铜绿假单胞菌MexAB-OprM、MexXY-OprM主动外排系统的耐药作用

摘要

Objective To investigate the role of active efflux systems MexAB-OprM and MexXY-OprM in the multidrug resistance of Pseudomonas aeruginosa. Methods Thirty-six strains of multidrug-resistant P. Aeruginosa were isolated from clinical specimens. The minimum inhibitory concentration (MIC) to cefotaxime was determined by agar dilution technique. The interference experiment was conducted in the presence of pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mRNA of mexA and mexX gene was analyzed by real-time RT-PCR to estimate the expression level of MexAB-OprM and MexXY-OprM efflux systems. The amplified products were analyzed by sequencing. The mechanism of overexpression was studied using Blast software. Results In the presence of CCCP, 24 of the 36 strains of P. Aeruginosa showed 4 times or higher increase of susceptibility to ciprofloxacin. The active efflux phenotype was identified in 66. 7% (24/36) of the strains. MexABOprM efflux system was overexpressed in 41.7% (15/36) of the strains. MexXY-OprM efflux system was overexpressed in 58. 3% (21/36) of the strains. Fifteen strains overexpressed both MexAB-OprM and MexXY-OprM efflux systems simultaneously. Mutation of mexR and mexZ with corresponding amino acid substitution was identified in 4 of the strains overexpressing mexA and mexX. Conclusions Overexpression of active efflux systems MexAB-OprM and MexXY-OprM in multidrug-resistant P. Aeruginosa is one of the mechanisms contributing to the multidrug resistance of the bacteria which is caused by the mutation of the regulatory genes mexR and mexZ, respectively.%目的 探讨MexAB-OprM、MexXY-OprM主动外排系统(外排泵)在多重耐药铜绿假单胞菌耐药机制中的作用.方法 选取36株临床分离的多重耐药铜绿假单胞菌,采用琼脂二倍稀释法,测定环丙沙星对铜绿假单胞菌的MIC及进行泵抑制剂碳酰氰基对-氯苯腙(CCCP)存在情况下的干预试验；用实时定量RT-PCR测定结构基因mexA、mexX的mRNA表达水平来判断MexAB-OprM、MexXY-OprM外排泵表达情况；用PCR法分别扩增其调控基因mexR、mexZ,并对其产物测序,用Blast软件在GenBank与已知序列比较,研究其过度表达的机制.结果 在CCCP作用下,36株铜绿假单胞菌中24株菌对环丙沙星的敏感性(MIC)由20～320 mg/L提高到2.5～40 mg/L,主动外排表型阳性率为66.7％ (24/36),15株菌高表达MexAB-OprM外排系统(41.7％),21株高表达MexXY-OprM外排系统(58.3％).15株高表达MexAB-OprM外排系统铜绿假单胞菌同时表达MexXY-OprM外排系统；12株干预试验阴性的铜绿假单胞菌均无mexA、mexX高表达；随机选择mexA、mexX高表达的4株细菌均发生mexR、mexZ基因突变,出现氨基酸替代.结论 主动外排系统MexAB-OprM、MexXY-OprM在多重耐药铜绿假单胞菌中过度表达是铜绿假单胞菌多重耐药的机制之一；外排泵MexAB-OprM、MexXY-OprM高表达分别与调控基因mexR、mexZ发生变异有关.