多重耐药铜绿假单胞菌外排泵基因表达与耐药表型和耐药程度的关系

目的建立实时荧光定量PCR法检测多重耐药铜绿假单胞菌(Pa)的外排泵基因表达量,探讨不同类型外排泵基因表达与耐药表型和耐药程度的关系。方法收集临床分离的多重耐药Pa共80株,分析其耐药表型;用实时荧光定量PCR检测不同类型外排泵基因的表达量,用外排泵抑制剂CCCP及PAβN进行外排泵基因筛查,并比较其结果。结果 64株(80%)检出了不同类型外排泵基因,包括单独表达MexA 30株(37.5%),MexC 12株(15.0%),MexX 8株(10.0%),同时表达MexA和MexC者10株(12.5%),同时表达MexA和MexX者4株(5.0%)。外排泵抑制剂筛查试验总阳性率分别为CCCP抑制法73.8%,PAβN抑制法72.5%。MexA的表达主要增强对美罗培南、三代头孢菌素、氟喹诺酮类、大环内酯类的耐药,且随表达量的增加而增强。MexC型主要增强对氨基糖苷类、氟喹诺酮类、三代头孢菌素、哌拉西林/他唑巴坦类的耐药性,且随表达量的增加而增强。MexX型外排泵的表达主要增强对氨基糖苷类、大环内酯类、氟喹诺酮类和氨曲南的耐药性。MexA和MexC型同时表达与MexA型一致,并增强了对氟喹诺酮类的耐药。MexA和MexX型同时表达与MexA型一致,并增强了对庆大霉素的耐药而降低了对氟喹诺酮类的耐药性。结论 Pa的外排泵基因的表达量与特定种类的药物耐药程度有关,外排泵基因的高表达参与并加剧了Pa的耐药性。

Correlation of the gene expression of efflux pumps with phenotypes and levels of antibiotic resistance in multidrug-resistant Pseudomonas aeruginosa

Objective To develop a real-time fluorescence quantitative PCR method for the detection of the gene expression of efflux pumps in multidrug-resistant Pseudomonas aeruginosa(Pa),and explore the correlation of the gene expression levels of different types of efflux pumps with the drug-resistant phenotypes and levels.Methods Eighty strains of multidrug-resistant Pa were isolated from the clinical samples,and the drug-resistant phenotypes were analyzed.The gene expression levels of different types of efflux pumps were determined by the real-time fluorescence quantitative PCR.The efflux pump inhibitors,e.g.,CCCP and PaβN,were used for screening efflux pump genes.Results The different efflux pump genes were detectable in 64 strains(80%) of Pa,including 30(37.5%) of MexA,12(15.0%) of MexC,8(10.0%) of MexX,10(12.5%) of MexA and MexC,and 4(5.0%) of MexX and MexA.The positive rates of CCCP and PAβN inhibiting the efflux pump gene expression were 73.8% and 72.5%,respectively.The expression of MexA was able to enhance the resistance of Pa to meropenem,the third generation cephalosporins,fluoroquinolones and macrolide,and the resistance level increased with the enhanced gene expression.The expression of MexC was able to strengthen the resistance of Pa to aminoglycosides,fluoroquinolones,the third generation cephalosporins and piperacillin/tazobactam,and the resistance level increased with the gene expression.The expression of MexX was able to increase the resistance of Pa to aminoglycosides,macrolides,fluoroquinolones and aztreonam.The coexpression of MexA and MexC,similar to the expression of MexA,also enhanced the resistance of Pa to fluoroquinolones,while the coexpression of MexA and MexX increased the resistance of Pa to gentamicin but reduced the resistance of Pa to fluoroquinolones.Conclusions The determination of the gene expression levels of efflux pumps in Pa may play an important role in evaluating the types and levels of drug resistance,and their expression levels are related to the resistant extent of scertain specific drugs,which demonstrate that the high level expression of the efflux pump genes may participate in and intensify the resistance of Pa to various drugs.