亚胺培南和阿米卡星双耐药铜绿假单胞菌的特性分析

Characterization of the Pseudomonas aeruginosa strains showing resistance to both imipenem and amikacin

目的通过分析亚胺培南(IPM)和阿米卡星(AMK)同时耐药的铜绿假单胞菌的特性,为探讨耐药机制、评估菌株临床危害提供实验数据。方法针对临床分离的25株铜绿假单胞菌,采用Etest试纸条法测定IPM、AMK的最小抑菌浓度(MIC);通过RT-qPCR检测外排泵MexY和膜孔蛋白OprD表达水平;通过结晶紫染色测定生物膜形成能力;采用氯仿萃取法测定绿脓菌素产生量。结果依据25株铜绿假单胞菌对IPM和AMK的敏感度不同将其分为GroupⅠ(5株,IPM敏感或中介、AMK敏感)、GroupⅡ(8株,IPM耐药、AMK敏感或中介)、GroupⅢ(12株,IPM、AMK均耐药)三组,其中对AMK耐药的菌株同时对IPM耐药。GroupⅢ菌株在生物膜形成能力和绿脓菌素产量上明显强于GroupⅠ和GroupⅡ菌株,差异有统计学意义(P<0.05)。与参考菌株ATCC27853比较,GroupⅢ菌株的外排泵mexY表达显著上调4～12倍、膜孔蛋白oprD表达显著下调70%～80%,差异有统计学意义(P<0.05)。结论AMK耐药的铜绿假单胞菌极可能对IPM耐药;生物膜形成能力、mexY和oprD的表达变化可能参与了铜绿假单胞菌对IPM和AMK的同时耐药。

Objective To provide experimental data for the understanding of antibiotic-resistance mechanism and evaluating the strains harmfulness by characterizing the Pseudomonas aeruginosa(P.aeruginosa)strains which are resistant to both imipenem(IPM)and amikacin(AMK).Methods A total of 25 P.aeruginosa isolates were collected from Meizhou People’s Hospital.E test was conducted to measure the minimum inhibitory concentration(MIC)of four different antibiotics,including IPM,AMK.The RNA levels of the efflux pump mexY and the membrane porin oprD were detected by RT-qPCR.Biofilm formation ability was measured by crystal violet staining assay.Chloroform extraction method was used to evaluate the production of pyocyanin.Results Twenty-five isolates were classified into three groups according to their susceptibility to IPM and AMK.GroupⅠcontains 5 strains which were susceptible or intermediate susceptible to IPM and susceptible to AMK;GroupⅡcontains 8 isolates which were resistant to IPM but intermediate susceptible or susceptible to AMK;GroupⅢcontains 12 isolates which were resistant to both antibiotics.Noticeably,in which,the isolates resistant to AMK were also resistant to IPM.The isolates showing resistance to both IPM and AMK displayed stronger abilities in biofilm formation and pyocyanin production than other isolates(P<0.05),meanwhile,they up-regulated the expression level of the efflux pump gene mexY by 4~12 folds and down-regulated the membrane porin gene oprD by 70%~80%significantly.Conclusions The P.aeruginosa isolates which were resistant to AMK were prone to be resistant to IPM.The underlying mechanisms might be related to the changes of biofilm formation ability and the expression of oprD and mexY.