3所综合性医院分离的多重耐药铜绿假单胞菌外排泵MexAB-OprM的表达分析  

Expression analysis of MexAB-OprM in multidrug-resistant Pseudomonas aeruginosa effluent pump isolated from 3 general hospitals

作　　者：冯金鑫 张瑞琴[2] FENG Jinxin;ZHANG Ruiqin(School of Pharmacy,Shanxi Medical University,Taiyuan 030001,China;Department of Pharmacy,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学药学院,太原030001 [2]山西医科大学第二医院药学部,太原030001

出　　处：《西北药学杂志》2021年第5期833-837,共5页Northwest Pharmaceutical Journal

基　　金：山西省科技厅面上项目(编号:201801D121221)。

摘　　要：目的分析多重耐药铜绿假单胞菌(PA)外排泵MexAB-OprM的表达,并探讨MexAB-OprM高表达与调控基因mexR、nalC和nalD突变的关系。方法利用肉汤稀释法进行头孢他啶、头孢哌酮舒巴坦、亚胺培南、环丙沙星、阿米卡星和氨曲南的敏感性试验,联合外排泵抑制剂苯丙氨酸-精氨酸-β萘酰胺(PAβN)进行外排泵的表型筛选试验;在表型筛选为阳性的PA中随机挑选7株,实时荧光定量-PCR(RT-PCR)法检测膜融合蛋白基因mexA的相对表达量;PCR扩增调控基因mexR、nalC和nalD,将扩增结果测序比对。结果耐环丙沙星、头孢他啶、氨曲南、头孢哌酮舒巴坦、亚胺培南和阿米卡星PA外排泵的表型筛选阳性率分别为96.8%、44.8%、41.4%、41.4%、40.0%、10.0%;以野生型铜绿假单胞菌(PAO1)为对照菌,mexA的相对表达量范围为1.56~10.03(2.29±0.60),属于外排泵的高表达;高表达菌株均发生mexR、nalC和nalD基因突变和氨基酸替换。结论环丙沙星的耐药以外排泵耐药机制为主,调控基因mexR、nalC和nalD的碱基突变导致了外排泵MexAB-OprM高表达,外排泵的高表达导致了铜绿假单胞菌的耐药。Objective To analyze the expression of MexAB-OprM in multidrug-resistant Pseudomonas aeruginosa,and to explore the relationship between the high expression of MexAB-OprM and the mutations of regulatory genes mexR,nalC and nalD.Methods The minimum inhibitory concentrations of ciprofloxacin,imipenem,amikacin,aztreonam,ceftazidime and cefperazone-sulbactam,with or without phenylalanine-arginine-βnaphthalene amide(PAβN),an efflux pump inhibitor,were determined by broth dilution method to select the positive strains for an overexpressed active efflux pump.Real-time fluorescence quantitative PCR test was used to detect the mRNA expression levels of membrane fusion proteins mexA;the regulatory genes mexR,nalC and nalD were amplified by PCR and their amplified protects were sequenced.Blast software was used to compare them with known sequences on Genbank.Results The phenotypic screening positive rates of ciprofloxacin,ceftazidime,amtronam,cefoperazone and sulbactam,imipenem and amikacin were 96.8%,44.8%,41.4%,41.4%,40.0%and 10.0%,respectively.With type Pseudomonas aeruginosa(PAO1)as the control strain,the relative expression level of mexA ranged from 1.56 to 10.03(2.29±0.60),which belonged to the high expression of efferent pump.mexR,nalC,nalD gene mutation and amino acid substitution were observed in all high expression strains.Conclusion The drug resistance mechanism of ciprofloxacin was mainly the exogenous pump resistance.The base mutation of the regulatory genes mexR,nalC and nalD led to the high expression of efflux pump MexAB-OprM,and the high expression of efflux pump led to the drug resistance of Pseudomonas aeruginosa.

关 键 词：多重耐药铜绿假单胞菌 耐药性 外排泵MexAB-OprM 基因 

分 类 号：R969.3[医药卫生—药理学]