchical Variable-Number Tandem Repeat Typing Scheme for Mycobacterium tuberculosis in China

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Luo, Tao; Yang, Chongguang; Pang, Yu; Zhao, Yanlin; Mei, Jian; Gao, Qian

2014-01-01

Molecular typing based on variable-number tandem repeats (VNTR) analysis is a promising tool for identifying transmission of Mycobacterium tuberculosis. However, the currently proposed 15- and 24-locus VNTR sets (VNTR-15/24) only have limited resolution and contain too many loci for large-scale typing in high burden countries. To develop an optimal typing scheme in China, we evaluated the resolution and robustness of 25 VNTR loci, using population-based collections of 1362 clinical isolates from six provinces across the country. The resolution of most loci showed considerable variations among regions. By calculating the average resolution of all possible combinations of 20 robust loci, we identified an optimal locus set with a minimum of 9 loci (VNTR-9) that could achieve comparable resolution of the standard VNTR-15. The VNTR-9 had consistently high resolutions in all six regions, and it was highly concordant with VNTR-15 for defining both clustered and unique genotypes. Furthermore, VNTR-9 was phylogenetically informative for classifying lineages/sublineages of M. tuberculosis. Three hypervariable loci (HV-3), VNTR 3232, VNTR 3820 and VNTR 4120, were proved important for further differentiating unrelated clustered strains based on VNTR-9. We propose the optimized VNTR-9 as first-line method and the HV-3 as second-line method for molecular typing of M. tuberculosis in China and surrounding countries. The development of hierarchical VNTR typing methods that can achieve high resolution with a small number of loci could be suitable for molecular epidemiology study in other high burden countries. PMID:24586989

Subtyping Salmonella enterica serovar enteritidis isolates from different sources by using sequence typing based on virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs).

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Liu, Fenyun; Kariyawasam, Subhashinie; Jayarao, Bhushan M; Barrangou, Rodolphe; Gerner-Smidt, Peter; Ribot, Efrain M; Knabel, Stephen J; Dudley, Edward G

2011-07-01

Salmonella enterica subsp. enterica serovar Enteritidis is a major cause of food-borne salmonellosis in the United States. Two major food vehicles for S. Enteritidis are contaminated eggs and chicken meat. Improved subtyping methods are needed to accurately track specific strains of S. Enteritidis related to human salmonellosis throughout the chicken and egg food system. A sequence typing scheme based on virulence genes (fimH and sseL) and clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-including multi-virulence-locus sequence typing (designated CRISPR-MVLST)-was used to characterize 35 human clinical isolates, 46 chicken isolates, 24 egg isolates, and 63 hen house environment isolates of S. Enteritidis. A total of 27 sequence types (STs) were identified among the 167 isolates. CRISPR-MVLST identified three persistent and predominate STs circulating among U.S. human clinical isolates and chicken, egg, and hen house environmental isolates in Pennsylvania, and an ST that was found only in eggs and humans. It also identified a potential environment-specific sequence type. Moreover, cluster analysis based on fimH and sseL identified a number of clusters, of which several were found in more than one outbreak, as well as 11 singletons. Further research is needed to determine if CRISPR-MVLST might help identify the ecological origins of S. Enteritidis strains that contaminate chickens and eggs.

Minim Typing – A Rapid and Low Cost MLST Based Typing Tool for Klebsiella pneumoniae

Science.gov (United States)

Andersson, Patiyan; Tong, Steven Y. C.; Bell, Jan M.; Turnidge, John D.; Giffard, Philip M.

2012-01-01

Here we report a single nucleotide polymorphism (SNP) based genotyping method for Klebsiella pneumoniae utilising high-resolution melting (HRM) analysis of fragments within the multilocus sequence typing (MLST) loci. The approach is termed mini-MLST or Minim typing and it has previously been applied to Streptococcus pyogenes, Staphylococcus aureus and Enterococcus faecium. Six SNPs were derived from concatenated MLST sequences on the basis of maximisation of the Simpsons Index of Diversity (D). DNA fragments incorporating these SNPs and predicted to be suitable for HRM analysis were designed. Using the assumption that HRM alleles are defined by G+C content, Minim typing using six fragments was predicted to provide a D = 0.979 against known STs. The method was tested against 202 K. pneumoniae using a blinded approach in which the MLST analyses were performed after the HRM analyses. The HRM-based alleles were indeed in accordance with G+C content, and the Minim typing identified known STs and flagged new STs. The tonB MLST locus was determined to be very diverse, and the two Minim fragments located herein contribute greatly to the resolving power. However these fragments are refractory to amplification in a minority of isolates. Therefore, we assessed the performance of two additional formats: one using only the four fragments located outside the tonB gene (D = 0.929), and the other using HRM data from these four fragments in conjunction with sequencing of the tonB MLST fragment (D = 0.995). The HRM assays were developed on the Rotorgene 6000, and the method was shown to also be robust on the LightCycler 480, allowing a 384-well high through-put format. The assay provides rapid, robust and low-cost typing with fully portable results that can directly be related to current MLST data. Minim typing in combination with molecular screening for antibiotic resistance markers can be a powerful surveillance tool kit. PMID:22428067

Exploring multilocus associations of inflammation genes and colorectal cancer risk using hapConstructor

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Abo Ryan

2010-12-01

Full Text Available Abstract Background In candidate-gene association studies of single nucleotide polymorphisms (SNPs, multilocus analyses are frequently of high dimensionality when considering haplotypes or haplotype pairs (diplotypes and differing modes of expression. Often, while candidate genes are selected based on their biological involvement in a given pathway, little is known about the functionality of SNPs to guide association studies. Investigators face the challenge of exploring multiple SNP models to elucidate which variants, independently or in combination, might be associated with a disease of interest. A data mining module, hapConstructor (freely-available in Genie software performs systematic construction and association testing of multilocus genotype data in a Monte Carlo framework. Our objective was to assess its utility to guide statistical analyses of haplotypes within a candidate region (or combined genotypes across candidate genes beyond that offered by a standard logistic regression approach. Methods We applied the hapConstructor method to a multilocus investigation of candidate genes involved in pro-inflammatory cytokine IL6 production, IKBKB, IL6, and NFKB1 (16 SNPs total hypothesized to operate together to alter colorectal cancer risk. Data come from two U.S. multicenter studies, one of colon cancer (1,556 cases and 1,956 matched controls and one of rectal cancer (754 cases and 959 matched controls. Results HapConstrcutor enabled us to identify important associations that were further analyzed in logistic regression models to simultaneously adjust for confounders. The most significant finding (nominal P = 0.0004; false discovery rate q = 0.037 was a combined genotype association across IKBKB SNP rs5029748 (1 or 2 variant alleles, IL6 rs1800797 (1 or 2 variant alleles, and NFKB1 rs4648110 (2 variant alleles which conferred an ~80% decreased risk of colon cancer. Conclusions Strengths of hapConstructor were: systematic identification of

Analysis of multilocus sequence typing and virulence characterization of Listeria monocytogenes isolates from Chinese retail ready-to-eat food

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Shi eWu

2016-02-01

Full Text Available Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST, virulence-associate genes, epidemic clones (ECs and sequence analysis of the important virulence factor: internalin A (inlA. The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs. With the exception of four new STs (ST804, ST805, ST806 and ST807, all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly and llsX were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80 of strains harbored the listeriolysin S genes (llsX. A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80 of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC within a novel PMSCs at position 326 (GAA→TAA. MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.

Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food.

Science.gov (United States)

Wu, Shi; Wu, Qingping; Zhang, Jumei; Chen, Moutong; Guo, Weipeng

2016-01-01

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.

Analysis of repeated measures data

CERN Document Server

Islam, M Ataharul

2017-01-01

This book presents a broad range of statistical techniques to address emerging needs in the field of repeated measures. It also provides a comprehensive overview of extensions of generalized linear models for the bivariate exponential family of distributions, which represent a new development in analysing repeated measures data. The demand for statistical models for correlated outcomes has grown rapidly recently, mainly due to presence of two types of underlying associations: associations between outcomes, and associations between explanatory variables and outcomes. The book systematically addresses key problems arising in the modelling of repeated measures data, bearing in mind those factors that play a major role in estimating the underlying relationships between covariates and outcome variables for correlated outcome data. In addition, it presents new approaches to addressing current challenges in the field of repeated measures and models based on conditional and joint probabilities. Markov models of first...

Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

Science.gov (United States)

Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

2015-01-01

CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers.

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Siew, Ging Yang; Ng, Wei Lun; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian ( Durio zibethinus ) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, H E  = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10 -3 . Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called "clones", "varieties", or "cultivars". Such matters have a direct impact on the regulation and management of durian genetic resources in the region.

Evolution in Australasian mangrove forests: multilocus phylogenetic analysis of the Gerygone warblers (Aves: Acanthizidae.

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Árpád S Nyári

Full Text Available The mangrove forests of Australasia have many endemic bird species but their evolution and radiation in those habitats has been little studied. One genus with several mangrove specialist species is Gerygone (Passeriformes: Acanthizidae. The phylogeny of the Acanthizidae is reasonably well understood but limited taxon sampling for Gerygone has constrained understanding of its evolution and historical biogeography in mangroves. Here we report on a phylogenetic analysis of Gerygone based on comprehensive taxon sampling and a multilocus dataset of thirteen loci spread across the avian genome (eleven nuclear and two mitochondrial loci. Since Gerygone includes three species restricted to Australia's coastal mangrove forests, we particularly sought to understand the biogeography of their evolution in that ecosystem. Analyses of individual loci, as well as of a concatenated dataset drawn from previous molecular studies indicates that the genus as currently defined is not monophyletic, and that the Grey Gerygone (G. cinerea from New Guinea should be transferred to the genus Acanthiza. The multilocus approach has permitted the nuanced view of the group's evolution into mangrove ecosystems having occurred on multiple occasions, in three non-overlapping time frames, most likely first by the G. magnirostris lineage, and subsequently followed by those of G. tenebrosa and G. levigaster.

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

NARCIS (Netherlands)

Siarkou, Victoria I.; Vorimore, Fabien; Vicari, Nadia; Magnino, Simone; Rodolakis, Annie; Pannekoek, Yvonne; Sachse, Konrad; Longbottom, David; Laroucau, Karine

2015-01-01

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94

Repeat interruptions in spinocerebellar ataxia type 10 expansions are strongly associated with epileptic seizures

Science.gov (United States)

McFarland, Karen N.; Liu, Jilin; Landrian, Ivette; Zeng, Desmond; Raskin, Salmo; Moscovich, Mariana; Gatto, Emilia M.; Ochoa, Adriana; Teive, Hélio A. G.; Rasmussen, Astrid; Ashizawa, Tetsuo

2014-01-01

Spinocerebellar ataxia type 10 (SCA10), an autosomal dominant neurodegenerative disorder, is the result of a non-coding, pentanucleotide repeat expansion within intron 9 of the Ataxin 10 gene. SCA10 patients present with pure cerebellar ataxia; yet, some families also have a high incidence of epilepsy. SCA10 expansions containing penta- and heptanucleotide interruption motifs, termed “ATCCT interruptions,” experience large contractions during germline transmission, particularly in paternal lineages. At the same time, these alleles confer an earlier age at onset which contradicts traditional rules of genetic anticipation in repeat expansions. Previously, ATCCT interruptions have been associated with a higher prevalence of epileptic seizures in one Mexican-American SCA10 family. In a large cohort of SCA10 families, we analyzed whether ATCCT interruptions confers a greater risk for developing seizures in these families. Notably, we find that the presence of repeat interruptions within the SCA10 expansion confers a 6.3-fold increase in the risk of an SCA10 patient developing epilepsy (6.2-fold when considering patients of Mexican ancestry only) and a 13.7-fold increase in having a positive family history of epilepsy (10.5-fold when considering patients of Mexican ancestry only). We conclude that the presence of repeat interruptions in SCA10 repeat expansion indicates a significant risk for the epilepsy phenotype and should be considered during genetic counseling. PMID:24318420

Identification of Variable-Number Tandem-Repeat (VNTR) Sequences in Legionella pneumophila and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme▿

Science.gov (United States)

Pourcel, Christine; Visca, Paolo; Afshar, Baharak; D'Arezzo, Silvia; Vergnaud, Gilles; Fry, Norman K.

2007-01-01

The utility of a genotypic typing assay for Legionella pneumophila was investigated. A multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) scheme using PCR and agarose gel electrophoresis is proposed based on eight minisatellite markers. Panels of well-characterized strains were examined in a multicenter analysis to validate the assay and to compare its performance to that of other genotyping assays. Excellent typeability, reproducibility, stability, and epidemiological concordance were observed. The MLVA type or profile is composed of a string of allele numbers, corresponding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. A database containing information from 99 L. pneumophila serogroup 1 strains and four strains of other serogroups and their MLVA profiles, which can be queried online, is available from http://bacterial-genotyping.igmors.u-psud.fr/. PMID:17251393

DEVELOPMENT OF A MULTIPLE-LOCUS VARIABLE NUMBER OF TANDEM REPEAT ANALYSIS (MLVA FOR HELICOBACTER PYLORI AND ITS APPLICATION TO HELICOBACTER PYLORI ISOLATES FROM ROSTOV REGION,RUSSIA

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Sorokin VM

2012-09-01

Full Text Available Stomach infection with Helicobacter pylori (H. pylori is the second most common infectious disease of humans. The severe pathological consequences of this infection include gastric and duodenal ulcer disease, the development of gastric mucosal atrophy, gastric carcinoma, and, more rarely, malignant tumors of the lymphoma. H. pylori infections cause very high morbidity and mortality and are of particular concern in developing countries, where H. pylori prevalences as high as 90% have been reported. The population of H. pylori shows a high genomic variability among isolates. And the polymorphism of repeat-units of genomics had participated the important process of evolution. A variety of molecular typing tools have been developed to access genetic relatedness in H. pylori isolates. However, there is still no standard genotyping system of this bacterium. The MLVA (Multi-Locus of Variable number of tandem repeat Analysis method is useful for performing phylogenetic analysis and is widely used in bacteria genotyping; however, there's little application in H. pylori analysis. This article is the first application of the MLVA method to investigate H. pylori isolates in Russia. MLVA of 4 VNTR loci with high discrimination power based on 10 candidates were performed on a collection of 22 strains of H. pylori which originated from Rostov region of Russia. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.

First worldwide proficiency study on variable-number tandem-repeat typing of Mycobacterium tuberculosis complex strains.

NARCIS (Netherlands)

Beer, J.L. de; Kremer, K.; Kodmon, C.; Supply, P.; Soolingen, D. van

2012-01-01

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency

Multi-locus estimates of population structure and migration in a fence lizard hybrid zone.

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Adam D Leaché

Full Text Available A hybrid zone between two species of lizards in the genus Sceloporus (S. cowlesi and S. tristichus on the Mogollon Rim in Arizona provides a unique opportunity to study the processes of lineage divergence and merging. This hybrid zone involves complex interactions between 2 morphologically and ecologically divergent subspecies, 3 chromosomal groups, and 4 mitochondrial DNA (mtDNA clades. The spatial patterns of divergence between morphology, chromosomes and mtDNA are discordant, and determining which of these character types (if any reflects the underlying population-level lineages that are of interest has remained impeded by character conflict. The focus of this study is to estimate the number of populations interacting in the hybrid zone using multi-locus nuclear data, and to then estimate the migration rates and divergence time between the inferred populations. Multi-locus estimates of population structure and gene flow were obtained from 12 anonymous nuclear loci sequenced for 93 specimens of Sceloporus. Population structure estimates support two populations, and this result is robust to changes to the prior probability distribution used in the Bayesian analysis and the use of spatially-explicit or non-spatial models. A coalescent analysis of population divergence suggests that gene flow is high between the two populations, and that the timing of divergence is restricted to the Pleistocene. The hybrid zone is more accurately described as involving two populations belonging to S. tristichus, and the presence of S. cowlesi mtDNA haplotypes in the hybrid zone is an anomaly resulting from mitochondrial introgression.

Antimicrobial susceptibility among clinical Nocardia species identified by multilocus sequence analysis.

Science.gov (United States)

McTaggart, Lisa R; Doucet, Jennifer; Witkowska, Maria; Richardson, Susan E

2015-01-01

Antimicrobial susceptibility patterns of 112 clinical isolates, 28 type strains, and 9 reference strains of Nocardia were determined using the Sensititre Rapmyco microdilution panel (Thermo Fisher, Inc.). Isolates were identified by highly discriminatory multilocus sequence analysis and were chosen to represent the diversity of species recovered from clinical specimens in Ontario, Canada. Susceptibility to the most commonly used drug, trimethoprim-sulfamethoxazole, was observed in 97% of isolates. Linezolid and amikacin were also highly effective; 100% and 99% of all isolates demonstrated a susceptible phenotype. For the remaining antimicrobials, resistance was species specific with isolates of Nocardia otitidiscaviarum, N. brasiliensis, N. abscessus complex, N. nova complex, N. transvalensis complex, N. farcinica, and N. cyriacigeorgica displaying the traditional characteristic drug pattern types. In addition, the antimicrobial susceptibility profiles of a variety of rarely encountered species isolated from clinical specimens are reported for the first time and were categorized into four additional drug pattern types. Finally, MICs for the control strains N. nova ATCC BAA-2227, N. asteroides ATCC 19247(T), and N. farcinica ATCC 23826 were robustly determined to demonstrate method reproducibility and suitability of the commercial Sensititre Rapmyco panel for antimicrobial susceptibility testing of Nocardia spp. isolated from clinical specimens. The reported values will facilitate quality control and standardization among laboratories. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

MultiLocus Sequence Analysis- and Amplified Fragment Length Polymorphism-based characterization of xanthomonads associated with bacterial spot of tomato and pepper and their relatedness to Xanthomonas species.

Science.gov (United States)

Hamza, A A; Robene-Soustrade, I; Jouen, E; Lefeuvre, P; Chiroleu, F; Fisher-Le Saux, M; Gagnevin, L; Pruvost, O

2012-05-01

MultiLocus Sequence Analysis (MLSA) and Amplified Fragment Length Polymorphism (AFLP) were used to measure the genetic relatedness of a comprehensive collection of xanthomonads pathogenic to solaneous hosts to Xanthomonas species. The MLSA scheme was based on partial sequences of four housekeeping genes (atpD, dnaK, efp and gyrB). Globally, MLSA data unambiguously identified strains causing bacterial spot of tomato and pepper at the species level and was consistent with AFLP data. Genetic distances derived from both techniques showed a close relatedness of (i) X. euvesicatoria, X. perforans and X. alfalfae and (ii) X. gardneri and X. cynarae. Maximum likelihood tree topologies derived from each gene portion and the concatenated data set for species in the X. campestris 16S rRNA core (i.e. the species cluster comprising all strains causing bacterial spot of tomato and pepper) were not congruent, consistent with the detection of several putative recombination events in our data sets by several recombination search algorithms. One recombinant region in atpD was identified in most strains of X. euvesicatoria including the type strain. Copyright © 2012 Elsevier GmbH. All rights reserved.

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

Science.gov (United States)

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

2011-01-01

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

Deciphering the genomic architecture of the stickleback brain with a novel multilocus gene-mapping approach.

Science.gov (United States)

Li, Zitong; Guo, Baocheng; Yang, Jing; Herczeg, Gábor; Gonda, Abigél; Balázs, Gergely; Shikano, Takahito; Calboli, Federico C F; Merilä, Juha

2017-03-01

Quantitative traits important to organismal function and fitness, such as brain size, are presumably controlled by many small-effect loci. Deciphering the genetic architecture of such traits with traditional quantitative trait locus (QTL) mapping methods is challenging. Here, we investigated the genetic architecture of brain size (and the size of five different brain parts) in nine-spined sticklebacks (Pungitius pungitius) with the aid of novel multilocus QTL-mapping approaches based on a de-biased LASSO method. Apart from having more statistical power to detect QTL and reduced rate of false positives than conventional QTL-mapping approaches, the developed methods can handle large marker panels and provide estimates of genomic heritability. Single-locus analyses of an F 2 interpopulation cross with 239 individuals and 15 198, fully informative single nucleotide polymorphisms (SNPs) uncovered 79 QTL associated with variation in stickleback brain size traits. Many of these loci were in strong linkage disequilibrium (LD) with each other, and consequently, a multilocus mapping of individual SNPs, accounting for LD structure in the data, recovered only four significant QTL. However, a multilocus mapping of SNPs grouped by linkage group (LG) identified 14 LGs (1-6 depending on the trait) that influence variation in brain traits. For instance, 17.6% of the variation in relative brain size was explainable by cumulative effects of SNPs distributed over six LGs, whereas 42% of the variation was accounted for by all 21 LGs. Hence, the results suggest that variation in stickleback brain traits is influenced by many small-effect loci. Apart from suggesting moderately heritable (h 2  ≈ 0.15-0.42) multifactorial genetic architecture of brain traits, the results highlight the challenges in identifying the loci contributing to variation in quantitative traits. Nevertheless, the results demonstrate that the novel QTL-mapping approach developed here has distinctive advantages

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China.

Science.gov (United States)

Yang, Xiaojuan; Yu, Shubo; Wu, Qingping; Zhang, Jumei; Wu, Shi; Rong, Dongli

2018-01-01

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes ( sea , seb , sec , sed , see , seg , seh , sei , sej ), the exfoliative toxin genes ( eta and etb ), the toxic shock syndrome toxin-1 gene ( tst ), and the Panton-Valentine leucocidin-encoding gene ( pvl ). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes ( sea - see ), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl , eta , etb , and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China

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Xiaojuan Yang

2018-03-01

Full Text Available The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST and polymerase chain reaction (PCR analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE genes (sea, seb, sec, sed, see, seg, seh, sei, sej, the exfoliative toxin genes (eta and etb, the toxic shock syndrome toxin-1 gene (tst, and the Panton-Valentine leucocidin-encoding gene (pvl. The isolates encompassed 26 different sequence types (STs, including four new STs (ST3482, ST3484, ST3485, ST3504, clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7% detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%, sea (33.3%, and seg (33.3%. The classical SE genes (sea–see, which contribute significantly to staphylococcal food poisoning (SFP, were detected in 72.5% of the S. aureus isolates. In addition, pvl, eta, etb, and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

The current MLVA typing scheme for Enterococcus faecium is less discriminatory than MLST and PFGE for epidemic-virulent, hospital-adapted clonal types

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Klare Ingo

2007-04-01

Full Text Available Abstract Background MLVA (multiple-locus variable-number tandem repeat analysis is a reliable typing technique introduced recently to differentiate also isolates of Enterococcus faecium. We used the established VNTR (variable number of tandem repeats scheme to test its suitability to differentiate 58 E. faecium isolates representing mainly outbreaks and clusters of infections and colonizations among patients from 31 German hospitals. All isolates were vancomycin-resistant (vanA type. Typing results for MLVA are compared with results of macrorestriction analysis in PFGE (pulsed-field gel electrophoresis and MLST (multi-locus sequence typing. Results All 51 but one hospital isolates from 1996–2006 were assigned to the clonal complex (CC of epidemic-virulent, hospital-adapted lineages (MLST CC-17; MLVA CC-1 and differed from isolates of sporadic infections and colonizations (n = 7; 1991–1995 and other non-hospital origins (n = 27. Typing of all 58 hospital VRE revealed MLVA as the least discriminatory method (Simpson's diversity index 0.847 when compared to MLST (0.911 and PFGE (0.976. The two most common MLVA types MT-1 (n = 16 and MT-159 (n = 14 combined isolates of several MLST types including also major epidemic, hospital-adapted, clonal types (MT-1: ST-17, ST-18, ST-280, ST-282; MT-159: ST-78, ST-192, ST-203. These data clearly indicate that non-related E. faecium could possess an identical MLVA type being especially critical when MLVA is used to elucidate supposed outbreaks with E. faecium within a single or among different hospitals. Stability of a given MLVA profile MT-12 (ST-117 during an outbreak over a period of five years was also shown. Conclusion MLVA is a suitable method to assign isolates of E. faecium into distinct clonal complexes. To investigate outbreaks the current MLVA typing scheme for E. faecium does not discriminate enough and cannot be recommended as a standard superior to PFGE.

[Molecular typing methods for Pasteurella multocida-A review].

Science.gov (United States)

Peng, Zhong; Liang, Wan; Wu, Bin

2016-10-04

Pasteurella multocida is an important gram-negative pathogenic bacterium that could infect wide ranges of animals. Humans could also be infected by P. multocida via animal bite or scratching. Current typing methods for P. multocida include serological typing methods and molecular typing methods. Of them, serological typing methods are based on immunological assays, which are too complicated for clinical bacteriological studies. However, the molecular methods including multiple PCRs and multilocus sequence typing (MLST) methods are more suitable for bacteriological studies of P. multocida in clinic, with their simple operation, high efficiency and accurate detection compared to the traditional serological typing methods, they are therefore widely used. In the current review, we briefly describe the molecular typing methods for P. multocida. Our aim is to provide a knowledge-foundation for clinical bacteriological investigation especially the molecular investigation for P. multocida.

Application of multi-locus analytical methods to identify interacting loci in case-control studies.

NARCIS (Netherlands)

Vermeulen, S.; Heijer, M. den; Sham, P.; Knight, J.

2007-01-01

To identify interacting loci in genetic epidemiological studies the application of multi-locus methods of analysis is warranted. Several more advanced classification methods have been developed in the past years, including multiple logistic regression, sum statistics, logic regression, and the

Features of Variable Number of Tandem Repeats in Yersinia pestis and the Development of a Hierarchical Genotyping Scheme.

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Yanjun Li

Full Text Available Variable number of tandem repeats (VNTRs that are widely distributed in the genome of Yersinia pestis proved to be useful markers for the genotyping and source-tracing of this notorious pathogen. In this study, we probed into the features of VNTRs in the Y. pestis genome and developed a simple hierarchical genotyping system based on optimized VNTR loci.Capillary electrophoresis was used in this study for multi-locus VNTR analysis (MLVA in 956 Y. pestis strains. The general features and genetic diversities of 88 VNTR loci in Y. pestis were analyzed with BioNumerics, and a "14+12" loci-based hierarchical genotyping system, which is compatible with single nucleotide polymorphism-based phylogenic analysis, was established.Appropriate selection of target loci reduces the impact of homoplasies caused by the rapid mutation rates of VNTR loci. The optimized "14+12" loci are highly discriminative in genotyping and source-tracing Y. pestis for molecular epidemiological or microbial forensic investigations with less time and lower cost. An MLVA genotyping datasets of representative strains will improve future research on the source-tracing and microevolution of Y. pestis.

Mapping of 34 minisatellite loci resolved by two-dimensional DNA typing

DEFF Research Database (Denmark)

Børglum, Anders; Nyegaard, Mette; Kvistgaard, AB

1997-01-01

Two-dimensional (2-D) DNA typing is based on electrophoretic separation of genomic DNA fragments in two dimensions according to independent criteria (size and base-pair sequence), followed by hybridization analysis using multilocus probes. The technique allows simultaneous visualization of several...... could be deduced, showing no evidence of clustering. In the analysis of spot patterns, use was made of a computerized image analysis system specifically designed for 2-D DNA typing. Since experimental variations between different separation patterns were automatically corrected for with this program......, rapid and reliable scorings could be obtained. The results presented demonstrate the availability of reliable genetic information throughout the 2-D separation pattern. Adding the use of semiautomated computerized pattern analysis, this study further substantiates the applicability of 2-D DNA typing...

Structure and Dynamics of RNA Repeat Expansions That Cause Huntington's Disease and Myotonic Dystrophy Type 1.

Science.gov (United States)

Chen, Jonathan L; VanEtten, Damian M; Fountain, Matthew A; Yildirim, Ilyas; Disney, Matthew D

2017-07-11

RNA repeat expansions cause a host of incurable, genetically defined diseases. The most common class of RNA repeats consists of trinucleotide repeats. These long, repeating transcripts fold into hairpins containing 1 × 1 internal loops that can mediate disease via a variety of mechanism(s) in which RNA is the central player. Two of these disorders are Huntington's disease and myotonic dystrophy type 1, which are caused by r(CAG) and r(CUG) repeats, respectively. We report the structures of two RNA constructs containing three copies of a r(CAG) [r(3×CAG)] or r(CUG) [r(3×CUG)] motif that were modeled with nuclear magnetic resonance spectroscopy and simulated annealing with restrained molecular dynamics. The 1 × 1 internal loops of r(3×CAG) are stabilized by one-hydrogen bond (cis Watson-Crick/Watson-Crick) AA pairs, while those of r(3×CUG) prefer one- or two-hydrogen bond (cis Watson-Crick/Watson-Crick) UU pairs. Assigned chemical shifts for the residues depended on the identity of neighbors or next nearest neighbors. Additional insights into the dynamics of these RNA constructs were gained by molecular dynamics simulations and a discrete path sampling method. Results indicate that the global structures of the RNA are A-form and that the loop regions are dynamic. The results will be useful for understanding the dynamic trajectory of these RNA repeats but also may aid in the development of therapeutics.

Multi-virulence-locus sequence typing of Staphylococcus lugdunensis generates results consistent with a clonal population structure and is reliable for epidemiological typing.

Science.gov (United States)

Didi, Jennifer; Lemée, Ludovic; Gibert, Laure; Pons, Jean-Louis; Pestel-Caron, Martine

2014-10-01

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Clonal diversity and population genetic structure of arbuscular mycorrhizal fungi (Glomus spp.) studied by multilocus genotyping of single spores

DEFF Research Database (Denmark)

Holtgrewe-Stukenbrock, Eva; Rosendahl, Søren

2005-01-01

A nested multiplex PCR (polymerase chain reaction) approach was used for multilocus genotyping of arbuscular mycorrhizal fungal populations. This method allowed us to amplify multiple loci from Glomus single spores in a single PCR amplification. Variable introns in the two protein coding genes Gm......FOX2 and GmTOR2 were applied as codominant genetic markers together with the LSU rDNA.   Genetic structure of Glomus spp. populations from an organically and a conventionally cultured field were compared by hierarchical sampling of spores from four plots in each field. Multilocus genotypes were...

