外排泵在替加环素耐药肠杆菌科细菌中的作用及调控机制研究

Carbapenem-resistant Enterobacteriaceae (CRE) have become one of the most important group of pathogens of nosocomial infections. Tigecycline, the first new glycylcycline antibiotic used for clinical application, is the primary choice for the treatment of CRE infection. During the last few years, emergence of tigecycline-resistant CRE has led to the situation that there are no drugs available in clinical treatment of those pathogens, which urges us to elucidate the resistant mechanisms. During our previous studies, we have isolated 11 strains of tigecycline-resistant CRE. Preliminary investigations have indicated that PAβN, an efflux pump inhibitor, was able to restore the susceptibility of the strains against tigecycline, suggesting that it is highly possible that the resistance of the CRE is resulted from the high expression of the efflux pumps. We propose to apply whole genome sequencing, transcriptome sequencing, real-time quantitative PCR, gene knock-out and recover, and other relevant methods to compare the expression and regulation of the efflux pumps among the tigecycline susceptible and resistant CRE strains, and to elucidate the function and regulatory mechanisms of various efflux pumps for the tigecycline resistance. The knowledge from the proposed studies will provide a theoretical basis for the control of the dissemination of tigecycline-resistant strains and experimental evidence for the development of new antimicrobial drugs for infections caused by tigecycline-resistant CRE.

碳青霉烯类耐药肠杆菌科细菌（CRE）已成为院内感染最重要的致病菌之一，替加环素作为首个应用于临床的新型甘氨酰环素类抗生素，是我国治疗CRE感染的最主要的抗菌药物。而近年来CRE对替加环素耐药使CRE感染陷入无药可治的困境，亟需明确其耐药机制。我们在前期研究已分离到11株替加环素耐药的CRE，外排泵抑制剂PAβN能恢复其敏感性，极可能通过外排泵高表达造成耐药。本研究拟采用全基因组测序、转录组测序、实时荧光定量RT-PCR，基因敲除和回补等方法比较耐药和敏感株中各类外排泵基因的表达情况及其调控基因的作用，阐明各类外排泵在替加环素耐药中的作用及调控机制。为控制替加环素耐药菌株的流行提供依据，为开发新抗菌药物提供实验基础。

碳青霉烯类耐药肠杆菌科细菌（CRE）已成为院内感染最重要的致病菌之一，替加环素作为首个应用于临床的新型甘氨酰环素类抗生素，是我国治疗CRE感染的最主要的抗菌药物。而近年来CRE对替加环素耐药使CRE感染陷入无药可治的困境，亟需明确其耐药机制。利用全基因组测序及转录组测序分别对替加环素诱导耐药株与临床敏感株之间主要外排泵基因序列差异，临床分离的替加环素耐药株与标准菌株之间基因表达差异进行分析，结果显示均无变化（包括前期分离的AcrB高表达的两株替加环素耐药肺炎克雷伯菌，有待进一步研究）。但是我们却发现了其中10株替加环素耐药的同一克隆的肺炎克雷伯菌，对其中一株进行全质粒测序发现，tet（A）基因发生了突变，并且与碳青霉烯耐药基因KPC-2位于同一质粒上，该基因突变不但介导了细菌对替加环素耐药，同时初步证实了替加环素的耐药可通过质粒传播。该研究结果不仅阐明了外排泵在替加环素耐药肠杆菌科细菌中的作用及其调控机制，更是发现了除外排泵以外的其他机制介导的替加环素耐药机制，为控制替加环素耐药菌株的流行提供基础，为后续开发新药提供了理论基础。