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。

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.