北京地区实验动物绿脓杆菌分离株MLVA分型

目的通过多位点可变数目串联重复序列分析（multiple-locus variable-number tandem-repeat analysis,MLVA）分型方法,研究北京地区实验动物绿脓杆菌分离株基因型和分布情况。方法选择13个可变数目重复序列（variable-number tandem-repeat,VNTR）位点,对实验动物及设施中检测出的141株绿脓杆菌的基因组DNA进行重复序列扩增,所得指纹图谱使用BioNumerics软件进行聚类分析,绘制系统发育树和最小生成树（minimum spanning tree,MST）。结果所采用的13个VNTR位点能够对全部分离株进行有效分型。141株绿脓杆菌主要被分为了3个基因群,56个基因型。各群所占比例分别为A群82.3%,B群占12.8%,C群占5.0%,辛普森多样性指数为0.763。同一区域内相邻实验动物单位的绿脓杆菌分离株同源关系较远。结论 MLVA方法对绿脓杆菌具有很好的分型能力,能够有效的追踪绿脓杆菌的来源。北京地区实验动物中绿脓杆菌分离株基因型多态性丰富,但无地域性同源关系。     

Evaluation of Brucella MLVA typing for human brucellosis

Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA-DNA homology >90%). The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis.     

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing

AIM: Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. METHODS AND RESULTS: Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. CONCLUSIONS: Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.     

Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay

The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson's index of diversity of D=0.98.     

A new typing technique for the Rickettsiales Ehrlichia ruminantium: multiple-locus variable number tandem repeat analysis

Ehrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis).Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19 years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains.The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies.     

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii

The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.     

Genetic diversity of Francisella tularensis subspecies holarctica strains isolated in Japan

The recently developed MLVA has high discriminatory power for the typing of individual strains or isolates of Francisella tularensis. In the present study, MLVA was applied to 33 Japanese F. tularensis subspecies holarctica strains to examine the genetic diversity of F. tularensis isolated. Among the seven VNTR loci analyzed, Ft-M2, Ft-M10, and Ft-M20 loci showed high genetic polymorphism in Japanese strains, whereas Ft-M3 was most variable in non-Japanese strains. These results provide novel extended information about the genomic diversity among the strains of F. tularensis ssp. holarctica distributed in Japan and enable determination of whether a given isolate is indigenous to Japan by examining these loci using MLVA.     

Population Structure of Bartonella henselae in Algerian Urban Stray Cats

Whole blood samples from 211 stray cats from Algiers, Algeria, were cultured to detect the presence of Bartonella species and to evaluate the genetic diversity of B. henselae strains by multiple locus VNTR analysis (MLVA). Bartonella henselae was the only species isolated from 36 (17%) of 211 cats. B. henselae genotype I was the predominant genotype (64%). MLVA typing of 259 strains from 30 bacteremic cats revealed 52 different profiles as compared to only 3 profiles using MLST. Of these 52 profiles, 48 (92.3%) were identified for the first time. One-third of the cats harbored one MLVA profile only. As there was a correlation between the age of cats and the number of MLVA profiles, we hypothesized that the single profile in these cats was the profile of the initial infecting strain. Two-third of the cats harbored 2 to 6 MLVA profiles simultaneously. The similarity of MLVA profiles obtained from the same cat, neighbor-joining clustering and structure-neighbor clustering indicate that such a diversity likely results from two different mechanisms occurring either independently or simultaneously: independent infections and genetic drift from a primary strain.     

