海南某三甲医院多重耐药铜绿假单胞菌MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM表达及与耐药表型的关系研究  

Expression of MexAB-OprM,MexCD-OprJ,MexEF-OprN and MexXY-OprM in multidrug-resistant Pseudomonas aeruginosa and their relationship with drug resistance phenotypes in a Third Class A Hospital in Hainan

作　　者：王生成 杨祚明 蔡潇阳 张素领 王晓峰 WANG Sheng-cheng;YANG Zuo-ming;CAI Xiao-yang;ZHANG Su-ling;WANG Xiao-feng(Department of Respiratory Medicine,Danzhou 571700,China;De partment of Clinical Laboratory,Danzhou People's Hospital,Danzhou 571700,China;Department of Oncology,Danzhou People's Hospital,Danzhou 571700,China;Department of Respiratory Medicine,Hainan Cancer Hospital,Haikou 570100,China)

机构地区：[1]儋州市人民医院呼吸内科,海南儋州571700 [2]儋州市人民医院检验科,海南儋州571700 [3]儋州市人民医院肿瘤内科,海南儋州571700 [4]海南省肿瘤医院呼吸内科,海南海口570100

出　　处：《实用药物与临床》2020年第3期247-252,共6页Practical Pharmacy and Clinical Remedies

基　　金：海南省医药卫生科研项目(1421000320A2004)。

摘　　要：目的探讨多重耐药铜绿假单胞菌(Pseudomonas aeruginosa,Pa)MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM的表达及其与耐药表型的关系。方法选取2016年9月至2018年10月儋州市人民医院微生物培养室分离的Pa 54株,采用琼脂稀释法测定14种抗菌药物的MIC,加外排泵抑制剂苯丙氨酸-精氨酸-β萘酰胺(PAβN)进行干预实验,实时荧光定量PCR检测膜融合蛋白mexA、mexC、mexE、mexX的mRNA表达水平,以判断MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM的表达情况,PCR扩增调控基因mexR、nfxB、mexT、mexZ对扩增产物进行测序,使用Blast软件在GenBank上与已知序列进行比对。结果RT-PCR检测发现,耐药菌株中18株(33.33%)Pa高表达MexAB-OprM外排系统,9株(16.67%)高表达MexCD-OprJ外排系统,12株(22.22%)高表达MexEF-OprN外排系统,14株(25.93%)高表达MexXY-OprM外排系统。抑制剂作用下18株MexAB-OprM外排泵高表达Pa株对头孢噻肟、庆大霉素、环丙沙星的MIC为2~64μg/ml,9株MexCD-OprJ高表达株中6株对头孢噻肟的敏感性MIC为2~128 mg/ml,12株MexEF-OprN中对环丙沙星、亚胺培南的敏感性MIC为4~128 mg/ml,14株MexXY-OprM为庆大霉素、环丙沙星高度耐药菌株。分别随机选择mexA、mexC、mexE、mexX高表达菌株中的4个菌株,mexR高表达菌株中发生3个mexR基因突变;4个nfxB高表达菌株中均发生82位的突变;4个mexT高表达菌株中均发生26位的突变;2个mexZ高表达菌株发生138位的突变,1个发生50位的突变。结论主动外排系统MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM过度表达是Pa多重耐药机制之一,同时与其调控基因mexR、nfxB、mexT、mexZ发生突变相关。Objective To investigate the expression of MexAB-OprM,MexCD-OprJ,MexEF-OprN and MexXY-OprM in multidrug-resistant Pseudomonas aeruginosa(Pa)and their relationship with drug resistance phenotype.Methods From September 2016 to October 2018,54 strains of Pa isolated from microorganism culture room of Danzhou People′s Hospital were selected,the MIC of 14 antimicrobial agents was determined by agar dilution method,the intervention experiment was carried out with the efflux pump inhibitor of phenylalanine-arginine-βnaphthalamide(PAβN).Real-time fluorescence quantitative PCR test was used to detect the mRNA expression levels of membrane fusion proteins mexA,mexC,mexE and mexX to determine the expression of MexAB-OprM,MexCD-OprJ,MexEF-OprN and MexXY-OprM;the regulatory genes mexR,nfxB,mexT and mexZ were amplified by PCR and their amplified products were sequenced.Blast software was used to compare them with known sequences on GenBank.Results RT-PCR showed that 18 strains(33.33%)of the drug-resistant strains were Pa-highly expressed MexAB-OprM efflux system,9 strains(16.67%)were highly expressed MexCD-OprJ efflux system,12 strains(22.22%)were highly expressed MexEF-OprN efflux system and 14 strains(25.93%)were highly expressed MexXY-OprM efflux system.Under the action of inhibitors,the MIC of 18 strains of MexAB-OprM efflux pumps high-expressed Pa strains against cefotaxime,gentamicin and ciprofloxacin was 2~64μg/ml;the sensitivity MIC of 6 strains of 9 MexCD-OprJ high-expressed strains to cefotaxime was 2~128μg/ml;the sensitivity MIC of 12 MexEF-OprN strains to ciprofloxacin and imipenem was 4~128μg/ml;14 strains of MexXY-OprM were highly resistant to gentamicin and ciprofloxacin.Four strains of mexA,mexC,mexE and mexX high expression strains were selected randomly and respectively,3 mexR gene mutations occurred in the mexR high-expressed strains;82-position mutations occurred in all the 4 nfxB high-expressed strains;26-position mutations occurred in all the 4 mexT high-expressed strains;138-position mutations occ

关 键 词：多重耐药铜绿假单胞菌 MEXAB-OPRM MexCD-OprJ MexEF-OprN MexXY-OprM 

分 类 号：R446.5[医药卫生—诊断学]