碳青霉烯类耐药铜绿假单胞菌外排泵MexAB-OprM的研究

Study on efflux pump MexAB-OprM in carbapenem-resistant Pseudomonas aeruginosa

目的研究铜绿假单胞菌对碳青霉烯类耐药与外排泵MexAB-OprM的相关性。方法琼脂稀释法测定亚胺培南和美罗培南对75株铜绿假单胞菌的最低抑菌浓度(MIC),联合外排泵抑制剂MC207110进行外排泵表型的筛选;用反转录-聚合酶链反应(RT-PCR)扩增MexAB-OprM融合蛋白结构基因mexA以及内参基因rpoD,并测定其mRNA的表达量,用PCR扩增MexAB-OprM相对高表达菌株的外排泵调节基因mexR、nalC和nalD,对扩增产物进行DNA双向测序;结果进行BLAST比对分析。结果 75株铜绿假单胞菌中外排泵表型阳性菌株13株,其中10株细菌的MexAB-OprM相对表达量增高,此高表达MexAB-OprM菌株的调节基因mexR、nalC及nalD均阳性。其中9株菌nalC均发生第71位氨基酸突变(甘氨酸→谷氨酸)、8株菌同时还有第209位氨基酸突变(丝氨酸→精氨酸),仅发现1株菌nalD第158位氨基酸突变(苏氨酸→异亮氨酸),8株菌mexR发生突变。结论外排泵MexAB-OprM的高表达在铜绿假单胞菌对碳青霉烯类耐药中可能起到重要作用。MexAB-OprM的高表达与其调节基因突变相关。

Objective To study the relationship between efflux pump MexAB‐OprM and carbapenem resistance of Pseudomonas aerginosa strains .Methods The minimum inhibitory concentrations of imipenem and meropenem were determined by agar dilution method for 75 strains of P .aerginosa in the absence or presence of MC207110 to screen the phenotypes of active efflux pump .Reverse transcriptase‐polymerase chain reaction （RT‐PCR） method was used to determine the mRNA expression level of mexA which encodes the membrane fusion protein in active efflux pump MexAB‐OprM and the reference （housekeeping） gene rpoD .PCR method was used to amplify the regulatory genes mexR ,nalC ,and nalD of active efflux pump MexAB‐OprM in the strains overexpressing the efflux pump . The PCR products were subject to DNA sequencing and BLAST analysis . Results Of the 75 P .aeruginosa strains ,13 （17 .3% ） were positive for efflux pump MexAB‐OprM .Overexpression of the efflux pump was identified in 10 of the 13 strains and associated with positive regulatory genes mexR ,nalC and nalD .A Gly71→Glu mutation in nalC was found in 9 strains ,and a Ser209→Arg mutation in nalC was identified in 8 strains .Only one strain had a Thr158→Ile mutation in nalD .Eight strains had mutation in mexR .Conclusions Overexpression of multidrug efflux pump MexAB‐OprM plays an important role in carbapenem resistance of P .aeruginosa .High level expression of MexAB‐OprM is related to the mutations of its regulatory genes .