Multilocus variable number tandem repeat analysis as a tool to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A

Aims:  To identify variable number tandem repeat (VNTR)-containing loci, and to use multilocus VNTR (MLVA) to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A isolated from diverse sources.
Methods and Results:  The whole genome sequence of Y. enterocolitica 8081 was analysed and eight VNTR loci with repeat sizes between 4 and 9 bp, and each containing more than four repeat copies were selected for MLVA typing of 88 strains of Y. enterocolitica . Of these, four loci were polymorphic and generated 26 MLVA genotypes among 81 strains of Y. enterocolitica biovar 1A. MLVA was found to be quite discriminatory (DI = 0·87). Cluster analysis and population modelling using minimum spanning tree (MST) clearly clustered Y. enterocolitica biovar 1A into two major groups.
Conclusions:  The MLVA is easy to perform and can be used to discern clonal relationship among strains of Y. enterocolitica . Also the phylogenetic relationships obtained with MLVA genotypes were in good agreement with those established by other typing methods.
Significance and Impact of the Study:  The MLVA method reported is relatively more discriminatory than the other genotyping methods and has the potential to be used as an epidemiological tool for the study of strains of Y. enterocolitica biovar 1A.     

Genotypic characterization of hospital Enterococcus faecalis strains using multiple-locus variable-number tandem-repeat analysis

Aims:  The level of genetic diversity and relationships between the specific genotypes and the distribution of virulence determinants among Enterococcus faecalis strains isolated from patients hospitalized in different wards of two hospitals were investigated.
Methods and Results:  Fifty-six clinical strains of E. faecalis , isolated from patients hospitalized in the period of 1999–2004 in several wards in Wrocław (Poland), were analysed by multiple-locus variable-number tandem-repeat analysis (MLVA). Analysis of seven genomic loci identified 40 novel genotypes among the analysed E. faecalis strains, with two major genomic groups, designated I and II, distinguished at a cut-off of 35%. With a similarity cut-off of 85·7%, the genotypes could be combined into 12 clusters (C1–C12), containing at least two isolates. The remaining 18 MLVA types were represented by a single isolate.
Conclusions:  Based on the data obtained by MLVA, it was found that (i) many E. faecalis isolates recovered from patients from the wards whose location allowed the potential transmission of micro-organisms, belonged to closely related MLVA types and (ii) possible relationships between specific E. faecalis genotype and the virulence factors lipase, haemolysin and esp gene can exist.
Significance and Impact of the Study:  Our study confirms that MLVA is a suitable method for the epidemiological study of E. faecalis and for the first time shows possible relationships between specific genotypes and such virulence determinants, i.e. lipase, haemolysin and esp gene.     

Hypervariable loci that enhance the discriminatory ability of newly proposed 15-loci and 24-loci variable-number tandem repeat typing method on Mycobacterium tuberculosis strains predominated by the Beijing family

The newly proposed 15- and 24-loci mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem repeat (VNTR) typing method was evaluated for its ability to differentiate 181 Mycobacterium tuberculosis Beijing family strains. Compared with the original 12-loci MIRU-VNTR typing method, the 15-loci system dramatically improved the discriminatory power for Beijing strains; however, large clusters that could be further differentiated by IS6110 restriction fragment length polymorphism (RFLP) were still obtained. The clonal stability and allelic diversity of a total of 31 VNTR loci were evaluated. VNTRs 3232, 3820, and 4120 were identified as the effective hypervariable VNTR set for the second-line typing of clustered strains following the 15-loci based scheme. Consequently, the discriminatory power of the new scheme (18 loci) equaled that of IS6110 RFLP.     

High-throughput typing method to identify a non-outbreak-involved Legionella pneumophila strain colonizing the entire water supply system in the town of Rennes, France

Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.     

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

Background

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.

Methodology/Principal Findings

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.

Conclusions/Significance

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.     

Fine-scale genetic diversity among Burkholderia pseudomallei soil isolates in northeast Thailand

Burkholderia pseudomallei soil isolates from northeast Thailand were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes. These close genetic relationships imply a recent colonization followed by localized expansion, similar to what occurs in an outbreak situation.     

Fine-Scale Genetic Diversity among Burkholderia pseudomallei Soil Isolates in Northeast Thailand

Burkholderia pseudomallei soil isolates from northeast Thailand were genotyped using multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes. These close genetic relationships imply a recent colonization followed by localized expansion, similar to what occurs in an outbreak situation.     

Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.     

