肠杆菌科细菌碳青霉烯酶质粒和MCR-1质粒整合潜力与机制研究

Infections with carbapenem resistant Enterobacteriaceae (CRE) are increasing rapidly and the main mechanisms of carbapenem resistance in Enterobacteriaceae are plasmid-mediated carbapenamases. Polymyxin is considered to be the last resort for the treatment of infections caused by CRE. The emergence of plasmid-mediated colistin resistance gene (mcr-1 gene) in CRE is tend to cause the integration and co-transmission of carbapenemase plasmid and MCR-1 plasmid, which poses great challenge for clinical CRE infections treatment. We have collected a large amount of mcr-1-positive Enterobacteriaceae and CRE as well as fifteen MCR-1 and carbapenamase plasmids co-hourboring Enterobacteriaceae nationwide. In current project, with the purpose of revealing the integration potential of carbapenemase and MCR-1 plasmids in Enterobacteriaceae, the complete plasmids sequence based analysis will be applied to investigate the homologous sequence that may mediate recombination between carbapenamase and MCR-1 plasmids, also to calculate the integration coefficiency between these two types of plasmids. The fitness cost of bacteria carrying both of these two types of plasmids will be also determined. The integrated plasmids will be obtained from bacteria that have been exposure to serial concentrated antibiotic pressure. To elucidate the integration mechanism between the two types of plasmids, the conservativeness and consistency of homologous fragments mediating recombination in the integrated plasmids sequences will be analyzed. Thereafter, in order to clarify the transmissible ability of integrated plasmids, their stability and conjugation ability will be also tested. All the results obtained in this study will provide a scientific basis for the prevention and treatment strategies of plasmid-mediated polymyxin resistant CRE infections.

碳青霉烯耐药肠杆菌科细菌（CRE）感染快速增加，质粒介导的碳青霉烯酶是CRE最重要的耐药机制。多粘菌素是目前CRE感染治疗的最后一道防线。质粒介导多粘菌素耐药基因mcr-1在CRE中的出现，极可能造成碳青霉烯酶质粒与MCR-1质粒整合并共传播，给CRE感染治疗带来严峻挑战。课题组前期已经收集大量临床分离的分别携带MCR-1质粒和碳青霉烯酶质粒以及15株同时携带MCR-1质粒和碳青霉烯酶质粒的肠杆菌科细菌。本研究将基于质粒全序列，分析两类质粒间可能介导重组的同源序列，计算两类质粒的整合系数，测定两类质粒共存时的适应性代价，揭示这两类质粒的整合潜力；通过在抗生素压力下，同时携带这两类质粒细菌的传代，获得整合质粒，寻找其序列中介导重组的同源片段，分析其保守性和一致性，阐明质粒整合机制；进而测定整合质粒的稳定性和接合能力，明确整合质粒的传播能力；为质粒介导多粘菌素耐药CRE感染防治提供科学依据。

碳青霉烯耐药肠杆菌科细菌（CRE）感染快速增加，在我国CRE对碳青霉烯类耐药最主要的机制是质粒介导的碳青霉烯酶。多黏菌素被认为是目前治疗CRE感染的最后一道防线。质粒介导的多黏菌素耐药基因mcr-1在CRE中的出现，引起CRE对多黏菌素耐药，并极可能造成碳青霉烯酶质粒与MCR-1质粒发生整合并共传播，给临床CRE感染的治疗带来严峻挑战。本研究首先通过质粒转化、质粒消除、分子克隆实验、生长速度试验等方法，明确碳青霉烯酶质粒和MCR-1质粒同时存在对大肠埃希菌带来的适应性代价要小于其单独存在时的适应性代价，而对肺炎克雷伯菌的适应性影响则与菌株背景相关；进而通过高通量测序、基因组学等方法，发现NDM质粒、KPC-2质粒等碳青霉烯酶质粒往往与MCR-1质粒同时存在于大肠埃希菌，而MCR-9质粒则与SIM质粒、KPC-2质粒、IMP-4质粒往往同时存在于阴沟肠杆菌复合体，同时我们也发现了少见的新型携带blaNDM-5基因的IncI1-I型质粒和IncY型携带mcr-1.1基因的质粒。本研究共获得3个整合质粒，通过比较基因组学的方法，明确整合的机制为质粒间的直接整合或转座子等移动元件的插入；通过接合实验，明确本课题获得的整合质粒，其水平转移能力较弱。本研究获得的质粒整合机制及整合质粒的传播特性将为多黏菌素耐药的CRE感染治疗及防控策略制定提供科学依据。