耐醋酸氯己定肺炎克雷伯菌的耐药机制及分子流行病学研究

Resistance mechanisms and molecular epidemiology of chlorhexidine acetate-resistant Klebsiella pneumoniae clinical isolates

目的研究临床分离肺炎克雷伯菌对醋酸氯己定消毒剂的耐药性、耐药机制及分子流行病学情况。方法收集2015年温州医科大学附属第一医院临床分离的肺炎克雷伯菌共332株,琼脂稀释法筛选醋酸氯己定耐药菌株并检测外排泵抑制剂羰基氰氯苯腙(CCCP)对醋酸氯己定最小抑菌浓度(MIC)的影响;聚合酶链反应(PCR)和实时荧光定量PCR(RT-qPCR)分别检测与醋酸氯己定抗性相关的外排泵基因cepA、qacE、qacΔE1的携带情况和表达情况;半定量结晶紫法检测生物膜形成能力;多位点序列分型(MLST)和脉冲场凝胶电泳(PFGE)分析醋酸氯己定耐药菌株间的同源性。结果25株醋酸氯己定耐药肺炎克雷伯菌在加入CCCP后的醋酸氯己定MIC值比加入之前的MIC值均降低4倍及以上;醋酸氯己定抗性基因cepA、qacE和qacΔE1的携带率分别为100%、40%和40%,且醋酸氯己定耐药组的上述基因表达水平均显著高于敏感组;醋酸氯己定耐药菌株的生物膜形成能力显著低于醋酸氯己定敏感菌株,其中,成膜能力阴性4株(16%),弱阳性20株(80%),阳性1株(4%);MLST和PFGE显示25株醋酸氯己定耐药菌株分别属于19种ST型和各种不同PFGE型。结论主动外排是肺炎克雷伯菌对醋酸氯己定消毒剂耐药的主要机制;醋酸氯己定耐药肺炎克雷伯菌具有不同程度的生物膜形成能力;耐药菌株的同源性较低。

Objective To investigate the chlorhexidine acetate-resistance in Klebsiella pneumoniae(K.pneumoniae)clinical isolates and to analyze the possible mechanisms and molecular epidemiology of resistant isolates.Methods A total of 332 K.pneumoniae clinical isolates were collected in the First Affiliated Hospital of Wenzhou Medical University in 2015.Standard agar dilution was used to screen chlorhexidine acetate-resistant isolates.The minimum inhibition concentrations(MIC)of chlorhexidine acetate to resistant isolates with and without the presence of carbonyl cyanide m-chlorophenyl hydrazone(CCCP),which was an efflux pump inhibitor,were analyzed.Efflux pump genes of cepA,qacE and qacΔE1 that carried by and expressed in those isolates were detected by polymerase chain reaction(PCR)and quantitative real-time PCR(RT-qPCR),respectively.The biofilm formation ability was measured by crystal violet staining.The homology among the chlorhexidine acetate-resistant isolates was investigated with multilocus sequence typing(MLST)and pulsed-field gel electrophoresis(PFGE).Results Twenty-five K.pneumoniae strains were resistant to chlorhexidine acetate.The MIC values of chlorhexidine acetate for them were reduced by at least four-fold in the presence of CCCP.Strains carrying the genes of cepA,qacE and qacΔE1 accounted for 100%,40%and 40%,respectively.The expression of the efflux pump genes in the chlorhexidine acetate-resistant isolates was higher than that in the susceptible isolates.The biofilm formation ability of the chlorhexidine acetate-resistant isolates was better than that of the susceptible isolates.Furthermore,negative,weak-positive and positive biofilm formation ability was observed in four(16%),20(80%)and one(4%)strains,respectively.The results of MLST and PFGE showed that the 25 chlorhexidine acetate-resistant isolates belonged to 19 different sequence types(ST)with diverse PFGE patterns.Conclusions This study suggested that active efflux was the main mechanism of chlorhexidine acetate resistance in K.pneumoniae.The 25 chlorhexidine acetate-resistant K.pneumoniae strains possessed different biofilm formation ability and shared low homology.