金黄色葡萄球菌新型肠毒素检测、表达、致病性以及食物基质对细菌生长及毒素基因表达影响的研究

The recent finding that there might be huge differences in the behavior of bacteria in the planktonic liquid state and immobilized bacteria found in food matrix brings into question about microbial risk assessment methods based on planktonic liquid cultures. The knowledge about the behavior of Staphylococcus aureus in food environments, for example, the expression of enterotoxin genes, the growth and survivability of cells on food matrix, is not fully understood at present. In order to improve the microbiologically safe food for human consumption, how environmental factors affect the expression of enterotoxins in foods should be taken into account to complement existing knowledge. In addition, Food safety is an important issue throughout the world. Staphylococcal food poisoning is one of the most common food-borne diseases in the world following the ingestion of staphylococcal enterotoxins (SEs). To date, 21 SEs have been identified. Whether these newly identified SEs could cause servious disease have not yet been done. As far as the above mentioned is concerned, several questions should be given special attention. Whether do the newly identified enterotoxins have the toxicity and potential risks to human being? How is the mode about the expression of the different staphylococcal enterotoxins genes at mRNA level during cell growth? What are the influences of food matrix on S. aureus growth, survivability and enterotoxin genes expression? How can we integrate them together in our study? What is more, our previous researches showed that the novel enterotoxins genes were detected with highly increased percentage compared with classical enterotoxins. Therefore, it is very urgent to embark on the related research in terms of detection, expression, and pathogenicity of the newly identified enterotoxins. For these reasons, in this proposal, we will focus on the isolation, identification, and genotyping of S. aureus strains from various food samples. The main targeted new enterotoxin genes will be cloned, expressed, and the products will be purified. The detection methods about the targeted enterotoxins by enzyme-linked immunosorbent assay (ELISA) will be developed. At the same time, we want to shed some light on the emetic and diarrheagenic activities of the targeted SEs. Further, we will investigate the temporal expression of different staphylococcal enterotoxins genes at mRNA level during cell growth. Moreover, we want to elucidate the influences of food matrix on S. aureus growth, survival and enterotoxin genes expression. The related data and results from this research can greatly enrich the current information and knowledge about the pathogenicity of Staphylococcal newly identified enterotoxins, and the influence of food matrix on S. aureus enterotoxin genes expression, which can shed new light on S.aureus food safety management and control system of our country.

细菌在液体纯培养状态和在食物基质中的生长行为存在不同,目前细菌在食物储藏条件下的生长行为未得到充分认识，不同食物基质对细菌毒力基因的表达，生长速度及胞外毒力形成等知识还需更新.另外,金黄色葡萄球菌引起的食源性疾病已经成为世界性食品安全与卫生问题,但其分泌的新型肠毒素是否具有致病性目前也未能给出确切答案.前期研究发现:新型肠毒素基因型高频出现.针对这些菌株的研究十分必要,因此,本课题拟从菌株分离、鉴定、分型及基因型检测开始,开展新型肠毒素基因克隆、重组蛋白表达与纯化;建立新型肠毒素ELISA检测方法;开展新型肠毒素致病性研究;开展新型肠毒素基因在mRNA水平上表达研究:开展新型肠毒素在不同食物基质中的表达及特异性挥发性成分识别研究.预期结果将揭示新型肠毒素的致病能力,以及不同培养基质与基因之间多因素对肠毒素表达与分泌的影响,为新型肠毒素的产生及控制提供科学依据.

金黄色葡萄球菌引起的食源性疾病已经成为世界性食品安全与卫生问题。前期研究发现新型肠毒素基因型高频出现，针对这些菌株的研究十分必要。本项目从菌株分离、鉴定、分型及基因型检测开始，开展新型肠毒素基因克隆、重组蛋白表达与纯化；建立新型肠毒素ELISA检测方法；开展新型肠毒素致病性研究；开展新型肠毒素基因在mRNA水平上表达研究；开展新型肠毒素在不同食物基质中的表达及特异性挥发性成分识别研究。为新型肠毒素的产生及控制提供科学依据。通过项目资助，按照计划任务书中的研究内容，认真完成上述所有四个方面的既定任务，并且超额完成任务。首先本研究开展了食品源金黄色葡萄球菌菌株的分离、分型、鉴定和所有分离菌株肠毒素基因型的检测；完成了5个新型肠毒素基因重组蛋白的构建和原核表达，以及重组蛋白的提取、纯化及鉴定；开展了新型肠毒素的致病性研究，建立新型肠毒素SEI、SEM、SEP的ELISA快速检测方法；通过动物模型构建，完成细胞增殖分析和细胞因子试验；完成不同种类的新型肠毒素基因在mRNA水平上表达研究；完成不同食物基质对肠毒素基因表达影响的研究；完成金黄色葡萄球菌在奶和肉中的生长趋势拟合研究；完成GC-MS对金黄色葡萄球菌污染不同食物的特异性挥发性物质分析。通过本项目资助，在国内外学术期刊上发表学术研究论文28篇（基金资助号标注），其中，中文文章20篇，SCI收录论文8篇，另外，目前还有一篇已接受SCI论文正在出版中（Journal of Dairy Science排版中）。项目资助期间获得授权专利1项（ZL201210136370.9），申报国家发明专利4项（CN201510659321.7、CN201510659178.1、CN201510421148.7、CN201510258560.1）。