Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157).
ABSTRACT Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.
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ABSTRACT: VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs) on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10(-05) to 1.8 × 10(-03) mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10(-03) mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.09/2013; 2013:390354. DOI:10.1155/2013/390354
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ABSTRACT: A published multiple-locus variable number of tandem-repeats analysis (MLVA) scheme was compared with pulsed-field gel electrophoresis (PFGE) for genotyping of 62 Escherichia coli O26 strains from humans, animals and food. The strains were isolated between 1947 and 2006 in eight countries on three continents and divided into 23 enterohaemorrhagic E. coli (EHEC), 33 enteropathogenic E. coli (EPEC), one enterotoxigenic E. coli (ETEC) and five avirulent strains. ETEC and avirulent E. coli serotyped as O26:H32. EHEC and EPEC O26 strains shared flagellar type H11 and the eae-beta gene, and divided into two clonal lineages by their arcA gene sequence and fermentation of rhamnose and dulcitol. The rhamnose/dulcitol-nonfermenting (RDF-), 'arcA allele 1' type comprised 22 EHEC and 15 EPEC strains. The rhamnose/dulcitol-fermenting (RDF+), 'arcA allele 2' type encompassed 17 EPEC and one EHEC strain. PFGE typing of the 62 O26 strains revealed 54 distinct patterns, whereas 29 profiles were obtained by MLVA. Like PFGE, MLVA divided RDF- and RDF+ O26:[H11] strains into two distinct clusters of related strains. The O26:H32 strains formed a separate PFGE cluster and two clusters by MLVA. MLVA was found as suitable, but more rapid and easier to standardize than PFGE for identifying genetically related E. coli O26 strains.FEMS Microbiology Letters 02/2010; 303(2):137-46. DOI:10.1111/j.1574-6968.2009.01874.x · 2.72 Impact Factor
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ABSTRACT: To perform a longitudinal study of the diversity of Escherichia coli O157 from a ruminant pasture/stream environment using multiple-locus variable-number tandem-repeat analysis (MLVA). Samples of faecal droppings from grazing ruminants and from an adjacent stream were tested longitudinally for E. coli O157 by enrichment and immunomagnetic separation (IMS). Using MLVA, 24 different profiles were identified from a total of 231 E. coli O157 isolates, of which 80 were included in a similarity analysis. Four main clusters with several subclusters were observed. Although there was close contact between sheep and cattle during the study period, E. coli O157 was surprisingly not detected from cattle faeces. The cluster analysis indicated both unrelated and closely related E. coli O157 strains. The choice of loci to target in MLVA is important for the subtyping result, as loci with high diversities are essential for discriminating between closely related isolates. There is a lack of data available on the use of MLVA to describe E. coli O157 diversity and changes over time in the animal reservoirs and the environment. Such data are needed in order to further develop MLVA as a typing method.Journal of Applied Microbiology 11/2008; 105(5):1344-53. DOI:10.1111/j.1365-2672.2008.03856.x · 2.39 Impact Factor