细菌外膜囊泡在黏菌素耐药性传递中的作用机制研究

The widespread use of colistin in agriculture inevitably increase the risk of emerging resistance. mcr-1, as well as mcr-2 to mcr-5, are plasmid-mediated and related to colistin resistance. The bacterial outer membrane vesicles (OMVs) can increase bacterial resistance to antibiotics by transfer resistance gene and protein between cells. Our previous study found that the mcr-1 gene was present in the outer membrane vesicles isolated from E. coli 08-85. Our hypothesis is that OMVs may play a role in the transfer of mcr genes and proteins. Therefore, in this project, the OMVs from colistin-resistant strains carrying mcr genes will be purified and transform the colistin sensitive strains. The changes of MIC against colistin between donors and transformers, mcr related genes and proteins in the OMVs of transformer, the delivery process dectected by high-resolution live cell fluorescence microscope，the conjugation efficacy of mutants with reduced production of OMVs will be performed to elucidate whether or not the colistin resistance based on the mcr genes and proteins is mediated through OMVs. We are hoping that this will provide a new insight in controlling of colistin resistance and the development of new drugs against colistin-resistant G- bacteria.

碳青霉烯酶的出现以及在农业领域的无序使用，使得黏菌素日益面临严峻的耐药性挑战。mcr-1及随后报道的mcr系列基因（mcr-2~mcr-5）与黏菌素耐药相关并可通过质粒快速传播。细菌外膜囊泡（OMVs）可通过传递耐药基因及蛋白等方式传递耐药性。课题组前期在一株多黏菌素耐药大肠埃希菌的OMVs中检测到mcr-1基因，推测OMVs可能介导mcr系列基因及蛋白在细菌间传递。因此，本课题将通过检测OMVs中mcr系列基因及蛋白、考察OMVs介导的mcr系列基因/蛋白及黏菌素耐药性在细菌间传递的效率、荧光标记MCR蛋白观察其动态传递过程、构建OMVs表达缺陷突变株验证其介导黏菌素耐药性传递机制等方法，研究OMVs在基于mcr系列基因及蛋白传递引起的黏菌素耐药性传播中的作用机制。期望通过本研究，为控制黏菌素耐药性传播、开发抗黏菌素耐药G-菌药物提供新思路。

碳青霉烯酶的出现以及在农业领域的无序使用，使得黏菌素面临日益严峻的耐药性挑战。mcr-1及随后报道的mcr系列基因与黏菌素耐药相关并可通过质粒快速传播，本项目主要研究细菌外膜囊泡(OMV)在mcr介导的黏菌素耐药性传递中的作用及机制。首先对临床分离菌进行了mcr系列基因筛选，获得mcr-1阳性大肠埃希菌5株及肺炎克雷伯菌1株，未检测到携带其他mcr基因菌株。随后通过PCR、Dotblot等方法对mcr-1阳性菌株的OMV进行mcr-1基因检测及遗传环境分析，发现5株大肠埃希菌OMV均携带mcr-1基因，其中两株菌OMV中mcr-1基因两端存在截短的ISApl1转座序列mcr-1 cassette，对这两株菌OMV进行Westernblot未检测到MCR-1蛋白。在细胞水平黏菌素耐药性传递实验中，发现OMV可以剂量依赖性的使黏菌素敏感菌获得耐药性，且mcr-1阳性菌OMV提供的黏菌素耐药性弱于mcr-1阴性菌OMV。在OMV介导的黏菌素耐药性机制研究中，通过PFGE方法在OMV中未检测到完整质粒；在获得耐药性受体菌中未检测到mcr-1基因，说明OMV可能不参与mcr-1基因传递。通过BC置换分析表明，OMV可以直接与黏菌素结合。对OMV及菌体外膜中lipid A结构鉴定及含量分析发现，mcr-1阳性菌OMV中存在pEtN修饰lipid A, 导致与黏菌素结合力减弱，且OMV中pEtN修饰lipid A的含量显著高于菌体外膜。综上，本项目首次报道了OMV在mcr-1介导的黏菌素耐药中的作用，发现大肠杆菌OMV通过“诱骗”作用直接结合黏菌素，使得作用于菌体的黏菌素浓度降低来参与黏菌素耐药，mcr-1阳性菌株OMV中因存在pEtN修饰lipid A而降低与黏菌素的结合，使敏感受体菌获得耐药性弱于mcr-1阴性菌株OMV。