Genetic Diversity of Neisseria meningitidis Serogroup C ST-4821 in China Based on Multiple-Locus Variable Number Tandem Repeat Analysis

Neisseria meningitidis sequence type (ST)-4821 was first reported in China in 2003, and a new hyper-virulent lineage has been designated as the ST-4821 complex. A large number of N. meningitidis ST-4821 strains have been identified in China since 2003; however, the microevolution characteristics of this complex are unclear. Different combinations of variable number of tandem repeats (VNTR) loci were used in multiple-locus VNTR analysis (MLVA) to analyze 118 N. meningitidis serogroup C ST-4821 strains isolated from seventeen provinces between 2003 and 2012. Additionally, MLVA with five VNTR loci was performed due to its high discriminatory power. One hundred and eighteen isolates were found to comprise 112 subtypes based on MLVA, and 16 outbreak-associated strains were clustered into one group. These data indicate a high level of diversity for N. meningitidis ST-4821 due to microevolution in the last decade. In addition, the results revealed high similarity between isolates from the same geographic origins, which is helpful when monitoring the spread of N. meningitidis serogroup C ST-4821 and will provide valuable information for the control and prevention of bacterial meningitis in China.     

Use of multiple‐locus variable number tandem repeat analysis and phage typing for subtyping ofSalmonella Enteritidis from sporadic human cases in the United States

Aims: To investigate the genetic diversity among S. Enteritidis isolates from different geographic regions to evaluate the relationship between phage types (PTs) and variable number tandem repeat analysis (VNTR) loci. Methods and Results: We performed multiple‐locus variable number tandem repeat analysis (MLVA) and phage typing on 245 S. Enteritidis isolates collected from sporadic human clinical cases in Michigan, Minnesota, New York, and Washington states between 2000 and 2007. Ninety‐four MLVA types and 22 different PTs were identified. Specific PTs were associated with a predominant allele for certain VNTR loci. Cluster analysis using a minimum‐spanning tree demonstrated two major clusters (I, II) and one minor cluster of isolates. PTs 8, 13a, 13 and 34 were significantly associated with MLVA cluster I. Phage types 1, 4, 6a, and 18 were significantly associated with MLVA cluster II. Conclusions: We found significant association between MLVA‐based clusters and PTs. Certain VNTR loci were associated with specific PTs and could serve as useful molecular markers for S. Enteritidis in epidemiological investigations. Significance and Impact of the Study: MLVA genotyping in combination with phage typing can be used for effective characterization of S. Enteritidis isolates. It can also be useful for tracing possible sources during investigations of sporadic and outbreak cases of S. Enteritidis.     

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.     

Multiple-locus variable-number of tandem-repeats analysis distinguishes Vibrio parahaemolyticus pandemic O3:K6 strains

A specific serotype of Vibrio parahaemolyticus, O3:K6, has recently been linked to epidemics of gastroenteritis in Southeast Asia, Japan, and North America. These pandemic O3:K6 strains appear to have recently spread across continents from a single origin to reach global coverage, based on profiling of strains by several molecular typing methods. In this study, variable-number tandem repeats (VNTR)-based fingerprinting was applied to clinical and environmental V. parahaemolyticus O3:K6 strains in an attempt to develop a molecular method with increased sensitivity for discriminating strains; the relative discriminatory powers were compared with ribotyping and pulsed-field gel electrophoresis (PFGE). All clinical strains tested were independent human isolates obtained from different outbreaks or from sporadic cases in Tokyo during the period from 1996 to 2003. Multiple-locus VNTR analysis (MLVA) was shown to have high resolution and reproducibility for typing of V. parahaemolyticus clones. MLVA analysis of 28 pandemic V. parahaemolyticus O3:K6 strains isolated from human cases produced 28 distinct VNTR patterns. The VNTR loci displayed between 2 and 15 alleles at each of eight loci with Nei's diversity index ranging from 0.35 and 0.91. These data demonstrated that MLVA is useful for individual strain typing of new O3:K6 strains, which appear to be closely related by other molecular methods.