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

Science.gov (United States)

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

2011-11-01

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

Emergence of Sequence Type 779 Methicillin-Resistant Staphylococcus aureus Harboring a Novel Pseudo Staphylococcal Cassette Chromosome mec (SCCmec)-SCC-SCCCRISPR Composite Element in Irish Hospitals

Science.gov (United States)

Kinnevey, Peter M.; Shore, Anna C.; Brennan, Grainne I.; Sullivan, Derek J.; Ehricht, Ralf; Monecke, Stefan; Slickers, Peter

2013-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods. PMID:23147725

Emergence of sequence type 779 methicillin-resistant Staphylococcus aureus harboring a novel pseudo staphylococcal cassette chromosome mec (SCCmec)-SCC-SCCCRISPR composite element in Irish hospitals.

LENUS (Irish Health Repository)

Kinnevey, Peter M

2013-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878\\/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods.

Use of the LUS in sequence allele designations to facilitate probabilistic genotyping of NGS-based STR typing results.

Science.gov (United States)

Just, Rebecca S; Irwin, Jodi A

2018-05-01

Some of the expected advantages of next generation sequencing (NGS) for short tandem repeat (STR) typing include enhanced mixture detection and genotype resolution via sequence variation among non-homologous alleles of the same length. However, at the same time that NGS methods for forensic DNA typing have advanced in recent years, many caseworking laboratories have implemented or are transitioning to probabilistic genotyping to assist the interpretation of complex autosomal STR typing results. Current probabilistic software programs are designed for length-based data, and were not intended to accommodate sequence strings as the product input. Yet to leverage the benefits of NGS for enhanced genotyping and mixture deconvolution, the sequence variation among same-length products must be utilized in some form. Here, we propose use of the longest uninterrupted stretch (LUS) in allele designations as a simple method to represent sequence variation within the STR repeat regions and facilitate - in the nearterm - probabilistic interpretation of NGS-based typing results. An examination of published population data indicated that a reference LUS region is straightforward to define for most autosomal STR loci, and that using repeat unit plus LUS length as the allele designator can represent greater than 80% of the alleles detected by sequencing. A proof of concept study performed using a freely available probabilistic software demonstrated that the LUS length can be used in allele designations when a program does not require alleles to be integers, and that utilizing sequence information improves interpretation of both single-source and mixed contributor STR typing results as compared to using repeat unit information alone. The LUS concept for allele designation maintains the repeat-based allele nomenclature that will permit backward compatibility to extant STR databases, and the LUS lengths themselves will be concordant regardless of the NGS assay or analysis tools

Visualization of pairwise and multilocus linkage disequilibrium structure using latent forests.

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Raphaël Mourad

Full Text Available Linkage disequilibrium study represents a major issue in statistical genetics as it plays a fundamental role in gene mapping and helps us to learn more about human history. The linkage disequilibrium complex structure makes its exploratory data analysis essential yet challenging. Visualization methods, such as the triangular heat map implemented in Haploview, provide simple and useful tools to help understand complex genetic patterns, but remain insufficient to fully describe them. Probabilistic graphical models have been widely recognized as a powerful formalism allowing a concise and accurate modeling of dependences between variables. In this paper, we propose a method for short-range, long-range and chromosome-wide linkage disequilibrium visualization using forests of hierarchical latent class models. Thanks to its hierarchical nature, our method is shown to provide a compact view of both pairwise and multilocus linkage disequilibrium spatial structures for the geneticist. Besides, a multilocus linkage disequilibrium measure has been designed to evaluate linkage disequilibrium in hierarchy clusters. To learn the proposed model, a new scalable algorithm is presented. It constrains the dependence scope, relying on physical positions, and is able to deal with more than one hundred thousand single nucleotide polymorphisms. The proposed algorithm is fast and does not require phase genotypic data.

Thrombospondin-1 type 1 repeats in a model of inflammatory bowel disease: transcript profile and therapeutic effects.

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Zenaida P Lopez-Dee

Full Text Available Thrombospondin-1 (TSP-1 is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS to induce colitis in wild-type (WT mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1 the three type 1 repeats of the TSP-1 (3TSR, (2 the second type 1 repeat (TSR2, or (3 TSR2 with the RFK sequence (TSR2+RFK. Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease.

Molecular characterization of Giardia psittaci by multilocus sequence analysis.

Science.gov (United States)

Abe, Niichiro; Makino, Ikuko; Kojima, Atsushi

2012-12-01

Multilocus sequence analyses targeting small subunit ribosomal DNA (SSU rDNA), elongation factor 1 alpha (ef1α), glutamate dehydrogenase (gdh), and beta giardin (β-giardin) were performed on Giardia psittaci isolates from three Budgerigars (Melopsittacus undulates) and four Barred parakeets (Bolborhynchus lineola) kept in individual households or imported from overseas. Nucleotide differences and phylogenetic analyses at four loci indicate the distinction of G. psittaci from the other known Giardia species: Giardia muris, Giardia microti, Giardia ardeae, and Giardia duodenalis assemblages. Furthermore, G. psittaci was related more closely to G. duodenalis than to the other known Giardia species, except for G. microti. Conflicting signals regarded as "double peaks" were found at the same nucleotide positions of the ef1α in all isolates. However, the sequences of the other three loci, including gdh and β-giardin, which are known to be highly variable, from all isolates were also mutually identical at every locus. They showed no double peaks. These results suggest that double peaks found in the ef1α sequences are caused not by mixed infection with genetically different G. psittaci isolates but by allelic sequence heterogeneity (ASH), which is observed in diplomonad lineages including G. duodenalis. No sequence difference was found in any G. psittaci isolates at the gdh and β-giardin, suggesting that G. psittaci is indeed not more diverse genetically than other Giardia species. This report is the first to provide evidence related to the genetic characteristics of G. psittaci obtained using multilocus sequence analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

Population biology of Streptococcus pneumoniae in West Africa: multilocus sequence typing of serotypes that exhibit different predisposition to invasive disease and carriage.

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Eric S Donkor

Full Text Available Little is known about the population biology of Streptococcus pneumoniae in developing countries, although the majority of pneumococcal infections occur in this setting. The aim of the study was to apply MLST to investigate the population biology of S. pneumoniae in West Africa.Seventy three invasive and carriage S. pneumoniae isolates from three West African countries including The Gambia, Nigeria and Ghana were investigated. The isolates covered seven serotypes (1, 3, 5, 6A, 11, 14, 23F and were subjected to multilocus sequence typing and antibiotic susceptibility testing.Overall, 50 different sequence types (STs were identified, of which 38% (29 were novel. The most common ST was a novel clone-ST 4012 (6.5%, and some clones including STs 913, 925, 1737, 2160 and 3310 appeared to be specific to the study region. Two STs including ST 63 and ST 4012 were associated with multiple serotypes indicating a history of serotype switching. ST 63 was associated with serotypes 3 and 23F, while ST 4012 was associated with serotypes 6A and 23. eBURST analyses using the stringent 6/7 identical loci definition grouped the 50 STs into 5 clonal complexes and 65 singletons, expressing a high level of genetic diversity among the isolates. Compared to the other serotypes, serotypes 1 and 5 isolates appeared to be more clonal. Internationally recognized antibiotic resistant clones of S. pneumoniae were generally absent in the population investigated and the only multidrug resistant isolate identified (1/66 belong to the Pneumocococcal Epidemiology Network clone ST 63.The pneumococcal population in West Africa is quite divergent, and serotypes that are common in invasive disease (such as serotypes 1 and 5 are more likely to be clonal than serotypes that are common in carriage.

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci Multiple Locus Variable-Number Tandem Repeats Analysis

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Ciervo Alessandra

2006-04-01

Full Text Available Abstract Background The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR Analysis (MLVA assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. Results Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. Conclusion In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the

Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats.

Science.gov (United States)

Armour, John A L; Palla, Raquel; Zeeuwen, Patrick L J M; den Heijer, Martin; Schalkwijk, Joost; Hollox, Edward J

2007-01-01

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.

Validation of Diagnostic Imaging Based on Repeat Examinations. An Image Interpretation Model

International Nuclear Information System (INIS)

Isberg, B.; Jorulf, H.; Thorstensen, Oe.

2004-01-01

Purpose: To develop an interpretation model, based on repeatedly acquired images, aimed at improving assessments of technical efficacy and diagnostic accuracy in the detection of small lesions. Material and Methods: A theoretical model is proposed. The studied population consists of subjects that develop focal lesions which increase in size in organs of interest during the study period. The imaging modality produces images that can be re-interpreted with high precision, e.g. conventional radiography, computed tomography, and magnetic resonance imaging. At least four repeat examinations are carried out. Results: The interpretation is performed in four or five steps: 1. Independent readers interpret the examinations chronologically without access to previous or subsequent films. 2. Lesions found on images at the last examination are included in the analysis, with interpretation in consensus. 3. By concurrent back-reading in consensus, the lesions are identified on previous images until they are so small that even in retrospect they are undetectable. The earliest examination at which included lesions appear is recorded, and the lesions are verified by their growth (imaging reference standard). Lesion size and other characteristics may be recorded. 4. Records made at step 1 are corrected to those of steps 2 and 3. False positives are recorded. 5. (Optional) Lesion type is confirmed by another diagnostic test. Conclusion: Applied on subjects with progressive disease, the proposed image interpretation model may improve assessments of technical efficacy and diagnostic accuracy in the detection of small focal lesions. The model may provide an accurate imaging reference standard as well as repeated detection rates and false-positive rates for tested imaging modalities. However, potential review bias necessitates a strict protocol

Revisiting the TALE repeat.

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Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

Germ-line CAG repeat instability causes extreme CAG repeat expansion with infantile-onset spinocerebellar ataxia type 2

DEFF Research Database (Denmark)

Vinther-Jensen, Tua; Ek, Jakob; Duno, Morten

2013-01-01

The spinocerebellar ataxias (SCA) are a genetically and clinically heterogeneous group of diseases, characterized by dominant inheritance, progressive cerebellar ataxia and diverse extracerebellar symptoms. A subgroup of the ataxias is caused by unstable CAG-repeat expansions in their respective ...... of paternal germ-line repeat sequence instability of the expanded SCA2 locus.European Journal of Human Genetics advance online publication, 10 October 2012; doi:10.1038/ejhg.2012.231....

The Evolution of Advanced Molecular Diagnostics for the Detection and Characterization of Mycoplasma pneumoniae.

Science.gov (United States)

Diaz, Maureen H; Winchell, Jonas M

2016-01-01

Over the past decade there have been significant advancements in the methods used for detecting and characterizing Mycoplasma pneumoniae, a common cause of respiratory illness and community-acquired pneumonia worldwide. The repertoire of available molecular diagnostics has greatly expanded from nucleic acid amplification techniques (NAATs) that encompass a variety of chemistries used for detection, to more sophisticated characterizing methods such as multi-locus variable-number tandem-repeat analysis (MLVA), Multi-locus sequence typing (MLST), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), single nucleotide polymorphism typing, and numerous macrolide susceptibility profiling methods, among others. These many molecular-based approaches have been developed and employed to continually increase the level of discrimination and characterization in order to better understand the epidemiology and biology of M. pneumoniae. This review will summarize recent molecular techniques and procedures and lend perspective to how each has enhanced the current understanding of this organism and will emphasize how Next Generation Sequencing may serve as a resource for researchers to gain a more comprehensive understanding of the genomic complexities of this insidious pathogen.

Typing of lymphogranuloma venereum Chlamydia trachomatis strains

NARCIS (Netherlands)

Christerson, Linus; de Vries, Henry J. C.; de Barbeyrac, Bertille; Gaydos, Charlotte A.; Henrich, Birgit; Hoffmann, Steen; Schachter, Julius; Thorvaldsen, Johannes; Vall-Mayans, Martí; Klint, Markus; Herrmann, Björn; Morré, Servaas A.

2010-01-01

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s,

Molecular characterization and multilocus genotypes of Enterocytozoon bieneusi among horses in southwestern China

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Lei Deng

2016-10-01

Full Text Available Abstract Background Enterocytozoon bieneusi is one of the most prevalent causative species of diarrhea and enteric diseases in various hosts. E. bieneusi has been identified in humans, mammals, birds, rodents and reptiles in China, but few studies have reported E. bieneusi in horses. Therefore, the present study was conducted to assess the prevalence, molecular characteristics and zoonotic potential of E. bieneusi among horses in southwestern China. Findings Three hundred and thirty-three fecal specimens were collected from horses on five farms in the Sichuan and Yunnan provinces of southwestern China. The prevalence of E. bieneusi was 22.5 % (75/333, as determined by nested polymerase chain reaction and sequencing analysis of the internal transcribed spacer region of the ribosomal RNA gene of E. bieneusi. Altogether, 10 genotypes were identified among the 75 E. bieneusi-positive samples: four of these genotypes were known (horse1, horse2, SC02 and D and six were novel (SCH1-4 and YNH1-2. Multilocus sequence typing using three microsatellites (MS1, MS3 and MS7 and one minisatellite (MS4 revealed three, two, three and three genotypes at these four loci, respectively. In phylogenetic analysis, all the genotypes of E. bieneusi obtained in this study were clustered into three distinct groups: D, SC02 and SCH1-3 were clustered into group 1 (zoonotic potential; SCH4 was clustered into group 2 (cattle-hosted; whereas horse2, YNH1 and YNH2 were clustered into group 6 (unclear zoonotic potential. Conclusions This is the first report of E. bieneusi among horses in southwestern China. This is also the first multilocus genotyping analysis using microsatellite and minisatellite markers of E. bieneusi in horses. The presence of genotype D, which was previously identified in humans, and genotypes SC02 and SCH1-3, which belong to potential zoonotic group 1, these results indicate that horses are a potential source of human E. bieneusi infections in China.

Test-retest repeatability of strength capacity, aerobic power and pericranial tenderness of neck and shoulder muscles in children - relevant for tension-type headache

DEFF Research Database (Denmark)

Tornøe, Birte; Andersen, Lars L; Skotte, J H

2013-01-01

Frequent or chronic tension-type headache in children is a prevalent and debilitating condition for the child, often leading to medication overuse. To explore the relationship between physical factors and tension-type headache in children, the quality of repeated measures was examined. The aim of...... of the present study was to determine the test-retest repeatability of parameters determining isometric neck and shoulder strength and stability, aerobic power, and pericranial tenderness in children....

Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea

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Lee Edwin KY

2009-01-01

Full Text Available Abstract Background Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of L. hongkongensis. In this study, a multilocus sequence typing (MLST system was developed for L. hongkongensis. The system was used to characterize 146 L. hongkongensis isolates, including 39 from humans and 107 from fish. Results Fragments (362 to 504 bp of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (ilvC to 45 (thiC]. All seven genes showed very low dn/ds ratios of ISA measurement showed significant linkage disequilibrium in isolates from both humans and fish. The ISA for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (acnB was detected in the split graphs. The P-value (P = 0 of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the rho, acnB and thiC loci, but the P-value (P = 1 of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial role in the evolution of L. hongkongensis. A website for L. hongkongensis MLST was set up and can be accessed at http://mlstdb.hku.hk:14206/MLST_index.html. Conclusion A highly reproducible and discriminative MLST system was developed for L. hongkongensis.

Comparative Study of IS6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

Science.gov (United States)

de Beer, Jessica L.; van Ingen, Jakko; de Vries, Gerard; Erkens, Connie; Sebek, Maruschka; Mulder, Arnout; Sloot, Rosa; van den Brandt, Anne-Marie; Enaimi, Mimount; Kremer, Kristin; Supply, Philip

2013-01-01

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a detailed evaluation on discriminatory power and agreement with findings in a cluster investigation was performed on 3,975 tuberculosis cases during the period of 2004 to 2008. The level of discrimination of the two typing methods did not differ substantially: RFLP typing yielded 2,733 distinct patterns compared to 2,607 in VNTR typing. The global concordance, defined as isolates labeled unique or identically distributed in clusters by both methods, amounted to 78.5% (n = 3,123). Of the remaining 855 cases, 12% (n = 479) of the cases were clustered only by VNTR, 7.7% (n = 305) only by RFLP typing, and 1.8% (n = 71) revealed different cluster compositions in the two approaches. A cluster investigation was performed for 87% (n = 1,462) of the cases clustered by RFLP. For the 740 cases with confirmed or presumed epidemiological links, 92% were concordant with VNTR typing. In contrast, only 64% of the 722 cases without an epidemiological link but clustered by RFLP typing were also clustered by VNTR typing. We conclude that VNTR typing has a discriminatory power equal to IS6110 RFLP typing but is in better agreement with findings in a cluster investigation performed on an RFLP-clustering-based cluster investigation. Both aspects make VNTR typing a suitable method for tuberculosis surveillance systems. PMID:23363841

Repeat Customer Success in Extension

Science.gov (United States)

Bess, Melissa M.; Traub, Sarah M.

2013-01-01

Four multi-session research-based programs were offered by two Extension specialist in one rural Missouri county. Eleven participants who came to multiple Extension programs could be called "repeat customers." Based on the total number of participants for all four programs, 25% could be deemed as repeat customers. Repeat customers had…

Repeatability of grasp recognition for robotic hand prosthesis control based on sEMG data.

Science.gov (United States)

Palermo, Francesca; Cognolato, Matteo; Gijsberts, Arjan; Muller, Henning; Caputo, Barbara; Atzori, Manfredo

2017-07-01

Control methods based on sEMG obtained promising results for hand prosthetics. Control system robustness is still often inadequate and does not allow the amputees to perform a large number of movements useful for everyday life. Only few studies analyzed the repeatability of sEMG classification of hand grasps. The main goals of this paper are to explore repeatability in sEMG data and to release a repeatability database with the recorded experiments. The data are recorded from 10 intact subjects repeating 7 grasps 12 times, twice a day for 5 days. The data are publicly available on the Ninapro web page. The analysis for the repeatability is based on the comparison of movement classification accuracy in several data acquisitions and for different subjects. The analysis is performed using mean absolute value and waveform length features and a Random Forest classifier. The accuracy obtained by training and testing on acquisitions at different times is on average 27.03% lower than training and testing on the same acquisition. The results obtained by training and testing on different acquisitions suggest that previous acquisitions can be used to train the classification algorithms. The inter-subject variability is remarkable, suggesting that specific characteristics of the subjects can affect repeatibility and sEMG classification accuracy. In conclusion, the results of this paper can contribute to develop more robust control systems for hand prostheses, while the presented data allows researchers to test repeatability in further analyses.

Prevalence of Chlamydia trachomatis Genotypes in Men Who Have Sex with Men and Men Who Have Sex with Women Using Multilocus VNTR Analysis-ompA Typing in Guangzhou, China.

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Xiaolin Qin

Full Text Available Chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infection in China. Although C. trachomatis genotypes can be discriminated by outer membrane protein gene (ompA sequencing, currently available methods have limited resolutions. This study used a high-resolution genotyping method, namely, multilocus variable number tandem-repeat analysis with ompA sequencing (MLVA-ompA, to investigate the local epidemiology of C. trachomatis infections among men who have sex with men (MSM and men who have sex with women (MSW attending a sexually transmitted diseases (STD clinic in Guangzhou, China.Rectal specimens from MSM and urethral specimens from MSW were collected between January 2013 and July 2014 at the Guangdong Provincial Center STD clinic. The specimens were sent to the laboratory for analyses. All specimens that were tested positive for C. trachomatis by the commercial nucleic acid amplification tests were genotyped by MLVA-ompA.Fifty-one rectal specimens from MSM and 96 urethral specimens from MSW were identified with C. trachomatis. One hundred and forty-four of the 147 specimens were fully genotyped by MLVA-ompA. Rectal specimens from MSM were divided into four ompA genotypes and urethral specimens from MSW into nine genotypes. No mixed infections were found among all specimens. The most frequent genotypes were D, G, J, E and F. All specimens were further divided into 46 types after ompA genotyping was combined with MLVA. Genotypes D-8.7.1 and G-3.4a.3 were the most frequent among MSM, whereas genotypes D-3.4a.4, E-8.5.1, F-8.5.1, and J-3.4a.2 were the most frequent subtypes among MSW. The discriminatory index D was 0.90 for MLVA, 0.85 for ompA, and 0.95 for MLVA-ompA.The most prevalent MLVA-ompA genotypes were significantly different between MSM and MSW from Guangzhou, China. Moreover, MLVA-ompA represented a more favorable degree of discrimination than ompA and could be a reliable complement for ompA for the routine

Comparative study of IS6110 restriction fragment length polymorphism and variable-number tandem-repeat typing of Mycobacterium tuberculosis isolates in the Netherlands, based on a 5-year nationwide survey

NARCIS (Netherlands)

de Beer, Jessica L.; van Ingen, Jakko; de Vries, Gerard; Erkens, Connie; Sebek, Maruschka; Mulder, Arnout; Sloot, Rosa; van den Brandt, Anne-Marie; Enaimi, Mimount; Kremer, Kristin; Supply, Philip; van Soolingen, Dick

2013-01-01

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a

Comparative Study of IS6110 Restriction Fragment Length Polymorphism and Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Isolates in the Netherlands, Based on a 5-Year Nationwide Survey

NARCIS (Netherlands)

Beer, J.L. de; Ingen, J. van; Vries, G. de; Erkens, C.; Sebek, M.; Mulder, A.; Sloot, R.; Brandt, A.M. van den; Enaimi, M.; Kremer, K.; Supply, P.; Soolingen, D. van

2013-01-01

In order to switch from IS6110 and polymorphic GC-rich repetitive sequence (PGRS) restriction fragment length polymorphism (RFLP) to 24-locus variable-number tandem-repeat (VNTR) typing of Mycobacterium tuberculosis complex isolates in the national tuberculosis control program in The Netherlands, a

Suppressing non-periodically repeating disturbances in mechanical servo systems

NARCIS (Netherlands)

Tousain, R.L.; Boissy, J.C.; Norg, M.L.; Steinbuch, M.; Bosgra, O.H.

1998-01-01

Non-periodically repeating (NPR) disturbances are fixed-shape disturbances that occur randomly in time. We can provide a control system with the capability to suppress this type of disturbance by adding in parallel to the input of the nominal feedback controller a learning look-up-table based

X-Chromosome short tandem repeat, advantages and typing ...

African Journals Online (AJOL)

Microsatellites of the X-chromosome have been increasingly studied in recent years as a useful tool in forensic analysis. This review describes some details of X-chromosomal short tandem repeat (STR) analysis. Among them are: microsatellites, amplification using polymerase chain reaction (PCR) of STRs, PCR product ...

Patient preferences for types of community-based cardiac rehabilitation programme.

Science.gov (United States)

Chia, Shermain; Wong, Xin Yi; Toon, Min Li; Seah, Yi; Yap, Angela Frances; Lim, Cindy; Tay, Hung Yong; Fong, Warren; Low, Lian Leng; Kwan, Yu Heng

2018-01-01

Cardiac rehabilitation (CR) improves mortality, morbidity and quality of life of cardiovascular patients. However, its uptake is poor especially in the hospitals due to long travel distances and office hours constraints. Community-based CR is a possible solution. To understand the type of community-based CR preferred and identify patient characteristics associated with certain programme combinations. A cross-sectional survey was administered to a randomised list of patients at risk for or with cardiovascular diseases at two community-based CR centres. Participants were presented with nine hypothetical choice sets and asked to choose only one of the two alternative programme combinations in each choice set. Attributes include support group presence, cash incentives, upfront deposit and out-of-pocket cost. The counts for each combination were tallied and corrected for repeats. Chi-square test and logistic regression were performed to understand the characteristics associated with the preferred CR combination. After correcting for repeats, patients most (85.2%) prefer CR programmes with new group activities, support group, cash rewards, deposit and out-of-pocket cost, and few exercise equipment with physiotherapist presence without the need for monitoring equipment. Patients with more than three bedrooms in their house are less likely (OR 0.367; CI 0.17 to 0.80; P=0.011) to choose the choice with no physiotherapist and few equipment available. This is the first study to explore patients' preferences for different types of community CR. Higher income patients prefer physiotherapist presence and are willing to settle for less equipment. Our study serves as a guide for designing future community-based CR programmes.

R-loops: targets for nuclease cleavage and repeat instability.

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Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Typing of Lymphogranuloma Venereum Chlamydia trachomatis Strains

Science.gov (United States)

Christerson, Linus; de Vries, Henry J.C.; de Barbeyrac, Bertille; Gaydos, Charlotte A.; Henrich, Birgit; Hoffmann, Steen; Schachter, Julius; Thorvaldsen, Johannes; Vall-Mayans, Martí; Klint, Markus; Morré, Servaas A.

2010-01-01

We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe. PMID:21029543

Investigation of genetic diversity and epidemiological characteristics of Pasteurella multocida isolates from poultry in southwest China by population structure, multi-locus sequence typing and virulence-associated gene profile analysis.

Science.gov (United States)

Li, Zhangcheng; Cheng, Fangjun; Lan, Shimei; Guo, Jianhua; Liu, Wei; Li, Xiaoyan; Luo, Zeli; Zhang, Manli; Wu, Juan; Shi, Yang

2018-04-25

Fowl cholera caused by Pasteurella multocida has always been a disease of global importance for poultry production. The aim of this study was to obtain more information about the epidemiology of avian P. multocida infection in southwest China and the genetic characteristics of clinical isolates. P. multocida isolates were characterized by biochemical and molecular-biological methods. The distributions of the capsular serogroups, the phenotypic antimicrobial resistance profiles, lipopolysaccharide (LPS) genotyping and the presence of 19 virulence genes were investigated in 45 isolates of P. multocida that were associated with clinical disease in poultry. The genetic diversity of P. multocida strains was performed by 16S rRNA and rpoB gene sequence analysis as well as multilocus sequence typing (MLST). The results showed that most (80.0%) of the P. multocida isolates in this study represented special P. multocida subspecies, and 71.1% of the isolates showed multiple-drug resistance. 45 isolates belonged to capsular types: A (100%) and two LPS genotypes: L1 (95.6%) and L3 (4.4%). MLST revealed two new alleles (pmi77 and gdh57) and one new sequence type (ST342). ST129 types dominated in 45 P. multocida isolates. Isolates belonging to ST129 were with the genes ompH+plpB+ptfA+tonB, whereas ST342 included isolates with fur+hgbA+tonB genes. Population genetic analysis and the MLST results revealed that at least one new ST genotype was present in the avian P. multocida in China. These findings provide novel insights into the epidemiological characteristics of avian P. multocida isolates in southwest China.

Multilocus Microsatellite Typing reveals intra-focal genetic diversity among strains of Leishmania tropica in Chichaoua Province, Morocco.

Science.gov (United States)

Krayter, Lena; Alam, Mohammad Zahangir; Rhajaoui, Mohamed; Schnur, Lionel F; Schönian, Gabriele

2014-12-01

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is a major public health threat. Strains of this species have been shown to display considerable serological, biochemical, molecular biological and genetic heterogeneity; and Multilocus Enzyme Electrophoresis (MLEE), has shown that in many countries including Morocco heterogenic variants of L. tropica can co-exist in single geographical foci. Here, the microsatellite profiles discerned by MLMT of nine Moroccan strains of L. tropica isolated in 2000 from human cases of CL from Chichaoua Province were compared to those of nine Moroccan strains of L. tropica isolated between 1988 and 1990 from human cases of CL from Marrakech Province, and also to those of 147 strains of L. tropica isolated at different times from different worldwide geographical locations within the range of distribution of the species. Several programs, each employing a different algorithm, were used for population genetic analysis. The strains from each of the two Moroccan foci separated into two phylogenetic clusters independent of their geographical origin. Genetic diversity and heterogeneity existed in both foci, which are geographically close to each other. This intra-focal distribution of genetic variants of L. tropica is not considered owing to in situ mutation. Rather, it is proposed to be explained by the importation of pre-existing variants of L. tropica into Morocco. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Optimization of sequence alignment for simple sequence repeat regions

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Ogbonnaya Francis C

2011-07-01

Full Text Available Abstract Background Microsatellites, or simple sequence repeats (SSRs, are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs. SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships. Findings To overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type. When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm. Conclusions The new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic

High-Resolution Typing Reveals Distinct Chlamydia trachomatis Strains in an At-Risk Population in Nanjing, China

NARCIS (Netherlands)

Bom, Reinier J. M.; van den Hoek, Anneke; Wang, Qianqiu; Long, Fuquan; de Vries, Henry J. C.; Bruisten, Sylvia M.

2013-01-01

We investigated Chlamydia trachomatis strains from Nanjing, China, and whether these strains differed from Amsterdam, the Netherlands. C. trachomatis type was determined with multilocus sequence typing. Most strains were specific to Nanjing, but some clustered with strains from Amsterdam. This

A Network-Based Algorithm for Clustering Multivariate Repeated Measures Data

Science.gov (United States)

Koslovsky, Matthew; Arellano, John; Schaefer, Caroline; Feiveson, Alan; Young, Millennia; Lee, Stuart

2017-01-01

The National Aeronautics and Space Administration (NASA) Astronaut Corps is a unique occupational cohort for which vast amounts of measures data have been collected repeatedly in research or operational studies pre-, in-, and post-flight, as well as during multiple clinical care visits. In exploratory analyses aimed at generating hypotheses regarding physiological changes associated with spaceflight exposure, such as impaired vision, it is of interest to identify anomalies and trends across these expansive datasets. Multivariate clustering algorithms for repeated measures data may help parse the data to identify homogeneous groups of astronauts that have higher risks for a particular physiological change. However, available clustering methods may not be able to accommodate the complex data structures found in NASA data, since the methods often rely on strict model assumptions, require equally-spaced and balanced assessment times, cannot accommodate missing data or differing time scales across variables, and cannot process continuous and discrete data simultaneously. To fill this gap, we propose a network-based, multivariate clustering algorithm for repeated measures data that can be tailored to fit various research settings. Using simulated data, we demonstrate how our method can be used to identify patterns in complex data structures found in practice.

Characterisation by multilocus sequence and porA and flaA typing of Campylobacter jejuni isolated from samples of dog faeces collected in one city in New Zealand.

