P15: A procedure for maintenance of the virulence plasmid (pYV) in Yersinia pestis under culture conditions and its application for growth studies

The pathogenicity of Yersinia pestis (YP) depends on the presence of a virulence plasmid (pYV).  The unstable nature of pYV in YP leads to the eventual outgrowth of pYV less cells due their higher growth rate.  Thus, it was necessary to develop procedures to monitor the presence of pYV during cultivation, storage, and laboratory manipulations.  The method involves streaking frozen stock culture on calcium-deficient Congo red (CR) magnesium oxalate agar (CR-MOX) and incubating at 37^oC for 24 h to isolate pYV-bearing clones.  The pYV colonies appeared as red pinpoint colonies expressing both low calcium response (Lcr) and CR-uptake whereas pYV negative colonies appeared as larger white or orange colonies.  The Lcr and CR positive clones were further confirmed as pYV positive by a PCR assay and by the expression of pYV-associated phenotypic virulence characteristics.  These pYV-bearing clones were inoculated in BHI broth and grown at 28^oC for 24 h for the preparation of frozen and working stock cultures.  Before frozen storage and preparation of working stock cultures, the culture was tested for the presence of pYV and its virulence associated phenotypes.  The Lcr-CR positive clones on CR-MOX were used as a working stock culture for 15 days.  To ensure the validity of this procedure for stability and maintenance of pYV in YP, the cells were examined and monitored for pYV stability during the subculturing in BHI broth and CR-MOX.  This technique allowed the retention of pYV and was successfully applied to a study of growth of YP in ground beef.