trm同源基因介导鲍曼不动杆菌替加环素耐药新机制研究

The widespread of Carbapenem resistant Acinetobacter baumannii (CRAB) has been a serious challenge for clinical anti-infection therapy. Tigecycline is an alternative drug for CRAB treatment with limited option. Recently, the tigecycline resistant strains have been isolated increasingly. The high expression of RND efflux pump is the main resistant mechanism of tigecycline, however it could not explain many phenomena of tigecycline resistant. In our previous studies, we found a new gene trm was responsible for tigecycline resistance by comparative genomics between the parent and tigecycline-resistant isolates. But the exact mechanism of this action is unclear. In this study, we will use gene knockout, gene complementary experiment, comparative transcriptomics, the methyl-sequencing, the protein expression and interaction experiment, UV spectrophotometry and HPLC to explore the new mechanism of tigecycline resistance mediated by trm in Acinetobacter baumannii, and to illuminate the role of differential expression genes related to trm in the process of tigecycline resistance, and to find a novel gene responsible for tigecycline resistance and its function. This study will enrich and improve the understanding of tigecycline resistance mechanisms, and provide a theoretical basis for the rational use of this antibiotic.

碳青霉烯耐药鲍曼不动杆菌（CRAB）的广泛流行给临床抗感染治疗带来严峻挑战。替加环素是治疗CRAB感染的少数可选药物之一。近年来，替加环素耐药菌株不断被分离。RND外排泵高表达是碳青霉烯耐药鲍曼不动杆菌替加环素的主要耐药机制，但该机制仍不能解释许多替加环素的耐药现象。在前期工作中，我们通过比较原始菌株和替加环素诱导耐药菌株的全基因组序列，发现替加环素耐药相关新基因trm，但该基因在替加环素耐药中的具体作用机制不清楚。该课题拟采用基因敲除、基因回补、转录组测序、甲基化测序、蛋白体外表达和相互作用、紫外分光光度测定和HPLC检测等方法，系统研究trm基因介导鲍曼不动杆菌替加环素耐药新机制和trm基因相关的差异表达基因在替加环素耐药中的作用，并发现替加环素耐药新基因，揭示新基因功能。该研究将丰富和完善对替加环素耐药机制的理解和认识，为临床合理应用替加环素提供理论依据。

碳青霉烯耐药鲍曼不动杆菌（CRAB）的广泛流行给临床抗感染治疗带来严峻挑战。替加环素是治疗CRAB感染的少数可选药物之一。近年来，替加环素耐药菌株不断被分离。该研究首先在替加环素敏感性降低的临床菌株中进行替加环素耐药机制研究，明确替加环素耐药鲍曼不动杆菌的分子分型为ST368和ST191，RND外排泵仍为替加环素的主要耐药机制，并发现adeR基因的E219K和adeS基因的A130D为替加环素耐药相关突变热点和IS插入序列ISAba1为插入突变热点。然后在鲍曼不动杆菌ATCC19606和ATCC17978中进行基因敲除和基因回补实验，在DNA水平研究trm基因及trm同源基因A1S_2858在替加环素耐药或敏感性降低中的作用。同时也研究了替加环素敏感性降低的临床分离鲍曼不动杆菌中trm基因的SNP分布特点, 临床菌株与ATCC19606菌株相比， trm基因存在SNP差异，但临床菌株的SNP大部分是一致的。在转录组水平研究了trm基因敲除菌株T2和亲本菌株ATCC19606比较转录组学，T2与ATCC19606相比，在转录组水平仅有5个基因的表达水平具有显著性差异，包括2个下调基因（A6739_RS02885和A6739_RS06905）和3个上调基因（A6739_RS03490、A6739_RS03480和A6739_RS03485），这些基因主要编码外排泵及外排泵调控基因。最后，利用蛋白质结构预测工具SWISS-MODEL对TRM蛋白进行空间结构预测。该研究将丰富和完善对替加环素耐药机制的理解和认识，为临床合理应用替加环素提供理论依据。