多粘菌素耐药基因（mcr-1）的分子演化、水平转移规律及机制研究

The polymyxins (colistin and polymyxin B) remain the last line of defense against many Gram-negative bacilli. However, a colistin resistance gene (mcr-1) has been reported in many countries since Nov. 2015, which attracts the world wide concerns. Recently, we have isolated mcr-1 encoding plasmid from Haihe River and the Yangtze River, respectively, and the evolutionary dynamics, the horizontal transfer discipline and conjugation mechanisms of the mcr-1 gene in water environment were unknown. In order to show the mcr-1 gene pollution in seven river basins, the amount of mcr-1 gene will be quantified by RT-qPCR, and the bacteria contained the mcr-1 gene will be isolated and identified. The evolutionary dynamics analysis will be performed based on the whole sequence mcr-1 encoding plasmid. In addition, to reveal the mechanisms of mcr-1gene horizontal transfer between the same species and cross-species, two categories of conjugation model mediated by mcr-1 encoding plasmid will be established, and the gene regulatory pathways of conjugation will be focus on. Our results will provide theoretical and technical support for the control of mcr-1 gene pollution in water environment.

多粘菌素被认为是对抗某些革兰氏阴性细菌引起严重感染的“最后一道防线”，然而2015年11月以来，人们在多个国家相继从禽畜、健康人群及患者体内发现了对多粘菌素耐药的新基因mcr-1，引起全球范围的关注。我们近期从海河、长江水样中分离到携带mcr-1基因的质粒，然而mcr-1基因在水环境中的演化、转移规律及机制不清楚。在前期研究的基础上，我们将利用分子生物学技术、细胞生物学技术和微生物学技术等对我国七大水系中mcr-1基因进行定量检测，摸清其污染状况，并对含有mcr-1基因的细菌进行分离、鉴定；在对携带mcr-1基因的质粒全序列以及结构进行分析的基础上，进行分子演化分析；建立两类质粒介导mcr-1基因的接合转移模型，并从接合转移的基因调控途径入手，揭示同种属和跨种属细菌间的转移机制，为控制mcr-1基因污染提供理论与技术支撑。

耐多粘菌素新基因mcr-1的发现，引起全球关注。最初的研究聚焦于人群、家禽和农产品中检测mcr-1基因，而与人们健康密切相关的水环境中的mcr-1基因污染情况，尚未见报道。已结题项目按照计划书的要求完成了各项研究内容，首先研究了我国七大水系中mcr-1基因污染情况，并对天津海河流域mcr-1基因污染情况进行了监测（每月1次，共半年），结果显示，海河已经存在不同程度的mcr-1基因污染，并发表SCI论文1篇；其次，从环境水样中分离到3个菌株，经过纯化后，进行质谱检测，Haihe-5，Haihe-13为大肠杆菌，Zhujiang-1为肺炎克雷伯菌；并检测它们了对16种抗生素的MIC，结果显示它们都是多重耐药细菌；提取质粒进行测序分析，结果显示都含有携带mcr-1基因的质粒，并对它们的序列进行了比对，初步获得了分子演化特征；同时发现位于菌株Haihe-13的plas1上的PmrE基因可能具有多粘菌素抗性，但是经过NCBI检索，未发现有相似的序列，随后对该基因进行克隆表达，证实了具有多粘菌素抗性，并申请发明专利1项。最后，以菌株Haihe-13为供体菌，E coli J53为受体菌建立接合对，结果显示，该mcr-1质粒的接合转移率约为10-6/recipients，并对接合过程相关基因配对基因trbB进行检测，初步探讨了质粒介导mcr-1基因接合转移规律及机制，相关研究还在进行中。