不同遗传背景的肺炎克雷伯菌替加环素耐药演变分子机制研究

The emergence of tigecycline-resistant K. pneumoniae has aggravated the complication of the clinical treatments. Our previous study found that for the two-thirds of tigecycline-resistant K. pneumoniae (TRKP), RamRA was the major factor accounting for tigecycline resistance. However, for the left one third of TRKP clinical isolates, some other underlying mechanisms were presumed to contributing to its resistance. Experimental markerless deletion strains of ramA gene, KP17∆ramA and clinical isolates acquired in our previous study will be used as the parental strains, to serially select different levels of tigecycline resistant mutants of K. pneumoniae when exposing to tigecycline with sub minimal inhibitory concentration (MIC). The stability of the mutants will be detected by serial passages without tigecycline selective pressure. The phenotypic modulation and fitness costs of tigecycline resistant mutants vs. parental strains will be examined by growth rates, and in vitro competition assays. Each mutant will be studied by the whole genome and transcriptome sequencing analysis to identify the de novo adaptive mutations and genes of evolving to tigecycline resistance in K. pneumoniae. For the candidate mutations and differential expression genes, RT-qPCR, gene knock-out and homologous recombination techniques will be used to illustrate their functions. And finally, it will be clarified the molecular evolution mechanisms contributing to tigecycline resistance development in K. pneumoniae. This promising project will provide the evidence for innovative strategies to combat or delay the trend of TRKP.

替加环素耐药肺炎克雷伯菌的出现使临床抗感染治疗陷入困难境地。我们前期研究发现，RamRA调控子在替加环素耐药肺炎克雷伯菌中起重要调控作用，但仍存在其他尚未阐明的机制。此外，携带blaKPC、blaNDM或mcr-1基因的不同遗传背景肺炎克雷伯菌，其替加环素耐药形成风险不明。为此，本项目拟从前期已获得的敲除子KP17∆ramA和临床菌株两方面入手，采用替加环素亚MIC浓度体外逐级筛选，获取不同水平的替加环素耐药突变子，并研究其稳定性。采用药敏试验、生长速率和体外竞争试验研究耐药突变子较亲体株的表型变化和适应性代价；采用全基因组和转录组测序，找到耐药突变子的重要突变位点和差异表达基因。通过RT-qPCR大规模临床菌株验证、基因敲除和等位基因替换等，验证差异基因的功能。最终阐明不同遗传背景肺炎克雷伯菌替加环素耐药演变分子进化机制，为防控替加环素耐药肺炎克雷伯菌的播散及延缓耐药趋势提供重要基础。

替加环素是治疗碳青霉烯耐药肺炎克雷伯菌（Carbapenem resistant Klebsiella pneumoniae，CRKP）的挽救性治疗措施之一，可水平转移的粘菌素耐药基因mcr-1的报道发现使临床治疗CRKP陷入困境。CRKP和携带mcr-1基因的粘菌素耐药肺炎克雷伯菌对替加环素耐药形成和发展不明；且肺炎克雷伯菌调控替加环素耐药的分子机制尚未完全阐明，可能存在其他替代通路调控替加环素耐药性形成和发展。为证实“不同遗传背景的肺炎克雷伯菌，替加环素耐药发展的分子进化轨迹不同；且可能存在其他替代通路参与调控替加环素耐药性形成”这一假说，我们以实验室构建的替加环素全局调控子RamA敲除子KP17∆ramA和携带不同耐药元件（blaKPC、blaNDM和mcr-1基因）的临床菌株，分别通过体外逐级筛选获得替加环素低水平耐药和高水平耐药的耐药突变群体。相比于CRKP，携带mcr-1基因的粘菌素耐药肺炎克雷伯菌更易于进化为替加环素高水平耐药突变群体。在我国流行的产KPC酶ST11型CRKP也易获得替加环素耐药表型，但其生长延迟相延长，其生长优势极易被其他优势生长菌所替代。不同遗传背景的肺炎克雷伯菌在获得耐药性的同时，部分群体可能通过产生补偿突变如细菌菌落形态变化以维持替加环素耐药群体的稳定性。体外筛选获得的替加环素耐药突变群体其生长速率较亲体株明显减慢，产生了一定的适应性代价，其对数生长期和平台期的竞争指数均<1 。而RamA敲除子获得的替加环素耐药突变群体其平台期的竞争指数较对数生长期明显降低。通过比较基因组学分析发现耐药突变群体基因突变位点数越多，耐药突变子的耐药水平越高，细菌群体生长曲线的延迟相越长；携带mcr-1、产blaKPC-2或blaNDM-1的肺炎克雷伯菌替加环素耐药进化轨迹不一致，且同一亲体株不同生物学重复群体也存在耐药进化不一致现象；替加环素耐药形成是细菌群体基因累积突变过程，RamR是调控替加环素耐药的重要核心调控子；替加环素耐药分子调控机制复杂，lon可能与RamR共同调控肺炎克雷伯菌替加环素耐药进化。本研究提示临床应谨慎、合理使用替加环素，为控制或延缓替加环素耐药形成和发展提供一定分子研究基础。