磷霉素联合多黏菌素E对产KPC酶肺炎克雷伯菌的协同杀菌作用及其机制研究

In recent years, the increasing prevalence of carbapenem-resistant Klebsiella pneumoniae strains poses a serious threat to patients. Fosfomycin and colistin, as the representatives of the old agents, had been demonstrated to be effective against multidrug resistance bacteria. However, fosfomycin and colistin monotherapy showed rapid selection of resistant variants, and the optimal values of pharmacokinetics-pharmacodynamics parameters for both drugs have not yet been clearly defined. The results of previous studies found the combination of fosfomycin and colistin showed synergistic activity and suppressed the emergence of resistant subpopulations. In addition, the activity of NADPH oxidase was increased, while the activity of superoxide dismutase and catalase were decreased. Therefore, it is of great value to clear how to promote rational utilization of fosfomycin combined with colistin against KPC-producing K. pneumoniae infections. To identify the maximum bactericidal scheme, we will examine the in vitro antibacterial effects by pharmacokinetics-pharmacodynamics analysis. Furthermore, the content of ROS formation, activity of oxidative stress key enzyme, permeability of cell membrane and cell wall, and DNA damage will be evaluated by molecular biological method. Moreover, we will do more experiments on transcriptomics analysis and gene knockout technology to clarify the mechanism of oxidative stress induced by the two drugs.

耐碳青霉烯类肺炎克雷伯菌的严峻形势给临床治疗带来了挑战。磷霉素和多黏菌素E作为“老药”代表被证实对多重耐药菌具有良好的抗菌活性，然而两药单用容易诱导快速耐药，且两药最佳药代动力学-药效学参数尚不完全明确。文献和我们近期的研究结果证实磷霉素联合多黏菌素E具有协同作用，两药联合后可明显降低耐药菌的出现，且产KPC酶肺炎克雷伯菌胞内的NADPH氧化酶活性增高，而超氧化物歧化酶和过氧化氢酶活性降低。因此研究如何合理使用磷霉素联合多黏菌素E治疗产KPC酶肺炎克雷伯菌感染具有十分重要的临床价值。本课题拟在前期基础上采用体外药代动力学-药效学研究确定两药联合的最大杀菌方案，使用分子生物学方法测定ROS生成含量、氧化应激关键酶的活性、细胞膜和细胞壁的通透性及DNA损伤。并采用转录组学分析与氧化应激相关功能基因的调控网络，使用基因敲除技术构建突变株，阐明两药联合后诱导的氧化应激反应机制。

背景及目的：耐碳青霉烯类肺炎克雷伯菌的严峻形势给临床治疗带来了挑战。本研究拟评估血流感染碳青霉烯耐药肺炎克雷伯菌（BSI-CRKP）的分子流行病学特征及产KPC酶肺炎克雷伯菌（KPC-Kp）感染危险因素，并通过体外实验评价磷霉素（FM）联合多黏菌素E（COL）的协同杀菌效果及作用机制。.方法：收集318株BSI-CRKP，检测药敏、毒力表型和基因型，并通过多位点序列分型分析其克隆相关性。采用回顾性配对病例对照研究分析KPC-Kp感染及导致死亡的危险因素。通过时间-杀菌曲线试验及突变率检测评价FM和COL单用及联合的抗菌效果、诱导耐药及细胞毒性。使用蒙特卡洛模拟（MCS）优化FM和COL的治疗方案。最后将时间-杀菌曲线试验与分子实验及转录组学研究相结合进一步明确FM联合COL的作用机制。.结果：在318株BSI-CRKP中，87.4%的菌株携带blaKPC-2基因，ST11型占66.6%。在多因素条件Logistic回归分析中，Pitt菌血症评分>4、胃管置入、连续性肾脏替代治疗和前期碳青霉烯类药物暴露与KPC-Kp感染相关。体外时间-杀菌曲线试验证实FM联合COL的抗菌效果、减少突变率及对Thp-1细胞的毒性优于单药。MCS发现对于肾功能正常患者，FM 8g q8h联合COL 9MIU负荷剂量，4.5MIU q12h的目标累积反应分数（CFR）接近80%。对于肾功能损伤患者，FM 6g/8g q8h联合COL负荷剂量方案可使CFR>90%。进一步研究发现FM联合COL作用于KPC-Kp后，在引起ROS累积同时可调控soxRS和氧化磷酸化水平清除ROS和修复损伤，并可通过抑制核糖体转录发挥杀菌作用。.结论：BSI-CRKP在我国不同地区存在克隆传播。针对KPC-Kp治疗，FM联合COL通过调控ROS累积和抑制核糖体蛋白转录介导协同杀菌作用，并可减少诱导耐药及细胞毒性，然而针对MIC和不同肾脏情况，两药的治疗方案应有所调整。

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