Science.gov (United States)

Mohan, V; Stevenson, M A; Marshall, J C; French, N P

2017-07-01

To investigate the prevalence of Campylobacter spp. and C. jejuni in dog faecal material collected from dog walkways in the city of Palmerston North, New Zealand, and to characterise the C. jejuni isolates by multilocus sequence typing (MLST) and porA and flaA antigen gene typing. A total of 355 fresh samples of dogs faeces were collected from bins provided for the disposal of dog faeces in 10 walkways in Palmerston North, New Zealand, between August 2008-July 2009. Presumptive Campylobacter colonies, cultured on modified charcoal cefoperazone deoxycholate plates, were screened for genus Campylobacter and C. jejuni by PCR. The C. jejuni isolates were subsequently characterised by MLST and porA and flaA typing, and C. jejuni sequence types (ST) were assigned. Of the 355 samples collected, 72 (20 (95% CI=16-25)%) were positive for Campylobacter spp. and 22 (6 (95% CI=4-9)%) were positive for C. jejuni. Of the 22 C. jejuni isolates, 19 were fully typed by MLST. Ten isolates were assigned to the clonal complex ST-45 and three to ST-52. The allelic combinations of ST-45/flaA 21/porA 44 (n=3), ST-45/flaA 22/porA 53 (n=3) and ST-52/ flaA 57/porA 905 (n=3) were most frequent. The successful isolation of C. jejuni from canine faecal samples collected from faecal bins provides evidence that Campylobacter spp. may survive outside the host for at least several hours despite requiring fastidious growth conditions in culture. The results show that dogs carry C. jejuni genotypes (ST-45, ST-50, ST-52 and ST-696) that have been reported in human clinical cases. Although these results do not provide any evidence either for the direction of infection or for dogs being a potential risk factor for human campylobacteriosis, dog owners are advised to practice good hygiene with respect to their pets to reduce potential exposure to infection.

Abnormal Base Excision Repair at Trinucleotide Repeats Associated with Diseases: A Tissue-Selective Mechanism

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Agathi-Vasiliki Goula

2013-07-01

Full Text Available More than fifteen genetic diseases, including Huntington’s disease, myotonic dystrophy 1, fragile X syndrome and Friedreich ataxia, are caused by the aberrant expansion of a trinucleotide repeat. The mutation is unstable and further expands in specific cells or tissues with time, which can accelerate disease progression. DNA damage and base excision repair (BER are involved in repeat instability and might contribute to the tissue selectivity of the process. In this review, we will discuss the mechanisms of trinucleotide repeat instability, focusing more specifically on the role of BER.

A repeatedly refuelable mediated biofuel cell based on a hierarchical porous carbon electrode

Science.gov (United States)

Fujita, Shuji; Yamanoi, Shun; Murata, Kenichi; Mita, Hiroki; Samukawa, Tsunetoshi; Nakagawa, Takaaki; Sakai, Hideki; Tokita, Yuichi

2014-05-01

Biofuel cells that generate electricity from renewable fuels, such as carbohydrates, must be reusable through repeated refuelling, should these devices be used in consumer electronics. We demonstrate the stable generation of electricity from a glucose-powered mediated biofuel cell through multiple refuelling cycles. This refuelability is achieved by immobilizing nicotinamide adenine dinucleotide (NAD), an electron-transfer mediator, and redox enzymes in high concentrations on porous carbon particles constituting an anode while maintaining their electrochemical and enzymatic activities after the immobilization. This bioanode can be refuelled continuously for more than 60 cycles at 1.5 mA cm-2 without significant potential drop. Cells assembled with these bioanodes and bilirubin-oxidase-based biocathodes can be repeatedly used to power a portable music player at 1 mW cm-3 through 10 refuelling cycles. This study suggests that the refuelability within consumer electronics should facilitate the development of long and repeated use of the mediated biofuel cells as well as of NAD-based biosensors, bioreactors, and clinical applications.

All-photonic quantum repeaters

Science.gov (United States)

Azuma, Koji; Tamaki, Kiyoshi; Lo, Hoi-Kwong

2015-01-01

Quantum communication holds promise for unconditionally secure transmission of secret messages and faithful transfer of unknown quantum states. Photons appear to be the medium of choice for quantum communication. Owing to photon losses, robust quantum communication over long lossy channels requires quantum repeaters. It is widely believed that a necessary and highly demanding requirement for quantum repeaters is the existence of matter quantum memories. Here we show that such a requirement is, in fact, unnecessary by introducing the concept of all-photonic quantum repeaters based on flying qubits. In particular, we present a protocol based on photonic cluster-state machine guns and a loss-tolerant measurement equipped with local high-speed active feedforwards. We show that, with such all-photonic quantum repeaters, the communication efficiency scales polynomially with the channel distance. Our result paves a new route towards quantum repeaters with efficient single-photon sources rather than matter quantum memories. PMID:25873153

SCCmecFinder, a Web-Based Tool for Typing of Staphylococcal Cassette Chromosome mec in Staphylococcus aureus Using Whole-Genome Sequence Data

DEFF Research Database (Denmark)

Kaya, Hülya; Hasman, Henrik; Larsen, Jesper

2018-01-01

Typing of methicillin-resistant Staphylococcus aureus (MRSA) is important in infection control and surveillance. The current nomenclature of MRSA includes the genetic background of the S. aureus strain determined by multilocus sequence typing (MLST) or equivalent methods like spa typing and typing...... of the genetic background and SCCmec, but so far, no bioinformatic tools for SCCmec typing have been available. Here, we report the development and evaluation of SCCmecFinder for characterization of the SCCmec element from S. aureus WGS data. SCCmecFinder is able to identify all SCCmec element types, designated...... a diverse collection of clinical isolates (n = 93) showed a high typeability level of 96.7%, which increased to 98.9% upon modification of the default settings. In conclusion, SCCmecFinder can be an alternative to more laborious SCCmec typing methods and is freely available at https...

Multilocus Sequence Analysis of Cercospora spp. from Different Host Plant Families

Directory of Open Access Journals (Sweden)

Floreta Fiska Yuliarni

2014-06-01

Full Text Available Identification of the genus Cercospora is still complicated due to the host preferences often being used as the main criteria to propose a new name. We determined the relationship between host plants and multilocus sequence variations (ITS rDNA including 5.8S rDNA, elongation factor 1-α, and calmodulin in Cercospora spp. to investigate the host specificity. We used 53 strains of Cercospora spp. infecting 12 plant families for phylogenetic analysis. The sequences of 23 strains of Cercospora spp. infecting the plant families of Asteraceae, Cucurbitaceae, and Solanaceae were determined in this study. The sequences of 30 strains of Cercospora spp. infecting the plant families of Fabaceae, Amaranthaceae, Apiaceae, Plumbaginaceae, Malvaceae, Cistaceae, Plantaginaceae, Lamiaceae, and Poaceae were obtained from GenBank. The molecular phylogenetic analysis revealed that the majority of Cercospora species lack host specificity, and only C. zinniicola, C. zeina, C. zeae-maydis, C. cocciniae, and C. mikaniicola were found to be host-specific. Closely related species of Cercospora could not be distinguished using molecular analyses of ITS, EF, and CAL gene regions. The topology of the phylogenetic tree based on the CAL gene showed a better topology and Cercospora species separation than the trees developed based on the ITS rDNA region or the EF gene.

Differential Regulation of Strand-Specific Transcripts from Arabidopsis Centromeric Satellite Repeats.

Directory of Open Access Journals (Sweden)

2005-12-01

Full Text Available Centromeres interact with the spindle apparatus to enable chromosome disjunction and typically contain thousands of tandemly arranged satellite repeats interspersed with retrotransposons. While their role has been obscure, centromeric repeats are epigenetically modified and centromere specification has a strong epigenetic component. In the yeast Schizosaccharomyces pombe, long heterochromatic repeats are transcribed and contribute to centromere function via RNA interference (RNAi. In the higher plant Arabidopsis thaliana, as in mammalian cells, centromeric satellite repeats are short (180 base pairs, are found in thousands of tandem copies, and are methylated. We have found transcripts from both strands of canonical, bulk Arabidopsis repeats. At least one subfamily of 180-base pair repeats is transcribed from only one strand and regulated by RNAi and histone modification. A second subfamily of repeats is also silenced, but silencing is lost on both strands in mutants in the CpG DNA methyltransferase MET1, the histone deacetylase HDA6/SIL1, or the chromatin remodeling ATPase DDM1. This regulation is due to transcription from Athila2 retrotransposons, which integrate in both orientations relative to the repeats, and differs between strains of Arabidopsis. Silencing lost in met1 or hda6 is reestablished in backcrosses to wild-type, but silencing lost in RNAi mutants and ddm1 is not. Twenty-four-nucleotide small interfering RNAs from centromeric repeats are retained in met1 and hda6, but not in ddm1, and may have a role in this epigenetic inheritance. Histone H3 lysine-9 dimethylation is associated with both classes of repeats. We propose roles for transcribed repeats in the epigenetic inheritance and evolution of centromeres.

Incremental Dynamic Analysis of Koyna Dam under Repeated Ground Motions

Science.gov (United States)

Zainab Nik Azizan, Nik; Majid, Taksiah A.; Nazri, Fadzli Mohamed; Maity, Damodar; Abdullah, Junaidah

2018-03-01

This paper discovers the incremental dynamic analysis (IDA) of concrete gravity dam under single and repeated earthquake loadings to identify the limit state of the dam. Seven ground motions with horizontal and vertical direction as seismic input considered in the nonlinear dynamic analysis based on the real repeated earthquake in the worldwide. All the ground motions convert to respond spectrum and scaled according to the developed elastic respond spectrum in order to match the characteristic of the ground motion to the soil type. The scaled was depends on the fundamental period, T1 of the dam. The Koyna dam has been selected as a case study for the purpose of the analysis by assuming that no sliding and rigid foundation, has been estimated. IDA curves for Koyna dam developed for single and repeated ground motions and the performance level of the dam identifies. The IDA curve of repeated ground motion shown stiffer rather than single ground motion. The ultimate state displacement for a single event is 45.59mm and decreased to 39.33mm under repeated events which are decreased about 14%. This showed that the performance level of the dam based on seismic loadings depend on ground motion pattern.

Staphylococcus aureus spa type t437

DEFF Research Database (Denmark)

Glasner, C; Pluister, G; Westh, H

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among...... included. Most isolates were shown to be monophyletic with 98% of the isolates belonging to the single MLVA complex 621, to which nearly all included isolates from China also belonged. More importantly, all MLST-typed isolates belonged to CC59. Our study implies that the European S. aureus t437 population...

Real-time whole-genome sequencing for routine typing, surveillance, and outbreak detection of verotoxigenic Escherichia coli

DEFF Research Database (Denmark)

Joensen, Katrine Grimstrup; Scheutz, Flemming; Lund, Ole

2014-01-01

suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing...

Human epidermal growth factor receptor 2 testing in invasive breast cancer: should histological grade, type and oestrogen receptor status influence the decision to repeat testing?

Science.gov (United States)

Rakha, Emad A; Pigera, Marian; Shin, Sandra J; D'Alfonso, Timothy; Ellis, Ian O; Lee, Andrew H S

2016-07-01

The recent American Society of Clinical Oncology/College of American Pathologists guidelines for human epidermal growth factor receptor 2 (HER2) testing in breast cancer recommend repeat testing based on tumour grade, tumour type, and hormone receptor status. The aim of this study was to test the value of these criteria. HER2 status was concordant in the core biopsies and excision specimens in 392 of 400 invasive carcinomas. The major reasons for discordance were amplification around the cut-off for positivity and tumour heterogeneity. Of 116 grade 3 carcinomas that were HER2-negative in the core biopsy, four were HER2-positive in the excision specimen. Three of these four either showed borderline negative amplification in the core biopsy or were heterogeneous. None of the 55 grade 1 carcinomas were HER2-positive. Review of repeat testing of HER2 in routine practice suggested that it may also be of value for multifocal tumours and if recommended by the person assessing the in-situ hybridization. Mandatory repeat HER2 testing of grade 3 HER2-negative carcinomas is not appropriate. This is particularly true if repeat testing is performed after borderline negative amplification in the core biopsy or in HER2-negative heterogeneous carcinomas. © 2015 John Wiley & Sons Ltd.

Genetic Relatedness Among Shiga Toxin-Producing Escherichia coli Isolated Along the Animal Food Supply Chain and in Gastroenteritis Cases in Qatar Using Multilocus Sequence Typing.

Science.gov (United States)

Palanisamy, Srikanth; Chang, YuChen; Scaria, Joy; Penha Filho, Rafael Antonio Casarin; Peters, Kenlyn E; Doiphode, Sanjay H; Sultan, Ali; Mohammed, Hussni O

2017-06-01

Pathogenic Escherichia coli has been listed among the most important bacteria associated with foodborne illnesses around the world. We investigated the genetic relatedness among Shiga toxin-producing E. coli (STEC) isolated along the animal food supply chain and from humans diagnosed with gastroenteritis in Qatar. Samples were collected from different sources along the food supply chain and from patients admitted to the hospital with complaints of gastroenteritis. All samples were screened for the presence of E. coli O157:H7 and non-O157 STEC using a combination of bacterial enrichment and molecular detection techniques. A proportional sampling approach was used to select positive samples from each source for further multilocus sequence typing (MLST) analysis. Seven housekeeping genes described for STEC were amplified by polymerase chain reaction, sequenced, and analyzed by MLST. Isolates were characterized by allele composition, sequence type (ST) and assessed for epidemiologic relationship within and among different sources. Nei's genetic distance was calculated at the allele level between sample pools in each site downstream. E. coli O157:H7 occurred at a higher rate in slaughterhouse and retail samples than at the farm or in humans in our sampling. The ST171, an ST common to enterotoxigenic E. coli and atypical enteropathogenic E. coli, was the most common ST (15%) in the food supply chain. None of the genetic distances among the different sources was statistically significant. Enterohemorrhagic E. coli pathogenic strains are present along the supply chain at different levels and with varying relatedness. Clinical isolates were the most diverse, as expected, considering the polyclonal diversity in the human microbiota. The high occurrence of these food adulterants among the farm products suggests that implementation of sanitary measures at that level might reduce the risk of human exposure.

Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material

NARCIS (Netherlands)

de Beer, Jessica L.; Akkerman, Onno W.; Schurch, Anita C.; Mulder, Arnout; van der Werf, Tjip S.; van der Zanden, Adri G. M.; van Ingen, Jakko; van Soolingen, Dick

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable

Repeated dose titration versus age-based method in electroconvulsive therapy: a pilot study

NARCIS (Netherlands)

Aten, J.J.; Oudega, M.L.; van Exel, E.; Stek, M.L.; van Waarde, J.A.

2015-01-01

In electroconvulsive therapy (ECT), a dose titration method (DTM) was suggested to be more individualized and therefore more accurate than formula-based dosing methods. A repeated DTM (every sixth session and dose adjustment accordingly) was compared to an age-based method (ABM) regarding treatment

Integrative taxonomy by molecular species delimitation: multi-locus data corroborate a new species of Balkan Drusinae micro-endemics.

Science.gov (United States)

Vitecek, Simon; Kučinić, Mladen; Previšić, Ana; Živić, Ivana; Stojanović, Katarina; Keresztes, Lujza; Bálint, Miklós; Hoppeler, Felicitas; Waringer, Johann; Graf, Wolfram; Pauls, Steffen U

2017-06-06

Taxonomy offers precise species identification and delimitation and thus provides basic information for biological research, e.g. through assessment of species richness. The importance of molecular taxonomy, i.e., the identification and delimitation of taxa based on molecular markers, has increased in the past decade. Recently developed exploratory tools now allow estimating species-level diversity in multi-locus molecular datasets. Here we use molecular species delimitation tools that either quantify differences in intra- and interspecific variability of loci, or divergence times within and between species, or perform coalescent species tree inference to estimate species-level entities in molecular genetic datasets. We benchmark results from these methods against 14 morphologically readily differentiable species of a well-defined subgroup of the diverse Drusinae subfamily (Trichoptera, Limnephilidae). Using a 3798 bp (6 loci) molecular data set we aim to corroborate a geographically isolated new species by integrating comparative morphological studies and molecular taxonomy. Our results indicate that only multi-locus species delimitation provides taxonomically relevant information. The data further corroborate the new species Drusus zivici sp. nov. We provide differential diagnostic characters and describe the male, female and larva of this new species and discuss diversity patterns of Drusinae in the Balkans. We further discuss potential and significance of molecular species delimitation. Finally we argue that enhancing collaborative integrative taxonomy will accelerate assessment of global diversity and completion of reference libraries for applied fields, e.g., conservation and biomonitoring.

MCMC multilocus lod scores: application of a new approach.

Science.gov (United States)

George, Andrew W; Wijsman, Ellen M; Thompson, Elizabeth A

2005-01-01

On extended pedigrees with extensive missing data, the calculation of multilocus likelihoods for linkage analysis is often beyond the computational bounds of exact methods. Growing interest therefore surrounds the implementation of Monte Carlo estimation methods. In this paper, we demonstrate the speed and accuracy of a new Markov chain Monte Carlo method for the estimation of linkage likelihoods through an analysis of real data from a study of early-onset Alzheimer's disease. For those data sets where comparison with exact analysis is possible, we achieved up to a 100-fold increase in speed. Our approach is implemented in the program lm_bayes within the framework of the freely available MORGAN 2.6 package for Monte Carlo genetic analysis (http://www.stat.washington.edu/thompson/Genepi/MORGAN/Morgan.shtml).

How do repeat suicide attempters differ from first timers? An exploratory record based analysis

Directory of Open Access Journals (Sweden)

Vikas Menon

2016-01-01

Full Text Available Background: Evidence indicates that repeat suicide attempters, as a group, may differ from 1st time attempters. The identification of repeat attempters is a powerful but underutilized clinical variable. Aims: In this research, we aimed to compare individuals with lifetime histories of multiple attempts with 1st time attempters to identify factors predictive of repeat attempts. Setting and Design: This was a retrospective record based study carried out at a teaching cum Tertiary Care Hospital in South India. Methods: Relevant data was extracted from the clinical records of 1st time attempters (n = 362 and repeat attempters (n = 61 presenting to a single Tertiary Care Center over a 4½ year period. They were compared on various sociodemographic and clinical parameters. The clinical measures included Presumptive Stressful Life Events Scale, Beck Hopelessness Scale, Coping Strategies Inventory – Short Form, and the Global Assessment of Functioning Scale. Statistical Analysis Used: First time attempters and repeaters were compared using appropriate inferential statistics. Logistic regression was used to identify independent predictors of repeat attempts. Results: The two groups did not significantly differ on sociodemographic characteristics. Repeat attempters were more likely to have given prior hints about their act (χ2 = 4.500, P = 0.034. In the final regression model, beck hopelessness score emerged as a significant predictor of repeat suicide attempts (odds ratio = 1.064, P = 0.020. Conclusion: Among suicide attempters presenting to the hospital, the presence of hopelessness is a predictor of repeat suicide attempts, independent of clinical depression. This highlights the importance of considering hopelessness in the assessment of suicidality with a view to minimize the risk of future attempts.

Clustered Regularly Interspaced Short Palindromic Repeats Are emm Type-Specific in Highly Prevalent Group A Streptococci.

Science.gov (United States)

Zheng, Po-Xing; Chan, Yuen-Chi; Chiou, Chien-Shun; Chiang-Ni, Chuan; Wang, Shu-Ying; Tsai, Pei-Jane; Chuang, Woei-Jer; Lin, Yee-Shin; Liu, Ching-Chuan; Wu, Jiunn-Jong

2015-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) are the bacterial adaptive immune system against foreign nucleic acids. Given the variable nature of CRISPR, it could be a good marker for molecular epidemiology. Group A streptococcus is one of the major human pathogens. It has two CRISPR loci, including CRISPR01 and CRISPR02. The aim of this study was to analyze the distribution of CRISPR-associated gene cassettes (cas) and CRISPR arrays in highly prevalent emm types. The cas cassette and CRISPR array in two CRISPR loci were analyzed in a total of 332 strains, including emm1, emm3, emm4, emm12, and emm28 strains. The CRISPR type was defined by the spacer content of each CRISPR array. All strains had at least one cas cassette or CRISPR array. More than 90% of the spacers were found in one emm type, specifically. Comparing the consistency between emm and CRISPR types by Simpson's index of diversity and the adjusted Wallace coefficient, CRISPR01 type was concordant to emm type, and CRISPR02 showed unidirectional congruence to emm type, suggesting that at least for the majority of isolates causing infection in high income countries, the emm type can be inferred from CRISPR analysis, which can further discriminate isolates sharing the same emm type.

RNA FISH for detecting expanded repeats in human diseases.

Science.gov (United States)

Urbanek, Martyna O; Krzyzosiak, Wlodzimierz J

2016-04-01

RNA fluorescence in situ hybridization (FISH) is a widely used technique for detecting transcripts in fixed cells and tissues. Many variants of RNA FISH have been proposed to increase signal strength, resolution and target specificity. The current variants of this technique facilitate the detection of the subcellular localization of transcripts at a single molecule level. Among the applications of RNA FISH are studies on nuclear RNA foci in diseases resulting from the expansion of tri-, tetra-, penta- and hexanucleotide repeats present in different single genes. The partial or complete retention of mutant transcripts forming RNA aggregates within the nucleoplasm has been shown in multiple cellular disease models and in the tissues of patients affected with these atypical mutations. Relevant diseases include, among others, myotonic dystrophy type 1 (DM1) with CUG repeats, Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) with CAG repeats, fragile X-associated tremor/ataxia syndrome (FXTAS) with CGG repeats, myotonic dystrophy type 2 (DM2) with CCUG repeats, amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) with GGGGCC repeats and spinocerebellar ataxia type 32 (SCA32) with GGCCUG. In this article, we summarize the results obtained with FISH to examine RNA nuclear inclusions. We provide a detailed protocol for detecting RNAs containing expanded CAG and CUG repeats in different cellular models, including fibroblasts, lymphoblasts, induced pluripotent stem cells and murine and human neuronal progenitors. We also present the results of the first single-molecule FISH application in a cellular model of polyglutamine disease. Copyright © 2015 Elsevier Inc. All rights reserved.

Accurate typing of short tandem repeats from genome-wide sequencing data and its applications.

Science.gov (United States)

Fungtammasan, Arkarachai; Ananda, Guruprasad; Hile, Suzanne E; Su, Marcia Shu-Wei; Sun, Chen; Harris, Robert; Medvedev, Paul; Eckert, Kristin; Makova, Kateryna D

2015-05-01

Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution. © 2015 Fungtammasan et al.; Published by Cold Spring Harbor Laboratory Press.

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

Science.gov (United States)

Arulandhu, Alfred J; Staats, Martijn; Hagelaar, Rico; Voorhuijzen, Marleen M; Prins, Theo W; Scholtens, Ingrid; Costessi, Adalberto; Duijsings, Danny; Rechenmann, François; Gaspar, Frédéric B; Barreto Crespo, Maria Teresa; Holst-Jensen, Arne; Birck, Matthew; Burns, Malcolm; Haynes, Edward; Hochegger, Rupert; Klingl, Alexander; Lundberg, Lisa; Natale, Chiara; Niekamp, Hauke; Perri, Elena; Barbante, Alessandra; Rosec, Jean-Philippe; Seyfarth, Ralf; Sovová, Tereza; Van Moorleghem, Christoff; van Ruth, Saskia; Peelen, Tamara; Kok, Esther

2017-10-01

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance. © The Authors 2017. Published by Oxford University Press.

Coagulase-Negative Staphylococci in Human Milk From Mothers of Preterm Compared With Term Neonates.

Science.gov (United States)

Soeorg, Hiie; Metsvaht, Tuuli; Eelmäe, Imbi; Metsvaht, Hanna Kadri; Treumuth, Sirli; Merila, Mirjam; Ilmoja, Mari-Liis; Lutsar, Irja

2017-05-01

Human milk is the preferred nutrition for neonates and a source of bacteria. Research aim: The authors aimed to characterize the molecular epidemiology and genetic content of staphylococci in the human milk of mothers of preterm and term neonates. Staphylococci were isolated once per week in the 1st month postpartum from the human milk of mothers of 20 healthy term and 49 preterm neonates hospitalized in the neonatal intensive care unit. Multilocus variable-number tandem-repeats analysis and multilocus sequence typing were used. The presence of the mecA gene, icaA gene of the ica-operon, IS 256, and ACME genetic elements was determined by PCR. The human milk of mothers of preterm compared with term neonates had higher counts of staphylococci but lower species diversity. The human milk of mothers of preterm compared with term neonates more often contained Staphylococcus epidermidis mecA (32.7% vs. 2.6%), icaA (18.8% vs. 6%), IS 256 (7.9% vs. 0.9%), and ACME (15.4% vs. 5.1%), as well as Staphylococcus haemolyticus mecA (90.5% vs. 10%) and IS 256 (61.9% vs. 10%). The overall distribution of multilocus variable-number tandem-repeats analysis (MLVA) types and sequence types was similar between the human milk of mothers of preterm and term neonates, but a few mecA-IS 256-positive MLVA types colonized only mothers of preterm neonates. Maternal hospitalization within 1 month postpartum and the use of an arterial catheter or antibacterial treatment in the neonate increased the odds of harboring mecA-positive staphylococci in human milk. Limiting exposure of mothers of preterm neonates to the hospital could prevent human milk colonization with more pathogenic staphylococci.

Molecular techniques for MRSA typing: current issues and perspectives

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P. A. Trindade

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

Molecular techniques for MRSA typing: current issues and perspectives

Directory of Open Access Journals (Sweden)

Trindade P. A.

2003-01-01

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

[Identification of novel variable number tandem repeat (VNTR) loci in Mycobacterium avium and development of an effective means of VNTR typing].

Science.gov (United States)

Kurokawa, Kazuhiro; Uchiya, Kei-Ichi; Yagi, Tetsuya; Takahashi, Hiroyasu; Niimi, Masaki; Ichikawa, Kazuya; Inagaki, Takayuki; Moriyama, Makoto; Nikai, Toshiaki; Hayashi, Yuta; Nakagawa, Taku; Ogawa, Kenji

2012-07-01

To make more effective use of variable number tandem repeat (VNTR) typing, we identified novel VNTR loci in Mycobacterium avium and used them for modified M. avium tandem repeat-VNTR (MATR-VNTR) typing. Analysis of a DNA sample extracted from a clinical isolate (strain HN135) with the FLX system genome sequencer (Roche Diagnostic System) led to discovery of several novel VNTR loci. The allelic diversity of the novel VNTR loci was evaluated for 71 clinical isolates and compared with the diversity of the MATR-VNTR loci. To improve efficacy of MATR-VNTR typing, we tested typing using 2 sets of loci selected from the newly identified loci and the MATR loci, i.e., one set containing 7 and another 16 loci. Hunter Gaston's discriminatory index (HGDI) was calculated for these sets. Six VNTR loci were newly identified, of which 5 showed a high diversity. The HGDI was 0.980 for the improved new typing using a set of 7 loci, and 0.995 for another set of 16 loci, while it was 0.992 for the conventional MATR-VNTR typing. VNTR typing with the set of the 7 loci enabled a rapid analysis, and another set of 16 loci enabled a precise analysis, as compared with conventional MATR-VNTR typing. A method that uses only VNTR loci with relatively high allelic diversity is considered to be a useful tool for VNTR typing of MAC isolates.

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

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Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

Science.gov (United States)

Alvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M

2013-01-01

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability

DEFF Research Database (Denmark)

Brurberg, May Bente; Elameen, Abdelhameed; Le, Ving Hong

2011-01-01

different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates...

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

Science.gov (United States)

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

2017-03-01

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

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans.

Science.gov (United States)

Boriollo, Marcelo Fabiano Gomes; Dias, Ricardo Antunes; Fiorini, João Evangelista; Oliveira, Nelma de Mello Silva; Spolidório, Denise Madalena Palomari; de Souza, Henrique Marques Barbosa; Figueira, Antonio Vargas de Oliveira; Pizzirani-Kleiner, Aline Aparecida

2010-09-01

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE)xS(SM)(EK)xS(SM)(SSRs)). Clustering analyses showed a mean of 9+/-12.4 isolates per cluster (3.8+/-8 isolates/taxon) for MLEE, 6.2+/-4.9 isolates per cluster (4+/-4.5 isolates/taxon) for SSRs, and 4.1+/-2.3 isolates per cluster (2.6+/-2.3 isolates/taxon) for EK. A total of 45 (13%), 39 (11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (S(J)) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented

Topological characteristics of helical repeat proteins

NARCIS (Netherlands)

Groves, M R; Barford, D

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem

Information technology-based approaches to reducing repeat drug exposure in patients with known drug allergies.

Science.gov (United States)

Cresswell, Kathrin M; Sheikh, Aziz

2008-05-01

There is increasing interest internationally in ways of reducing the high disease burden resulting from errors in medicine management. Repeat exposure to drugs to which patients have a known allergy has been a repeatedly identified error, often with disastrous consequences. Drug allergies are immunologically mediated reactions that are characterized by specificity and recurrence on reexposure. These repeat reactions should therefore be preventable. We argue that there is insufficient attention being paid to studying and implementing system-based approaches to reducing the risk of such accidental reexposure. Drawing on recent and ongoing research, we discuss a number of information technology-based interventions that can be used to reduce the risk of recurrent exposure. Proven to be effective in this respect are interventions that provide real-time clinical decision support; also promising are interventions aiming to enhance patient recognition, such as bar coding, radiofrequency identification, and biometric technologies.

