新外排泵（KpgD/KpgE）介导的肺炎克雷伯菌对替加环素耐药机制及其调控研究

Published studies have demonstrated that tigecycline resistance mainly conferred by the overproduction of AcrAB efflux pump in Klebsiella pneumoniae. In our previous study, the mechanisms of tigecycline-resitant K. pneumoniae have been explored, and the results suggested the presence of alternative resistance mechanisms in these strains. This study aims to: 1) in vitro obtain tigecycline-resistant K. pneumoniae strains by tigecycline induced experiments, and then explore the tigecycline resistance mechanisms of these mutants (done), 2) detect the difference of gene expression (mainly focus on the efflux pump related genes) between tigecycline-susceptible K. pneumoniae parental strains and their derivatives using RNA-seq (done), and confirm the novel efflux pump gene mediating tigecycline resistance by gene knockout, 3) compare the difference in the local regulatory sequence of the novel tigecycline resistance gene between tigecycline-resistant and -susceptible strains , and then carry out gene clone, and gene knockout to know the role of the local regulators; confirm the global regulators (such as RamA) of the novel efflux pump genes through gene knockout, and finally identify the regulatory pathways of the novel resistance mechanism. Further study on the tigecycline resistance mechanisms will be helpful for clinical rational use of this agent, and provide clues for the development of new drugs.

现有研究提示肺炎克雷伯菌对替加环素的耐药主要由多药外排泵AcrAB过表达引起。但申请人前期对替加环素耐药肺炎克雷伯菌耐药机制研究发现，有部分菌株的耐药机制不能用现有机制解释，提示存在新耐药机制。本研究：①通过替加环素梯度诱导的方法，在体外获得替加环素耐药株，并对耐药株行现有机制验证（已完成）；②对现有机制不能解释的替加环素耐药菌株及其亲本，通过转录组测序发现差异表达基因，筛选出可能参与替加环素耐药的新外排泵基因（已完成），拟对候选基因行基因敲除和回补，确定介导替加环素耐药的新基因；③拟比较新外排泵基因周边调控序列在敏感株及其诱导耐药株之间的差异，通过基因克隆、基因敲除和回补等方法，阐述新基因的局部调控通路；拟对整体调控子如RamA行基因敲除，确定新外排泵基因的整体调控通路，最终阐明新耐药基因的调控通路。通过对替加环素耐药机制的深入研究将为该药的临床合理应用提供依据，为新药的研发提供思路。

近年来，全球范围内肺炎克雷伯菌耐药形式严峻，多重耐药肺炎克雷伯菌引起的感染病死率高，治疗药物非常有限。替加环素是为数不多的几个可选药物之一，但是，在临床使用过程中易发生耐药。目前认为外排泵AcrAB高表达是介导肺炎克雷伯菌对替加环素耐药主要机制，该外排泵主要受整体转录调控子RamA调控。我们前期从1000余株临床分离肺炎克雷伯菌中筛选到到26株替加环素不敏感肺炎克雷伯菌，发现约1/3菌株的替加环素耐药机制不能用现有机制解释。在本项目中，我们通过体外梯度替加环素诱导耐药肺炎克雷伯菌株，并对这些菌株进行现有机制的验证，发现75%的替加环素耐药菌株不能用现有机制解释。但诱导菌株均有转录调控子RamA的转录高表达，进一步研究发现几乎所有菌株的ramR均发生了错义突变。通过其中替加环素高耐药水平（＞128 mg/L）的菌株进行转录组测序和分析，发现外排泵kpgD/kpgE呈高表达。用同源重组的方法分别对kpgD、kpgE、acrB、oqxB、ramA基因敲除和/或回补，对敲除和/或回补株进行替加环素及其他多种药物最低抑菌浓度（MIC）测定和转录水平表达，结果发现RamA可以调控acrB和oqxB高表达，进而介导呋喃妥因、喹诺酮类、头霉素类多种抗菌药物耐药。acrB敲除株的替加环素MIC水平较亲本显著降低，oqxB、kpgD、kpgE基因敲除株的替加环素MIC较亲本无变化。通过对K2606及其子代K2606-4、K2606-16全基因组测序和转录组测序分析，发现外排泵tetA基因在两个子代肺炎克雷伯菌中均发生突变。本项目首次证实转录调控子RamA可以调控外排泵OqxAB介导多种抗菌药物耐药，进一步完善了RamA的调控通路。进一步明确AcrAB是介导肺炎克雷伯菌对替加环素耐药的重要因素，外排泵TetA可能参与肺炎克雷伯菌对替加环素耐药，外排泵OqxAB、KpgD、KpgE不参与替加环素耐药。本课题对临床合理使用替加环素提供了重要理论依据。