耐多黏菌素肺炎克雷伯菌的分子流行病学及毒力相关研究

Molecular epidemiology and virulence characteristics of polymyxin-resistant Klebsiella pneumonia

目的分析耐多黏菌素肺炎克雷伯菌的耐药机制、分子流行病学和毒力特征。方法2011年至2016年温州医科大学附属第一医院住院患者的各类临床标本中共分离到1376株肺炎克雷伯菌。采用琼脂稀释法筛选多黏菌素耐药菌株;采用聚合酶链反应检测多黏菌素耐药相关基因,采用实时荧光定量聚合酶链反应检测耐药基因的mRNA相对表达水平;采用脉冲场凝胶电泳、多位点序列分型和大蜡螟幼虫感染模型分别研究耐药菌株的分子流行病学和毒力特征。结果从1376株肺炎克雷伯菌菌株中,共筛选出14株(1.02%)耐多黏菌素肺炎克雷伯菌。10株耐多黏菌素菌株的MgrB发生氨基酸替换(K2E、F28C),9株菌株的PhoQ发生氨基酸替换(D150G)。所有耐药菌株中均未检测到mcr、crrB等基因,与标准菌株相比,耐药菌株的pmrH和pmrD的mRNA相对表达水平增加。分子流行病学结果显示,14株耐药株分为9种序列型;大蜡螟幼虫感染模型表明耐多黏菌素肺炎克雷伯菌具有更高的毒力。结论耐多黏菌素肺炎克雷伯菌多存在mgrB和phoQ基因突变,且mgrB基因突变可能在菌株毒力的变化中起关键作用;本研究中的多黏菌素耐药菌株之间同源性较低。

Objective To analyze the molecular epidemiology and virulence characteristics of polymyxin-resistant Klebsiella pneumoniae(K.pneumoniae).Methods From 2011 to 2016,1376 strains of K.pneumoniae were isolated from various clinical specimens of hospitalized patients in First Affiliated Hospital of Wenzhou Medical University.Agar dilution method was used to screen out the polymyxin-resistant strains.Polymerase chain reaction(PCR)was used to detect the genes related to polymyxin resistance,and real-time fluorescence quantitative PCR was used to detect the relative mRNA expression level of drug resistant genes.Pulsed-field gel electrophoresis,multilocus sequence typing and Galleria mellonella larvae infection model were performed to analyze the molecular epidemiological and virulent characteristics.Results A total of 14 strains(1.02%)of polymyxin-resistant K.pneumoniae were detected among 1376 K.pneumoniae isolates.Subsequent sequencing identified mutations leading to amino-acid changes(K2E,F28C)in MgrB of 10 isolates and D150G in PhoQ of nine isolates,and genes such as mcr and crrB were not detected in all drug-resistant strains.Compared with standard strains,the relative expression levels of pmrH and pmrD mRNA of these drug resistant strains were increased.Analysis of the molecular epidemiology indicated that the 14 drug-resistant strains were divided into nine clones.Galleria mellonella larvae infection model revealed polymyxin-resistant K.pneumoniae isolates had higher virulence.Conclusions Polymyxin-resistant K.pneumoniae has mutations in mgrB and phoQ genes,and mgrB mutation may play a key role in the change of virulence profiles.The homology among the polymyxin-resistant K.pneumoniae stains in this study is low.