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

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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

2013-02-01

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

First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains

Science.gov (United States)

de Beer, Jessica L.; Kremer, Kristin; Ködmön, Csaba; Supply, Philip

2012-01-01

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future. PMID:22170917

Repeatability and Reproducibility of Fibre-Based Nanogenerator Synthesized by Electrospinning Machine

International Nuclear Information System (INIS)

Suyitno; Huda, Sholiehul; Arifin, Zainal; Hadi, Syamsul; Lambang, Raymundus Lullus

2014-01-01

Zinc oxide fibres-based nanogenerators synthesized easily by electrospinning machine are promising to harvest electricity from mechanical energy. However, the repeatability and reproducibility were two major factors needed to be investigated to minimize product failure and to determine the feasibility of mass production of nanogenerators. The green fibres of zinc oxide were produced by electrospinning machine of zinc acetate and polyvinyl alcohol solution at a flow rate of 4 μL/min followed by sintering at temperature 550°C with heating rate 240°C/h. Each 10 nanogenerators was tested by three trained operators with three times of repetition at compressive load 0.5 kg. The nanogenerators revealed the maximum output voltage ranging from 203 to 217 mV. The value of repeatability and reproducibility of nanogenerators was approximately 24.29% showing that nanogenerators were still acceptable to be mass-produced. The relatively low reproducibility was mainly due to the operators, so that the checklist needed to be made easier and simpler for all the variables affecting to the quality of the fibres. Reducing the value of the repeatability and reproducibility is interesting to study further by creating a rotating collector so that the thickness and orientation of fibres can be arranged better

Typing Candida albicans oral isolates from healthy Brazilian schoolchildren using multilocus enzyme electrophoresis reveals two highly polymorphic taxa

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Marcelo Fabiano Gomes Boriollo

2011-09-01

Full Text Available The genetic diversity of C. albicans oral isolates from 75 healthy schoolchildren from eight schools located in different geographic areas of Piracicaba city, São Paulo state, Brazil, was established using isoenzymes marker (Multilocus Enzyme Electrophoresis - MLEE and cluster analysis. Patterns of monoclonal and polyclonal oral colonization by C. albicans within and between groups of schoolchildren were identified. However, significant divergence between the observed and the expected genotypic frequencies (Hardy-Weinberg equilibrium test was not detected in the geographically adjacent groups, suggesting the hypothesis that populations of healthy schoolchildren do not correspond to the selection factor (differential survival of strains. Two highly polymorphic and distantly genetically related taxa (A and B were identified within the total population of yeasts, each contained subgroups (A1, A2, A3, A4, B1 and B2 and clusters of moderately related strains (from I to X, suggesting the existence of strains restricted or not to certain groups of geographically limited, healthy students. However, the coexistence of identical strains in healthy schoolchildren from the same school (geographically related reinforces the hypothesis of oral transmission, where the sources of propagation could be explored. Furthermore, this could also be used in current and retrospective analyses of C. albicans isolated from immunocompetent and immunocompromised people, in order to detect commensal or potentially pathogenic yeast groups, predominantly in candidiasis, and in the development of strategies to prevent transmission or human propagation.

Typing Candida albicans oral isolates from healthy brazilian schoolchildren using multilocus enzyme electrophoresis reveals two highly polymorphic taxa

Science.gov (United States)

Boriollo, Marcelo Fabiano Gomes; Spolidorio, Denise Madalena Palomari; Barros, Letizia Monteiro; Bassi, Rodrigo Carlos; Garcia, José Antonio Dias; Costa, Ana Maria Duarte Dias; Rosa, Edvaldo Antonio Ribeiro; Höfling, José Francisco

2011-01-01

The genetic diversity of C. albicans oral isolates from 75 healthy schoolchildren from eight schools located in different geographic areas of Piracicaba city, São Paulo state, Brazil, was established using isoenzymes marker (Multilocus Enzyme Electrophoresis – MLEE) and cluster analysis. Patterns of monoclonal and polyclonal oral colonization by C. albicans within and between groups of schoolchildren were identified. However, significant divergence between the observed and the expected genotypic frequencies (Hardy-Weinberg equilibrium test) was not detected in the geographically adjacent groups, suggesting the hypothesis that populations of healthy schoolchildren do not correspond to the selection factor (differential survival) of strains. Two highly polymorphic and distantly genetically related taxa (A and B) were identified within the total population of yeasts, each contained subgroups (A1, A2, A3, A4, B1 and B2) and clusters of moderately related strains (from I to X), suggesting the existence of strains restricted or not to certain groups of geographically limited, healthy students. However, the coexistence of identical strains in healthy schoolchildren from the same school (geographically related) reinforces the hypothesis of oral transmission, where the sources of propagation could be explored. Furthermore, this could also be used in current and retrospective analyses of C. albicans isolated from immunocompetent and immunocompromised people, in order to detect commensal or potentially pathogenic yeast groups, predominantly in candidiasis, and in the development of strategies to prevent transmission or human propagation. PMID:24031720

Quantum repeated games revisited

International Nuclear Information System (INIS)

Frąckiewicz, Piotr

2012-01-01

We present a scheme for playing quantum repeated 2 × 2 games based on Marinatto and Weber’s approach to quantum games. As a potential application, we study the twice repeated Prisoner’s Dilemma game. We show that results not available in the classical game can be obtained when the game is played in the quantum way. Before we present our idea, we comment on the previous scheme of playing quantum repeated games proposed by Iqbal and Toor. We point out the drawbacks that make their results unacceptable. (paper)

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley.

Science.gov (United States)

Bull, Carolee T; Clarke, Christopher R; Cai, Rongman; Vinatzer, Boris A; Jardini, Teresa M; Koike, Steven T

2011-07-01

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.

Comparison of three types of full-body compression garments on throwing and repeat-sprint performance in cricket players.

Science.gov (United States)

Duffield, Rob; Portus, Marc

2007-07-01

To compare the effects of three types of full-body compression garments (Skins, Adidas and Under Armour) on repeat-sprint and throwing performance in cricket players. Following familiarisation, 10 male cricket players performed four randomised exercise sessions (3 garments and a control). Each session involved a 30 min repeat-sprint exercise protocol comprising 20 m sprints every minute, separated by submaximal exercise. Throwing tests included a pre-exercise and a postexercise maximal distance test and accuracy throwing tests. During each session, measures of heart rate, skin temperature, change in body mass, rate of perceived exertion and perceived muscle soreness were recorded. Capillary blood samples were analysed before and after exercise for lactate, pH, O(2) saturation and O(2) partial pressure, and 24 h after exercise for creatine kinase (CK). Ratings of perceived muscle soreness were also obtained 24 h after exercise. No significant differences (p>0.05) were evident in repeat-sprint performance (10 m, 20 m time or total submaximal distance covered) or throwing performance (maximum distance or accuracy). No significant differences (p>0.05) were observed in heart rate, body mass change or blood measures during exercise. Significant differences (p0.05). No benefit was noted when wearing compression garments for repeat-sprint or throwing performance; however, the use of the garments as a recovery tool, when worn after exercise, may be beneficial to reduce postexercise trauma and perceived muscle soreness.

Characterization of European Yersinia enterocolitica 1A strains using restriction fragment length polymorphism and multilocus sequence analysis.

Science.gov (United States)

Murros, A; Säde, E; Johansson, P; Korkeala, H; Fredriksson-Ahomaa, M; Björkroth, J

2016-10-01

Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5. Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica. © 2016 The Society for Applied Microbiology.

Antarctic Ice Sheet Slope and Aspect Based on Icesat's Repeat Orbit Measurement

Science.gov (United States)

Yuan, L.; Li, F.; Zhang, S.; Xie, S.; Xiao, F.; Zhu, T.; Zhang, Y.

2017-09-01

Accurate information of ice sheet surface slope is essential for estimating elevation change by satellite altimetry measurement. A study is carried out to recover surface slope of Antarctic ice sheet from Ice, Cloud and land Elevation Satellite (ICESat) elevation measurements based on repeat orbits. ICESat provides repeat ground tracks within 200 meters in cross-track direction and 170 meters in along-track direction for most areas of Antarctic ice sheet. Both cross-track and along-track surface slopes could be obtained by adjacent repeat ground tracks. Combining those measurements yields a surface slope model with resolution of approximately 200 meters. An algorithm considering elevation change is developed to estimate the surface slope of Antarctic ice sheet. Three Antarctic Digital Elevation Models (DEMs) were used to calculate surface slopes. The surface slopes from DEMs are compared with estimates by using in situ GPS data in Dome A, the summit of Antarctic ice sheet. Our results reveal an average surface slope difference of 0.02 degree in Dome A. High resolution remote sensing images are also used in comparing the results derived from other DEMs and this paper. The comparison implies that our results have a slightly better coherence with GPS observation than results from DEMs, but our results provide more details and perform higher accuracy in coastal areas because of the higher resolution for ICESat measurements. Ice divides are estimated based on the aspect, and are weakly consistent with ice divides from other method in coastal regions.

Large-scale studies of the HphI insulin gene variable-number-of-tandem-repeats polymorphism in relation to Type 2 diabetes mellitus and insulin release

DEFF Research Database (Denmark)

Hansen, S K; Gjesing, A P; Rasmussen, S K

2004-01-01

The class III allele of the variable-number-of-tandem-repeats polymorphism located 5' of the insulin gene (INS-VNTR) has been associated with Type 2 diabetes and altered birthweight. It has also been suggested, although inconsistently, that the class III allele plays a role in glucose-induced ins......The class III allele of the variable-number-of-tandem-repeats polymorphism located 5' of the insulin gene (INS-VNTR) has been associated with Type 2 diabetes and altered birthweight. It has also been suggested, although inconsistently, that the class III allele plays a role in glucose...

Boston Type 1 Keratoprosthesis versus Repeat Donor Keratoplasty for Corneal Graft Failure: A Systematic Review and Meta-analysis.

Science.gov (United States)

Ahmad, Sumayya; Mathews, Priya M; Lindsley, Kristina; Alkharashi, Majed; Hwang, Frank S; Ng, Sueko M; Aldave, Anthony J; Akpek, Esen Karamursel

2016-01-01

To compare repeat penetrating keratoplasty (PK) with Boston type I keratoprosthesis (KPro) implantation for full-thickness donor corneal graft failure. Previous donor graft failure is a common indication for both PK and KPro implantation. Selection of the surgical procedure is entirely dependent on the surgeon because there are no studies available for guidance. Therefore, a systematic review was undertaken to examine vision, device retention, graft clarity, and postoperative glaucoma and infection outcomes after repeat PK versus KPro implantation. Articles with data regarding repeat PK published between 1990 and 2014 were identified in PubMed, EMBASE, the Latin American and Caribbean Health Sciences Literature Database, and the Cochrane Central Register of Controlled Trials and were reviewed. Results were compared with a retrospective review of consecutive, nonrandomized, longitudinal case series of KPro implantations performed at 5 tertiary care centers in the United States. Visual acuity at 2 years was the primary outcome measure. The proportion of clear grafts in the repeat PK group, device retention in the KPro group, and the development of postoperative glaucoma and infection were secondary outcome measures. The search strategy identified 17 128 articles in the PK analysis. After screening, 26 studies (21 case series and 5 cohort studies) were included in the review. Pooled analysis of the 26 unique studies demonstrated a 42% (95% confidence interval [CI], 30%-56%) likelihood of maintaining 20/200 or better at 2 years after repeat PK, compared with an 80% (95% CI, 68%-88%) probability with KPro implantation. The probability of maintaining a clear graft at 5 years was 47% (95% CI, 40%-54%) after repeat PK, whereas the probability of retention of the KPro at 5 years was 75% (95% CI, 64%-84%). The rate of progression of glaucoma at 3 years was 25% (95% CI, 10%-44%) after repeat PK and 30% in the KPro cohort. These results demonstrate favorable outcomes of KPro

Interstitial telomere-like repeats in the Arabidopsis thaliana genome.

Science.gov (United States)

Uchida, Wakana; Matsunaga, Sachihiro; Sugiyama, Ryuji; Kawano, Shigeyuki

2002-02-01

Eukaryotic chromosomal ends are protected by telomeres, which are thought to play an important role in ensuring the complete replication of chromosomes. On the other hand, non-functional telomere-like repeats in the interchromosomal regions (interstitial telomeric repeats; ITRs) have been reported in several eukaryotes. In this study, we identified eight ITRs in the Arabidopsis thaliana genome, each consisting of complete and degenerate 300- to 1200-bp sequences. The ITRs were grouped into three classes (class IA-B, class II, and class IIIA-E) based on the degeneracy of the telomeric repeats in ITRs. The telomeric repeats of the two ITRs in class I were conserved for the most part, whereas the single ITR in class II, and the five ITRs in class III were relatively degenerated. In addition, degenerate ITRs were surrounded by common sequences that shared 70-100% homology to each other; these are named ITR-adjacent sequences (IAS). Although the genomic regions around ITRs in class I lacked IAS, those around ITRs in class II contained IAS (IASa), and those around five ITRs in class III had nine types of IAS (IASb, c, d, e, f, g, h, i, and j). Ten IAS types in classes II and III showed no significant homology to each other. The chromosomal locations of ITRs and IAS were not category-related, but most of them were adjacent to, or part of, a centromere. These results show that the A. thaliana genome has undergone chromosomal rearrangements, such as end-fusions and segmental duplications.

Empirical Statistical Power for Testing Multilocus Genotypic Effects under Unbalanced Designs Using a Gibbs Sampler

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Chaeyoung Lee

2012-11-01

Full Text Available Epistasis that may explain a large portion of the phenotypic variation for complex economic traits of animals has been ignored in many genetic association studies. A Baysian method was introduced to draw inferences about multilocus genotypic effects based on their marginal posterior distributions by a Gibbs sampler. A simulation study was conducted to provide statistical powers under various unbalanced designs by using this method. Data were simulated by combined designs of number of loci, within genotype variance, and sample size in unbalanced designs with or without null combined genotype cells. Mean empirical statistical power was estimated for testing posterior mean estimate of combined genotype effect. A practical example for obtaining empirical statistical power estimates with a given sample size was provided under unbalanced designs. The empirical statistical powers would be useful for determining an optimal design when interactive associations of multiple loci with complex phenotypes were examined.

Comparison of a Variable-Number Tandem-Repeat (VNTR) Method for Typing Mycobacterium avium with Mycobacterial Interspersed Repetitive-Unit-VNTR and IS1245 Restriction Fragment Length Polymorphism Typing▿ †

OpenAIRE

Inagaki, Takayuki; Nishimori, Kei; Yagi, Tetsuya; Ichikawa, Kazuya; Moriyama, Makoto; Nakagawa, Taku; Shibayama, Takami; Uchiya, Kei-ichi; Nikai, Toshiaki; Ogawa, Kenji

2009-01-01

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of ...

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

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Desirée Villahermosa

2017-05-01

Full Text Available Defective mismatch repair (MMR in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6, which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1, which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3 recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.

A framework for assessing the concordance of molecular typing methods and the true strain phylogeny of Campylobacter jejuni and C. coli using draft genome sequence data

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Catherine Dianna Carrillo

2012-05-01

Full Text Available Tracking of sources of sporadic cases of campylobacteriosis remains challenging, as commonly used molecular typing methods have limited ability to unambiguously link genetically related strains. Genomics has become increasingly prominent in the public health response to enteric pathogens as methods enable characterization of pathogens at an unprecedented level of resolution. However, the cost of sequencing and expertise required for bioinformatic analyses remains prohibitive, and these comprehensive analyses are limited to a few priority strains. Although several molecular typing methods are currently widely used for epidemiological analysis of campylobacters, it is not clear how accurately these methods reflect true strain relationships. To address this, we analyzed 104 publically available whole genome sequences (WGS of C. jejuni and C. coli. In addition to in silico determination of multi-locus sequence (MLST, fla and porA type, as well as comparative genomic fingerprint (CGF, we inferred a reference phylogeny based on conserved core genome elements. Molecular typing data were compared to the reference phylogeny for concordance using the Adjusted Wallace Coefficient (AWC with confidence intervals. Although MLST targets the sequence variability in core genes and CGF targets insertions/deletions of accessory genes, both methods are based on multilocus analysis and provided better estimates of true phylogeny than methods based on single loci (porA, fla. A more comprehensive WGS dataset including additional genetically related strains, both epidemiologically linked and unlinked, will be necessary to assess performance of methods for outbreak investigations and surveillance activities. Analyses of the strengths and weaknesses of widely used typing methodologies in inferring true strain relationships will provide guidance in the interpretation of this data for epidemiological purposes.

Constructs for the expression of repeating triple-helical protein domains

International Nuclear Information System (INIS)

Peng, Yong Y; Werkmeister, Jerome A; Vaughan, Paul R; Ramshaw, John A M

2009-01-01

The development of novel scaffolds will be an important aspect in future success of tissue engineering. Scaffolds will preferably contain information that directs the cellular content of constructs so that the new tissue that is formed is closely aligned in structure, composition and function to the target natural tissue. One way of approaching this will be the development of novel protein-based constructs that contain one or more repeats of functional elements derived from various proteins. In the present case, we describe a strategy to make synthetic, recombinant triple-helical constructs that contain repeat segments of biologically relevant domains. Copies of a DNA fragment prepared by PCR from human type III collagen have been inserted in a co-linear contiguous fashion into the yeast expression vector YEpFlag-1, using sequential addition between selected restriction sites. Constructs containing 1, 2 and 3 repeats were designed to maintain the (Gly-X-Y) repeat, which is essential for the formation of an extended triple helix. All constructs gave expressed protein, with the best being the 3-repeat construct which was readily secreted. This material had the expected composition and N-terminal sequence. Incubation of the product at low temperature led to triple-helix formation, shown by reaction with a conformation dependent monoclonal antibody.

Constructs for the expression of repeating triple-helical protein domains

Energy Technology Data Exchange (ETDEWEB)

Peng, Yong Y; Werkmeister, Jerome A; Vaughan, Paul R; Ramshaw, John A M, E-mail: jerome.werkmeister@csiro.a [CSIRO Molecular and Health Technologies, Bag 10, Clayton South, VIC 3169 (Australia)

2009-02-15

The development of novel scaffolds will be an important aspect in future success of tissue engineering. Scaffolds will preferably contain information that directs the cellular content of constructs so that the new tissue that is formed is closely aligned in structure, composition and function to the target natural tissue. One way of approaching this will be the development of novel protein-based constructs that contain one or more repeats of functional elements derived from various proteins. In the present case, we describe a strategy to make synthetic, recombinant triple-helical constructs that contain repeat segments of biologically relevant domains. Copies of a DNA fragment prepared by PCR from human type III collagen have been inserted in a co-linear contiguous fashion into the yeast expression vector YEpFlag-1, using sequential addition between selected restriction sites. Constructs containing 1, 2 and 3 repeats were designed to maintain the (Gly-X-Y) repeat, which is essential for the formation of an extended triple helix. All constructs gave expressed protein, with the best being the 3-repeat construct which was readily secreted. This material had the expected composition and N-terminal sequence. Incubation of the product at low temperature led to triple-helix formation, shown by reaction with a conformation dependent monoclonal antibody.

Multilocus phylogeny and antifungal susceptibility of Aspergillus section Circumdati from clinical samples and description of A. pseudosclerotiorum sp. nov.

Science.gov (United States)

A multilocus phylogenetic study was carried out to assess the species distribution in a set of 34 clinical isolates of Aspergillus section Circumdati from the USA and their in vitro antifungal susceptibility were determined against eight antifungal drugs. The genetic markers used were ITS, BenA, CaM...

Mating type genes in the genus Neofusicoccum: Mating strategies and usefulness in species delimitation.

Science.gov (United States)

Lopes, Anabela; Phillips, Alan J L; Alves, Artur

2017-04-01

The genus Neofusicoccum includes species with wide geographical and plant host distribution, some of them of economic importance. The genus currently comprises 27 species that are difficult to identify based on morphological features alone. Thus, species differentiation is based on phylogenetic species recognition using multigene genealogies. In this study, we characterised the mating type genes of Neofusicoccum species. Specific primers were designed to amplify and sequence MAT genes in several species and a PCR-based mating type diagnostic assay was developed. Homothallism was the predominant mating strategy among the species tested. Furthermore, the potential of mating type gene sequences for species delimitation was evaluated. Phylogenetic analyses were performed on both MAT genes and compared with multigene genealogies using sequences of the ribosomal internal transcribed spacer region, translation elongation factor 1-alpha and beta-tubulin. Phylogenies based on mating type genes could discriminate between the species analysed and are in concordance with the results obtained with the more conventional multilocus phylogenetic analysis approach. Thus, MAT genes represent a powerful tool to delimit cryptic species in the genus Neofusicoccum. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Fluorescence In Situ Hybridization (FISH-Based Karyotyping Reveals Rapid Evolution of Centromeric and Subtelomeric Repeats in Common Bean (Phaseolus vulgaris and Relatives

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Aiko Iwata-Otsubo

2016-04-01

Full Text Available Fluorescence in situ hybridization (FISH-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus. Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2–4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species.

Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

Directory of Open Access Journals (Sweden)

Yanhao Lai

Full Text Available Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA, an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER. We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.

Belief-Based Equilibria in the Repeated Prisoners' Dilemma with Private Monitoring

OpenAIRE

V. Bhaskar; Ichiro Obara

2000-01-01

We analyze the infinitely repeated prisoners' dilemma with imperfect private monitoring and discounting. The main contribution of this paper is to construct ``belief-based'' strategies, where a player's continuation strategy is a function only of his beliefs. This simplifies the analysis considerably, and allows us to explicitly construct sequential equilibria for such games, thus enabling us to invoke the one-step deviation principle of dynamic programming. By doing so, we prove that one can...

Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST

NARCIS (Netherlands)

O'Donnell, K.; Humber, R.A.; Geiser, D.M.; Kang, S.; Robert, V.; Park, B.; Crous, P.W.; Johnston, P.; Aoki, T.; Rooney, A.P.; Rehner, S.A.

2012-01-01

We constructed several multilocus DNA sequence datasets to assess the phylogenetic diversity of insecticolous fusaria, especially focusing on those housed at the Agricultural Research Service Collection of Entomopathogenic Fungi (ARSEF), and to aid molecular identifications of unknowns via the

Discrepancy variation of dinucleotide microsatellite repeats in eukaryotic genomes

Directory of Open Access Journals (Sweden)

HUAN GAO

2009-01-01

Full Text Available To address whether there are differences of variation among repeat motif types and among taxonomic groups, we present here an analysis of variation and correlation of dinucleotide microsatellite repeats in eukaryotic genomes. Ten taxonomic groups were compared, those being primates, mammalia (excluding primates and rodentia, rodentia, birds, fish, amphibians and reptiles, insects, molluscs, plants and fungi, respectively. The data used in the analysis is from the literature published in the Journal of Molecular Ecology Notes. Analysis of variation reveals that there are no significant differences between AC and AG repeat motif types. Moreover, the number of alleles correlates positively with the copy number in both AG and AC repeats. Similar conclusions can be obtained from each taxonomic group. These results strongly suggest that the increase of SSR variation is almost linear with the increase of the copy number of each repeat motif. As well, the results suggest that the variability of SSR in the genomes of low-ranking species seem to be more than that of high-ranking species, excluding primates and fungi.

Development of analog watch with minute repeater

Science.gov (United States)

Okigami, Tomio; Aoyama, Shigeru; Osa, Takashi; Igarashi, Kiyotaka; Ikegami, Tomomi

A complementary metal oxide semiconductor with large scale integration was developed for an electronic minute repeater. It is equipped with the synthetic struck sound circuit to generate natural struck sound necessary for the minute repeater. This circuit consists of an envelope curve drawing circuit, frequency mixer, polyphonic mixer, and booster circuit made by using analog circuit technology. This large scale integration is a single chip microcomputer with motor drivers and input ports in addition to the synthetic struck sound circuit, and it is possible to make an electronic system of minute repeater at a very low cost in comparison with the conventional type.

Species identification and molecular typing of human Brucella isolates from Kuwait.

Science.gov (United States)

Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

simple sequence repeats (EST-SSR)

African Journals Online (AJOL)

Yomi

2012-01-19

Jan 19, 2012 ... 212 primer pairs selected, based on repeat patterns of n≥8 for di-, tri-, tetra- and penta-nucleotide repeat ... Cluster analysis revealed a high genetic similarity among the sugarcane (Saccharum spp.) breeding lines which could reduce the genetic gain in ..... The multiple allele characteristic of SSR com-.

Assessing species boundaries using multilocus species delimitation in a morphologically conserved group of neotropical freshwater fishes, the Poecilia sphenops species complex (Poeciliidae.

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Justin C Bagley

Full Text Available Accurately delimiting species is fundamentally important for understanding species diversity and distributions and devising effective strategies to conserve biodiversity. However, species delimitation is problematic in many taxa, including 'non-adaptive radiations' containing morphologically cryptic lineages. Fortunately, coalescent-based species delimitation methods hold promise for objectively estimating species limits in such radiations, using multilocus genetic data. Using coalescent-based approaches, we delimit species and infer evolutionary relationships in a morphologically conserved group of Central American freshwater fishes, the Poecilia sphenops species complex. Phylogenetic analyses of multiple genetic markers (sequences of two mitochondrial DNA genes and five nuclear loci from 10/15 species and genetic lineages recognized in the group support the P. sphenops species complex as monophyletic with respect to outgroups, with eight mitochondrial 'major-lineages' diverged by ≥2% pairwise genetic distances. From general mixed Yule-coalescent models, we discovered (conservatively 10 species within our concatenated mitochondrial DNA dataset, 9 of which were strongly supported by subsequent multilocus Bayesian species delimitation and species tree analyses. Results suggested species-level diversity is underestimated or overestimated by at least ~15% in different lineages in the complex. Nonparametric statistics and coalescent simulations indicate genealogical discordance among our gene tree results has mainly derived from interspecific hybridization in the nuclear genome. However, mitochondrial DNA show little evidence for introgression, and our species delimitation results appear robust to effects of this process. Overall, our findings support the utility of combining multiple lines of genetic evidence and broad phylogeographical sampling to discover and validate species using coalescent-based methods. Our study also highlights the

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

OpenAIRE

Siarkou, Victoria I.; Vorimore, Fabien; Vicari, Nadia; Magnino, Simone; Rodolakis, Annie; Pannekoek, Yvonne; Sachse, Konrad; Longbottom, David; Laroucau, Karine

2015-01-01

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previo...

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

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Marjolein van Gent

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

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

Science.gov (United States)

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

2011-01-01

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

New multilocus sequence typing of MRSA in São Paulo, Brazil

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M.S. Carmo

2011-10-01

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

Diverse Genotypes of Yersinia pestis Caused Plague in Madagascar in 2007.

Science.gov (United States)

Riehm, Julia M; Projahn, Michaela; Vogler, Amy J; Rajerison, Minoaerisoa; Andersen, Genevieve; Hall, Carina M; Zimmermann, Thomas; Soanandrasana, Rahelinirina; Andrianaivoarimanana, Voahangy; Straubinger, Reinhard K; Nottingham, Roxanne; Keim, Paul; Wagner, David M; Scholz, Holger C

2015-06-01

Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks.

A Modified Jonckheere Test Statistic for Ordered Alternatives in Repeated Measures Design

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Hatice Tül Kübra AKDUR

2016-09-01

Full Text Available In this article, a new test based on Jonckheere test [1] for  randomized blocks which have dependent observations within block is presented. A weighted sum for each block statistic rather than the unweighted sum proposed by Jonckheereis included. For Jonckheere type statistics, the main assumption is independency of observations within block. In the case of repeated measures design, the assumption of independence is violated. The weighted Jonckheere type statistic for the situation of dependence for different variance-covariance structure and the situation based on ordered alternative hypothesis structure of each block on the design is used. Also, the proposed statistic is compared to the existing test based on Jonckheere in terms of type I error rates by performing Monte Carlo simulation. For the strong correlations, circular bootstrap version of the proposed Jonckheere test provides lower rates of type I error.

Relationship between income and repeat criminal victimization in Brazil

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Marcelo Justus

2015-09-01

Full Text Available This study analyzes the effect of income on repeat criminal victimization in Brazil using data from the 2009 National Household Sample Survey and its special supplement on victimization and access to justice. Two count-data models were estimated for four types of crime: theft, robbery, attempted theft/robbery, and physical assault. A positive nonlinear effect of income on repeat victimization for the three types of property crimes and a negative nonlinear effect of income on physical assault were observed.

Glycoprotein I of herpes simplex virus type 1 contains a unique polymorphic tandem-repeated mucin region

DEFF Research Database (Denmark)

Norberg, Peter; Olofsson, Sigvard; Tarp, Mads Agervig

2007-01-01

Glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) contains a tandem repeat (TR) region including the amino acids serine and threonine, residues that can be utilized for O-glycosylation. The length of this TR region was determined for 82 clinical HSV-1 isolates and the results revealed......-glycosylation not only for the two most commonly expressed N-acetyl-d-galactosamine (GalNAc)-T1 and -T2 transferases, but also for the GalNAc-T3, -T4 and -T11 transferases. Immunoblotting of virus-infected cells showed that gI was exclusively O-glycosylated with GalNAc monosaccharides (Tn antigen). A polymorphic mucin...

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

Science.gov (United States)

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

2015-12-01

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

The development of the micro-solid propellant thruster array with improved repeatability

International Nuclear Information System (INIS)

Seo, Daeban; Kwon, Sejin; Lee, Jongkwang

2012-01-01

This paper presents the development of a micro-solid propellant thruster array with improved repeatability. The repeatability and low performance variation of each thruster unit with a high ignition success rate is essential in micro-solid propellant thruster array. To date, the study on the improvement of the repeatability has not yet been reported. As the first step for this study, we propose a new type of micro igniter, using a glass wafer called the heater-contact micro igniter. This igniter is also designed to improve the ignition characteristics of a glass-based micro igniter. The prototype of the igniter array is designed and fabricated to establish its fabrication process and to conduct its performance evaluation. Through the firing test, the performance of the heater-contact micro igniter is verified. The 5 × 5 sized micro-solid propellant thruster array is designed and fabricated applying the developed heater-contact igniter. The measured average thrust of each thruster unit is 2.542 N, and calculated standard deviation is 0.369 N. The calculated average total impulse and its standard deviation are 0.182 and 0.04 mNs, respectively. Based on these results, the improvement of repeatability is verified. Finally, the ignition control system of the micro-thruster array is developed. (paper)

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

Science.gov (United States)

Inoue, D; Santiago, P; Horne, W C; Baron, R

1997-10-03

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

Molecular characterization and multi-locus genotypes of Enterocytozoon bieneusi from captive red kangaroos (Macropus Rufus in Jiangsu province, China.

