Identification and Characterization of Yersinia from Food and Environmental Sources

Yersinia genus includes currently 18 species: Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia bercovieri, Yersinia mollaretii, Yersinia rohdei, Yersinia ruckeri, Yersinia aldovae, Yersinia aleksiciae, Yersinia massiliensis, Yersinia similis, Yersinia entomophaga, Yersinia nurmii, Yersinia pekkanenii and Yersinia wautersii. The history of the genus Yersinia can be dated back to 1883. In 1965, the genus consisted of only three species: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis. Since then the taxonomy of the genus has been under tremendous change over the years, and especially the taxonomy of Y. enterocolitica, of which many new species have been separated from. Still Y. enterocolitica is a group of very heterogeneous bacteria, which can be divided into 6 biotypes and about 30 serotypes and into pathogenic and non-pathogenic strains. This variability makes the identification of Y. enterocolitica very challenging. In the thesis, two Yersinia species; Y. nurmii and Y. pekkanenii are described. These species were characterized by polyphasic taxonomic methods, including of 16S rRNA gene analysis, multilocus sequence analysis (MLSA) of housekeeping genes glnA, gyrB, recA and HSP60, DNA-DNA hybridization studies, 16S and 23S rRNA gene restriction fragment length polymorphism (RFLP), and phenotyping. Y. nurmii was isolated from broiler meat packaged under modified atmosphere, and Y. pekkanenii from water, soil and lettuce samples. In the 16S rRNA gene analysis and the 16S and 23S rRNA gene RFLP analysis, these two species grouped with other Yersinia, but in separate clusters. In the phylogenetic analysis of separate or concatenated housekeeping genes, the species formed unique monophyletic groups in all phylogenetic trees constructed. Y. nurmii had a phenotypic profile most similar to that of Y. ruckeri. Y. pekkanenii could not be differentiated from Y. pseudotuberculosis using phenotypic tests. Methods of polyphasic taxonomy were also used to estimate the taxonomic position of European Y. enterocolitica strains of non-pathogenic biotype 1A. Y. enterocolitica has been divided into two subspecies; Y. enterocolitica subsp. enterocolitica and Y. enterocolitica subsp. palearctica. Both subspecies consist of pathogenic and non-pathogenic biotypes. In this thesis, 212 Y. enterocolitica strains were characterized by numerical analysis of HindIII ribopatterns (16S and 23S rRNA gene RFLP). These strains consisted of 162 strains of biotype 1A and 50 strains of biotypes 2 to 4 isolated from different sources in Europe during years 1997-2013. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). The biotype 1A strains studied were found to form a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and Y. enterocolitica subsp. palearctica, with suggestion of the existence of a subspecies formed by non-pathogenic Y. enterocolitica biotype 1A strains. Finally, while studying Enterobacteriaceae in cold-stored (6 °C), modified atmosphere-packaged (MAP) pig cheek (musculus masseter) and hind leg meat (musculus semimembranosus), it was noticed that the pathogenic Y. enterocolitica subsp. palearctica bioserotype 4/O:3 multiplied into high numbers from a non-detectable level in (MAP) pig cheek meat. The Enterobacteriaceae isolated in this study were identified by 16S and 23S rRNA gene RFLP using the HindIII enzyme. Y. enterocolitica bioserotype 4/O:3 is the most common pathogenic bioserotype word-wide, and it can be transmitted to humans through raw or undercooked pork. Usually the growth of Enterobacteriaceae is inhibited by a modified atmosphere with 20% or more CO2 at refrigerated temperatures. However, in this study, high numbers of Y. enterocolitica bioserotype 4/O:3 was observed in MAP cold-stored pig cheek meat, with a concentration of 30% CO2 and 70% O2 in the packages.