Yersinia enterocolitica and Yersinia pseudotuberculosis, the two enteropathogenic Yersinia species for humans are worldwide distributed and are causing frequently diarrhea to inhabitants of temperate and cold countries. Yersinia enterocolitica is a major cause of foodborne disease, by consumption of contaminated pork meat further associated substantial economic cost. However, investigating enteropathogenic Yersinia is hardly performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stools samples. Moreover, current isolation procedures are time consuming and expensive, thus leading to underestimated incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (mAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of mAbs were selected by testing their specificity and affinity toward enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of mAbs were selected to develop highly sensitive enzyme immunoassays (EIA) and lateral flow immunoassays (LFI or dipsticks) convenient for rapid diagnostic purpose. The limit of detection of the EIAs ranged from 3.2 x 10(3) cfu/ml to 8.8 x 10(4) cfu/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and ranged for the LFIs from 10(5) cfu/ml to 10(6) cfu/ml. A similar limit of detection was observed for artificially contaminated human feces.
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 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 multi-locus sequence typing-based scheme that can speciate Yersinia strains to a level of resolution equal to whole genome sequencing. Our assay is designed so that it is able to accurately sub-type the important human pathogenic species Y. 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 the scheme to be an important molecular typing system to allow accurate and reproducible speciation of Yersinia isolates, a process often inconsistent in non-specialist laboratories. Additionally our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica.
The genus Yersinia has been used as a model system to study pathogen evolution. Using whole-genome sequencing of all Yersinia species, we delineate the gene complement of the whole genus and define patterns of virulence evolution. Multiple distinct ecological specializations appear to have split pathogenic strains from environmental, nonpathogenic lineages. This split demonstrates that contrary to hypotheses that all pathogenic Yersinia species share a recent common pathogenic ancestor, they have evolved independently but followed parallel evolutionary paths in acquiring the same virulence determinants as well as becoming progressively more limited metabolically. Shared virulence determinants are limited to the virulence plasmid pYV and the attachment invasion locus ail. These acquisitions, together with genomic variations in metabolic pathways, have resulted in the parallel emergence of related pathogens displaying an increasingly specialized lifestyle with a spectrum of virulence potential, an emerging theme in the evolution of other important human pathogens.
The genus Yersinia contains three species pathogenic for humans, one of which is the enteropathogen Yersinia pseudotuberculosis. A recent analysis by Multi Locus Sequence Typing (MLST) of the 'Y. pseudotuberculosis complex' revealed that this complex comprises three distinct populations: the Y. pestis/Y. pseudotuberculosis group, the recently described species Yersinia similis, and a third not yet characterized population designated 'Korean Group', because most strains were isolated in Korea. The aim of this study was to perform an in depth phenotypic and genetic characterization of the three populations composing the Y. pseudotuberculosis complex (excluding Y. pestis, which belonged to the Y. pseudotuberculosis cluster in the MLST analysis). Using a set of strains representative of each group, we found that the three populations had close metabolic properties, but were nonetheless distinguishable based on D-raffinose and D-melibiose fermentation, and on pyrazinamidase activity. Moreover, high-resolution electrospray mass spectrometry highlighted protein peaks characteristic of each population. Their 16S rRNA gene sequences shared high identity (?99.5%), but specific nucleotide signatures for each group were identified. Multi-Locus Sequence Analysis also identified three genetically closely related but distinct populations. Finally, an Average Nucleotide Identity (ANI) analysis performed after sequencing the genomes of a subset of strains of each group also showed that intragroup identity (average for each group ?99%) was higher than intergroup diversity (94.6-97.4%). Therefore, all phenotypic and genotypic traits studied concurred with the initial MLST data indicating that the Y. pseudotuberculosis complex comprises a third and clearly distinct population of strains forming a novel Yersinia species that we propose to designate Yersinia wautersii sp. nov. The isolation of some strains from humans, the detection of virulence genes (on the pYV and pVM82 plasmids, or encoding the superantigen ypmA) in some isolates, and the absence of pyrazinamidase activity (a hallmark of pathogenicity in the genus Yersinia) argue for the pathogenic potential of Y. wautersii.
We sequenced the genome of a clinical isolate of Yersinia enterocolitica (IP10393) from France. This strain belongs to bioserotype 4/O:3, which is the most common pathogenic subgroup worldwide. The draft genome has a size of 4,463,212 bp and a G+C content of 47.0%, and it is predicted to contain 4,181 coding sequences.
The species Yersinia intermedia is a member of the genus Yersinia which belongs to the Enterobacteriaceae family. This species is divided into eight biotypes, according to Brenners biotyping scheme. This scheme relies on five tests (utilization of Simmons citrate and acid production from d-melibiose, d-raffinose, alpha-methyl-d-glucoside [alphaMG], and l-rhamnose). The collection of the French Yersinia Reference Laboratory (Institut Pasteur, Paris, France) contained 44 strains that were originally identified as Y. intermedia but whose characteristics did not fit into the biotyping scheme. These 44 strains were separated into two biochemical groups: variant 1 (positive for acid production from l-rhamnose and alphaMG and positive for Simmons citrate utlization) and variant 2 (positive for acid production from l-rhamnose and alphaMG). These atypical strains could correspond to new biotypes of Y. intermedia, to Y. frederiksenii strains having the atypical property of fermenting alphaMG, or to new Yersinia species. These strains did not exhibit growth or phenotypic properties different from those of Y. intermedia and Y. frederiksenii and did not harbor any of the virulence traits usually found in pathogenic species. DNA-DNA hybridizations performed between one strain each of variants 1 and 2 and the Y. intermedia and Y. frederiksenii type strains demonstrated that these variants do belong to the Y. intermedia species. We thus propose that Brenners biotyping scheme be updated by adding two new biotypes: 9 (for variant 1) and 10 (for variant 2) to the species Y. intermedia.
Enteropathogenic Yersinia are among the most frequent agents of human diarrhea in temperate and cold countries. However, the incidence of yersiniosis is largely underestimated because of the peculiar growth characteristics of pathogenic Yersinia, which make their isolation from poly-contaminated samples difficult. The use of specific procedures for Yersinia isolation is required, but is expensive and time consuming, and therefore is not systematically performed in clinical pathology laboratories. A means to circumvent this problem would be to use a single procedure for the isolation of all bacterial enteropathogens. Since the Statens Serum Institut enteric medium (SSI) has been reported to allow the growth at 37°C of most gram-negative bacteria, including Yersinia, our study aimed at evaluating its performances for Yersinia isolation, as compared to the commonly used Yersinia-specific semi-selective Cefsulodin-Irgasan-Novobiocin medium (CIN) incubated at 28°C. Our results show that Yersinia pseudotuberculosis growth was strongly inhibited on SSI at 37°C, and therefore that this medium is not suitable for the isolation of this species. All Yersinia enterocolitica strains tested grew on SSI, while some non-pathogenic Yersinia species were inhibited. The morphology of Y. enterocolitica colonies on SSI allowed their differentiation from various other gram-negative bacteria commonly isolated from stool samples. However, in artificially contaminated human stools, the recovery of Y. enterocolitica colonies on SSI at 37°C was difficult and was 3 logs less sensitive than on CIN at 28°C. Therefore, despite its limitations, the use of a specific procedure (CIN incubated at 28°C) is still required for an efficient isolation of enteropathogenic Yersinia from stools.
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