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Zhijun Zhong

Full Text Available Enterocytozoon bieneusi is the most common pathogen of microsporidian species infecting humans worldwide. Although E. bieneusi has been found in a variety of animal hosts, information on the presence of E. bieneusi in captive kangaroos in China is limited. The present study was aimed at determining the occurrence and genetic diversity of E. bieneusi in captive kangaroos. A total of 61 fecal specimens (38 from red kangaroos and 23 from grey kangaroos were collected from Nanjing Hongshan Forest Zoo and Hongshan Kangaroo Breeding Research Base, Jiangsu province, China. Using the nested PCR amplification ITS gene of rRNA of E. bieneusi, totally 23.0% (14/61 of tested samples were PCR-positive with three genotypes (i.e. one known genotype, CHK1, and two novel genotypes, CSK1 and CSK2. Multi-locus sequence typing using three microsatellites (MS1, MS3, and MS7 and one minisatellite (MS4 revealed one, five, two, and one types at these four loci, respectively. In phylogenetic analysis, the two genotypes, CHK1 and CSK1, were clustered into a new group of unknown zoonotic potential, and the novel genotype CSK2 was clustered into a separate clade with PtEb and PtEbIX. To date, this is the first report on the presence of E. bieneusi in captive red kangaroos in Jiangsu province, China. Furthermore, a high degree of genetic diversity was observed in the E. bieneusi genotype and seven MLGs (MLG1-7 were found in red kangaroos. Our findings suggest that infected kangaroo may act as potential reservoirs of E. bieneusi and be source to transmit infections to other animal.

Evaluation of a highly discriminating multiplex multi-locus variable-number of tandem-repeats (MLVA) analysis for Vibrio cholerae.

Science.gov (United States)

Olsen, Jaran S; Aarskaug, Tone; Skogan, Gunnar; Fykse, Else Marie; Ellingsen, Anette Bauer; Blatny, Janet M

2009-09-01

Vibrio cholerae is the etiological agent of cholera and may be used in bioterror actions due to the easiness of its dissemination, and the public fear for acquiring the cholera disease. A simple and highly discriminating method for connecting clinical and environmental isolates of V. cholerae is needed in microbial forensics. Twelve different loci containing variable numbers of tandem-repeats (VNTRs) were evaluated in which six loci were polymorphic. Two multiplex reactions containing PCR primers targeting these six VNTRs resulted in successful DNA amplification of 142 various environmental and clinical V. cholerae isolates. The genetic distribution inside the V. cholerae strain collection was used to evaluate the discriminating power (Simpsons Diversity Index=0.99) of this new MLVA analysis, showing that the assay have a potential to differentiate between various strains, but also to identify those isolates which are collected from a common V. cholerae outbreak. This work has established a rapid and highly discriminating MLVA assay useful for track back analyses and/or forensic studies of V. cholerae infections.

Application of Variable-Number Tandem-Repeat Typing To Discriminate Ralstonia solanacearum Strains Associated with English Watercourses and Disease Outbreaks

Science.gov (United States)

Bryant, Ruth; Bew, Janice; Conyers, Christine; Stones, Robert; Alcock, Michael; Elphinstone, John

2013-01-01

Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks. PMID:23892739

Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing

NARCIS (Netherlands)

Huyen, Mai N. T.; Kremer, Kristin; Lan, Nguyen T. N.; Buu, Tran N.; Cobelens, Frank G. J.; Tiemersma, Edine W.; de Haas, Petra; van Soolingen, Dick

2013-01-01

In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of

Multilocus and SSU rRNA gene phylogenetic analyses of available cyanobacterial genomes, and their relation to the current taxonomic system

Czech Academy of Sciences Publication Activity Database

Mareš, Jan

2018-01-01

Roč. 811, č. 1 (2018), s. 19-34 ISSN 0018-8158 R&D Projects: GA ČR(CZ) GA15-11912S Institutional support: RVO:67985939 Keywords : 16S rRNA * Cyanobacterial orders * Multilocus phylogeny Subject RIV: EH - Ecology, Behaviour OBOR OECD: Ecology Impact factor: 2.056, year: 2016

Differential Distribution of Type II CRISPR-Cas Systems in Agricultural and Nonagricultural Campylobacter coli and Campylobacter jejuni Isolates Correlates with Lack of Shared Environments.

Science.gov (United States)

Pearson, Bruce M; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H M

2015-09-02

CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Ion torrent personal genome machine sequencing for genomic typing of Neisseria meningitidis for rapid determination of multiple layers of typing information.

Science.gov (United States)

Vogel, Ulrich; Szczepanowski, Rafael; Claus, Heike; Jünemann, Sebastian; Prior, Karola; Harmsen, Dag

2012-06-01

Neisseria meningitidis causes invasive meningococcal disease in infants, toddlers, and adolescents worldwide. DNA sequence-based typing, including multilocus sequence typing, analysis of genetic determinants of antibiotic resistance, and sequence typing of vaccine antigens, has become the standard for molecular epidemiology of the organism. However, PCR of multiple targets and consecutive Sanger sequencing provide logistic constraints to reference laboratories. Taking advantage of the recent development of benchtop next-generation sequencers (NGSs) and of BIGSdb, a database accommodating and analyzing genome sequence data, we therefore explored the feasibility and accuracy of Ion Torrent Personal Genome Machine (PGM) sequencing for genomic typing of meningococci. Three strains from a previous meningococcus serogroup B community outbreak were selected to compare conventional typing results with data generated by semiconductor chip-based sequencing. In addition, sequencing of the meningococcal type strain MC58 provided information about the general performance of the technology. The PGM technology generated sequence information for all target genes addressed. The results were 100% concordant with conventional typing results, with no further editing being necessary. In addition, the amount of typing information, i.e., nucleotides and target genes analyzed, could be substantially increased by the combined use of genome sequencing and BIGSdb compared to conventional methods. In the near future, affordable and fast benchtop NGS machines like the PGM might enable reference laboratories to switch to genomic typing on a routine basis. This will reduce workloads and rapidly provide information for laboratory surveillance, outbreak investigation, assessment of vaccine preventability, and antibiotic resistance gene monitoring.

Experimental and modelling buckling of wood-based columns under repeated loading

Directory of Open Access Journals (Sweden)

Nafa Z.

2010-06-01

Full Text Available Collapse of timber constructions can appear under the effect of load that exceeds the resistance of a carrying element or under the effect of a geometrical instability like buckling. In addition, loading can be constant or varying for example loads due to wind or earthquakes. The aim of this paper is to study the behaviour and the lifetime of columns in wood or based-wood material such as glulam (GL or laminated veneer lumber (LVL under repeated loading leading to buckling.

White light scanner-based repeatability of 3-dimensional digitizing of silicon rubber abutment teeth impressions.

Science.gov (United States)

Jeon, Jin-Hun; Lee, Kyung-Tak; Kim, Hae-Young; Kim, Ji-Hwan; Kim, Woong-Chul

2013-11-01

The aim of this study was to evaluate the repeatability of the digitizing of silicon rubber impressions of abutment teeth by using a white light scanner and compare differences in repeatability between different abutment teeth types. Silicon rubber impressions of a canine, premolar, and molar tooth were each digitized 8 times using a white light scanner, and 3D surface models were created using the point clouds. The size of any discrepancy between each model and the corresponding reference tooth were measured, and the distribution of these values was analyzed by an inspection software (PowerInspect 2012, Delcamplc., Birmingham, UK). Absolute values of discrepancies were analyzed by the Kruskal-Wallis test and multiple comparisons (α=.05). The discrepancy between the impressions for the canine, premolar, and molar teeth were 6.3 µm (95% confidence interval [CI], 5.4-7.2), 6.4 µm (95% CI, 5.3-7.6), and 8.9 µm (95% CI, 8.2-9.5), respectively. The discrepancy of the molar tooth impression was significantly higher than that of other tooth types. The largest variation (as mean [SD]) in discrepancies was seen in the premolar tooth impression scans: 26.7 µm (95% CI, 19.7-33.8); followed by canine and molar teeth impressions, 16.3 µm (95% CI, 15.3-17.3), and 14.0 µm (95% CI, 12.3-15.7), respectively. The repeatability of the digitizing abutment teeth's silicon rubber impressions by using a white light scanner was improved compared to that with a laser scanner, showing only a low mean discrepancy between 6.3 µm and 8.9 µm, which was in an clinically acceptable range. Premolar impression with a long and narrow shape showed a significantly larger discrepancy than canine and molar impressions. Further work is needed to increase the digitizing performance of the white light scanner for deep and slender impressions.

Multilocus phylogeny of the avian family Alaudidae (larks) reveals complex morphological evolution, non-monophyletic genera and hidden species diversity.

Science.gov (United States)

Alström, Per; Barnes, Keith N; Olsson, Urban; Barker, F Keith; Bloomer, Paulette; Khan, Aleem Ahmed; Qureshi, Masood Ahmed; Guillaumet, Alban; Crochet, Pierre-André; Ryan, Peter G

2013-12-01

The Alaudidae (larks) is a large family of songbirds in the superfamily Sylvioidea. Larks are cosmopolitan, although species-level diversity is by far largest in Africa, followed by Eurasia, whereas Australasia and the New World have only one species each. The present study is the first comprehensive phylogeny of the Alaudidae. It includes 83.5% of all species and representatives from all recognised genera, and was based on two mitochondrial and three nuclear loci (in total 6.4 kbp, although not all loci were available for all species). In addition, a larger sample, comprising several subspecies of some polytypic species was analysed for one of the mitochondrial loci. There was generally good agreement in trees inferred from different loci, although some strongly supported incongruences were noted. The tree based on the concatenated multilocus data was overall well resolved and well supported by the data. We stress the importance of performing single gene as well as combined data analyses, as the latter may obscure significant incongruence behind strong nodal support values. The multilocus tree revealed many unpredicted relationships, including some non-monophyletic genera (Calandrella, Mirafra, Melanocorypha, Spizocorys). The tree based on the extended mitochondrial data set revealed several unexpected deep divergences between taxa presently treated as conspecific (e.g. within Ammomanes cinctura, Ammomanes deserti, Calandrella brachydactyla, Eremophila alpestris), as well as some shallow splits between currently recognised species (e.g. Certhilauda brevirostris-C. semitorquata-C. curvirostris; Calendulauda barlowi-C. erythrochlamys; Mirafra cantillans-M. javanica). Based on our results, we propose a revised generic classification, and comment on some species limits. We also comment on the extraordinary morphological adaptability in larks, which has resulted in numerous examples of parallel evolution (e.g. in Melanocorypha mongolica and Alauda leucoptera [both

Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013.

Science.gov (United States)

Tian, Guo-Zhong; Cui, Bu-Yun; Piao, Dong-Ri; Zhao, Hong-Yan; Li, Lan-Yu; Liu, Xi; Xiao, Pei; Zhao, Zhong-Zhi; Xu, Li-Qing; Jiang, Hai; Li, Zhen-Jun

2017-05-02

Brucellosis was a common human and livestock disease caused by Brucella strains, the category B priority pathogens by the US Center for Disease Control (CDC). Identified as a priority disease in human and livestock populations, the increasing incidence in recent years in China needs urgent control measures for this disease but the molecular background important for monitoring the epidemiology of Brucella strains at the national level is still lacking. A total of 600 Brucella isolates collected during 60 years (from 1953 to 2013) in China were genotyped by multiple locus variable-number tandem repeat analysis (MLVA) and the variation degree of MLVA11 loci was calculated by the Hunter Gaston Diversity Index (HGDI) values. The charts and map were processed by Excel 2013, and cluster analysis and epidemiological distribution was performed using BioNumerics (version 5.1). The 600 representative Brucella isolates fell into 104 genotypes with 58 singleton genotypes by the MLVA11 assay, including B. melitensis biovars 2 and 3 (five main genotypes), B. abortus biovars 1 and 3 (two main genotypes), B. suis biovars 1 and 3 (three main genotypes), and B. canis (two main genotypes) respectively. While most B. suis biovar 1 and biovar 3 were respectively found in northern provinces and southern provinces, B. melitensis and B. abortus strains were dominant in China. Canine Brucellosis was only found in animals without any human cases reported. Eight Brucellosis epidemic peaks emerged during the 60 years between 1953 and 2013: 1955 - 1959, 1962 - 1969, 1971 - 1975, 1977 - 1983, 1985 - 1989, 1992 - 1997, 2000 - 2008 and 2010 - 2013 in China. Brucellosis has its unique molecular epidemiological patterns with specific spatial and temporal distribution according to MLVA. IDOP-D-16-00101.

Aspergillus Section Fumigati Typing by PCR-Restriction Fragment Polymorphism▿

OpenAIRE

Staab, Janet F.; Balajee, S. Arunmozhi; Marr, Kieren A.

2009-01-01

Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from...

A study on radiographic repeat rate data of several hospitals in Jeddah

International Nuclear Information System (INIS)

Al-Malki, M.A.; Abulfaraj, W.H.; Bhuiyan, S.I.; Kinsara, A.A.

2003-01-01

Radiographic repeat rate data in diagnostic radiology in King Fahad Hospital (KFH), King Abdulaziz Hospital (KAH), and Maternity and Children Hospital (MCH) in Jeddah, Saudi Arabia, have been studied. The study provided valuable information to suggest preventive measures to reduce repeats. The variables included in the study are exposure techniques, examination types, total number of films used, number of films repeated, the film sizes, gender, the age groups of the patients, and reason for repetition. The total number of examinations in all three hospitals is 6001 using 8887 films on 5412 patients. The average repeat rate was 7.93%, where the individual hospital repeat rates were 9.57% in the MCH, 7.84% in KAH and 7.44% in KFH. The repeat rate for children and infants was found to be undesirable. The quality assurance (QA) programme can effectively reduce the unnecessary exposure and can identify the cause of the exposure. The overexposure, underexposure and position fault were the foremost contributors for repeats and constitute 32.91%, 28.94% and 22.98% of the total respectively. The QA study identified that human error and equipment malfunction are the major contributors to these causes of repeats. The highest repetition rate was for pelvis, 13.64%, followed by skull, 11.59%, and abdomen, 10.41%. It is estimated that the total area of wasted film in all three hospitals is 74.3 m 2 . As per the average repeat rate, the cost of repeat films in the entire kingdom per year has been projected to be about US$1.82 million (SR 6.83 million) in the government hospitals only. Based on the findings of this study a set of recommendations have been prescribed for the radiology department to reduce the repeat rate and to improve the safety culture. (author)

A study on radiographic repeat rate data of several hospitals in Jeddah

Energy Technology Data Exchange (ETDEWEB)

Al-Malki, M.A.; Abulfaraj, W.H.; Bhuiyan, S.I.; Kinsara, A.A

2003-07-01

Radiographic repeat rate data in diagnostic radiology in King Fahad Hospital (KFH), King Abdulaziz Hospital (KAH), and Maternity and Children Hospital (MCH) in Jeddah, Saudi Arabia, have been studied. The study provided valuable information to suggest preventive measures to reduce repeats. The variables included in the study are exposure techniques, examination types, total number of films used, number of films repeated, the film sizes, gender, the age groups of the patients, and reason for repetition. The total number of examinations in all three hospitals is 6001 using 8887 films on 5412 patients. The average repeat rate was 7.93%, where the individual hospital repeat rates were 9.57% in the MCH, 7.84% in KAH and 7.44% in KFH. The repeat rate for children and infants was found to be undesirable. The quality assurance (QA) programme can effectively reduce the unnecessary exposure and can identify the cause of the exposure. The overexposure, underexposure and position fault were the foremost contributors for repeats and constitute 32.91%, 28.94% and 22.98% of the total respectively. The QA study identified that human error and equipment malfunction are the major contributors to these causes of repeats. The highest repetition rate was for pelvis, 13.64%, followed by skull, 11.59%, and abdomen, 10.41%. It is estimated that the total area of wasted film in all three hospitals is 74.3 m{sup 2}. As per the average repeat rate, the cost of repeat films in the entire kingdom per year has been projected to be about US$1.82 million (SR 6.83 million) in the government hospitals only. Based on the findings of this study a set of recommendations have been prescribed for the radiology department to reduce the repeat rate and to improve the safety culture. (author)

The last half-repeat of transcription activator-like effector (TALE) is dispensable and thereby TALE-based technology can be simplified.

Science.gov (United States)

Zheng, Chong-Ke; Wang, Chun-Lian; Zhang, Xiao-Ping; Wang, Fu-Jun; Qin, Teng-Fei; Zhao, Kai-Jun

2014-09-01

To activate the expression of host genes that contribute to pathogen growth, pathogenic Xanthomonas bacteria inject their transcription activator-like effectors (TALEs) into plant cells and the TALEs bind to target gene promoters by the central repeat region consisting of near-perfect 34-amino-acid repeats (34-aa repeats). Based on the recognition codes between the 34-aa repeats and the targeted nucleotides, TALE-based technologies, such as designer TALEs (dTALEs) and TALE nucleases (TALENs), have been developed. Amazingly, every natural TALE invariantly has a truncated last half-repeat (LHR) at the end of the 34-aa repeats. Consequently, all the reported dTALEs and TALENs also harbour their LHRs. Here, we show that the LHRs in dTALEs are dispensable for the function of gene activation by both transient expression assays in Nicotiana benthamiana and gene-specific targeting in the rice genome, indicating that TALEs might originate from a single progenitor. In the light of this finding, we demonstrate that dTALEs can be constructed through two simple steps. Moreover, the activation strengths of dTALEs lacking the LHR are comparable with those of dTALEs harbouring the LHR. Our results provide new insights into the origin of natural TALEs, and will facilitate the simplification of the design and assembly of TALE-based tools, such as dTALEs and TALENs, in the near future. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Agreement and repeatability of vascular reactivity estimates based on a breath-hold task and a resting state scan.

Science.gov (United States)

Lipp, Ilona; Murphy, Kevin; Caseras, Xavier; Wise, Richard G

2015-06-01

FMRI BOLD responses to changes in neural activity are influenced by the reactivity of the vasculature. By complementing a task-related BOLD acquisition with a vascular reactivity measure obtained through breath-holding or hypercapnia, this unwanted variance can be statistically reduced in the BOLD responses of interest. Recently, it has been suggested that vascular reactivity can also be estimated using a resting state scan. This study aimed to compare three breath-hold based analysis approaches (block design, sine-cosine regressor and CO2 regressor) and a resting state approach (CO2 regressor) to measure vascular reactivity. We tested BOLD variance explained by the model and repeatability of the measures. Fifteen healthy participants underwent a breath-hold task and a resting state scan with end-tidal CO2 being recorded during both. Vascular reactivity was defined as CO2-related BOLD percent signal change/mmHg change in CO2. Maps and regional vascular reactivity estimates showed high repeatability when the breath-hold task was used. Repeatability and variance explained by the CO2 trace regressor were lower for the resting state data based approach, which resulted in highly variable measures of vascular reactivity. We conclude that breath-hold based vascular reactivity estimations are more repeatable than resting-based estimates, and that there are limitations with replacing breath-hold scans by resting state scans for vascular reactivity assessment. Copyright © 2015. Published by Elsevier Inc.

Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains▿

Science.gov (United States)

Macheras, Edouard; Roux, Anne-Laure; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby; Bodmer, Thomas; Cambau, Emmanuelle; Gaillard, Jean-Louis; Heym, Beate

2011-01-01

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536T, M. massiliense CIP 108297T, and M. bolletii CIP 108541T) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering

Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.

Science.gov (United States)

Macheras, Edouard; Roux, Anne-Laure; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby; Bodmer, Thomas; Cambau, Emmanuelle; Gaillard, Jean-Louis; Heym, Beate

2011-02-01

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the

spa typing for epidemiological surveillance of Staphylococcus aureus

NARCIS (Netherlands)

Hallin, Marie; Friedrich, Alexander W; Struelens, Marc J; Caugant, Dominique A.

2009-01-01

The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of Staphylococcus aureus. The X region is constituted of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method

Genetic Diversity of the Mycobacterium tuberculosis Beijing Family Based on SNP and VNTR Typing Profiles in Asian Countries

Science.gov (United States)

Chen, Yih-Yuan; Chang, Jia-Ru; Huang, Wei-Feng; Kuo, Shu-Chen; Su, Ih-Jen; Sun, Jun-Ren; Chiueh, Tzong-Shi; Huang, Tsi-Shu; Chen, Yao-Shen; Dou, Horng-Yunn

2012-01-01

The Mycobacterium tuberculosis (MTB) Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China) were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI) of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6) and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3) could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955) to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST) data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82%) and Thailand (92%). Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred. PMID:22808061

Genetic diversity of the Mycobacterium tuberculosis Beijing family based on SNP and VNTR typing profiles in Asian countries.

Directory of Open Access Journals (Sweden)

Yih-Yuan Chen

Full Text Available The Mycobacterium tuberculosis (MTB Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6 and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3 could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955 to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82% and Thailand (92%. Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred.

On the crustal bias of repeat stations in Romania

Directory of Open Access Journals (Sweden)

Venera Dobrica

2013-03-01

Full Text Available A magnetic induction model has been applied to recordings obtained in 2010 during the field campaigns for geomagnetic measurements at the 26 repeat stations of the Romanian secular variation network. The model is based on the observation that a variable external magnetic field induces a response of the Earth's interior not only by electromagnetic induction, but also by magnetic induction in the magnetic rocks above the Curie temperature. The model computes coefficients of a linear relationship between recorded values of a certain geomagnetic element (X, Y, Z, or F at the repeat station and recorded X, Y, Z values at a reference station (in this case, SUA observatory. Coefficients depend on magnetic permeabilities of rocks beneath the station and stand as a proxy for the anomaly bias characterizing the site. Maps of the lateral variation of this type of information were obtained and discussed.

Mechanisms of global diversification in the marine species Madeiran Storm-petrel Oceanodroma castro and Monteiro's Storm-petrel O. monteiroi: Insights from a multi-locus approach.

Science.gov (United States)

Silva, Mauro F; Smith, Andrea L; Friesen, Vicki L; Bried, Joël; Hasegawa, Osamu; Coelho, M Manuela; Silva, Mónica C

2016-05-01

The evolutionary mechanisms underlying the geographic distribution of gene lineages in the marine environment are not as well understood as those affecting terrestrial groups. The continuous nature of the pelagic marine environment may limit opportunities for divergence to occur and lineages to spatially segregate, particularly in highly mobile species. Here, we studied the phylogeography and historical demography of two tropically distributed, pelagic seabirds, the Madeiran Storm-petrel Oceanodroma castro, sampled in the Azores, Madeira, Galapagos and Japan, and its sister species Monteiro's Storm-petrel O. monteiroi (endemic to the Azores), using a multi-locus dataset consisting of 12 anonymous nuclear loci and the mitochondrial locus control region. Both marker types support the existence of four significantly differentiated genetic clusters, including the sampled O. monteiroi population and three populations within O. castro, although only the mitochondrial locus suggests complete lineage sorting. Multi-locus coalescent analyses suggest that most divergence events occurred within the last 200,000years. The proximity in divergence times precluded robust inferences of the species tree, in particular of the evolutionary relationships of the Pacific populations. Despite the great potential for dispersal, divergence among populations apparently proceeded in the absence of gene flow, emphasizing the effect of non-physical barriers, such as those driven by the paleo-oceanographical environments, philopatry and local adaptation, as important mechanisms of population divergence and speciation in highly mobile marine species. In view of the predicted climate change impacts, future changes in the demography and evolutionary dynamics of marine populations might be expected. Copyright © 2016 Elsevier Inc. All rights reserved.

Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance, a Novel Antimicrobial Resistance Multilocus Typing Scheme for Tracking Global Dissemination of N. gonorrhoeae Strains.

Science.gov (United States)

Demczuk, W; Sidhu, S; Unemo, M; Whiley, D M; Allen, V G; Dillon, J R; Cole, M; Seah, C; Trembizki, E; Trees, D L; Kersh, E N; Abrams, A J; de Vries, H J C; van Dam, A P; Medina, I; Bharat, A; Mulvey, M R; Van Domselaar, G; Martin, I

2017-05-01

A curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeae was developed and is publicly accessible (https://ngstar.canada.ca). The N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes ( penA , mtrR , porB , ponA , gyrA , parC , and 23S rRNA) associated with resistance to β-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penA sequence, combining the historical nomenclature for penA types I to XXXVIII with novel nucleotide sequence designations; the full mtrR sequence and a portion of its promoter region; portions of ponA , porB , gyrA , and parC ; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) ( n = 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs ( n = 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 ( n = 100; 13.0%), ST-42 and ST-91 ( n = 45; 5.9%), ST-64 ( n = 44; 5.72%), and ST-139 ( n = 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 ( n = 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 ( n = 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 ( n = 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 ( n = 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeae strains. © Crown copyright 2017.

New polymorphisms within the variable number tandem repeat (VNTR) 7 locus of Mycobacterium avium subsp. paratuberculosis.

Science.gov (United States)

Fawzy, Ahmad; Zschöck, Michael; Ewers, Christa; Eisenberg, Tobias

2016-06-01

Variable number tandem repeat (VNTR) is a frequently employed typing method of Mycobacterium avium paratuberculosis (MAP) isolates. Based on whole genome sequencing in a previous study, allelic diversity at some VNTR loci seems to over- or under-estimate the actual phylogenetic variance among isolates. Interestingly, two closely related isolates on one farm showed polymorphism at the VNTR 7 locus, raising concerns about the misleading role that it might play in genotyping. We aimed to investigate the underlying basis of VNTR 7-polymorphism by analyzing sequence data for published genomes and field isolates of MAP and other M. avium complex (MAC) members. In contrast to MAP strains from cattle, strains from sheep displayed an "imperfect" repeat within VNTR 7, which was identical to respective allele types in other MAC genomes. Subspecies- and strain-specific single nucleotide polymorphisms (SNPs) and two novel (16 and 56 bp) repeats were detected. Given the combination of the three existing repeats, there are at least five different patterns for VNTR 7. The present findings highlight a higher polymorphism and probable instability of VNTR 7 locus that needs to be considered and challenged in future studies. Until then, sequencing of this locus in future studies is important to correctly assign the underlying allele types.(1). Copyright © 2016 Elsevier Ltd. All rights reserved.

Multilocus analyses of an Antarctic fish species flock (Teleostei, Notothenioidei, Trematominae): Phylogenetic approach and test of the early-radiation event

Czech Academy of Sciences Publication Activity Database

Janko, Karel; Marshall, C.; Musilová, Zuzana; Van Houdt, J.; Couloux, A.; Cruaud, C.; Lecointre, G.

2011-01-01

Roč. 60, č. 3 (2011), s. 305-316 ISSN 1055-7903 R&D Projects: GA AV ČR KJB600450903; GA MŠk LC06073 Institutional research plan: CEZ:AV0Z50450515 Keywords : Species tree versus gene tree * Multilocus phylogeny * Diversification rate Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.609, year: 2011

Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing.

NARCIS (Netherlands)

Huyen, M.N.; Kremer, K.; Lan, N.T.; Buu, T.N.; Cobelens, F.G.; Tiemersma, E.W.; Haas, P. de; Soolingen, D. van

2013-01-01

BACKGROUND: In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard

A Comparison of Molecular Typing Methods Applied to Enterobacter cloacae complex: hsp60 Sequencing, Rep-PCR, and MLST

Directory of Open Access Journals (Sweden)

Roberto Viau

2017-02-01

Full Text Available Molecular typing using repetitive sequenced-based PCR (rep-PCR and hsp60 sequencing were applied to a collection of diverse Enterobacter cloacae complex isolates. To determine the most practical method for reference laboratories, we analyzed 71 E. cloacae complex isolates from sporadic and outbreak occurrences originating from 4 geographic areas. While rep-PCR was more discriminating, hsp60 sequencing provided a broader and a more objective geographical tracking method similar to multilocus sequence typing (MLST. In addition, we suggest that MLST may have higher discriminative power compared to hsp60 sequencing, although rep-PCR remains the most discriminative method for local outbreak investigations. In addition, rep-PCR can be an effective and inexpensive method for local outbreak investigation.

Assessment of MultiLocus Sequence Analysis As a Valuable Tool for the Classification of the Genus Salinivibrio

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Clara López-Hermoso

2017-06-01

Full Text Available The genus Salinivibrio includes obligatory halophilic bacteria and is commonly isolated from hypersaline habitats and salted food products. They grow optimally between 7.5 and 10% salts and are facultative anaerobes. Currently, this genus comprises four species, one of them, S. costicola, with three subspecies. In this study we isolated and characterized an additional 70 strains from solar salterns located in different locations. Comparative 16S rRNA gene sequence analysis identified these strains as belonging to the genus Salinivibrio but could not differentiate strains into species-like groups. To achieve finer phylogenetic resolution, we carried out a MultiLocus Sequence Analysis (MLSA of the new isolates and the type strains of the species of Salinivibrio based on the individual as well as concatenated sequences of four housekeeping genes: gyrB, recA, rpoA, and rpoD. The strains formed four clearly differentiated species-like clusters called phylogroups. All of the known type and subspecies strains were associated with one of these clusters except S. sharmensis. One phylogroup had no previously described species coupled to it. Further DNA–DNA hybridization (DDH experiments with selected representative strains from these phylogroups permitted us to validate the MLSA study, correlating the species level defined by the DDH (70% with a 97% cut-off for the concatenated MLSA gene sequences. Based on these criteria, the novel strains forming phylogroup 1 could constitute a new species while strains constructing the other three phylogroups are members of previously recognized Salinivibrio species. S. costicola subsp. vallismortis co-occurs with S. proteolyticus in phylogroup 4, and separately from other S. costicola strains, indicating its need for reclassification. On the other hand, genome fingerprinting analysis showed that the environmental strains do not form clonal populations and did not cluster according to their site of cultivation. In

Robot-Assisted End-Effector-Based Stair Climbing for Cardiopulmonary Exercise Testing: Feasibility, Reliability, and Repeatability.

