重要梭状芽孢杆菌及其代谢物谱的集成鉴定技术及风险评估

The problem of Clostridium contamination in infant foods has caused great public concerns. Currently, methods for isolating and identifying Clostridium from samples are inadequate in China except that for Clostridium botulinum. Clostridium sporogenes, Clostridium baratii and Clostridium buryricum are three common bacteria that are isolated from powdered infant formula, Not only can they release toxins that are different from botulinum neurotoxin, but also they can produce botulinum neurotoxins due to the acceptance of botulinum neurotoxins(BONTs) genes that are transferred by Clostridium botulinum, which thus further causes toxic reactions of human or animals. Therefore, this research has put focuses on the following aspects: (1) through infants survey and food sampling, the situations of Clostridium contamination in infants and powdered infant formula will be estimated. (2) The Synergistic-separation methodology for Clostridium from samples will be optimized and standardized. (3) Combining traditional and new identification methods, an integrated identification technology for Clostridium and its metabolites will be established. (4) The risk assessment of Clostridium contamination on infant health will be assessed. In conclusion, this research has great significance on the precaution of infant illness that are caused by Clostridium, and also on the development of advanced contamination-blocking technology and scientific control strategy.

婴幼儿食品中梭状芽孢杆菌污染逐渐被人们所关注，目前我国除肉毒梭菌外，极少研究其他梭菌，尚无完整、配套的菌株分离及鉴定标准化程序。生孢梭菌、巴氏梭菌及丁酸梭菌等重要梭状芽孢杆菌作为婴幼儿配方粉中常见污染菌，本身既可产生不同于肉毒毒素的其他有毒代谢产物，又可接受肉毒梭菌平行转移的肉毒毒素毒力基因而产生肉毒毒素，引起人或动物中毒反应。因此本项目重点开展以下几方面研究：（1）通过人群调查和采样方式了解婴幼儿人群及婴幼儿配方粉中重要梭状芽孢杆菌污染状况；（2）样品中重要梭状芽孢杆菌协同分离方法的优化及标准化；（3）结合传统与新型鉴定方法，建立相互补充的梭状芽胞杆菌及其代谢物谱集成鉴定技术；（4）重要梭状芽孢杆菌的暴露对婴幼儿健康影响风险评估。完成本项目，对重要梭状芽孢杆菌在婴幼儿人群中可能引起的疾病暴发及时预警、发展先进的污染阻断技术和科学控制措施等，具有十分重要的意义。

生胞梭菌、丁酸梭菌等重要梭状芽胞杆菌在食品和环境中广泛存在，可在婴儿人群肠道中定植进而致病。本项目通过人群病例对照研究方式对婴儿人群进行流行病学调查，并采集婴儿生物样品、食品样品、环境样品和市售婴儿配方粉样品进行梭状芽胞杆菌分离、鉴定、毒素检测及溯源分析等，同时对我国婴儿人群梭状芽胞杆菌暴露对婴儿人群健康进行风险评估。研究结果显示：（1）本研究建立了不同样品中梭状芽胞杆菌协同分离技术、集成鉴定体系、肉毒毒素毒力基因四重荧光PCR快检技术及PFGE溯源分型技术等四种技术体系。（2）人群研究结果显示，在501名婴儿人群中共采集样品813份，分离出梭状芽胞杆菌246株，总检出率为30.3%（246/813），其中检出率较高分别为生胞梭菌7.7%（68/813）、双酶梭菌6.2%（50/813）和丁酸梭菌4.6%（37/813）。（3）梭状芽胞杆菌分离率最高的样品为粪便样品37.3%，接下来依次为市售婴儿配方粉22.3%、环境涂抹样品19.5%和食品样品11.1%。（4）人群各分组间梭状芽胞杆菌分离情况经比较分析可见，实验组人群检出率（46.1%）稍高于对照组（41.6%）；农村地区（48.6%）检出率高于城镇地区（40.8%）；混合/人工喂养（57.5%）组检出率明显高于母乳喂养（29.6%）组； 7-12月龄组（63.4%）检出率明显高于0-6月龄组（36.5%）。（5）所有梭状芽胞杆菌分离株均未有肉毒毒素毒力基因检出。（6）生胞梭菌有毒代谢产物疑为异吲哚-4丙酸和苯丙酸。本项目的开展，不仅首次获得了我国婴儿人群梭状芽胞杆菌暴露水平，为相关部门制定有关政策提供基础数据，更可为与梭状芽胞杆菌感染相关疾病，如肉毒中毒、新生儿坏死性小肠结肠炎等提供有效实验室检测和临床治疗技术支持，预防和保护我国婴儿人群健康。