我国新发现的A-B+型艰难梭菌的基因组多态性及进化特征研究

Clostridium difficile (C. difficile) has emerged as a major nosocomial pathogen, with the expanding of its range of infection and host, which has been listed in routine surveillance pathogens. Based on the results of virulence detection by ELISA, pathetic C. difficile are divided into two toxin types, A-B+ and A+B+. To face the new thereat from C. difficile, a serial of genomic and molecular research have been performed. However, most of these studies is focused on toxin type A+B+, and few of them are about strain A-B+ (which is found more popular in China and other Asia areas). Furthermore, the strain with A-B+ present stronger drug resistance to clindamycin, and a few of these strains shows higher virulence. A-B+ strains have more diversity in China. In Europe and US, we only found ST37, however, in our previous MLST analysis of 104 Chinese strains we found additional 6 MLST type (ST-15,48,100,117,118,119) in A-B+ toxin type. The Pathogenicity Locus (PaLoc) sequence is also differing to all available genomes sequences, suggested large genome diversity in China. Hence, in our study, we try to carry out population structure and evolution study among different A-B+ strains. Genome resequencing, cell toxin experiment and animal experiment will be used in finding new PaLoc region, to determine the C. difficile evolution relationship. All these studies will provide new molecular diagnostics target for C. difficile toxin detection, and open avenues for the development of new epidemic tools for C. difficile surveillance.

艰难梭菌是新发现的、重要的院内感染病原菌，其感染范围和宿主范围不断扩大，已列入欧美国家常规监测。根据其毒素存在状况，致病菌株主要分为两类：A-B+型和A+B+型。欧美以A+B+菌株为主，因而主要对这一菌型开展了较多研究。而A-B+型是我国艰难梭菌的突出型别，存在高度遗传变异性，国外仅发现的ST37型，中国还存在6种MLST型别（ST-15,48,100,117,118,119型），致病编码区的序列与已知序列差异较大，且具有更强的克林霉素耐药性，部分毒力也得到了增强。因此，本研究拟采用比较基因组学和分子杂交技术，结合细胞毒素测定与动物肠袢实验，对A-B+型艰难梭菌的不同型别临床分离株进行研究，系统的了解不同序列型别间的进化关系、致病相关基因及耐药区域，为A-B+型菌株致病力变化规律、毒素分型和耐药机制研究提供基础，对开展艰难梭菌感染的流行病学调查、监测和防治具有重要的应用和理论价值。

在本项目中课题组按计划完成了各项任务。艰难梭菌是一种新发现的、重要的院内感染病原菌，为革兰氏染色阳性、厌氧芽孢杆菌，可导致抗生素相关腹泻。随着研究开展，目前已成为导致国内外院内感染的最重要病原菌之一，在欧美排首位。毒素A-B+型艰难梭菌是我国的突出型别，存在高度遗传变异性。课题组以10株毒素为A-B+菌株为起始研究目标，结合tcdA/B全基因水平研究，病原全基因组测序和分子分型等流行病学研究，研究对不同分子型别艰难梭菌的基因组差异，探讨了艰难梭菌获得毒力和耐药基因的途径和机制。在样本量逐步扩大到100余株的情况下，结合细胞毒素实验，对细菌的毒力进行了定性研究，拓展了研究宽度和深度，为我们更好地理解tcdA/B基因的多态性特征与进化、毒力的关系，对今后的流行病学监测和临床快速监测提供了依据。项目过程中，我们针对艰难梭菌基因组差异大得特征，还对艰难梭菌的检测引物进行了优化，并进行了广泛应用。伴随测序技术的进展，我们仍然在延续项目的进展，利用三代测序技术或更多地全基因组成像技术，以期获得更为准确的中国特有的A-B+的全基因图谱，对更深入的开展艰难梭菌感染的流行病学调查、监测和防治具有重要的应用和理论价值。