婴幼儿肠道艰难梭菌的定植特征及其耐药机制研究

Clostridium difficile infection (CDI) is the main cause of antibiotic-associated diarrhea (AAD), and the children may be the incubated media and transmission host of toxic C. difficile strain. Our previous study showed there had a high carrier rate of asymptomatic C. difficile in the gut of healthy infants, which severely affected the structure of the host intestinal microbiota. In this project, we will firstly isolate intestinal C. difficile from children (diarrhea and non-diarrhea) less than 2 years-old, compare the evolution and genetic relationship of the C. difficile isolated from children and adults, and demonstrate the children-to-adults transmission mechanism of the same toxic strain. Then, the intestinal flora composition of the target population will be analyzed by NGS, and the biomarker (distinctive microflora, short-chain fatty acids) which could differentiate the asymptomatic colonized and non-colonized, AAD and non-AAD children; finally, after the analysis of drug resistance, the antibiotic resistance-related genes chip detecting and the whole genome re-sequencing, the characteristics and mechanisms of multiple resistance in C. difficile isolates will be explored and discussed. This research is very meaningful to resolve the interaction and effects of carriage of different toxic C. difficile strains against the host intestinal microflora, and could provide research basis for prevention of AAD and CDI, control the emerging and spreading of multi-antibiotic resistance, and developing new anti-microbial drug.

艰难梭菌感染(CDI)是抗生素相关腹泻(AAD)的主因，儿童可能是致病株的潜伏媒介和传播宿主。本课题组前期研究发现健康婴幼儿肠道中艰难梭菌携带率较高，影响其肠道菌群种类和丰度。本项目拟以0-2岁幼儿（健康、腹泻）为目标人群，通过比较艰难梭菌的携带率、多态性，幼儿分离株与成人致病株的分子溯源、分离株的蛋白组学和动物感染的分析，准确认识婴幼儿肠道艰难梭菌的定植特征，初步确定同型菌株在儿童与成人间的传播规律；通过菌群二代测序和粪便短链脂肪酸测定，比较不同人群的肠道菌群结构，获得快速诊断CDI、AAD的生物标志物—肠道特征菌群和短链脂肪酸；通过分离株耐药相关基因芯片检测和全基因组重测序，阐明婴幼儿肠道艰难梭菌的多重耐药现状、特点及其发生机制。研究有助于解析艰难梭菌携带与儿童肠道菌群的互作，可为预防AAD和CDI、控制耐药菌的人群传播、开发新型抑菌药物等提供研究数据，具有重要的科学意义和应用价值。

依托母婴队列，开展了膳食因素对健康的母婴肠道菌群结构及传递的影响分析。研究收集了60对母婴的膳食资料及生物标本（孕晚期妇女粪便及其子代胎便），对母婴肠道菌群进行测序并分析菌群结构及其垂直传播特征，初步探讨膳食营养素对菌群结构的影响。结果显示孕晚期妇女肠道菌群以双歧杆菌科、毛螺菌科、肠杆菌科为主；菌群垂直传递中子代以肠球菌科、双歧杆菌科、肠杆菌科为主，顺产新生儿遗传自母亲的菌群比例和数量均高于剖宫产新生儿；新生儿肠道中的6种SCFAs含量无性别和生产方式的差异，母亲肠道中丙酸、正丁酸相对丰度高于子代，而乙酸、异丁酸相对丰度低于子代（P<0.05）；母亲肠道SIgA浓度高于子代，男婴SIgA浓度高于女婴（P<0.05）。对于长链多不饱和脂肪酸，未摄入的孕妇及其子代的肠道内正戊酸含量均高于摄入的孕妇及其子代（P=0.001、P=0.043）。本研究的孕期妇女能量、钙、不溶性膳食纤维和脂肪酸的摄入量分层均不能改变菌群的母婴垂直传递特征。孕晚期妇女的不溶膳食纤维摄入量与自身肠道的肠杆菌、钙摄入量和自身肠道的双歧杆菌的相对丰度正相关；孕晚期妇女的钙摄入量与其子代肠道中肠杆菌的相对丰度负相关，长链不饱和脂肪酸摄入与其子代肠道中瘤胃菌科的相对丰度正相关。.艰难梭菌感染是抗生素相关腹泻的主因，儿童可能是致病株的潜伏媒介和传播宿主。收集了6岁以下的健康非腹泻儿童的粪便标本221份，分离29株可疑艰难梭菌菌株，16s rRNA全长基因测序比对分析，确认15株艰难梭菌。完成了6株艰难梭菌全基因组测序、毒力因子、分子分型、耐药表型、耐药特征检测与分析，结果显示儿童肠道分离的厌氧梭菌基因组上携带毒力基因、抗生素耐药基因序列，但对昆虫和细胞的毒力实验结果显示其毒性普遍较低，耐药谱简单，暂未发现对万古霉素和美罗培南耐药的分离株。对分离和未分离出厌氧梭菌的儿童粪便标本进行肠道菌群测定，发现携带厌氧梭菌对肠道菌群丰度和种类有显著的影响，特别是双歧杆菌属的细菌。其在儿童与成人之间的传递机制仍在探索中。.通过标准品加正丁醇内参法建立一种肠道菌群生物标志物-SCFA的快速检测方法。确定了一种从粪便标本直接检测艰难梭菌tpi高保守区，对阳性样本重点做细菌分离培养的快速初筛方法。适合于队列研究中人群随访样本的高通量检测，具有一定的推广价值。