启动子及插入序列在肺炎克雷伯菌blaKPC基因表达中的作用研究

The emergence of Carbapenem-resistant Klebsiella pneumoniae has posed a great challenge to clinical therapy. Our previous study found that the mechanism of Klebsiella pneumoniae on carbapenem resistance is mainly caused by carbapenemase encoded by the blaKPC gene. In addition, KPC-producing Klebsiella pneumoniae showed different levels of resistance to carbapenem, but the mechanism is unknown. Therefore, we collected Klebsiella pneumoniae strains from various regions of China. Different carbapenem resistant levels of clinical strains and their transformants were compared with blaKPC gene expression, using plasmid extraction, cloning and qRT-PCR. The genetic environment of blaKPC gene was investigated by two-generation sequencing and primer extension assays. The specific mechanism of blaKPC gene transcription and regulation were analyzed by ChIP-seq and electrophoretic mobility shift assay. The effect of different promoter and insertion sequence on the expression of blaKPC gene was verified through the lacZ report plasmid fusion test. This promising project will provide the molecular mechanism of different carbapenem resistant levels of Klebsiella pneumoniae, and important clues for clinical treatment of drug-resistant bacteria.

碳青霉烯耐药肺炎克雷伯菌的出现给临床抗感染治疗带来了巨大挑战。我们前期研究发现，肺炎克雷伯菌对碳青霉烯酶耐药的机制主要由产blaKPC基因所编码的碳青霉烯酶所导致。此外，携带blaKPC的肺炎克雷伯菌对碳青霉烯存在不同水平的耐药，其形成机制不明。为此，本项目拟从多中心收集的肺炎克雷伯菌株对blaKPC基因的表达及blaKPC上游基因环境的分析入手，采用药敏试验、质粒转化、qRT-PCR技术，获取不同碳青霉烯耐药水平的临床菌株及转化子，并研究其blaKPC的表达水平变化。采用二代测序、引物延伸实验，测定blaKPC基因上游的基因环境。通过ChIP-Seq和凝胶阻滞试验，明确参与blaKPC基因转录及调控具体机制。通过lacZ报告质粒融合试验，验证不同启动子及插入序列对于KPC基因的表达的影响。最终阐明我国携带blaKPC基因的不同耐药水平菌株发生的分子机制，为临床抗击耐药菌提供重要线索。

碳青霉烯酶耐药的肺炎克雷伯菌（CRKP）是临床上重要的耐药菌。我国多中心碳青霉烯耐药的肠杆菌科细菌耐药监测显示，产KPC型碳青霉烯酶介导的CRKP是我国的主要流行株。为了探究携带blaKPC基因的肺炎克雷伯菌对碳青霉烯存在不同水平的耐药形成机制，本研究收集了我国多中心碳青霉烯耐药的肺炎克雷伯菌（CRKP）1801株，以CLSI推荐的参考方法进行了药敏试验。选取不同遗传背景携带blaKPC基因的CRKP进行了质粒接合试验，结果显示，接合子在获得blaKPC耐药基因的情况下并没有显示出原有的高水平耐药特征。对已完成全基因组测序的51株不同碳青霉烯酶耐药水平的肺炎克雷伯菌进行分析。OmpK35的缺失、OmpK36的135GD136氨基酸插入联合产KPC是高水平碳青霉烯酶耐药的ST11型CRKP的主要原因。通过评估多中心CRKP对头孢他定-阿维巴坦体外药敏试验结果显示，头孢他定-阿维巴坦对产KPC型CRKP具有较好的体外药敏活性。同时，通过分析KPC基因表达水平及拷贝数、OmpK36、OmpK37表达水平发现，阿维巴坦的耐药是blaKPC基因高表达与OmpK36及OmpK37的低水平表达协同结果，而blaKPC基因高表达与blaKPC基因的高拷贝数相关。推测恢复OmpK36的生物学功能对于降低产KPC型CRKP的耐药水平具有积极作用。