湖南省铜绿假单胞菌四种外排泵在多重耐药菌株中表达的分布及作用  

Distribution and effects of gene expression of four efflux pumps in Pseudomonas aeruginosa with multi-drug resistance in Hunan province

作　　者：蔡兴东[1] 胡成平[1] 李玮[1] 顾其华[1] 

机构地区：[1]中南大学湘雅医院呼吸内科,长沙410008

出　　处：《中华微生物学和免疫学杂志》2010年第2期130-134,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的探讨湖南省多重耐药（multi—drug resistance，MDR）铜绿假单胞菌（Pseudomonas aeruginosa，Pa）中4种外排泵表达的分布和作用，以及相关调控基因的突变情况。方法收集2008年湖南省临床首次分离非重复多重耐药几株40株，以外排泵抑制剂苯丙氨酸-精氨酸-β萘酰胺及4种药敏纸片进行外排泵表型初步筛选，PCR扩增膜融合蛋白基因及相关外排泵调控基因并测序；以18组非多重耐药菌株为对照研究分析Pa外排泵基因表达在多重耐药中的作用。结果表型筛选MDR组菌株MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY—OprM阳性率分别为45．0％（18／40）、30．0％（12／40）、42．5％（17／40）、12．5％（5／40）；RT-PCR显示mexA、mexC、mexE和mexX表达阳性率分别为100％（58／58）、22．5％（9／40）、45．0％（18／40）和22．5％（9／40）。Real—time PCR半定量分析菌株超表达mexA和mexX阳性率分别为55．0％（22／40）和22．5％（9／40）。非多重耐药组中，mexA与mexX real—time PCR超表达阳性率均为0（0／18）。mexC和mexERT-PCR表达阳性率分别为11．1％（2／18）、38．9％（7／18）。两组相比，mexA与mexX超表达差异有统计学意义（P〈0．001，P=0．045）。随机挑选超表达mexA菌株Pa20出现mexR的164GT→GAG、126Val→Glu替代；超表达mexX菌株Pa34出现mexZ的164GCGG→AG、55Ala→Glu。结论湖南省多重耐药Pn株大多存在主动外排的耐药机制，其耐药与外排基因Mex—AB-OprM和MexXY-OprM超表达有关。主动外排泵MexAB—OprM和MexXY-OprM超表达大多存在其调控基因突变。Abstract：Objective To investigate the effects and distribution of 4 efflux pumps and correlated mutation of regulatory gene in multi-drug resistance Pseudomonas aeruginosa （Pa） in Hunan province. Methods Forty non-duplicated clinical strains of multiple-drug-resistant Pseudomonas aeruginosa were collected in Hunan in 2008, then the phenotype was screened with efflux pumps inhibitor phenylalanine-L-β-naphthylamide and 4 antimicrobial susceptibility test discs. Genes of the efflux pump membrane fusion protein were amplified by PCR, and correlated efflux pump regulatory genes were amplified and sequenced to analyze the role of efflux pump gene expression in multi-drug resistance compared to that of 18 strains with non-multi-drug resistance. Re-suits The positivity rates of MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM were 45.0% （18/40）, 30.0% （12/40）, 42.5% （17/40） and 12.5% （5/40） respectively with phenotype screening in multi-drug resistance group. The positivity rates of mexA, mexC, mexE and mexX were 100% （58/58）, 22.5% （9/40）, 45.0% （18/40） and 22.5% （9/40） with RT-PCR. The overexpressed positivity rates of mexA and mexX were 55.0% （22/40） and 22.5% （9/40） respectively by semi-quantitative analysis with real-time PCR. However, no over-expression of mexA and mexX in non-multi-drug resistance group with real-time PCR. The positive expression rates of mexC and mexE with RT-PCR were 11.1% （2/18）, 38.9% （7/18） in non-multi-drug resistance group. The difference of overexpression of mexA and mexX was significant（P〈0.001, P=0.045） between two groups. The strain Pa20 with mexA overexpressian displayed gene mutations in mexR（164GTC→GAG） and amino acid substitution（126Val→Glu）, the strain Pa34 with overexpression of mexX had 164GCG→GAG,55Ala→GIu. Conclusion Most of Pa with multi-drug resistance contained the resistant mechanism of efflux pumps and most-ly due to the overexpression of MexAB-OprM and MexXY-OprM in Hunan. There are mostly regulato

关 键 词：铜绿假单胞菌 多重耐药 外排泵 外排泵抑制剂 

分 类 号：R378[医药卫生—病原生物学]