Isolation of pathogenic Yersinia enterocolitica in Turkey and expression of lipopolysaccharide genes in vivo

Yersinia enterocolitica is a food-borne pathogen that is very rarely encountered in Turkey. In this work, several human, porcine and environmental samples collected from Izmir region in Turkey were examined for the presence of Y. enterocolitica using different cultivation and enrichment methods. A total of nine pathogenic Y. enterocolitica strains were isolated; five strains from pig stool and manure samples and four strains from waste water samples. On the other hand, no Y. enterocolitica was isolated from human diarrheal stool samples (n=102) and from 12 gulf, canal, municipal pool and well water samples. Biochemical and serological characterization of the 9 Y. enterocolitica strains revealed that they belonged to three different bioserotypes: 4/O:3, 2/O:9, and 2/O:5,27. All the strains were deemed pathogenic based on based on ystA, ystB, yadA, virF/lcrF and ail virulence factors specific PCR and phenotypic pathogenicity analysis. Detection of pathogenic Y. enterocolitica strains from the pig and waste water samples from the Izmir region indicates that Y. enterocolitica is a potential risk for public health. Lipopolisaccharide is one of the most significant virulence determinants of Y. enterocolitica strains. Temperature regulates most if not all virulence factor genes of Y. enterocolitica including lipopolysaccharide and working of the expression of LPS genes in vivo during infection is essential to get understanding and solving on the interactions between a bacterium and host. Reporter genes are used to monitor transcription indirectly by inserting genes that encode an assayable protein under the control of promoters of interest. In our project, we constructed luciferase reporter strains of Y. enterocolitica serotype O:3. We first constructed a strain YeO3-LuxCDE where the luxCDE genes direct the biosynthesis of luciferase substrate. Then the luciferase-encoding luxAB cassette was introduced into the YeO3-LuxCDE genome fused downstream of the O-antigen and outer core gene cluster promoters. While the outer core reporter strain was expressed at 37°C than at room temperature, the O-antigen reporter strain was expressed at room temperature than 37°C. Balb/c mice were infected by these reporter strains to determine in which tissues the luxAB genes were expressed during infection. The measurements were done by IVIS spectrum and indirect mRNA expression was screened until 5. days of post-infections. The outer core and O-antigen were expressed in intestinal system especially followed by the infection and it was observed that the reporter strains spread even bone marrow after 5. days-post infection and the importance of LPS was emphasized in pathogenesis. Keywords: Yersinia enterocolitica, pig farm, wastewater, LPS, diarrhea, PCR, LuxCDABE, IVIS spectrum, Outer core, O-antijen, in vivo gene expression