不同类型mcr-1质粒对肠杆菌科细菌适应性和毒力的影响及其机制研究

Polymyxin is considered to be the last resort for the treatment of infections caused by carbapenem resistant Enterobacteriaceae (CRE). The emergence of plasmid mediated colistin resistance gene, mcr-1, makes it possible for the widely horizontal transmission of polymyxin resistance in Enterobacteriaceae possible and likely to cause a rapid increase in the polymyxin resistance extremely. Previous studies have shown that after obtaining mcr-1 plasmid, the changes of fitness and virulence of bacteria were variant. The variances are probably due to the different types of plasmids and bacteria. We have already collected multiple types of mcr-1 plasmids from clinical isolates. We will transfer different types of clinical mcr-1 plasmids (common types and two less common types), mcr-1 recombined plasmid and corresponding plasmid vector into different kinds of Enterobacteriaceae to study the effect of mcr-1 plasmids on the fitness and virulence of Enterobacteriaceae. Transformed bacteria with altered fitness or virulence will be selected for further evaluation. We are going to focus on the effect of mcr-1 gene’s expression level on fitness and virulence, comparison of the transcriptome before and after obtaining mcr-1 gene, AMPs susceptibility alteration and transmission electron microscopy study of the bacterial morphology and so on. We hope to clarify the mechanisms of changes of fitness and virulence and then provide a scientific basis for anti-infection treatments and prevention and control strategy formulation.

多粘菌素是目前治疗碳青霉烯耐药肠杆菌科细菌感染的最后一道防线。质粒介导多粘菌素耐药基因mcr-1的出现使肠杆菌科细菌多粘菌素耐药广泛水平传播成为可能，造成多粘菌素耐药性的快速增加。现有研究发现细菌获得mcr-1质粒后，其适应性和毒力变化存在很大差异。产生上述差异的原因极可能与mcr-1质粒类型和菌种有关。课题组前期研究已从临床分离菌中获得了多种类型mcr-1质粒。本研究将把不同类型的临床mcr-1质粒（常见类型和2种少见类型）及mcr-1重组质粒和空白质粒载体分别转入不同肠杆菌科细菌中，明确不同类型的mcr-1质粒对不同肠杆菌科细菌适应性和毒力的影响。选取适应性或毒力有明显差异的菌株，通过明确获得mcr-1质粒前后细菌转录组学变化、抗菌肽敏感性差异、透射电镜扫描图像差异及mcr-1表达量对细菌生长速度和毒力的影响等，阐明细菌适应性和毒力变化的机制，为抗感染治疗和防控策略制定提供科学依据。

质粒介导的多黏菌素耐药基因mcr-1，使肠杆菌科细菌多黏菌素耐药广泛传播成为可能，近些年更有多种新的mcr基因型被发现和报道。以往研究中，细菌获得mcr基因后，其适应性代价和毒力存在不同水平的变化。本课题基于适应性代价方面的研究得出，在大肠埃希菌中，mcr-1基因带来的适应性代价最小，mcr-9基因最大，而在肺炎克雷伯菌中mcr-8基因的适应性代价更小，在阴沟肠杆菌中mcr-9带来的适应性代价比mcr-10带来的更小。并且，在临床分离肺炎克雷伯菌株中，携带mcr-1基因的IncHI2/IncHI2A型质粒的生长竞争能力弱于携带mcr-8的IncFIA/IncFII型质粒。在毒力水平方面，本研究通过细胞黏附和吞噬实验发现，mcr-8基因给肺炎克雷伯菌带来的细胞毒力水平要强于mcr-1基因，而大蜡螟幼虫毒力实验没有统计学差异。扫描电镜结果显示mcr基因带来适应性代价的同时，导致细菌生长过程中易发生破碎。通过比较基因组学研究发现，调控蛋白家族基因arcA或arcB等基因的突变可能是携带mcr-1的质粒发生适应性代价变化的原因。除mcr基因和菌种外，本研究发现，mcr-1基因与blaKPC-2基因共存时，导致的肠杆菌科细菌适应性代价要小于两者单独存在时的代价，这可能与mcr-1表达量降低相关。本课题研究结果表明mcr基因带来的细菌适应性和毒力变化与mcr基因种类及肠杆菌科细菌菌种相关，mcr-1引起大肠埃希菌的适应性代价最小，而在肺炎克雷伯菌中，mcr-8可能引起较小的适应性代价和毒力增强。并且，blaKPC-2基因的存在可能减少mcr-1引起肠杆菌科细菌的适应性代价。通过明确mcr基因引起肠杆菌科细菌适应性代价和毒力变化并探索其机制，有助于应对携带mcr基因的肠杆菌科细菌的传播，为多黏菌素耐药菌感染防控策略提供科学依据。