Development Of Molecular Identification And Detection Methods Of Vibrio Parahaemolyticus And Salmonella Based On O Serogroup、Comparative Genomics And Transcriptomics Of Two Escherichia Coli Strains

ⅠFoodborn pathogens including Vibrio parahaemolyticus and Salmonella are great threat for food safety and health. V. parahaemolyticus is a human pathogen that is widely disseminated in estuarine, marine and coastal environments throughout the world, and is recognized as the leading cause of marine food-borne illness worldwide. Salmonella is widely distributed in nature, and it is transmitted via contaminated food, including meat, poultry, eggs, dairy products, and fresh produces, such as tomatoes and lettuces, thereby gaining entry into almost every aspect of the human food chain. It can cause human illnesses such as typhoid fever, paratyphoid fever, and other salmonelloses. Pathogens infections have been characterized by causal associations with multiple, diverse serotypes, and serotype determination of foodborn pathogens is important for disease assessment, infection control, and epidemiological surveillance.In this study, the O-serogroup genetic determinants (OGDs) of all13V. parahaemolyticus O serogroups were identified. A PCR assay based on the O-serogroup specific genes was developed for the identification and detection of all13V. parahaemolyticus O-serogroups. The assay was tested against41target strains and21strains of other species. A double-blind test including105environmental specimens was also performed, and was found to be highly specific and reproducible, with detection sensitivity of1ng of genomic DNA. It was demonstrated that V. parahaemolyticus at the level of104CFU/ml in mock water specimens and the enrichment culture of samples inoculated with at the level of1CFU/ml were detected. As few as2to18CFUs (initial inoculums) of V. parahaemolyticus was detectable in1g oyster sample after enrichment using this PCR method.A microarray system that targets the O antigen-specific genes of Salmonella was developed for simultaneously detection and identification all46Salmonella O serogroups. Of these,40serogroups can be confidently identified, and the remaining6, in three pairs (serogroups067and B, E1and E4, and serogroups A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against293Salmonella strains,186Shigella strains, representative Escherichia coli strains, and10strains of other bacterial species. The assay correctly identified288(98%) of the Salmonella strains. The detection sensitivity was determined to be50ng genomic DNA per sample. By testing simulated samples in a tomato background,2to8CFU per gram inoculated could be detected after enrichment. These two molecular protocol developed in this study were suitable for rapid detection and identification of pathogens from clinical and environmental samples, with the potential for application in epidemiologic investigations and other food safety applications.ⅡUnderstanding bacterial adaptation is a great challenge for scientists and medical doctors to battle infectious diseases. Bacterial cells have a high level of mutation rate and can adapt to the dynamic host environments by selecting mutants which are more fit to the condition. Thus, a systematic investigation of the whole gene expression profiles of clinical isolates would be needed for modern diagnostic and treatment of infectious diseases.In this study we report the complete genome of two Escherichia coli O156:H25strains CB647and PT199, which were isolated from cattle and sheep respectively. Their ability to colonize to human intestinal epithelial cell HT-29has significant differences, while only CB647strain had been found in illness human. We also studied the transcriptome of the two strains in vitro and after3h attachment with HT-29cell. Our study presents the global bacterial gene expression profile of the two strains during infection of human intestinal epithelial cell and characterized their interactions; provides answers about the role of different animal species as reservoirs for human pathogenic types; elucidates genes and mechanisms which are important for human pathogenicity.