Science.gov (United States)

Stoller, Oliver; Schindelholz, Matthias; Hunt, Kenneth J

2016-01-01

Neurological impairments can limit the implementation of conventional cardiopulmonary exercise testing (CPET) and cardiovascular training strategies. A promising approach to provoke cardiovascular stress while facilitating task-specific exercise in people with disabilities is feedback-controlled robot-assisted end-effector-based stair climbing (RASC). The aim of this study was to evaluate the feasibility, reliability, and repeatability of augmented RASC-based CPET in able-bodied subjects, with a view towards future research and applications in neurologically impaired populations. Twenty able-bodied subjects performed a familiarisation session and 2 consecutive incremental CPETs using augmented RASC. Outcome measures focussed on standard cardiopulmonary performance parameters and on accuracy of work rate tracking (RMSEP-root mean square error). Criteria for feasibility were cardiopulmonary responsiveness and technical implementation. Relative and absolute test-retest reliability were assessed by intraclass correlation coefficients (ICC), standard error of the measurement (SEM), and minimal detectable change (MDC). Mean differences, limits of agreement, and coefficients of variation (CoV) were estimated to assess repeatability. All criteria for feasibility were achieved. Mean V'O2peak was 106±9% of predicted V'O2max and mean HRpeak was 99±3% of predicted HRmax. 95% of the subjects achieved at least 1 criterion for V'O2max, and the detection of the sub-maximal ventilatory thresholds was successful (ventilatory anaerobic threshold 100%, respiratory compensation point 90% of the subjects). Excellent reliability was found for peak cardiopulmonary outcome measures (ICC ≥ 0.890, SEM ≤ 0.60%, MDC ≤ 1.67%). Repeatability for the primary outcomes was good (CoV ≤ 0.12). RASC-based CPET with feedback-guided exercise intensity demonstrated comparable or higher peak cardiopulmonary performance variables relative to predicted values, achieved the criteria for V'O2max

Robot-Assisted End-Effector-Based Stair Climbing for Cardiopulmonary Exercise Testing: Feasibility, Reliability, and Repeatability.

Directory of Open Access Journals (Sweden)

Oliver Stoller

Full Text Available Neurological impairments can limit the implementation of conventional cardiopulmonary exercise testing (CPET and cardiovascular training strategies. A promising approach to provoke cardiovascular stress while facilitating task-specific exercise in people with disabilities is feedback-controlled robot-assisted end-effector-based stair climbing (RASC. The aim of this study was to evaluate the feasibility, reliability, and repeatability of augmented RASC-based CPET in able-bodied subjects, with a view towards future research and applications in neurologically impaired populations.Twenty able-bodied subjects performed a familiarisation session and 2 consecutive incremental CPETs using augmented RASC. Outcome measures focussed on standard cardiopulmonary performance parameters and on accuracy of work rate tracking (RMSEP-root mean square error. Criteria for feasibility were cardiopulmonary responsiveness and technical implementation. Relative and absolute test-retest reliability were assessed by intraclass correlation coefficients (ICC, standard error of the measurement (SEM, and minimal detectable change (MDC. Mean differences, limits of agreement, and coefficients of variation (CoV were estimated to assess repeatability.All criteria for feasibility were achieved. Mean V'O2peak was 106±9% of predicted V'O2max and mean HRpeak was 99±3% of predicted HRmax. 95% of the subjects achieved at least 1 criterion for V'O2max, and the detection of the sub-maximal ventilatory thresholds was successful (ventilatory anaerobic threshold 100%, respiratory compensation point 90% of the subjects. Excellent reliability was found for peak cardiopulmonary outcome measures (ICC ≥ 0.890, SEM ≤ 0.60%, MDC ≤ 1.67%. Repeatability for the primary outcomes was good (CoV ≤ 0.12.RASC-based CPET with feedback-guided exercise intensity demonstrated comparable or higher peak cardiopulmonary performance variables relative to predicted values, achieved the criteria for V'O2

Tetranucleotide repeat polymorphism at the human prostatic acid phosphatase (ACPP) gene

Energy Technology Data Exchange (ETDEWEB)

Polymeropoulos, M H; Xiao, Hong; Rath, D S; Merril, C R [National Inst. of Mental Health Neuroscience Center, Washington, DC (United States)

1991-09-11

The polymorphic (AAAT){sub n} repeat begins at base pair 2342 of the human prostatic acid phosphatase gene on chromosome 3q21-qter. The polymorphism can be typed using the polymerase chain reaction (PCR) as described previously. The predicted length of the amplified sequence was 275 bp. Co-dominant segregation was observed in two informative families. The human prostatic acid phosphatase gene has been assigned to chromosome 3q21-qter.

Heralded quantum repeater based on the scattering of photons off single emitters in one-dimensional waveguides

Energy Technology Data Exchange (ETDEWEB)

Song, Guo-Zhu; Zhang, Mei; Ai, Qing; Yang, Guo-Jian [Department of Physics, Applied Optics Beijing Area Major Laboratory, Beijing Normal University, Beijing 100875 (China); Alsaedi, Ahmed; Hobiny, Aatef [NAAM-Research Group, Department of Mathematics, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia); Deng, Fu-Guo, E-mail: fgdeng@bnu.edu.cn [Department of Physics, Applied Optics Beijing Area Major Laboratory, Beijing Normal University, Beijing 100875 (China); NAAM-Research Group, Department of Mathematics, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589 (Saudi Arabia)

2017-03-15

We propose a heralded quantum repeater based on the scattering of photons off single emitters in one-dimensional waveguides. We show the details by implementing nonlocal entanglement generation, entanglement swapping, and entanglement purification modules with atoms in waveguides, and discuss the feasibility of the repeater with currently achievable technology. In our scheme, the faulty events can be discarded by detecting the polarization of the photons. That is, our protocols are accomplished with a fidelity of 100% in principle, which is advantageous for implementing realistic long-distance quantum communication. Moreover, additional atomic qubits are not required, but only a single-photon medium. Our scheme is scalable and attractive since it can be realized in solid-state quantum systems. With the great progress on controlling atom-waveguide systems, the repeater may be very useful in quantum information processing in the future.

A novel Hartman Shack-based topography system: repeatability and agreement for corneal power with Scheimpflug+Placido topographer and rotating prism auto-keratorefractor.

Science.gov (United States)

Prakash, Gaurav; Srivastava, Dhruv; Choudhuri, Sounak

2015-12-01

The purpose of this study is to analyze the repeatability and agreement of corneal power using a new Hartman type topographer in comparison to Scheimpflug+Placido and autorefractor devices. In this cross sectional, observational study performed at the cornea services of a specialty hospital, 100 normal eyes (100 consecutive candidates) without any previous ocular surgery or morbidity except refractive error were evaluated. All candidates underwent three measurements each on a Full gradient, Hartman type topographer (FG) (iDesign, AMO), Scheimpflug+Placido topographer (SP) (Sirius, CSO) and rotating prism auto-keratorefractor (AR) (KR1, Nidek). The parameters assessed were flat keratometry (K1), steep keratometry (K2), steep axis (K2 axis), mean K, J 0 and J 45. Intra-device repeatability and inter-device agreement were evaluated. On repeatability analysis, the intra-device means were not significantly different (ANOVA, p > 0.05). Intraclass correlations (ICC) were >0.98 except for J 0 and J 45. In terms of intra-measurement standard deviation (Sw), the SP and FG groups fared better than AR group (p 0.05, R (2) 0.05). ICC ranged from 0.92 to 0.99 (p < 0.001). Regression fits on Bland-Altman plots suggested no clinically significant effect of average values over difference in means. The repeatability of Hartman type topographer in normal eyes is comparable to SP combination device and better than AR. The agreement between the three devices is good. However, we recommend against interchanging these devices between follow-ups or pooling their data.

New paradigms for Salmonella source attribution based on microbial subtyping.

Science.gov (United States)

Mughini-Gras, Lapo; Franz, Eelco; van Pelt, Wilfrid

2018-05-01

Microbial subtyping is the most common approach for Salmonella source attribution. Typically, attributions are computed using frequency-matching models like the Dutch and Danish models based on phenotyping data (serotyping, phage-typing, and antimicrobial resistance profiling). Herewith, we critically review three major paradigms facing Salmonella source attribution today: (i) the use of genotyping data, particularly Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA), which is replacing traditional Salmonella phenotyping beyond serotyping; (ii) the integration of case-control data into source attribution to improve risk factor identification/characterization; (iii) the investigation of non-food sources, as attributions tend to focus on foods of animal origin only. Population genetics models or simplified MLVA schemes may provide feasible options for source attribution, although there is a strong need to explore novel modelling options as we move towards whole-genome sequencing as the standard. Classical case-control studies are enhanced by incorporating source attribution results, as individuals acquiring salmonellosis from different sources have different associated risk factors. Thus, the more such analyses are performed the better Salmonella epidemiology will be understood. Reparametrizing current models allows for inclusion of sources like reptiles, the study of which improves our understanding of Salmonella epidemiology beyond food to tackle the pathogen in a more holistic way. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mediating Role of the Reward Network in the Relationship between the Dopamine Multilocus Genetic Profile and Depression

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Liang Gong

2017-09-01

Full Text Available Multiple genetic loci in the dopamine (DA pathway have been associated with depression symptoms in patients with major depressive disorder (MDD. However, the neural mechanisms underlying the polygenic effects of the DA pathway on depression remain unclear. We used an imaging genetic approach to investigate the polygenic effects of the DA pathway on the reward network in MDD. Fifty-three patients and 37 cognitively normal (CN subjects were recruited and underwent resting-state functional magnetic resonance imaging (R-fMRI scans. Multivariate linear regression analysis was employed to measure the effects of disease and multilocus genetic profile scores (MGPS on the reward network, which was constructed using the nucleus accumbens (NAc functional connectivity (NAFC network. DA-MGPS was widely associated within the NAFC network, mainly in the inferior frontal cortex, insula, hypothalamus, superior temporal gyrus, and occipital cortex. The pattern of DA-MGPS effects on the fronto-striatal pathway differed in MDD patients compared with CN subjects. More importantly, NAc-putamen connectivity mediates the association between DA MGPS and anxious depression traits in MDD patients. Our findings suggest that the DA multilocus genetic profile makes a considerable contribution to the reward network and anxious depression in MDD patients. These results expand our understanding of the pathophysiology of polygenic effects underlying brain network abnormalities in MDD.

The D4Z4 macrosatellite repeat acts as a CTCF and A-type lamins-dependent insulator in facio-scapulo-humeral dystrophy.

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Alexandre Ottaviani

2009-02-01

Full Text Available Both genetic and epigenetic alterations contribute to Facio-Scapulo-Humeral Dystrophy (FSHD, which is linked to the shortening of the array of D4Z4 repeats at the 4q35 locus. The consequence of this rearrangement remains enigmatic, but deletion of this 3.3-kb macrosatellite element might affect the expression of the FSHD-associated gene(s through position effect mechanisms. We investigated this hypothesis by creating a large collection of constructs carrying 1 to >11 D4Z4 repeats integrated into the human genome, either at random sites or proximal to a telomere, mimicking thereby the organization of the 4q35 locus. We show that D4Z4 acts as an insulator that interferes with enhancer-promoter communication and protects transgenes from position effect. This last property depends on both CTCF and A-type Lamins. We further demonstrate that both anti-silencing activity of D4Z4 and CTCF binding are lost upon multimerization of the repeat in cells from FSHD patients compared to control myoblasts from healthy individuals, suggesting that FSHD corresponds to a gain-of-function of CTCF at the residual D4Z4 repeats. We propose that contraction of the D4Z4 array contributes to FSHD physio-pathology by acting as a CTCF-dependent insulator in patients.

Genetic Relatedness of Staphylococcus haemolyticus in Gut and Skin of Preterm Neonates and Breast Milk of Their Mothers.

Science.gov (United States)

Soeorg, Hiie; Metsvaht, Hanna Kadri; Keränen, Evamaria Elisabet; Eelmäe, Imbi; Merila, Mirjam; Ilmoja, Mari-Liis; Metsvaht, Tuuli; Lutsar, Irja

2018-04-02

Staphylococcus haemolyticus is a common colonizer and cause of late-onset sepsis (LOS) in preterm neonates. By describing genetic relatedness, we aimed to determine whether mother's breast milk (BM) is a source of S. haemolyticus colonizing neonatal gut and skin and/or causing LOS. S. haemolyticus was isolated from stool and skin swabs of 49 BM-fed preterm neonates admitted to neonatal intensive care unit, 20 healthy BM-fed term neonates and BM of mothers once a week and typed by multilocus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Virulence-related genes were determined by PCR. Compared with term neonates S. haemolyticus colonized more commonly gut (35% vs 89.9%; pskin (50% vs 91.8%; pskin with MLVA-types indistinguishable from those in BM. Most frequent MLVA-types belonged to sequence type 3 or 42, comprised 71.1-78.4% of isolates from preterm neonates/mothers and caused all seven LOS episodes. LOS-causing strain colonized the gut of 4/7 and the skin of 5/7 neonates, but not BM, prior to onset of LOS. S. haemolyticus colonizing gut and skin or causing LOS in preterm neonates rarely originate from BM, but are mecA-positive strains adapted to hospital environment.

Repeated dose titration versus age-based method in electroconvulsive therapy: a pilot study.

Science.gov (United States)

Aten, Jan Jaap; Oudega, Mardien; van Exel, Eric; Stek, Max L; van Waarde, Jeroen A

2015-06-01

In electroconvulsive therapy (ECT), a dose titration method (DTM) was suggested to be more individualized and therefore more accurate than formula-based dosing methods. A repeated DTM (every sixth session and dose adjustment accordingly) was compared to an age-based method (ABM) regarding treatment characteristics, clinical outcome, and cognitive functioning after ECT. Thirty-nine unipolar depressed patients dosed using repeated DTM and 40 matched patients treated with ABM were compared. Montgomery-Åsberg Depression Rating Scale (MADRS) and Mini-Mental State Examination (MMSE) were assessed at baseline and at the end of the index course, as well as the total number of ECT sessions. Both groups were similar regarding age, sex, psychotic features, mean baseline MADRS, and median baseline MMSE. At the end of the index course, the two methods showed equal outcome (mean end MADRS, 11.6 ± 8.3 in DTM and 9.5 ± 7.6 in ABM (P = 0.26); median end MMSE, 28 (25-29) and 28 (25-29.8), respectively (P = 0.81). However, the median number of all ECT sessions differed 16 (11-22) in DTM versus 12 (10-14.8) in ABM; P = 0.02]. Using regression analysis, dosing method and age were independently associated with the total number of ECT sessions, with less sessions needed in ABM (P = 0.02) and in older patients (P = 0.001). In this comparative cohort study, ABM and DTM showed equal outcome for depression and cognition. However, the median ECT course duration in repeated DTM appeared longer. Additionally, higher age was associated with shorter ECT courses regardless of the dosing method. Further prospective studies are needed to confirm these findings.

Practical Issues in Evidence-Based Use of Performance Supplements: Supplement Interactions, Repeated Use and Individual Responses.

Science.gov (United States)

Burke, Louise M

2017-03-01

Current sports nutrition guidelines recommend that athletes only take supplements following an evidence-based analysis of their value in supporting training outcomes or competition performance in their specific event. While there is sound evidence to support the use of a few performance supplements under specific scenarios (creatine, beta-alanine, bicarbonate, caffeine, nitrate/beetroot juice and, perhaps, phosphate), there is a lack of information around several issues needed to guide the practical use of these products in competitive sport. First, there is limited knowledge around the strategy of combining the intake of several products in events in which performance benefits are seen with each product in isolation. The range in findings from studies involving combined use of different combinations of two supplements makes it difficult to derive a general conclusion, with both the limitations of individual studies and the type of sporting event to which the supplements are applied influencing the potential for additive, neutral or counteractive outcomes. The repeated use of the same supplement in sports involving two or more events within a 24-h period is of additional interest, but has received even less attention. Finally, the potential for individual athletes to respond differently, in direction and magnitude, to the use of a supplement seems real, but is hard to distinguish from normal day to day variability in performance. Strategies that can be used in research or practice to identify whether individual differences are robust include repeat trials, and the collection of data on physiological or genetic mechanisms underpinning outcomes.

Atomic force microscopy-based repeated machining theory for nanochannels on silicon oxide surfaces

Energy Technology Data Exchange (ETDEWEB)

Wang, Z.Q., E-mail: wangzhiqian@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, CAS, Shenyang 110016 (China); Graduate University of the Chinese Academy of Sciences, Beijing 100049 (China); Jiao, N.D. [State Key Laboratory of Robotics, Shenyang Institute of Automation, CAS, Shenyang 110016 (China); Tung, S. [Department of Mechanical Engineering, University of Arkansas, Fayetteville, AR 72701 (United States); Dong, Z.L. [State Key Laboratory of Robotics, Shenyang Institute of Automation, CAS, Shenyang 110016 (China)

2011-02-01

The atomic force microscopy (AFM)-based repeated nanomachining of nanochannels on silicon oxide surfaces is investigated both theoretically and experimentally. The relationships of the initial nanochannel depth vs. final nanochannel depth at a normal force are systematically studied. Using the derived theory and simulation results, the final nanochannel depth can be predicted easily. Meanwhile, if a nanochannel with an expected depth needs to be machined, a right normal force can be selected simply and easily in order to decrease the wear of the AFM tip. The theoretical analysis and simulation results can be effectively used for AFM-based fabrication of nanochannels.

Multilocus sequence typing and phylogenetic analysis of Propionibacterium acnes

DEFF Research Database (Denmark)

Kilian, Mogens; Scholz, Christian F. P.; Lomholt, Hans B.

2012-01-01

Propionibacterium acnes is a commensal of human skin but is also implicated in the pathogenesis of acne vulgaris, in biofilm-associated infections of medical devices and endophthalmitis, and in infections of bone and dental root canals. Recent studies associate P. acnes with prostate cancer...... schemes were compared with reference to a phylogenetic tree based on 78 P. acnes genomes and their gene contents. Further support for a basically clonal population structure of P. acnes and a scenario of the global spread of epidemic clones of P. acnes was obtained. Compared to the Belfast scheme...

MLVA and MLST typing of Brucella from Qinghai, China.

Science.gov (United States)

Ma, Jun-Ying; Wang, Hu; Zhang, Xue-Fei; Xu, Li-Qing; Hu, Gui-Ying; Jiang, Hai; Zhao, Fang; Zhao, Hong-Yan; Piao, Dong-Ri; Qin, Yu-Min; Cui, Bu-Yun; Lin, Gong-Hua

2016-04-13

The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans.

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms.

Science.gov (United States)

Vergara-Jaque, Ariela; Fenollar-Ferrer, Cristina; Kaufmann, Desirée; Forrest, Lucy R

2015-01-01

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also

Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

Directory of Open Access Journals (Sweden)

Cristina eFenollar Ferrer

2015-09-01

Full Text Available Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to either the outside or inside of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (asymmetry of these systems has been successfully used as a bioinformatic tool, called repeat-swap modeling to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that

The repeatability and reproducibility of the BioRID IIg in a repeatable laboratory seat based on a production car seat.

Science.gov (United States)

Hynd, David; Depinet, Paul; Lorenz, Bernd

2013-01-01

The United Nations Economic Commission for Europe Informal Group on GTR No. 7 Phase 2 are working to define a build level for the BioRID II rear impact (whiplash) crash test dummy that ensures repeatable and reproducible performance in a test procedure that has been proposed for future legislation. This includes the specification of dummy hardware, as well as the development of comprehensive certification procedures for the dummy. This study evaluated whether the dummy build level and certification procedures deliver the desired level of repeatability and reproducibility. A custom-designed laboratory seat was made using the seat base, back, and head restraint from a production car seat to ensure a representative interface with the dummy. The seat back was reinforced for use in multiple tests and the recliner mechanism was replaced by an external spring-damper mechanism. A total of 65 tests were performed with 6 BioRID IIg dummies using the draft GTR No.7 sled pulse and seating procedure. All dummies were subject to the build, maintenance, and certification procedures defined by the Informal Group. The test condition was highly repeatable, with a very repeatable pulse, a well-controlled seat back response, and minimal observed degradation of seat foams. The results showed qualitatively reasonable repeatability and reproducibility for the upper torso and head accelerations, as well as for T1 Fx and upper neck Fx . However, reproducibility was not acceptable for T1 and upper neck Fz or for T1 and upper neck My . The Informal Group has not selected injury or seat assessment criteria for use with BioRID II, so it is not known whether these channels would be used in the regulation. However, the ramping-up behavior of the dummy showed poor reproducibility, which would be expected to affect the reproducibility of dummy measurements in general. Pelvis and spine characteristics were found to significantly influence the dummy measurements for which poor reproducibility was

Simple sequence repeat (SSR)-based genetic variability among ...

African Journals Online (AJOL)

The objective of this study was to compare if simple sequence repeat (SSR) markers could correctly identify peanut genotypes with difference in specific leaf weight (SLW) and relative water content (RWC). Four peanut genotypes and two water regimes (FC and 1/3 available water; 1/3 AW) were arranged in factorial ...

Molecular epidemiology of Neisseria gonorrhoeae strains circulating in Indonesia using multi-locus variable number tandem repeat analysis (MLVA) and Neisseria gonorrhoeae multi-antigen sequence typing (NG-NAST) techniques

NARCIS (Netherlands)

Hananta, I. Putu Yuda; van Dam, Alje Pieter; Schim van der Loeff, Maarten Franciscus; Dierdorp, Mirjam; Wind, Carolien Marleen; Soebono, Hardyanto; de Vries, Henry John Christiaan; Bruisten, Sylvia Maria

2018-01-01

Background: Control of gonorrhea in resource-limited countries, such as Indonesia, is mostly unsuccessful. Examining Neisseria gonorrhoeae (Ng) transmission networks using strain typing might help prioritizing public health interventions. Methods: In 2014, urogenital Ng strains were isolated from

The SCA1 (Spinocerebellar ataxia type 1 and MJD (Machado-Joseph disease CAG repeats in normal individuals: segregation analysis and allele frequencies

Directory of Open Access Journals (Sweden)

Cláudia Emília Vieira Wiezel

2003-01-01

Full Text Available Spinocerebellar ataxia type 1 (SCA1 and Machado-Joseph disease (MJD/SCA3 are autosomal dominant neurodegenerative diseases caused by expansions of a CAG trinucleotide repeat in the SCA1 and MJD genes. These expanded sequences are unstable upon transmission, leading to an intergeneration increase in the number of repeats (dynamic mutation. The transmission of the CAG repeat was studied in normal mother-father-child trios, referred for paternity testing (SCA1, n = 367; MJD, n = 879. No segregation distortion was detected. The CAG allele frequencies were determined in 330 unrelated individuals (fathers from couples tested for paternity. The allele frequency distributions did not differ from those previously reported for European populations. The estimated values for the statistic parameters indicating diversity at the SCA1 locus did not differ much from those reported previously for other STRs in the Brazilian population, while those for the MJD locus were close to or higher than the maximum values of previous reports. This shows that SCA1 and MJD are highly informative loci for applications in genetic and population studies and for forensic analysis.

Evaluation of the HOOF-Print assay for typing Brucella abortus strains isolated from cattle in the United States: results with four performance criteria

Directory of Open Access Journals (Sweden)

Ewalt Darla R

2005-06-01

Full Text Available Abstract Background A fundamental question that arises during epidemiological investigations of bacterial disease outbreaks is whether the outbreak strain is genetically related to a proposed index strain. Highly discriminating genetic markers for characterizing bacterial strains can help in clarifying the genetic relationships among strains. Under the auspices of the European Society of Clinical Microbiology and Infectious Diseases, the European Study Group for Epidemiological Markers (ESGEM established guidelines for evaluating the performance of typing systems based of a number of criteria. Recently, HOOF-Print genotype analysis, a new method for typing Brucella abortus strains based on hypervariability at eight tandem repeat loci, was described. This paper evaluates the HOOF-Print assay by four of the criteria set out by the ESGEM: typeability, reproducibility, power of discrimination, and concordance with other typing methods. Results The HOOF-Print Assay was evaluated with a test population composed of 97 unrelated field isolates and 6 common laboratory strains of B. abortus. Both typeability and reproducibility of the assay were excellent. Allele diversity and frequency varied widely among the eight loci, ranging from 1 to 13 alleles. The power of discrimination, measured by the Hunter-Gaston discrimination index (HGDI, varied by locus ranging from 0 to 0.89, where a maximal value of 1.0 indicates discrimination of all strains. The HGDI values calculated for subgroups sorted by biovar were similar to the values determined for the whole population. None of the individual loci achieved the recommended HGDI threshold of 0.95, but the HGDI of the composite profiles was 0.99 (93 unique genotypes from 97 field strains evaluated, well above the recommended threshold. By comparison, the HGDI value for biovar typing was 0.61 in a test population biased with disproportionate numbers of the less common biovars. Cluster analysis based on HOOF

Multi-Repeated Projection Lithography for High-Precision Linear Scale Based on Average Homogenization Effect

Directory of Open Access Journals (Sweden)

Dongxu Ren

2016-04-01

Full Text Available A multi-repeated photolithography method for manufacturing an incremental linear scale using projection lithography is presented. The method is based on the average homogenization effect that periodically superposes the light intensity of different locations of pitches in the mask to make a consistent energy distribution at a specific wavelength, from which the accuracy of a linear scale can be improved precisely using the average pitch with different step distances. The method’s theoretical error is within 0.01 µm for a periodic mask with a 2-µm sine-wave error. The intensity error models in the focal plane include the rectangular grating error on the mask, static positioning error, and lithography lens focal plane alignment error, which affect pitch uniformity less than in the common linear scale projection lithography splicing process. It was analyzed and confirmed that increasing the repeat exposure number of a single stripe could improve accuracy, as could adjusting the exposure spacing to achieve a set proportion of black and white stripes. According to the experimental results, the effectiveness of the multi-repeated photolithography method is confirmed to easily realize a pitch accuracy of 43 nm in any 10 locations of 1 m, and the whole length accuracy of the linear scale is less than 1 µm/m.

Global diversity of the Ganoderma lucidum complex (Ganodermataceae, Polyporales) inferred from morphology and multilocus phylogeny.

Science.gov (United States)

Zhou, Li-Wei; Cao, Yun; Wu, Sheng-Hua; Vlasák, Josef; Li, De-Wei; Li, Meng-Jie; Dai, Yu-Cheng

2015-06-01

Species of the Ganoderma lucidum complex are used in many types of health products. However, the taxonomy of this complex has long been chaotic, thus limiting its uses. In the present study, 32 collections of the complex from Asia, Europe and North America were analyzed from both morphological and molecular phylogenetic perspectives. The combined dataset, including an outgroup, comprised 33 ITS, 24 tef1α, 24 rpb1 and 21 rpb2 sequences, of which 19 ITS, 20 tef1α, 20 rpb1 and 17 rpb2 sequences were newly generated. A total of 13 species of the complex were recovered in the multilocus phylogeny. These 13 species were not strongly supported as a single monophyletic lineage, and were further grouped into three lineages that cannot be defined by their geographic distributions. Clade A comprised Ganoderma curtisii, Ganoderma flexipes, Ganoderma lingzhi, Ganoderma multipileum, Ganoderma resinaceum, Ganoderma sessile, Ganoderma sichuanense and Ganoderma tropicum, Clade B comprised G. lucidum, Ganoderma oregonense and Ganoderma tsugae, and Clade C comprised Ganoderma boninense and Ganoderma zonatum. A dichotomous key to the 13 species is provided, and their key morphological characters from context, pores, cuticle cells and basidiospores are presented in a table. The taxonomic positions of these species are briefly discussed. Noteworthy, the epitypification of G. sichuanense is rejected. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of rearing environment and population origin on responses to repeated behavioural trials in cane toads (Rhinella marina).

Science.gov (United States)

Gruber, Jodie; Whiting, Martin J; Brown, Gregory; Shine, Richard

2018-05-02

Behavioural response to repeated trials in captivity can be driven by many factors including rearing environment, population of origin, habituation to captivity/trial conditions and an individual's behavioural type (e.g., bold versus shy). We tested the effect of rearing environment (captive raised common-garden versus wild-caught) and population origin (range-edge versus range-front) on the responses of invasive cane toads (Rhinella marina) to repeated exploration and risk-taking assays in captivity. We found that behavioural responses to identical assays performed on two occasions were complex and showed few consistent patterns based on rearing environment or population of origin. However, behavioural traits were repeatable across Trial Blocks when all sample populations were grouped together, indicating general consistency in individual toad behaviour across repeated behavioural assays. Our findings exemplify the complexity and unpredictability of behavioural responses and their effects on the repeatability and interpretation of behavioural traits across repeated behavioural assays in captivity. To meaningfully interpret the results from repeated behavioural assays, we need to consider how multiple factors may affect behavioural responses to these tests and importantly, how these responses may affect the repeatability of behavioural traits across time. Copyright © 2018. Published by Elsevier B.V.

Skeletal muscle fiber type composition and performance during repeated bouts of maximal, concentric contractions

Science.gov (United States)

Colliander, E. B.; Dudley, G. A.; Tesch, P. A.

1988-01-01

Force output and fatigue and recovery patterns were studied during intermittent short-term exercise. 27 men performed three bouts of 30 maximal unilateral knee extensions on 2 different occasions. Blood flow was maintained or occluded during recovery periods (60 s). Blood flow was restricted by inflating a pneumatic cuff placed around the proximal thigh. Muscle biopsies from vastus lateralis were analyzed for identification of fast twitch (FT) and slow twitch (ST) fibers and relative FT area. Peak torque decreased during each bout of exercise and more when blood flow was restricted during recovery. Initial peak torque (IPT) and average peak torque (APT) decreased over the three exercise bouts. This response was 3 fold greater without than with blood flow during recovery. IPT and APT decreased more in individuals with mainly FT fibers than in those with mainly ST fibers. It is suggested that performance during repeated bouts of maximal concentric contractions differs between individuals with different fiber type composition. Specifically, in high intensity, intermittent exercise with emphasis on anaerobic energy release a high FT composition may not necessarily be advantageous for performance.

Evolution and polymorphism in the multilocus Levene model with no or weak epistasis.

