Molecular Epidemiology And Study On Virulence Factor Of Escherichia Coli O157

E. coli0157:H7is a main serotype of EHEC, which is an important foodborne pathogen to human. The infection of E. coli O157:H7can lead to mild diarrhea, hemorrhagic colitis (HC), hemolytic uremic syndrome(HUS), thrombosis thrombocytopenic purpura(TTP) and many diseases, even death. The prevalence and outbreak of EHEC0157:H7infection has been widely reported in China and many foreign countries since its’first report in1982in the United States. It became a hot issue of the World organization of public health and food safety organizations. This study was developed for a better understanding of the prevalence of EHEC O157:H7in Shanghai and surrounding areas, which also created a platform for our lab to research EHEC O157:H7and made important theories basis for the future applied research and pathogenesis exploration. The exact works in this study were shown below:1. To clarify the prevalence of EHEC O157:H7in Shanghai and surrounding areas,collect feces samples from pig farms, cattle farms, chicken farms, and meat samples from some markets in shanghai and surrounding regions. Sorbitol MacConkey (SMAC) plate separation and PCR detection of specific gene of rfbE methods were applied to isolate and identify EHEC O157:H7strains. Serum agglutination assay and gene sequencing were used to confirm the obtained strains. The virulence genes of the strains were detected by PCR method, the crystal violet staining was used to determine their ability to form biofilm. Multilocus sequence analysis (MLST) and random primers polymorphism analysis (RAPD) were developed to classify the obtained strains by molecular character-istics. Triplex PCR method was applied for a phylogenetic clustering analysis and understanding their genetic relationship.We isolated and obtained22EHEC O157strains during July2010to December2011, with12isolates conserved in our lab previously. According to PCR detection of flagellin gene fliC of H7,12strains were identified as EHEC O157:H7serotype, and other10strains were identified as EHEC O157:NM.6of total strains were found to possess4virulence genes,3strains had2virulence genes,5strains had2virulence genes,7strains had1virulence gene, and only2strains contained no virulence gene. EHEC26, EHEC27and EHEC40were found to show strong biofilm-forming ability. EHEC001and EHEC028showed mild biofilm-forming ability, EHEC002, EHEC008and EHEC030only showed weak formation of biofilm capacity, and the remaining14strains showed no biofilm-forming ability.The total strains can be divided into5clusters by RAPD analysis method, with EHEC036,037,038,039,041and042belong to cluster Ⅰ, EHEC026,027,040and044belong to cluster Ⅱ, EHEC001,025,030,043and045belong to cluster Ⅲ, EHEC002,005,015,046and047belong to cluster Ⅳ, EHEC008and028belong to cluster Ⅴ. The phylogenetic clustering analysis suggested the main strains belong to type D(59.1%),22.72%of total groups belong to type A,13.64%belong to type B2and4.55%belong to B1. The 22obtained strains were classified as13subtype by MLST clustering analysis method, and most strains belonged to ST11、 ST88and ST641subtype.3new strains were found during our study, which can not be found in the reported subtypes submitted to database.2. Prokaryotic clone for further study of EHEC O157easier detection methods and applications, using gene expression technology, its specific gene rfbE successfully cloning, expression and purification of the gene encoding the protein.3. To analyze whether Z1965gene affect the virulence and biological functions of EHEC O157strains,this study successfully contructed a EHEC0157:H7Z1965gene deletion strain by Red recombination system.