致泻性大肠埃希菌杂合株的种群和进化特征研究

Diarrhoeagenic Escherichia coli (DEC) carrying virulence factors from multiple pathovars that cannot be categorized is called DEC hybrid strain. DEC hybrids represented by EHAEC O104:H4 have greater virulence and transmission competitiveness, leading to serious consequences. Therefore, elucidating the population and evolutionary characteristics of these hybrid strains is of extremely important epidemiological significance. Based on the present studies, we plan to explore the phenotypic and drug resistance characteristics of DEC hybrids accumulated in the surveillance of long-term diarrheal diseases in our region, and to evaluate virulence characteristics of hybrid strains by six kinds of in-vitro toxin assays, including screening for 46 virulence genes, cell adhesion and cytotoxicity tests. Meanwhile, we will extract and integrate public data resources, establish a comprehensive database of DEC hybrids for the first time, use MLST and WGS-based population genomics analysis, combine with the above population characteristics, in order to construct the cluster relationship and phylogenetic tree of DEC hybrids, elucidate the population structure and evolutionary relationship of DEC hybrids that have not been solved in the past, and explore specific or key genes related to the easy transfer or integration of the virulence genes of hybrid strains. The implementation of this project will provide an important molecular basis for the identification, traceability, prevention and control, and the exploration of transmission mechanisms of DEC hybrid strains.

携带多种跨类别的分子标志而不能归类的致泻性大肠埃希菌（DEC）称为杂合株，以EHAEC O104:H4为代表的DEC杂合株可具有更强的毒力和传播竞争力，进而导致严重后果，因此阐明这类杂合株的种群和进化特征具有极其重要的流行病学意义。申请者拟在已有研究的基础上，研究本区域长期腹泻症候群监测中积累的DEC杂合株的表型和耐药特征，并通过筛查46种毒力基因，细胞粘附和细胞毒性试验等6种体外毒力试验评估杂合株毒力特征。同时提取整合公共数据资源，首次建立一个全面的DEC杂合株数据库，并综合运用MLST和基于WGS的群体基因组学分析等方法，结合上述种群特征构建DEC杂合株的聚类关系和系统发育图谱，阐明以往研究未曾解决的DEC杂合株种群结构和进化关联，挖掘与杂合株毒力基因易转移或整合相关的特异或关键基因。此项目的实施将为DEC杂合株的分类鉴定、溯源、防控以及探索其传播机制提供重要的分子基础。

致泻性大肠埃希菌（Diarrheagenic E. coli，DEC）是世界上最重要的食源性致病菌之一，也是我国东南沿海地区首要的急性腹泻致病菌。DEC依其毒力因子、致病机制及遗传学特性一般可分为5类，而携带多种跨类别的分子标志而不能归类的DEC称为杂合株，可具有更强的毒力和传播竞争，因此监测并分析本地区DEC和杂合株的特征具有重要意义。本项目在前期研究基础上，开展腹泻症候群病原谱监测研究，分析DEC菌株生物学特征，探索DEC混合感染和杂合株的鉴定路径，对2009-2020年间分离的疑似杂合株重新分纯筛选，结果表明本地区腹泻症候群中DEC仍处于细菌性病原的首位，2018-2020年未检出致病力较强的STEC。不同型别DEC混合感染的较常见，在阳性中的占比为13-25%，但是未发现DEC杂合株（后续将通过更敏感的深度测序的方法鉴定）。DEC菌株特征监测时，首次鉴定和菌株保存时应挑选单个菌落，阳性的菌落应多次连续划线分离单个克隆。项目初步建立了一个DEC杂合株的基本信息库。总之本课题的实施为DEC杂合株的分类鉴定、溯源、变异规律研究提供了重要的信息资源及分子证据。