鲍曼不动杆菌舒巴坦耐药新机制研究

Acientobacter baumannii has emerged as one of the most important pathogens associated with nosocomial infection. Sulbactam, one of the most effective antimicrobial agents against A. baumannii infection, is confronted with difficulties of increasing resistant rate in recent years. Studies on the resistant mechanisms are few except for the mutation of ftsI gene and high expression of TEM-1-type β-lactamase. However, both published studies as well as our previous findings supported that the above mechanisms showed few contributions to clinical sulbactam-resistant strains and were irrelevant with the resistant levels of sulbactam. So there most likely have some novel unrevealed mechanisms associated with the sulbactam resistance. By using high-throughput genome sequencing technology on the induced sulbactam resistant strains of ATCC17978 and ZJ06, our previous findings revealed that the mutations of ftsZ and ponA gene might be contributed to the sulbactam resistance. This study will verify the functions of the mutant ftsI gene and the high expression of TEM-1-type β-lactamase in clinical strains and concentrate on the novel mutant genes. We prefer to further discover some novel differential expressed genes by comparing the differences of transcriptome data between ATCC17978/ ZJ06 and their induced resistant strains, reveal the functions of mutant genes (ftsZ and ponA) and the novel differential expressed genes in the induced resistant strains by gene knockout and complementation experiment. We will further clarify the functions of the mutant genes and the novel differential expressed genes in clinical A. baumannii strains with different sulbactam MICs levels. Overall, we hope to systematically identify the resistant mechanisms of sulbactam to A. baumannii and finally provide evidence on the control of clinical sulbactam resistant-A. baumannii strains.

鲍曼不动杆菌已成为医院感染的主要病原菌之一，舒巴坦作为治疗鲍曼不动杆菌感染的重要抗菌药物，近年来耐药率明显增加，但其耐药机制的研究很少。ftsI基因突变及TEM-1酶高表达是目前已知的最主要耐药机制，但此机制只能解释临床分离菌的少数耐药现象，本课题前期亦发现此机制与舒巴坦耐药水平无明显相关性，极有可能存在新的机制。利用基因组重测序及比较基因组学技术，前期已在ATCC17978及ZJ06舒巴坦体外诱导耐药株中发现了ftsZ及ponA基因的突变可能与舒巴坦耐药相关。本课题拟在完成已知耐药机制在临床分离菌舒巴坦耐药中作用的基础上，通过基因敲除及回补技术明确ftsZ及ponA突变基因的功能；进一步运用转录组测序技术在诱导耐药菌中发现新的差异表达基因，明确其耐药相关功能，并在临床分离菌中进行验证，从而系统阐明鲍曼不动杆菌舒巴坦的耐药机制，为临床舒巴坦耐药鲍曼不动杆菌的防治提供基础。

鲍曼不动杆菌是医院感染的主要病原菌之一，对常用抗菌药物的多重及广泛耐药现象使之成为临床抗感染治疗的严重威胁。舒巴坦对鲍曼不动杆菌具有良好的天然抗菌活性，是临床上治疗鲍曼不动杆菌感染重要武器。但是，近年来随着舒巴坦复合制剂在临床上的广泛使用，鲍曼不动杆菌对其耐药率逐年上升，遗憾的是耐药机制相关研究很少，耐药产生的原因未完全阐明。本研究以不同遗传背景的鲍曼不动杆菌临床菌株为研究对象，并根据舒巴坦药敏MICs值对菌株进行分组，运用PCR、qPCR、蛋白表达、全基因组测序、基因敲除及回补技术等多种分子生物学技术和方法，运用全基因组学、比较基因组学以及蛋白组学等生物信息分析工具和方法，从基因水平、转录水平以及蛋白表达水平等多层次、多水平全面阐明了β-内酰胺酶在鲍曼不动杆菌对舒巴坦耐药产生中的重要作用。本研究揭示了TEM酶表达量与舒巴坦MICs的中度相关性；首次报道了鲍曼不动杆菌中染色体中存在blaTEM-1D基因多拷贝串联现象，提示染色体上的耐药基因可以通过IS介导的基因复制方式来增加基因的拷贝数，从而增加酶的表达量，介导细菌对舒巴坦更高水平的耐药。其二，通过对ampC基因的深入研究，发现AmpC酶与舒巴坦耐药产生的相关性，提示ISAba1能调控ampC表达量，从而介导产生舒巴坦耐药。其三，发现blaOXA-23基因的携带与否与舒巴坦耐药与否存在呈正相关，进一步验证了OXA-23酶对舒巴坦的强大水解作用，这也是鲍曼不动杆菌对舒巴坦耐药的最主要机制。