【作者】 郭丹；

【导师】 冯露；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 Ⅰ食源性致病菌严重危害着食品安全和人类健康,沙门氏菌和副溶血弧菌是两种常见的食源性致病菌。副溶血弧菌广泛分布在世界各地的河口、沿海岸和海洋,是海产品引发食源性疾病最重要的因素。沙门氏菌存在于多种食物中,包括肉、蛋类、奶制品、蔬菜等,可以通过这些食物进入人体,引起伤寒发热、沙门氏菌病等疾病爆发。致病菌感染与细菌血清型有着重要的关系,对致病菌血清型的鉴定对疾病评估、感染的控制和流行病学调查有着重要意义。本研究完成了副溶血弧菌O血清型决定簇基因序列的破译,筛选出副溶血弧菌所有13种O血清型的特异基因,建立了副溶血弧菌O血清型分型的多重PCR分子分型方法。这一方法经41株副溶血弧菌、21株其它种属细菌验证,具有很好的特异性。对从上海地区海产品中分离到的105份环境样品进行双盲实验检测,均得到正确的结果。灵敏度实验表明,该检测方法可以检测出1ng的基因组DNA,未经富集培养的细菌可以在104CFU/ml的水平被检测到,1CFU/ml的细菌经过富集培养也完全可以得到正确鉴定。每克牡蛎制备的模拟实际样品中含有2到8个CFU的细菌初始量即可通过我们的PCR体系检测到。另外我们建立了针对沙门氏菌所有46种O血清型的基因芯片检测方法,可以成功区分除了相似度特别高的6种血清型(沙门氏菌067和B,沙门氏菌E1和E4,沙门氏菌A和D1)外的所有40种O血清型。该芯片方法经293株沙门氏菌、186株大肠杆菌和志贺氏菌以及10株其它种属细菌验证,具有很好的特异性。灵敏度实验表明在50ng的基因组水平该芯片可以得到稳定有效的检测结果。模拟西红柿样品实验表明,每克样品中2到8个CFU的细菌即可被芯片方法鉴定。因此我们建立的两种食源性致病菌分子检测方法可以取代传统的血清型方法,应用到临床和环境样品检测中去。Ⅱ细菌在适应宿主环境的过程中会产生大量的突变,以此来更好地适应环境。系统地研究致病菌的基因表达和进化差异,对了解细菌适应宿主的机制和致病机制有着重要作用。我们研究了两株不同来源的大肠杆菌0156：H25菌株PT199和CB647,它们分别分离自羊和牛,而且只有CB647菌株会对人致病。它们对人结肠癌上皮细胞HT-29的吸附能力有明显差别。我们完成了PT199和CB647的全基因组测序,还有另外10株分离自病人、牛、羊和肉不同来源的大肠杆菌0156：H25菌株,通过比较基因组学分析揭示了它们的遗传差异和变化。同时研究了PT199和CB647菌株在与HT-29细胞吸附前后的基因表达变化和差异。通过对遗传差异和基因表达差异的综合分析,发现了它们可能与宿主特异性及致病性差异有关的基因,进行进一步的深入研究。更多还原

【Abstract】 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.更多还原