Science.gov (United States)

Bürger, Reinhard

2010-09-01

Evolution and the maintenance of polymorphism under the multilocus Levene model with soft selection are studied. The number of loci and alleles, the number of demes, the linkage map, and the degree of dominance are arbitrary, but epistasis is absent or weak. We prove that, without epistasis and under mild, generic conditions, every trajectory converges to a stationary point in linkage equilibrium. Consequently, the equilibrium and stability structure can be determined by investigating the much simpler gene-frequency dynamics on the linkage-equilibrium manifold. For a haploid species an analogous result is shown. For weak epistasis, global convergence to quasi-linkage equilibrium is established. As an application, the maintenance of multilocus polymorphism is explored if the degree of dominance is intermediate at every locus and epistasis is absent or weak. If there are at least two demes, then arbitrarily many multiallelic loci can be maintained polymorphic at a globally asymptotically stable equilibrium. Because this holds for an open set of parameters, such equilibria are structurally stable. If the degree of dominance is not only intermediate but also deme independent, and loci are diallelic, an open set of parameters yielding an internal equilibrium exists only if the number of loci is strictly less than the number of demes. Otherwise, a fully polymorphic equilibrium exists only nongenerically, and if it exists, it consists of a manifold of equilibria. Its dimension is determined. In the absence of genotype-by-environment interaction, however, a manifold of equilibria occurs for an open set of parameters. In this case, the equilibrium structure is not robust to small deviations from no genotype-by-environment interaction. In a quantitative-genetic setting, the assumptions of no epistasis and intermediate dominance are equivalent to assuming that in every deme directional selection acts on a trait that is determined additively, i.e., by nonepistatic loci with

A joint model for repeated events of different types and multiple longitudinal outcomes with application to a follow-up study of patients after kidney transplant

NARCIS (Netherlands)

Musoro, Jammbe Z.; Geskus, Ronald B.; Zwinderman, Aeilko H.

2015-01-01

This paper presents an extension of the joint modeling strategy for the case of multiple longitudinal outcomes and repeated infections of different types over time, motivated by postkidney transplantation data. Our model comprises two parts linked by shared latent terms. On the one hand is a

Alu repeats as markers for forensic DNA analyses

Energy Technology Data Exchange (ETDEWEB)

Batzer, M.A.; Alegria-Hartman, M. [Lawrence Livermore National Lab., CA (United States); Kass, D.H. [Louisiana State Univ., New Orleans, LA (United States)] [and others

1994-01-01

The Human-Specific (HS) subfamily of Alu sequences is comprised of a group of 500 nearly identical members which are almost exclusively restricted to the human genome. Individual subfamily members share an average of 98.9% nucleotide identity with the HS subfamily consensus sequence, and have an average age of 2.8 million years. We have developed a Polymerase Chain Reaction (PCR) based assay using primers complementary to the 5 inch and 3 inch unique flanking DNA sequences from each HS Alu that allow the locus to be assayed for the presence or absence of the Alu repeat. The dimorphic HS Alu sequences probably inserted in the human genome after the radiation of modem humans (within the last 200,000-one million years) and represent a unique source of information for human population genetics and forensic DNA analyses. These sites can be developed into Dimorphic Alu Sequence Tagged Sites (DASTS) for the Human Genome Project. HS Alu family member insertions differ from other types of polymorphism (e.g. Variable Number of Tandem Repeat [VNTR] or Restriction Fragment Length Polymorphism [RFLP]) in that polymorphisms due to Alu insertions arise as a result of a unique event which has occurred only one time in the human population and spread through the population from that point. Therefore, individuals that share HS Alu repeats inherited these elements from a common ancestor. Most VNTR and RFLP polymorphisms may arise multiple times in parallel within a population.

REPETITION AS A SPECIAL TYPE OF SYNTACTIC RELATIONS IN REPRESENTED SPEECH: BASED ON THE PROSE BY MARINA TSVETAEVA

Directory of Open Access Journals (Sweden)

Olga Pavlovna Puchinina

2017-03-01

Full Text Available The author studies the peculiarities of repetition, being one of the ways to build syntactic relations in the represented speech structure, based on the prose by Marina Tsvetaeva. The article describes the different types of repetitions, their distinctive features and frequency of usage in the studied tests. The author describes different types of repetitions in the studied cases of represented speech from the point of view of their position. Repetition occurs at the beginning, end of sentences, beginning and end of a statement or paragraph, end of a statement and beginning of the next one; lexical items may be repeated in the middle of a statement. The morphology of repetitions, i.e., the way by what parts of speech the repeated words and structures are expressed, is of interest from the point of view of functional grammar. The author notes that Tsvetaeva repeats different parts of speech: conjunctions, prepositions, particles, nouns, pronouns, adverbs, numerals, verbs, modal words or a combination of two words. Moreover, due to her special intention, Tsvetaeva intensifies repetition through particular phonetic devices, such as alliteration, rhyme and rhythm, which make her prosaic works sound poetic. Purpose. The article is devoted to the topic of rendering another person’s speech, as it continues to be one of the most important issues of modern linguistics. The subject of analysis is repetition and its different types in the structure of represented speech on the material of prose texts by Marina Tsvetaeva. The author’s aims is to reveal the way these types of repetition (lexical, syntactic, semantic ones function in the structure of represented speech and what effect is achieved with their help. Methodology. The research has been conducted using the continuous sampling method and the quantitative estimation method, aimed to identify the frequency of using different types of repetition and repeated parts of speech and constructions in the

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

DEFF Research Database (Denmark)

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

2016-01-01

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

Characterization of the Emerging Salmonella 4,[5],12:i:- in Danish Animal Production

DEFF Research Database (Denmark)

Argüello, Hector; Sørensen, Gitte; Carvajal, Ana

2014-01-01

Abstract The monophasic Salmonella variant with the antigenic formula Salmonella 4,[5],12:i:- has emerged in the last decade as one of the main serotypes related to human salmonellosis. In the present study, a collection of 94 isolates of the S. 4,12:i:- and S. 4,5,12:i:- coming from Danish farm ...... in Danish food animal production with well-characterized clones that are described by previous studies, demonstrating the emergence and spread of this serotype in Denmark....... animals, swine (86), cattle (7), and poultry (1), with well-defined identification was further typed by polymerase chain reaction serotyping, phage typing, and molecular typing (polymerase chain reaction and multilocus variable-number tandem-repeat analysis [MLVA]). Moreover, the determination...

Statistical Validation for Clinical Measures: Repeatability and Agreement of Kinect™-Based Software.

Science.gov (United States)

Lopez, Natalia; Perez, Elisa; Tello, Emanuel; Rodrigo, Alejandro; Valentinuzzi, Max E

2018-01-01

The rehabilitation process is a fundamental stage for recovery of people's capabilities. However, the evaluation of the process is performed by physiatrists and medical doctors, mostly based on their observations, that is, a subjective appreciation of the patient's evolution. This paper proposes a tracking platform of the movement made by an individual's upper limb using Kinect sensor(s) to be applied for the patient during the rehabilitation process. The main contribution is the development of quantifying software and the statistical validation of its performance, repeatability, and clinical use in the rehabilitation process. The software determines joint angles and upper limb trajectories for the construction of a specific rehabilitation protocol and quantifies the treatment evolution. In turn, the information is presented via a graphical interface that allows the recording, storage, and report of the patient's data. For clinical purposes, the software information is statistically validated with three different methodologies, comparing the measures with a goniometer in terms of agreement and repeatability. The agreement of joint angles measured with the proposed software and goniometer is evaluated with Bland-Altman plots; all measurements fell well within the limits of agreement, meaning interchangeability of both techniques. Additionally, the results of Bland-Altman analysis of repeatability show 95% confidence. Finally, the physiotherapists' qualitative assessment shows encouraging results for the clinical use. The main conclusion is that the software is capable of offering a clinical history of the patient and is useful for quantification of the rehabilitation success. The simplicity, low cost, and visualization possibilities enhance the use of the software Kinect for rehabilitation and other applications, and the expert's opinion endorses the choice of our approach for clinical practice. Comparison of the new measurement technique with established

Characterization of Salmonella Typhimurium isolates from domestically acquired infections in Finland by phage typing, antimicrobial susceptibility testing, PFGE and MLVA.

Science.gov (United States)

Lienemann, Taru; Kyyhkynen, Aino; Halkilahti, Jani; Haukka, Kaisa; Siitonen, Anja

2015-07-02

Salmonella enterica spp. enterica serotype Typhimurium (STM) is the most common agent of domestically acquired salmonellosis in Finland. Subtyping methods which allow the characterization of STM are essential for effective laboratory-based STM surveillance and for recognition of outbreaks. This study describes the diversity of Finnish STM isolates using phage typing, antimicrobial susceptible testing, pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA), and compares the discriminatory power and the concordance of these methods. A total of 375 sporadic STM isolates were analysed. The isolates were divided into 31 definite phage (DT) types, dominated by DT1 (47 % of the isolates), U277 (9 % of the isolates) and DT104 (8 % of the isolates). Of all the isolates, 62 % were susceptible to all the 12 antimicrobials tested and 11 % were multidrug resistant. Subtyping resulted in 83 different XbaI-PFGE profiles and 111 MLVA types. The three most common XbaI-PFGE profiles (STYM1, STYM7 and STYM8) and one MLVA profile with three single locus variants accounted for 56 % and 49 % of the STM isolates, respectively. The studied isolates showed a genetic similarity of more than 70 % by XbaI-PFGE. In MLVA, 71 % of the isolates lacked STTR6 and 77 % missed STTR10p loci. Nevertheless, the calculated Simpson's diversity index for XbaI-PFGE was 0.829 (95 % CI 0.792-0.865) and for MLVA 0.867 (95 % CI 0.835-0.898). However, the discriminatory power of the 5-loci MLVA varied among the phage types. The highest concordance of the results was found between XbaI-PFGE and phage typing (adjusted Wallace coefficient was 0.833 and adjusted Rand coefficient was 0.627). In general, the calculated discriminatory power was higher for genotyping methods (MLVA and XbaI-PFGE) than for phenotyping methods (phage typing). Overall, comparable diversity indices were calculated for PFGE and MLVA (both DI > 0.8). However, MLVA was phage type dependent

Test-retest repeatability of strength capacity, aerobic power and pericranial tenderness of neck and shoulder muscles in children - relevant for tension-type headache

Directory of Open Access Journals (Sweden)

Tornøe B

2013-08-01

Full Text Available Birte Tornøe,1,2,5,6 Lars L Andersen,3 Jørgen H Skotte,3 Rigmor Jensen,4 Gunvor Gard,1 Liselotte Skov,2 Inger Hallström1 1Department of Health Sciences, Lund University, Scania, Sweden; 2Children's Headache Clinic, Department of Pediatrics, University of Copenhagen, Herlev Hospital, Herlev, Denmark; 3National Research Centre for the Working Environment, Copenhagen, Denmark; 4Danish Headache Center, Department of Neurology, University of Copenhagen, Glostrup Hospital, Glostrup, Denmark; 5Department of Physiotherapy and Occupational Therapy, University of Copenhagen, Glostrup Hospital, Glostrup, Denmark; 6Department of Physiotherapy, Medical Department, University of Copenhagen, Herlev Hospital, Herlev, Denmark Background: Frequent or chronic tension-type headache in children is a prevalent and debilitating condition for the child, often leading to medication overuse. To explore the relationship between physical factors and tension-type headache in children, the quality of repeated measures was examined. The aim of the present study was to determine the test-retest repeatability of parameters determining isometric neck and shoulder strength and stability, aerobic power, and pericranial tenderness in children. Methods: Twenty-five healthy children, 9 to 18 years of age, participated in test-retest procedures within a 1-week interval. A computerized padded force transducer was used for testing. The tests included the isometric maximal voluntary contraction and force steadiness of neck flexion and extension, and the isometric maximal voluntary contraction and rate of force of the dominant shoulder. Pericranial tenderness was recorded by means of standardized manual palpation, and a submaximal cycle ergometer test predicted maximal oxygen uptake (VO2 max. The measurements were evaluated in steps, using the intraclass correlation coefficient (ICC; changes in the mean between the two test occasions; the levels of agreement, visualized in Bland

[Comparative analysis of clustered regularly interspaced short palindromic repeats (CRISPRs) loci in the genomes of halophilic archaea].

Science.gov (United States)

Zhang, Fan; Zhang, Bing; Xiang, Hua; Hu, Songnian

2009-11-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a widespread system that provides acquired resistance against phages in bacteria and archaea. Here we aim to genome-widely analyze the CRISPR in extreme halophilic archaea, of which the whole genome sequences are available at present time. We used bioinformatics methods including alignment, conservation analysis, GC content and RNA structure prediction to analyze the CRISPR structures of 7 haloarchaeal genomes. We identified the CRISPR structures in 5 halophilic archaea and revealed a conserved palindromic motif in the flanking regions of these CRISPR structures. In addition, we found that the repeat sequences of large CRISPR structures in halophilic archaea were greatly conserved, and two types of predicted RNA secondary structures derived from the repeat sequences were likely determined by the fourth base of the repeat sequence. Our results support the proposal that the leader sequence may function as recognition site by having palindromic structures in flanking regions, and the stem-loop secondary structure formed by repeat sequences may function in mediating the interaction between foreign genetic elements and CAS-encoded proteins.

Characterization of methicillin-resistant Staphylococcus aureus Sequence Type 398

DEFF Research Database (Denmark)

Christiansen, Mette Theilgaard

Staphylococcus aureus is an opportunistic pathogen that colonizes the nares and skin surfaces of several animal species, including man. S. aureus can cause a wide variety of infections ranging from superficial soft tissue and skin infections to severe and deadly systemic infections. Traditionally S....... aureus and methicillin-resistant Staphylococcus aureus (MRSA) have been associated with hospitals, but during the past decades MRSA has emerged in the community and now a new branch of MRSA has been found in association with livestock (LA-MRSA). A specific lineage (multilocus sequence type 398 (ST398...

Repeated morphine treatment influences operant and spatial learning differentially

Institute of Scientific and Technical Information of China (English)

Mei-Na WANG; Zhi-Fang DONG; Jun CAO; Lin XU

2006-01-01

Objective To investigate whether repeated morphine exposure or prolonged withdrawal could influence operant and spatial learning differentially. Methods Animals were chronically treated with morphine or subjected to morphine withdrawal. Then, they were subjected to two kinds of learning: operant conditioning and spatial learning.Results The acquisition of both simple appetitive and cued operant learning was impaired after repeated morphine treatment. Withdrawal for 5 weeks alleviated the impairments. Single morphine exposure disrupted the retrieval of operant memory but had no effect on rats after 5-week withdrawal. Contrarily, neither chronic morphine exposure nor 5-week withdrawal influenced spatial learning task of the Morris water maze. Nevertheless, the retrieval of spatial memory was impaired by repeated morphine exposure but not by 5-week withdrawal. Conclusion These observations suggest that repeated morphine exposure can influence different types of learning at different aspects, implicating that the formation of opiate addiction may usurp memory mechanisms differentially.

Streptococcus suis, an important pig pathogen and emerging zoonotic agent—an update on the worldwide distribution based on serotyping and sequence typing

Science.gov (United States)

Goyette-Desjardins, Guillaume; Auger, Jean-Philippe; Xu, Jianguo; Segura, Mariela; Gottschalk, Marcelo

2014-01-01

Streptococcus suis is an important pathogen causing economic problems in the pig industry. Moreover, it is a zoonotic agent causing severe infections to people in close contact with infected pigs or pork-derived products. Although considered sporadic in the past, human S. suis infections have been reported during the last 45 years, with two large outbreaks recorded in China. In fact, the number of reported human cases has significantly increased in recent years. In this review, we present the worldwide distribution of serotypes and sequence types (STs), as determined by multilocus sequence typing, for pigs (between 2002 and 2013) and humans (between 1968 and 2013). The methods employed for S. suis identification and typing, the current epidemiological knowledge regarding serotypes and STs and the zoonotic potential of S. suis are discussed. Increased awareness of S. suis in both human and veterinary diagnostic laboratories and further establishment of typing methods will contribute to our knowledge of this pathogen, especially in regions where complete and/or recent data is lacking. More research is required to understand differences in virulence that occur among S. suis strains and if these differences can be associated with specific serotypes or STs. PMID:26038745

[Clustered regularly interspaced short palindromic repeats (CRISPR) site in Bacillus anthracis].

Science.gov (United States)

Gao, Zhiqi; Wang, Dongshu; Feng, Erling; Wang, Bingxiang; Hui, Yiming; Han, Shaobo; Jiao, Lei; Liu, Xiankai; Wang, Hengliang

2014-11-04

To investigate the polymorphism of clustered regularly interspaced short palindromic repeats (CRISPR) in Bacillu santhracis and the application to molecular typing based on the polymorphism of CRISPR in B. anthracis. We downloaded the whole genome sequence of 6 B. anthracis strains and extracted the CRISPR sites. We designed the primers of CRISPR sites and amplified the CRISPR fragments in 193 B. anthracis strains by PCR and sequenced these fragments. In order to reveal the polymorphism of CRISPR in B. anthracis, wealigned all the extracted sequences and sequenced results by local blasting. At the same time, we also analyzed the CRISPR sites in B. cereus and B. thuringiensis. We did not find any polymorphism of CRISPR in B. anthracis. The molecular typing approach based on CRISPR polymorphism is not suitable for B. anthracis, but it is possible for us to distinguish B. anthracis from B. cereus and B. thuringiensis.

Crystal structure of the second fibronectin type III (FN3) domain from human collagen α1 type XX.

Science.gov (United States)

Zhao, Jingfeng; Ren, Jixia; Wang, Nan; Cheng, Zhong; Yang, Runmei; Lin, Gen; Guo, Yi; Cai, Dayong; Xie, Yong; Zhao, Xiaohong

2017-12-01

Collagen α1 type XX, which contains fibronectin type III (FN3) repeats involving six FN3 domains (referred to as the FN#1-FN#6 domains), is an unusual member of the fibril-associated collagens with interrupted triple helices (FACIT) subfamily of collagens. The results of standard protein BLAST suggest that the FN3 repeats might contribute to collagen α1 type XX acting as a cytokine receptor. To date, solution NMR structures of the FN#3, FN#4 and FN#6 domains have been determined. To obtain further structural evidence to understand the relationship between the structure and function of the FN3 repeats from collagen α1 type XX, the crystal structure of the FN#2 domain from human collagen α1 type XX (residues Pro386-Pro466; referred to as FN2-HCXX) was solved at 2.5 Å resolution. The crystal structure of FN2-HCXX shows an immunoglobulin-like fold containing a β-sandwich structure, which is formed by a three-stranded β-sheet (β1, β2 and β5) packed onto a four-stranded β-sheet (β3, β4, β6 and β7). Two consensus domains, tencon and fibcon, are structural analogues of FN2-HCXX. Fn8, an FN3 domain from human oncofoetal fibronectin, is the closest structural analogue of FN2-HCXX derived from a naturally occurring sequence. Based solely on the structural similarity of FN2-HCXX to other FN3 domains, the detailed functions of FN2-HCXX and the FN3 repeats in collagen α1 type XX cannot be identified.

Prevalence and molecular typing of Vibrio parahaemolyticus isolated from seafood in Shanghai using multilocus sequence typing (MLST)

Science.gov (United States)

Vibrio parahaemolyticus is a gram-negative bacterium that inhabits coastal and marine environments. Thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and the type III secretion system are considered the potential virulent factors of pathogenic V. parahaemolyticus. The frequency of str...

Fault-tolerant quantum repeater with atomic ensembles and linear optics

International Nuclear Information System (INIS)

Chen Zengbing; Zhao Bo; Chen Yuao; Schmiedmayer, Joerg; Pan Jianwei

2007-01-01

We present a detailed analysis of a robust quantum repeater architecture building on the original Duan-Lukin-Cirac-Zoller (DLCZ) protocol [L.M. Duan et al. Nature (London) 414, 413 (2001)]. The architecture is based on two-photon Hong-Ou-Mandel-type interference which relaxes the long-distance interferometric stability requirements by about seven orders of magnitude, from subwavelength for the single photon interference required by DLCZ to the coherence length of the photons, thereby removing the weakest point in the DLCZ scheme. Our proposal provides an exciting possibility for robust and realistic long-distance quantum communication

DNA-based genetic markers for Rapid Cycling Brassica rapa (Fast Plants type designed for the teaching laboratory.

Directory of Open Access Journals (Sweden)

Eryn E. Slankster

2012-06-01

Full Text Available We have developed DNA-based genetic markers for rapid-cycling Brassica rapa (RCBr, also known as Fast Plants. Although markers for Brassica rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP based markers and 14 variable number tandem repeat (VNTR-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a nongenic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology.

Loss and recovery of Arabidopsis-type telomere repeat sequences 5'-(TTTAGGG)(n)-3' in the evolution of a major radiation of flowering plants.

OpenAIRE

Adams, S. P.; Hartman, T. P.; Lim, K. Y.; Chase, M. W.; Bennett, M. D.; Leitch, I. J.; Leitch, A. R.

2001-01-01

Fluorescent in situ hybridization and Southern blotting were used for showing the predominant absence of the Arabidopsis-type telomere repeat sequence (TRS) 5'-(TTTAGGG)(n)-3' (the 'typical' telomere) in a monocot clade which comprises up to 6300 species within Asparagales. Initially, two apparently disparate genera that lacked the typical telomere were identified. Here, we used the new angiosperm phylogenetic classification for predicting in which other related families such telomeres might ...

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level.

Science.gov (United States)

Ahlstrom, Christina; Barkema, Herman W; Stevenson, Karen; Zadoks, Ruth N; Biek, Roman; Kao, Rowland; Trewby, Hannah; Haupstein, Deb; Kelton, David F; Fecteau, Gilles; Labrecque, Olivia; Keefe, Greg P; McKenna, Shawn L B; De Buck, Jeroen

2015-03-08

Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne's disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates. Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including "bison type" isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1-2 to 239-240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd. The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne's disease control. VNTR typing often failed to

Microevolution and History of the Plague Bacillus, Yersinia pestis

National Research Council Canada - National Science Library

Achtman, Mark

2004-01-01

.... The microevolution of Y. pestis was therefore investigated by three different multilocus molecular methods, targeting genomewide synonymous SNPs, variation in number of tandem repeats, and insertion of IS100 insertion elements...

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

LENUS (Irish Health Repository)

Shore, Anna C

2010-05-01

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

Multilocus Genetic Characterization of Lactobacillus fermentum Isolated from Ready-to-Eat Canned Food.

Science.gov (United States)

Sulaiman, Irshad M; Jacobs, Emily; Simpson, Steven; Kerdahi, Khalil

2017-06-01

The primary mission of the U.S. Food and Drug Administration is to enforce the Food, Drug, and Cosmetic Act and regulate food, drug, and cosmetic products. Thus, this agency monitors the presence of pathogenic microorganisms in these products, including canned foods, as one of the regulatory action criteria and also ensures that these products are safe for human consumption. This study was carried out to investigate the effectiveness of pathogen control and integrity of ready-to-eat canned food containing Black Bean Corn Poblano Salsa. A total of nine unopened and recalled canned glass jars from the same lot were examined initially by conventional microbiologic protocols that involved a two-step enrichment, followed by streaking on selective agar plates, for the presence of gram-positive and gram-negative bacteria. Of the eight subsamples examined for each sample, all subsamples of one of the containers were found positive for the presence of slow-growing rod-shaped, gram-positive, facultative anaerobic bacteria. The recovered isolates were subsequently sequenced at rRNA and gyrB loci. Afterward, multilocus sequence typing (MLST) was performed characterizing 11 additional known MLST loci (clpX, dnaA, dnaK, groEL, murC, murE, pepX, pyrG, recA, rpoB, and uvrC). Analyses of the nucleotide sequences of rRNA, gyrB, and 11 MLST loci confirmed these gram-positive bacteria recovered from canned food to be Lactobacillus fermentum . Thus, the DNA sequencing of housekeeping MLST genes can provide species identification of L. fermentum and can be used in the canned food monitoring program of public health importance.

Reclassification of Borrelia spp. isolated in South Korea using Multilocus Sequence Typing.

Science.gov (United States)

Park, Kyung-Hee; Choi, Yeon-Joo; Kim, Jeoungyeon; Park, Hye-Jin; Song, Dayoung; Jang, Won-Jong

2018-05-31

Using Borrelia isolated from South Korea, we evaluated by MLST and three intergenic genes (16S rRNA, ospA, and 5S-23S IGS) typing to analyze the relationship between host and vector and molecular background. Using the MLST analysis, we identified B. afzelii, B. yangtzensis, B. garinii, and B. bavariensis. This study was first report of the identification of B. yangtzensis using the MLST in South Korea.

A multilocus phylogeny reveals deep lineages within African galagids (Primates: Galagidae)

Science.gov (United States)

2014-01-01

Background Bushbabies (Galagidae) are among the most morphologically cryptic of all primates and their diversity and relationships are some of the most longstanding problems in primatology. Our knowledge of galagid evolutionary history has been limited by a lack of appropriate molecular data and a paucity of fossils. Most phylogenetic studies have produced conflicting results for many clades, and even the relationships among genera remain uncertain. To clarify galagid evolutionary history, we assembled the largest molecular dataset for galagos to date by sequencing 27 independent loci. We inferred phylogenetic relationships using concatenated maximum-likelihood and Bayesian analyses, and also coalescent-based species tree methods to account for gene tree heterogeneity due to incomplete lineage sorting. Results The genus Euoticus was identified as sister taxon to the rest of the galagids and the genus Galagoides was not recovered as monophyletic, suggesting that a new generic name for the Zanzibar complex is required. Despite the amount of genetic data collected in this study, the monophyly of the family Lorisidae remained poorly supported, probably due to the short internode between the Lorisidae/Galagidae split and the origin of the African and Asian lorisid clades. One major result was the relatively old origin for the most recent common ancestor of all living galagids soon after the Eocene-Oligocene boundary. Conclusions Using a multilocus approach, our results suggest an early origin for the crown Galagidae, soon after the Eocene-Oligocene boundary, making Euoticus one of the oldest lineages within extant Primates. This result also implies that one – or possibly more – stem radiations diverged in the Late Eocene and persisted for several million years alongside members of the crown group. PMID:24694188

Repeated detection of European bat lyssavirus type 2 in dead bats found at a single roost site in the UK.

Science.gov (United States)

Banyard, Ashley C; Johnson, N; Voller, K; Hicks, D; Nunez, A; Hartley, M; Fooks, A R

2009-01-01

In August 2007, European bat lyssavirus type 2 (EBLV-2) was isolated from a Daubenton's bat found at Stokesay Castle. In September 2008, another bat from the same vicinity of Stokesay Castle also tested positive for EBLV-2. This is the first occurrence of repeated detection of EBLV-2 from a single site. Here, we report the detection of low levels of viral RNA in various bat organs by qRT-PCR and detection of viral antigen by immunohistochemistry. We also report sequence data from both cases and compare data with those derived from other EBLV-2 isolations in the UK.

Overt attention and context factors: the impact of repeated presentations, image type, and individual motivation.

Directory of Open Access Journals (Sweden)

Kai Kaspar

Full Text Available The present study investigated the dynamic of the attention focus during observation of different categories of complex scenes and simultaneous consideration of individuals' memory and motivational state. We repeatedly presented four types of complex visual scenes in a pseudo-randomized order and recorded eye movements. Subjects were divided into groups according to their motivational disposition in terms of action orientation and individual rating of scene interest.Statistical analysis of eye-tracking data revealed that the attention focus successively became locally expressed by increasing fixation duration; decreasing saccade length, saccade frequency, and single subject's fixation distribution over images; and increasing inter-subject variance of fixation distributions. The validity of these results was supported by verbal reports. This general tendency was weaker for the group of subjects who rated the image set as interesting as compared to the other group. Additionally, effects were partly mediated by subjects' motivational disposition. Finally, we found a generally strong impact of image type on eye movement parameters. We conclude that motivational tendencies linked to personality as well as individual preferences significantly affected viewing behaviour. Hence, it is important and fruitful to consider inter-individual differences on the level of motivation and personality traits within investigations of attention processes. We demonstrate that future studies on memory's impact on overt attention have to deal appropriately with several aspects that had been out of the research focus until now.

Combined use of a new SNP-based assay and multilocus SSR markers to assess genetic diversity of Xylella fastidiosa subsp. pauca infecting citrus and coffee plants.

Science.gov (United States)

Montes-Borrego, Miguel; Lopes, Joao R S; Jiménez-Díaz, Rafael M; Landa, Blanca B

2015-03-01

Two haplotypes of Xylella fastidiosa subsp. pauca (Xfp) that correlated with their host of origin were identified in a collection of 90 isolates infecting citrus and coffee plants in Brazil, based on a single-nucleotide polymorphism in the gyrB sequence. A new single-nucleotide primer extension (SNuPE) protocol was designed for rapid identification of Xfp according to the host source. The protocol proved to be robust for the prediction of the Xfp host source in blind tests using DNA from cultures of the bacterium, infected plants, and insect vectors allowed to feed on Xfp-infected citrus plants. AMOVA and STRUCTURE analyses of microsatellite data separated most Xfp populations on the basis of their host source, indicating that they were genetically distinct. The combined use of the SNaPshot protocol and three previously developed multilocus SSR markers showed that two haplotypes and distinct isolates of Xfp infect citrus and coffee in Brazil and that multiple, genetically different isolates can be present in a single orchard or infect a single tree. This combined approach will be very useful in studies of the epidemiology of Xfp-induced diseases, host specificity of bacterial genotypes, the occurrence of Xfp host jumping, vector feeding habits, etc., in economically important cultivated plants or weed host reservoirs of Xfp in Brazil and elsewhere. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Identification and characterization of short tandem repeats in the Tibetan macaque genome based on resequencing data.

Science.gov (United States)

Liu, San-Xu; Hou, Wei; Zhang, Xue-Yan; Peng, Chang-Jun; Yue, Bi-Song; Fan, Zhen-Xin; Li, Jing

2018-07-18

The Tibetan macaque, which is endemic to China, is currently listed as a Near Endangered primate species by the International Union for Conservation of Nature (IUCN). Short tandem repeats (STRs) refer to repetitive elements of genome sequence that range in length from 1-6 bp. They are found in many organisms and are widely applied in population genetic studies. To clarify the distribution characteristics of genome-wide STRs and understand their variation among Tibetan macaques, we conducted a genome-wide survey of STRs with next-generation sequencing of five macaque samples. A total of 1 077 790 perfect STRs were mined from our assembly, with an N50 of 4 966 bp. Mono-nucleotide repeats were the most abundant, followed by tetra- and di-nucleotide repeats. Analysis of GC content and repeats showed consistent results with other macaques. Furthermore, using STR analysis software (lobSTR), we found that the proportion of base pair deletions in the STRs was greater than that of insertions in the five Tibetan macaque individuals (Pgenome showed good amplification efficiency and could be used to study population genetics in Tibetan macaques. The neighbor-joining tree classified the five macaques into two different branches according to their geographical origin, indicating high genetic differentiation between the Huangshan and Sichuan populations. We elucidated the distribution characteristics of STRs in the Tibetan macaque genome and provided an effective method for screening polymorphic STRs. Our results also lay a foundation for future genetic variation studies of macaques.

