DETECTION OF YERSINIA PESTIS BY COMPARISON OF VIRULENCE PLASMID (PYV/PCD)‐ASSOCIATED PHENOTYPES IN YERSINIA SPECIES*

Abstract

Consumption of meat contaminated with Yersinia pestis can cause oro‐pharyngeal plague in humans. Existing microbiological media designed for selective detection of Y. pestis in food are not satisfactory for that purpose. Expression of genetic determinants in Yersinia species including low calcium response (Lcr), colony size, crystal violet (CV) binding, Congo red (CR) uptake, autoagglutination (AA) and hydrophobicity (HP) were compared. Lcr and CV binding were detectable within 24 h at 37C in Yersinia enterocolitica and Yersinia pseudotuberculosis but at 48 h in Y. pestis. Colony size, AA, and HP characteristics were expressed in Y. pseudotuberculosis and Y. enterocolitica, but not in Y. pestis. CR uptake in Y. pestis was demonstrated only on calcium‐deficient CR‐magnesium oxalate (CR‐MOX) tryptic soy agar but was expressed on both CR‐MOX and low‐calcium agarose medium for Y. pseudotuberculosis and Y. enterocolitica. CR‐uptake phenotype by Y. pestis can be used for the detection of this pathogen in foods. PRACTICAL APPLICATIONS Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis can cause diverse human diseases. Y. enterocolitica and Y. pseudotuberculosis can cause intestinal distress when consumed in contaminated food. Y. pestis, the causative agent of bubonic plague in humans, is also found in food and can cause febrile illness in humans. Thus, food can have a significant role in the dissemination of human plague. There is a concern for risk assessors about the presence of Y. pestis in food. A virulence plasmid of 70 kb is directly involved with virulence of these pathogens. Until now, separating Y. enterocolitica and Y. pseudotuberculosis from Y. pestis required sophisticated molecular biology and immunological techniques. This problem was eliminated by analyses of virulence plasmid encoded phenotypes as simple and reliable targets for the rapid detection of Y. pestis. These classical microbiological assays should greatly assist both regulatory agencies and the food industry in the detection of Y. pestis. Further, these techniques should also greatly assist medical and public health laboratories to identify this pathogen.

Clonality and Antibiotic Susceptibility ofYersinia enterocoliticaIsolated from U.S. Market Weight Hogs

“…The pYV-less (YEP À ) strains were obtained from large, flat white colonies that emerged spontaneously from swine fecal and GERO:3 YEP þ cultures grown at 378C on Congo redbrain heart infusion agarose as described (Bhaduri and Sommers, 2008). The absence of pYV in these YEP À strains was confirmed by a polymerase chain reaction (PCR) assay targeting virF gene as described (Bhaduri, 2003).…”

“…Clinical isolates of all serotypes implicated in human disease harbor a 70-kb plasmid that contributes to virulence of the bacteria and is referred to as the virulence plasmid (pYV) (Bhaduri, 2001(Bhaduri, , 2002Skurnik et al, 2002;Bhaduri and Sommers, 2008). Elements encoded by the chromosome are also necessary for virulence (chromosomally encoded virulence factors [CEVF]) (Carniel et al, 2000;Revell and Miller, 2001).…”

Virulence Plasmid (pYV)-Associated Expression of Phenotypic Virulent Determinants in PathogenicYersiniaSpecies: A Convenient Method for Monitoring the Presence of pYV under Culture Conditions and Its Application for Isolation/Detection ofYersinia pestisin Food

“…That the expression of CR-uptake on CR-MOX is specifically encoded by pYV was further confirmed using a number of derivatives of clinical strains of Y. pestis (CDC A1122, CO99.3015, Yokohama, P12, D1, D3, D5, D7, D9, D13, D17) containing Pgm but lacking the pYV [7, 10]. These Pgm + /pYV − strains did not bind CR on CR-MOX.…”

“…A derivative of a clinical Y. pestis (KIM5: Kurdistan Iran man) strain lacking the chromosomal 102-kb Pgm locus (pigmentation), but harboring all three virulence plasmids (pYV, pFra/pMT1, and F1) [7, 9], was used for our study. The Pgm locus is only present in Y .…”

“…enterocolitica (serotype O:3; strain GER) and Y . pseudotuberculosis (serotype O:1b; strain PB1/+) were also used in our study [7, 13]. The presence of pYV in Y .…”