肺炎克雷伯菌耐替加环素的分子机制研究

Tigecycline is one of the last resorts against multidrug-resistant Klebsiella pneumoniae, however, in the tigecycline-resistant Klebsiella pneumoniae is increased globally in recent years. Unfortunately, the mechanism is still unclear. Through the preliminary work of this study, we have accomplished whole-genome sequencing of the tigecycline resistant strains,and found that the chromosome coding several OqxAB-like and AcrAB-like multidrug efflux pump systems, we have confirmed the expression level of oqxA and arcA is related to tigecycline resistance by Real-time PCR, but the regulatory mechanism is unknown. The focus of this project is to investigate the different drug susceptibility Klebsiella pneumoniae and find variable factors that related to resistance mechanism, and then verify them by using high-throughput sequencing, comparative genomics analysis, and RNA-seq. In this project, we'll identify the differential of expressed genes by analysis of the SNPs of whole genome and general trend of locus mutation; investigate the transcriptional regulation and regulatory networks of the OqxAB and AcrAB efflux pump system; validate the mechanism in different levels of resistant strains by Real-time RT-PCR analysis; finally, identify the biological function of the resistance genes by knockout experiment. The aim of our research is to reveal the mechanism of resistance to tigecycline of Klebsiella pneumoniae, so that we could provide the theoretical basis for the prevention of dissemination of tigecycline-resistant Klebsiella pneumoniae, and the exploration of new drug targets.

替加环素是治疗碳青霉烯类抗生素耐药肺炎克雷伯菌的主要药物，但近年来全球替加环素耐药菌株不断增加，其耐药机制尚不清楚。本课题完成了替加环素耐药菌株的全基因组测序，发现染色体编码多个OqxAB和ArcAB外排泵，通过Real-time RT-PCR证实oqxA和acrA表达水平与耐药相关，但其调控机制不明；本课题拟在此基础上对不同替加环素敏感性的肺炎克雷伯菌采用高通量测序、比较基因组学分析、转录组测序等技术，以从全局研究耐药机制，通过找变量再靶向验证的策略，分析全基因组水平的SNPs，鉴别差异表达的基因，研究OqxAB和AcrAB的转录调控及其协同调控网络，进一步通过Real-time RT-PCR在不同耐药水平的菌株中验证，最后通过基因敲除实验鉴定耐药基因的生物学功能。通过此研究，系统阐明肺炎克雷伯菌对替加环素耐药的形成机制，为预防替加环素耐药菌株的播散和药物新靶点的探索提供理论基础。

我国临床肺炎克雷伯菌分离株对替加环素耐药性逐渐提高，而其对替加环素耐药的机制依旧不明。本课题针对某三甲医院分离的替加环素非敏感菌株开展流行病学研究，选取了其中的32株替加环素耐药肺炎克雷伯菌作为研究对象，建立了PFGE，MLST，比较基因组学，RNA-seq以及RT-PCR研究方法，系统地探讨了外排泵在肺炎克雷伯菌替加环素耐药机制中的作用。我们的结果表明本研究所覆盖的耐替加环素肺炎克雷伯菌的MLST与PFGE的分型结果基本一致，而ST37是耐药菌株的主要流行克隆。通过对典型替加环素耐药菌株5422的比较基因组研究发现其与K. pneumoniae ATCC BAA-2146的亲缘关系最近，而且5422菌株的基因组中编码了16组外排泵。RNA-seq与RT-PCR实验证实5422菌株的替加环素耐药与AcrAB和OqxAB外排泵的表达有重要的关联，在高浓度的替加环素胁迫作用下，这两个外排泵系统的表达量大幅提高，与抗生素浓度成正比关系。我们的研究为部分肺炎克雷伯菌对替加环素耐药的机制提供了合理解释，并为预防替加环素耐药菌株的播散和药物新靶点的探索提供理论基础。