AgdbNet – antigen sequence database software for bacterial typing

Directory of Open Access Journals (Sweden)

Maiden Martin CJ

2006-06-01

Full Text Available Abstract Background Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. Results Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. Conclusion The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated.

A vaccine of L2 epitope repeats fused with a modified IgG1 Fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types.

Science.gov (United States)

Chen, Xue; Liu, Hongyang; Zhang, Ting; Liu, Yanchun; Xie, Xixiu; Wang, Zhirong; Xu, Xuemei

2014-01-01

Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17-36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development.

Role of memory errors in quantum repeaters

International Nuclear Information System (INIS)

Hartmann, L.; Kraus, B.; Briegel, H.-J.; Duer, W.

2007-01-01

We investigate the influence of memory errors in the quantum repeater scheme for long-range quantum communication. We show that the communication distance is limited in standard operation mode due to memory errors resulting from unavoidable waiting times for classical signals. We show how to overcome these limitations by (i) improving local memory and (ii) introducing two operational modes of the quantum repeater. In both operational modes, the repeater is run blindly, i.e., without waiting for classical signals to arrive. In the first scheme, entanglement purification protocols based on one-way classical communication are used allowing to communicate over arbitrary distances. However, the error thresholds for noise in local control operations are very stringent. The second scheme makes use of entanglement purification protocols with two-way classical communication and inherits the favorable error thresholds of the repeater run in standard mode. One can increase the possible communication distance by an order of magnitude with reasonable overhead in physical resources. We outline the architecture of a quantum repeater that can possibly ensure intercontinental quantum communication

Combined effects of repeated oral hygiene motivation and type of toothbrush on orthodontic patients: a blind randomized clinical trial.

Science.gov (United States)

Marini, Ida; Bortolotti, Francesco; Parenti, Serena Incerti; Gatto, Maria Rosaria; Bonetti, Giulio Alessandri

2014-09-01

To investigate the effects on plaque index (PI) scores of manual or electric toothbrush with or without repeated oral hygiene instructions (OHI) and motivation on patients wearing fixed orthodontic appliances. One month after the orthodontic fixed appliance bonding on both arches, 60 patients were randomly assigned to four groups; groups E1 (n  =  15) and E2 (n  =  15) received a powered rotating-oscillating toothbrush, and groups M1 (n  =  15) and M2 (n  =  15) received a manual toothbrush. Groups E1 and M1 received OHI and motivation at baseline (T0) and after 4, 8, 12, 16, and 20 weeks (T4, T8, T12, T16, and T20, respectively) by a Registered Dental Hygienist; groups E2 and M2 received OHI and motivation only at baseline. At each time point a blinded examiner scored plaque of all teeth using the modified Quigley-Hein PI. In all groups the PI score decreased significantly over time, and there were differences among groups at T8, T12, T16, and T20. At T8, PI scores of group E1 were lower than those of group E2, and at T12, T16, and T20, PI scores of groups M1 and E1 were lower compared to those of groups M2 and E2. A linear mixed model showed that the effect of repeated OHI and motivation during time was statistically significant, independently from the use of manual or electric toothbrush. The present results showed that repeated OHI and motivation are crucial in reducing PI score in orthodontic patients, independent of the type of toothbrush used.

TRStalker: an efficient heuristic for finding fuzzy tandem repeats.

Science.gov (United States)

Pellegrini, Marco; Renda, M Elena; Vecchio, Alessio

2010-06-15

Genomes in higher eukaryotic organisms contain a substantial amount of repeated sequences. Tandem Repeats (TRs) constitute a large class of repetitive sequences that are originated via phenomena such as replication slippage and are characterized by close spatial contiguity. They play an important role in several molecular regulatory mechanisms, and also in several diseases (e.g. in the group of trinucleotide repeat disorders). While for TRs with a low or medium level of divergence the current methods are rather effective, the problem of detecting TRs with higher divergence (fuzzy TRs) is still open. The detection of fuzzy TRs is propaedeutic to enriching our view of their role in regulatory mechanisms and diseases. Fuzzy TRs are also important as tools to shed light on the evolutionary history of the genome, where higher divergence correlates with more remote duplication events. We have developed an algorithm (christened TRStalker) with the aim of detecting efficiently TRs that are hard to detect because of their inherent fuzziness, due to high levels of base substitutions, insertions and deletions. To attain this goal, we developed heuristics to solve a Steiner version of the problem for which the fuzziness is measured with respect to a motif string not necessarily present in the input string. This problem is akin to the 'generalized median string' that is known to be an NP-hard problem. Experiments with both synthetic and biological sequences demonstrate that our method performs better than current state of the art for fuzzy TRs and that the fuzzy TRs of the type we detect are indeed present in important biological sequences. TRStalker will be integrated in the web-based TRs Discovery Service (TReaDS) at bioalgo.iit.cnr.it. Supplementary data are available at Bioinformatics online.

DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling

Directory of Open Access Journals (Sweden)

Pauline Ogrodzki

2017-09-01

Full Text Available The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST, capsular profiling of the K-antigen and colanic acid (CA biosynthesis regions, and CRISPR-cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC 1 and 4, sequence types (ST 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis, C. dublinensis and C. universalis. The majority of CRISPR-cas types across the genus was the I-E (Ecoli type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors.

DNA-Sequence Based Typing of the Cronobacter Genus Using MLST, CRISPR-cas Array and Capsular Profiling.

Science.gov (United States)

Ogrodzki, Pauline; Forsythe, Stephen J

2017-01-01

The Cronobacter genus is composed of seven species, within which a number of pathovars have been described. The most notable infections by Cronobacter spp. are of infants through the consumption of contaminated infant formula. The description of the genus has greatly improved in recent years through DNA sequencing techniques, and this has led to a robust means of identification. However some species are highly clonal and this limits the ability to discriminate between unrelated strains by some methods of genotyping. This article updates the application of three genotyping methods across the Cronobacter genus. The three genotyping methods were multilocus sequence typing (MLST), capsular profiling of the K -antigen and colanic acid (CA) biosynthesis regions, and CRISPR- cas array profiling. A total of 1654 MLST profiled and 286 whole genome sequenced strains, available by open access at the PubMLST Cronobacter database, were used this analysis. The predominance of C. sakazakii and C. malonaticus in clinical infections was confirmed. The majority of clinical strains being in the C. sakazakii clonal complexes (CC) 1 and 4, sequence types (ST) 8 and 12 and C. malonaticus ST7. The capsular profile K2:CA2, previously proposed as being strongly associated with C. sakazakii and C. malonaticus isolates from severe neonatal infections, was also found in C. turicensis , C. dublinensis and C. universalis . The majority of CRISPR- cas types across the genus was the I-E (Ecoli) type. Some strains of C. dublinensis and C. muytjensii encoded the I-F (Ypseudo) type, and others lacked the cas gene loci. The significance of the expanding profiling will be of benefit to researchers as well as governmental and industrial risk assessors.

A Method against Interrupted-Sampling Repeater Jamming Based on Energy Function Detection and Band-Pass Filtering

Directory of Open Access Journals (Sweden)

Hui Yuan

2017-01-01

Full Text Available Interrupted-sampling repeater jamming (ISRJ is a new kind of coherent jamming to the large time-bandwidth linear frequency modulation (LFM signal. Many jamming modes, such as lifelike multiple false targets and dense false targets, can be made through setting up different parameters. According to the “storage-repeater-storage-repeater” characteristics of the ISRJ and the differences in the time-frequency-energy domain between the ISRJ signal and the target echo signal, one new method based on the energy function detection and band-pass filtering is proposed to suppress the ISRJ. The methods mainly consist of two parts: extracting the signal segments without ISRJ and constructing band-pass filtering function with low sidelobe. The simulation results show that the method is effective in the ISRJ with different parameters.

Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers.

Science.gov (United States)

Gao, Chunsheng; Xin, Pengfei; Cheng, Chaohua; Tang, Qing; Chen, Ping; Wang, Changbiao; Zang, Gonggu; Zhao, Lining

2014-01-01

Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of expressed sequence tag simple sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.

Selection pressure on human STR loci and its relevance in repeat expansion disease

KAUST Repository

Shimada, Makoto K.; Sanbonmatsu, Ryoko; Yamaguchi-Kabata, Yumi; Yamasaki, Chisato; Suzuki, Yoshiyuki; Chakraborty, Ranajit; Gojobori, Takashi; Imanishi, Tadashi

2016-01-01

Short Tandem Repeats (STRs) comprise repeats of one to several base pairs. Because of the high mutability due to strand slippage during DNA synthesis, rapid evolutionary change in the number of repeating units directly shapes the range of repeat

Repeating and non-repeating fast radio bursts from binary neutron star mergers

Science.gov (United States)

Yamasaki, Shotaro; Totani, Tomonori; Kiuchi, Kenta

2018-04-01

Most fast radio bursts (FRB) do not show evidence of repetition, and such non-repeating FRBs may be produced at the time of a merger of binary neutron stars (BNS), provided that the BNS merger rate is close to the high end of the currently possible range. However, the merger environment is polluted by dynamical ejecta, which may prohibit the radio signal from propagating. We examine this by using a general-relativistic simulation of a BNS merger, and show that the ejecta appears about 1 ms after the rotation speed of the merged star becomes the maximum. Therefore there is a time window in which an FRB signal can reach outside, and the short duration of non-repeating FRBs can be explained by screening after ejecta formation. A fraction of BNS mergers may leave a rapidly rotating and stable neutron star, and such objects may be the origin of repeating FRBs like FRB 121102. We show that a merger remnant would appear as a repeating FRB on a time scale of ˜1-10 yr, and expected properties are consistent with the observations of FRB 121102. We construct an FRB rate evolution model that includes these two populations of repeating and non-repeating FRBs from BNS mergers, and show that the detection rate of repeating FRBs relative to non-repeating ones rapidly increases with improving search sensitivity. This may explain why only the repeating FRB 121102 was discovered by the most sensitive FRB search with Arecibo. Several predictions are made, including the appearance of a repeating FRB 1-10 yr after a BNS merger that is localized by gravitational waves and subsequent electromagnetic radiation.

CRISPR-cas loci profiling of Cronobacter sakazakii pathovars.

Science.gov (United States)

Ogrodzki, Pauline; Forsythe, Stephen James

2016-12-01

Cronobacter sakazakii sequence types 1, 4, 8 and 12 are associated with outbreaks of neonatal meningitis and necrotizing enterocolitis infections. However clonality results in strains which are indistinguishable using conventional methods. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)-cas loci profiling for epidemiological investigations. Seventy whole genomes of C. sakazakii strains from four clonal complexes which were widely distributed temporally, geographically and origin of source were profiled. All strains encoded the same type I-E subtype CRISPR-cas system with a total of 12 different CRISPR spacer arrays. This study demonstrated the greater discriminatory power of CRISPR spacer array profiling compared with multilocus sequence typing, which will be of use in source attribution during Cronobacter outbreak investigations.

Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples.

Directory of Open Access Journals (Sweden)

Yibing Wang

2011-02-01

Full Text Available Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV and the genitourinary genotypes D-K. Although these genotypes can be discriminated by outer membrane protein gene (ompA sequencing or multi-locus sequence typing (MLST, neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.We evaluated variable number tandem repeat (VNTR and ompA sequencing (now called multi-locus VNTR analysis and ompA or "MLVA-ompA" to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43% were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59% were fully genotyped by MLVA-ompA. Only one mixed infection (E & D in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives.

Science.gov (United States)

Brun, Sophie; Madrid, Hugo; Gerrits Van Den Ende, Bert; Andersen, Birgitte; Marinach-Patrice, Carine; Mazier, Dominique; De Hoog, G Sybren

2013-01-01

The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens. Copyright © 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Multilocus enzyme electrophoresis on agarose gel as an aid to the identification of entomopathogenic Bacillus sphaericus strains.

Science.gov (United States)

Zahner, V; Rabinovitch, L; Cavados, C F; Momen, H

1994-04-01

Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types). Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype. Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40). The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C. 3.4.13.9) were polymorphic. The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group. This latter group appears to be distinct from other strains of these species. All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

Towards accurate de novo assembly for genomes with repeats

NARCIS (Netherlands)

Bucur, Doina

2017-01-01

De novo genome assemblers designed for short k-mer length or using short raw reads are unlikely to recover complex features of the underlying genome, such as repeats hundreds of bases long. We implement a stochastic machine-learning method which obtains accurate assemblies with repeats and

In vivo activation of human immunodeficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice

OpenAIRE

Morrey, John D; Bourn, S M; Bunch, T D; Jackson, M K; Sidwell, R W; Barrows, L R; Daynes, R A; Rosen, C A

1991-01-01

UV irradiation has been shown to activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in cell culture; however, only limited studies have been described in vivo. UV light has been categorized as UV-A (400 to 315 nm), -B (315 to 280 nm), or -C (less than 280 nm); the longer wavelengths are less harmful but more penetrative. Highly penetrative UV-A radiation constitutes the vast majority of UV sunlight reaching the earth's surface but is normally harmless. UV-B ir...

Alu repeats as markers for human population genetics

Energy Technology Data Exchange (ETDEWEB)

Batzer, M.A.; Alegria-Hartman, M. [Lawrence Livermore National Lab., CA (United States); Bazan, H. [Louisiana State Univ., New Orleans, LA (United States). Medical Center] [and others

1993-09-01

The Human-Specific (HS) subfamily of Alu sequences is comprised of a group of 500 nearly identical members which are almost exclusively restricted to the human genome. Individual subfamily members share an average of 97.9% nucleotide identity with each other and an average of 98.9% nucleotide identity with the HS subfamily consensus sequence. HS Alu family members are thought to be derived from a single source ``master`` gene, and have an average age of 2.8 million years. We have developed a Polymerase Chain Reaction (PCR) based assay using primers complementary to the 5 in. and 3 in. unique flanking DNA sequences from each HS Alu that allows the locus to be assayed for the presence or absence of an Alu repeat. Individual HS Alu sequences were found to be either monomorphic or dimorphic for the presence or absence of each repeat. The monomorphic HS Alu family members inserted in the human genome after the human/great ape divergence (which is thought to have occurred 4--6 million years ago), but before the radiation of modem man. The dimorphic HS Alu sequences inserted in the human genome after the radiation of modem man (within the last 200,000-one million years) and represent a unique source of information for human population genetics and forensic DNA analyses. These sites can be developed into Dimorphic Alu Sequence Tagged Sites (DASTS) for the Human Genome Project as well. HS Alu family member insertion dimorphism differs from other types of polymorphism (e.g. Variable Number of Tandem Repeat [VNTR] or Restriction Fragment Length Polymorphism [RFLP]) because individuals share HS Alu family member insertions based upon identity by descent from a common ancestor as a result of a single event which occurred one time within the human population. The VNTR and RFLP polymorphisms may arise multiple times within a population and are identical by state only.

Use of portable exposure meters for comparing mobile phone base station radiation in different types of areas in the cities of Basel and Amsterdam.

Science.gov (United States)

Urbinello, Damiano; Huss, Anke; Beekhuizen, Johan; Vermeulen, Roel; Röösli, Martin

2014-01-15

Radiofrequency electromagnetic fields (RF-EMF) are highly variable and differ considerably within as well as between areas. Exposure assessment studies characterizing spatial and temporal variation are limited so far. Our objective was to evaluate sources of data variability and the repeatability of daily measurements using portable exposure meters (PEMs). Data were collected at 12 days between November 2010 and January 2011 with PEMs in four different types of urban areas in the cities of Basel (BSL) and Amsterdam (AMS). Exposure from mobile phone base stations ranged from 0.30 to 0.53 V/m in downtown and business areas and in residential areas from 0.09 to 0.41 V/m. Analysis of variance (ANOVA) demonstrated that measurements from various days were highly reproducible (measurement duration of approximately 30 min) with only 0.6% of the variance of all measurements from mobile phone base station radiation being explained by the measurement day and only 0.2% by the measurement time (morning, noon, afternoon), whereas type of area (30%) and city (50%) explained most of the data variability. We conclude that mobile monitoring of exposure from mobile phone base station radiation with PEMs is useful due to the high repeatability of mobile phone base station exposure levels, despite the high spatial variation. © 2013.

REPEATABILITY OF FRUIT QUALITY TRAITS OF CACTUS PEAR

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VALTÂNIA XAVIER NUNES

2017-12-01

Full Text Available ABSTRACT Repeatability analysis has been used to study traits in several crops, assisting in the definition of the minimum number needed to evaluate genotypes more efficiently and with less time and resource consumption. So far, however, no repeatability studies on cactus pear have been found in the literature. The objective of this study was to determine the coefficient of repeatability for cactus pear fruits traits and the minimum number of evaluations (fruit that can provide acceptable accuracy for the prediction of the true value. The experiment was conducted at the Federal Institute of Bahia/Campus Guanambi, with 150 fruits collected from three municipalities in the state of Bahia. The coefficients of repeatability were estimated by the methods of analysis of variance, principal components based on the covariance (PCCV and correlation (PCC matrices, and structural analysis based on the correlation matrix (SA. The analysis of variance showed that, except for fruit diameter, the effect of the production site (municipality was significant for all traits evaluated. The PCCV method was proven the most suitable for studying the repeatability of quality traits of cactus pear fruits. Seven fruits were required to determine, with 90% confidence, the traits length, diameter, fruit firmness, skin thickness, number of seeds, fruit mass, bark mass, pulp mass, pH, titratable acidity, soluble solids, SS/AT ratio, and pulp yield.

Selection of body sway parameters according to their sensitivity and repeatability

Directory of Open Access Journals (Sweden)

Nejc Sarabon

2010-03-01

Full Text Available For the precise evaluation of body balance, static type of tests performed on a force plate are the most commonly used ones. In these tests, body sway characteristics are analyzed based on the model of inverted pendulum and looking at the center of pressure (COP movement in time. Human body engages different strategies to compensate for balance perturbations. For this reason, there is a need to identify parameters which are sensitive to specific balance changes and which enable us to identify balance sub-components. The aim of our study was to investigate intra-visit repeatability and sensibility of the 40 different body sway parameters. Twenty-nine subjects participated in the study. They performed three different balancing tasks of different levels of difficulty, three repetitions each. The hip-width parallel stance and the single leg stance, both with open eyes, were used as ways to compare different balance intensities due to biomechanical changes. Additionally, deprivation of vision was used in the third balance task to study sensitivity to sensory system changes. As shown by intraclass correlation coefficient (ICC, repeatability of cumulative parameters such as COP, maximal amplitude and frequency showed excellent repeatability (ICC>0,85. Other parameters describing sub-dynamics through single repetition proved to have unsatisfying repeatability. Parameters most sensitive to increased intensity of balancing tasks were common COP, COP in medio-lateral and in antero-posterior direction, and maximal amplitues in the same directions. Frequency of oscilations has proved to be sensitive only to deprivation of vision. As shown in our study, cumulative parameters describing the path which the center of pressure makes proved to be the most repeatable and sensitive to detect different increases of balancing tasks enabling future use in balance studies and in clinical practice.

Reduction and technical simplification of testing protocol for walking based on repeatability analyses: An Interreg IVa pilot study

Directory of Open Access Journals (Sweden)

Nejc Sarabon

2010-12-01

Full Text Available The aim of this study was to define the most appropriate gait measurement protocols to be used in our future studies in the Mobility in Ageing project. A group of young healthy volunteers took part in the study. Each subject carried out a 10-metre walking test at five different speeds (preferred, very slow, very fast, slow, and fast. Each walking speed was repeated three times, making a total of 15 trials which were carried out in a random order. Each trial was simultaneously analysed by three observers using three different technical approaches: a stop watch, photo cells and electronic kinematic dress. In analysing the repeatability of the trials, the results showed that of the five self-selected walking speeds, three of them (preferred, very fast, and very slow had a significantly higher repeatability of the average walking velocity, step length and cadence than the other two speeds. Additionally, the data showed that one of the three technical methods for gait assessment has better metric characteristics than the other two. In conclusion, based on repeatability, technical and organizational simplification, this study helped us to successfully define a simple and reliable walking test to be used in the main study of the project.

Multilocus Sequence Typing (MLST) and Phylogenetic Analysis of Propionibacterium acnes

DEFF Research Database (Denmark)

Kilian, Mogens; Scholz, Christian; Lomholt, Hans B

2011-01-01

Propionibacterium acnes is a commensal of human skin but is also implicated in the pathogenesis of acne vulgaris and in biofilm-associated infections of medical devices and endophthalmitis, and in infections of bone and dental root canals. Recent studies associate P. acnes with prostate cancer...... with reference to a phylogenetic tree based on 78 P. acnes genomes and their gene contents. Further support for a basically clonal population structure of P. acnes and a scenario of global spread of epidemic clones of P. acnes was obtained. Compared with the Belfast scheme, the Aarhus MLST scheme (http...

Addictive behaviors and addiction-prone personality traits: associations with a dopamine multilocus genetic profile.

Science.gov (United States)

Davis, Caroline; Loxton, Natalie J

2013-07-01

The purpose of this study was to examine reward-related genetic risk for addictive behaviors in a healthy community sample (n=217) of men and women. We tested a mediation model predicting that a quantitative multilocus genetic profile score - reflecting the additive effects of alleles known to confer relatively increased dopamine signaling in the ventral striatum - would relate positively to a composite measure of addictive behaviors, and that this association would be mediated by personality traits consistently associated with addiction disorders. Our model was strongly supported by the data, and accounted for 24% of the variance in addictive behaviors. These data suggest that brain reward processes tend to exert their influence on addiction risk by their role in the development of relatively stable personality traits associated with addictive behaviors. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular epidemiology and multilocus sequence analysis of potentially zoonotic Giardia spp. from humans and dogs in Jamaica.

Science.gov (United States)

Lee, Mellesia F; Cadogan, Paul; Eytle, Sarah; Copeland, Sonia; Walochnik, Julia; Lindo, John F

2017-01-01

Giardia spp. are the causative agents of intestinal infections in a wide variety of mammals including humans and companion animals. Dogs may be reservoirs of zoonotic Giardia spp.; however, the potential for transmission between dogs and humans in Jamaica has not been studied. Conventional PCR was used to screen 285 human and 225 dog stool samples for Giardia targeting the SSU rDNA gene followed by multilocus sequencing of the triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), and β-giardin (bg) genes. Prevalence of human infections based on PCR was 6.7 % (19/285) and canine infections 19.6 % (44/225). Nested PCR conducted on all 63 positive samples revealed the exclusive presence of assemblage A in both humans and dogs. Sub-assemblage A-II was responsible for 79.0 % (15/19) and 70.5 % (31/44) of the infections in humans and dogs, respectively, while sub-assemblage A-I was identified at a rate of 15.8 % (3/19) and 29.5 % (13/44) in humans and dogs, respectively. The predominance of a single circulating assemblage among both humans and dogs in Jamaica suggests possible zoonotic transmission of Giardia infections.

Clonal spread of blaOXA-72-carrying Acinetobacter baumannii sequence type 512 in Taiwan.

Science.gov (United States)

Kuo, Han-Yueh; Hsu, Po-Jui; Chen, Jiann-Yuan; Liao, Po-Cheng; Lu, Chia-Wei; Chen, Chang-Hua; Liou, Ming-Li

2016-07-01

This is the first report to show an insidious outbreak of armA- and blaOXA-72-carrying Acinetobacter baumannii sequence type 512 (ST512) at a study hospital in northern Taiwan. Multilocus sequence typing revealed that this was a ST512 clone. All of the isolates with ST512 carried a novel 12,056-bp repGR2 in combination with a repGR12-type plasmid. This plasmid, designated pAB-ML, had one copy of the blaOXA-72 gene that was flanked by XerC/XerD-like sites and conferred resistance to carbapenems. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

SCCmecFinder, a Web-Based Tool for Typing of Staphylococcal Cassette Chromosome mec in Staphylococcus aureus Using Whole-Genome Sequence Data.

Science.gov (United States)

Kaya, Hülya; Hasman, Henrik; Larsen, Jesper; Stegger, Marc; Johannesen, Thor Bech; Allesøe, Rosa Lundbye; Lemvigh, Camilla Koldbæk; Aarestrup, Frank Møller; Lund, Ole; Larsen, Anders Rhod

2018-01-01

Typing of methicillin-resistant Staphylococcus aureus (MRSA) is important in infection control and surveillance. The current nomenclature of MRSA includes the genetic background of the S. aureus strain determined by multilocus sequence typing (MLST) or equivalent methods like spa typing and typing of the mobile genetic element staphylococcal cassette chromosome mec (SCC mec ), which carries the mecA or mecC gene. Whereas MLST and spa typing are relatively simple, typing of SCC mec is less trivial because of its heterogeneity. Whole-genome sequencing (WGS) provides the essential data for typing of the genetic background and SCC mec , but so far, no bioinformatic tools for SCC mec typing have been available. Here, we report the development and evaluation of SCC mec Finder for characterization of the SCC mec element from S. aureus WGS data. SCC mec Finder is able to identify all SCC mec element types, designated I to XIII, with subtyping of SCC mec types IV (2B) and V (5C2). SCC mec elements are characterized by two different gene prediction approaches to achieve correct annotation, a Basic Local Alignment Search Tool (BLAST)-based approach and a k -mer-based approach. Evaluation of SCC mec Finder by using a diverse collection of clinical isolates ( n = 93) showed a high typeability level of 96.7%, which increased to 98.9% upon modification of the default settings. In conclusion, SCC mec Finder can be an alternative to more laborious SCC mec typing methods and is freely available at https://cge.cbs.dtu.dk/services/SCCmecFinder. IMPORTANCE SCC mec in MRSA is acknowledged to be of importance not only because it contains the mecA or mecC gene but also for staphylococcal adaptation to different environments, e.g., in hospitals, the community, and livestock. Typing of SCC mec by PCR techniques has, because of its heterogeneity, been challenging, and whole-genome sequencing has only partially solved this since no good bioinformatic tools have been available. In this

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

Directory of Open Access Journals (Sweden)

Macedo Andrea M

2001-01-01

Full Text Available Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

Preventing Repeat Teen Births PSA (:60)

Centers for Disease Control (CDC) Podcasts

This 60 second public service announcement is based on the April 2013 CDC Vital Signs report, which discusses repeat teen births and ways teens, parents and guardians, health care providers, and communities can help prevent them.

Multilocus phylogenetic reconstruction of the Clavariaceae (Agaricales) reveals polyphyly of agaricoid members.

Science.gov (United States)

Birkebak, Joshua M; Adamčík, Slavomír; Looney, Brian P; Matheny, P Brandon

2016-09-01

The genus Camarophyllopsis contains species with lamellate (agaricoid) basidiomes in the family Clavariaceae (Agaricales), a group otherwise dominated by club-like (clavarioid) or branched (coralloid) forms. Previous studies have suggested that species classified in Camarophyllopsis occur in two independent lineages. We reconstructed a multilocus phylogeny of the Clavaria-Camarophyllopsis-Clavicorona clade in the Clavariaceae using RNA polymerase II second largest subunit (rpb2), nuclear ribosomal 28S, and nuclear ribosomal ITS1-5.8S-ITS2 regions data and detected three independent groups of agaricoid fungi, including the genera Camarophyllopsis, Hodophilus, and Lamelloclavaria gen. nov, which distinctly differ in their pileipellis structure. In all, nine major lineages within the Clavaria-Camarophyllopsis-Clavicorona clade were recovered: Clavaria sensu stricto, Camarophyllopsis sensu stricto, Hodophilus, the Clavaria pullei clade, the Clavaria fumosa clade, Lamelloclavaria gen. nov., the Clavaria atrofusca clade, Holocoryne (= Clavaria sect. Holocoryne), and Clavicorona Clavaria is paraphyletic and represented by five clades. Additional gene sampling is necessary to determine and confirm relatedness of these lineages before splitting Clavaria into additional genera. © 2016 by The Mycological Society of America.

Clonal structure in Ichthyobacterium seriolicida, the causative agent of bacterial haemolytic jaundice in yellowtail, Seriola quinqueradiata, inferred from molecular epidemiological analysis.

Science.gov (United States)

Matsuyama, T; Fukuda, Y; Sakai, T; Tanimoto, N; Nakanishi, M; Nakamura, Y; Takano, T; Nakayasu, C

2017-08-01

Bacterial haemolytic jaundice caused by Ichthyobacterium seriolicida has been responsible for mortality in farmed yellowtail, Seriola quinqueradiata, in western Japan since the 1980s. In this study, polymorphic analysis of I. seriolicida was performed using three molecular methods: amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST) and multiple-locus variable-number tandem repeat analysis (MLVA). Twenty-eight isolates were analysed using AFLP, while 31 isolates were examined by MLST and MLVA. No polymorphisms were identified by AFLP analysis using EcoRI and MseI, or by MLST of internal fragments of eight housekeeping genes. However, MLVA revealed variation in repeat numbers of three elements, allowing separation of the isolates into 16 sequence types. The unweighted pair group method using arithmetic averages cluster analysis of the MLVA data identified four major clusters, and all isolates belonged to clonal complexes. It is likely that I. seriolicida populations share a common ancestor, which may be a recently introduced strain. © 2016 John Wiley & Sons Ltd.

Typing Method for the QUB11a Locus of Mycobacterium tuberculosis: IS6110 Insertions and Tandem Repeat Analysis

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Eriko Maeda-Mitani

2016-01-01

Full Text Available QUB11a is used as a locus for variable number of tandem repeats (VNTR analysis of Mycobacterium tuberculosis Beijing lineage. However, amplification of QUB11a occasionally produces large fragments (>1,400 bp that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110 insertion may complicate VNTR analysis of large QUB11a fragments in M. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110 insertion in the QUB11a locus of M. tuberculosis using capillary electrophoresis analysis and BsmBI digestion. All 29 large QUB11a fragments (>1,200 bp investigated contained IS6110 insertions and varied in the number of repeats (18 patterns and location of IS6110 insertions. This method allows VNTR analysis with high discrimination.