粘液型多重耐药鲍曼不动杆菌毒力特点及形成机制研究

As a conditioned pathogen, multidrug-resistant Acinetobacter baumannii (MDR-AB) has spread worldwide which used to be thought of low virulence. However, in recent decades there is an upward trend in highly virulent MDR-AB, which has caused a great concern. In our previous study we found that the isolation rate of hypermucoid MDR-AB (HMMDR-AB) had been rising and some non-mucoid MDR-AB(NMMDR-AB) presented a hypermucoid phenotype after anti-infective treatment. As the hypermucoid phenotype is closely related to bacterial capsule, one of the classic virulence factors, it is suggested that HMMDR-AB pose high virulence. However, the details of HMMDR-AB virulence characteristics and related mechanisms remain unclear. For this study we have preliminary collected HMMDR-AB and NMMDR-AB derived from blood and sterile body fluids including strains isolated from the same patient before and after anti-infective treatment. Through assessment of capsule polysaccharide, in-vitro virulence, building animal infection models, genomic sequencing and comparative genomic analysis combined with gene knockout and complement, we aim to make clear whether HMMDR-AB poses high virulence and the role of capsule polysaccharide in bacterial virulence as well as clarify the mechanisms of MDR-AB virulence and detect new polysaccharide biosynthesis regulatory genes and new virulence genes. This study could enrich the knowledge of MDR-AB pathogenic mechanism and provide new clues to treatment of MDR-AB infections.

多重耐药鲍曼不动杆菌（MDR-AB）作为条件致病菌广泛流行，既往认为低毒力，近年来有从低毒力演变为高毒力的趋势。课题组前期研究发现粘液型MDR-AB（HMMDR-AB）分离率不断上升，并在抗菌药物治疗过程中MDR-AB由非粘液型转变为粘液型的现象。细菌的高粘液表型与经典的毒力因子荚膜多糖密切相关，提示HMMDR-AB具有高毒力，但其确切毒力特点和形成机制仍不清楚。本课题组以血液与无菌体液来源的包括同一患者抗菌药物治疗前后分离的粘液型与非粘液型MDR-AB菌株为主要研究对象，拟通过荚膜多糖表达量分析、体外毒力评估、动物感染模型、细菌全基因组测序和比较基因组分析结合基因敲除及回补等手段，揭示HMMDR-AB是否具有高毒力以及荚膜多糖与细菌毒力的相关性，阐明HMMDR-AB毒力形成机制并发现新荚膜多糖调控基因和新毒力基因。研究结果将丰富MDR-AB致病机制的认识，极可能为治疗其感染提供新的思路。

鲍曼不动杆菌是一种非常重要的医院内致病菌，近年来其耐药菌株数量不断增加。鲍曼不动杆菌菌落可以有光滑或无光泽的外观，但也可表现出粘液样，粘液型鲍曼不动杆菌分离率近年来越来越高。本研究从56例鲍曼不动杆菌感染患者中分离出60株鲍曼不动杆菌，所有患者均为粘液型鲍曼不动杆菌感染，其中4例患者除粘液型外，还分离出非粘液型鲍曼不动杆菌。本研究中通过观察菌落和荚膜的形态，用结晶紫染色法测定生物被膜形成能力对不同粘液表型的菌株进行了比较。对所有菌株进行全基因组测序（WGS），然后进行核心基因组多位点序列分型（cgMLST）和耐药基因分析。最后，选取部分菌株进行大蜡螟感染模型的毒力测定。本课题组在粘液型鲍曼不动杆菌中观察到比非粘液型鲍曼不动杆菌更大的荚膜；但发现粘液表型与菌株产生的生物膜数量无关。鲍曼不动杆菌几乎全部为多重耐药菌株，含有多种耐药基因。粘液型鲍曼不动杆菌的ST型以ST191和ST195为主，其中ST191菌株毒力较强，ST195菌株毒力较弱。粘液型鲍曼不动杆菌对大多数抗生素耐药，部分菌株具有较高的毒力，临床应引起重视。此外，在本课题研究过程中发现，通过对亚胺培南压力下传代的不动杆菌属细菌进行基因组测序比较，产OXA-499的不动杆菌属细菌在低浓度亚胺培南压力下可由碳青霉烯不敏感进化为碳青霉烯耐药，blaOXA-499启动子区域的单碱基突变参与了耐药进化。提示在临床治疗过程中，需注意监测碳青霉烯不敏感菌株的药敏变化，以免治疗失败。