质粒介导多粘菌素耐药基因mcr-1转移机制研究

Increasingly prominent infection of multiple drug-resistant bacteria has brought serious clinical challenges to anti-infection treatment. Colistin is the last resort of multiple drug-resistant gram-negative bacteria infection treatment. However, as emergence of the plasmid mediated colistin resistance gene mcr-1, the last defense line of antimicrobial agents has risks in destruction. Since the mcr-1 gene was first discovered in 2015, bacteria carrying mcr-1 have been found in many countries and regions and spread rapidly. Moreover, this gene further showed a trend of spread to other bacteria. At present, most studies focus on popular characteristic and clinical prognosis of strain carrying mcr-1. However, the mcr-1 transfer mechanism has not yet been elucidated. Object of this research is 91 clinical E. coli isolates carrying mcr-1, their transfer ability are identified by the conjugation and transformation experiment. The transfer mechanism of mcr-1 is explored by the methods of restriction enzyme digestion cloning and gene insertion site distribution and frequency detection. The epidemic outbreak of strain carrying mcr-1 is further evaluated by the fitness cost of different types of plasmid. It is helpful to understand the disseminated essence of colistin resistant bacteria and provide the basis for its prevention and control strategies through research on transfer mechanism of strain producing mcr-1.

日益严重的多重耐药菌感染给临床抗感染治疗带来了严峻挑战。多粘菌素是目前治疗多重耐药革兰阴性菌感染的“最后防线”。随着质粒介导多粘菌素耐药基因mcr-1的出现，抗菌药物“最后一道防线”面临被突破风险。自2015年mcr-1基因首次被发现以来，携带该基因的细菌已在全球多个国家和地区迅速流行，且该基因呈进一步向其他细菌播散趋势。目前，多数研究仅关注携带mcr-1菌株流行特点及临床感染预后，但其转移机制尚未阐明。本课题拟从临床感染菌株分离91株携带mcr-1基因大肠埃希菌为研究对象，通过接合、转化实验明确携带mcr-1菌株质粒的转移能力；应用基因插入位点分布测序及插入频率检测等方法，阐明该基因转移机制；通过测定携带mcr-1不同类型质粒菌株的适应性代价，明确携带该基因菌株感染爆发流行的可能。针对产MCR-1菌株转移机制的研究，有助于了解多粘菌素耐药细菌播散的本质，为寻求其防控策略提供基础。

日益严重的多重耐药菌感染给临床抗感染治疗带来了严峻挑战。多粘菌素是目前治疗多重耐药革兰阴性菌感染的“最后防线”。随着质粒介导多粘菌素耐药基因mcr-1的出现，抗菌药物“最后一道防线”面临被突破风险。自2015年mcr-1基因首次被发现以来，携带该基因的细菌已在全球多个国家和地区迅速流行，且该基因呈进一步向其他细菌播散趋势。目前，多数研究仅关注携带mcr-1菌株流行特点，但其转移机制尚未阐明。本课题拟前期从临床感染菌株分离91株携带mcr-1基因大肠埃希菌为研究对象，通过质粒杂交定位、接合、转化实验，明确83%菌株携带mcr-1 质粒的具有转移能力；质粒基因组测序分型进一步表明mcr-1 质粒主要分为IncI2、IncX4及IncHI2型；通过对ISApl1插入元件测序分析结果显示，66.8% 的细菌丢失了ISApl1结构，11.4%的细菌包含2个或以上拷贝的ISApl1插入序列；质粒全基因组序列分析发现，序列大的IncHI2型mcr-1质粒（大小>200kb）通常含有多个复制子类型，是由小的质粒相互整合从而形成；质粒的进化分析显示mcr-1基因的传播主要依赖于质粒而不是转座子，通过全基因测序及插入位点测序明确富含ATTACCACATTTTAAGAATATTATTAATCT区域为转座元件△ISApl1-pap2-mcr-1序列插入热点区域，在其转移中发挥重要作用。通过测定携带mcr-1不同类型质粒菌株的适应性代价，发现IncI2型质粒具有最低的生长适应代价（约18%），明确该类型质粒是导致mcr-1基因广泛传播的重要原因。此外，本课题还发现共产MCR-1与NDM-5、NDM16临床分离的大肠埃希菌，提示耐药基因在菌株中有进一步整合的趋势，临床需与重视。针对产MCR-1菌株转移机制的研究，有助于了解多粘菌素耐药细菌播散的本质，为寻求其防控策略提供基础。. 此外，本项目联合培养博士研究生2名，另有在读1名博士生和 1名硕士生参与了研究。