左氧氟沙星体外诱导铜绿假单胞菌外排泵耐药和交叉耐药机制研究  

Investigation of induced resistance and cross-resistance by levofloxacin in vitro and the resistant mechanism of efflux pumps in Pseudomonas aeruginosa

作　　者：王碧浪 金秀萍 王家学 WANG Bi-lang;JIN Xiu-ping;WANG Jia-xue(General Hospital of the Medical Community,Keqiao District Hospital of Traditional Chinese Medicine,Shaoxing,Zhejiang 312030,China;不详)

机构地区：[1]绍兴市柯桥区中医医院医共体总院,浙江绍兴312030 [2]杭州医学院检验医学院,浙江杭州310053

出　　处：《中国卫生检验杂志》2021年第6期680-683,共4页Chinese Journal of Health Laboratory Technology

基　　金：浙江省医学重点学科建设计划项目(2015-13);2021年浙江省医药卫生科技计划面上项目(2021KY640)。

摘　　要：目的研究左氧氟沙星(LEV)体外诱导铜绿假单胞菌(PA)外排泵耐药和交叉耐药的机制。方法多步诱导法进行铜绿假单胞菌体外LEV诱导耐药试验,倍比稀释法测定诱导成功菌株诱导前后对LEV、环丙沙星(CIP)、司帕沙星(SPX)和加替沙星(GAT)的最小抑菌浓度(MIC)。用外排泵抑制剂(PaβN)筛选主动外排表型阳性菌株,PCR扩增诱导成功菌株外排泵MexAB-Opr M相关基因mex B、opr M和mexR,mexR基因扩增产物测序分析。结果 12株铜绿假单胞菌诱导出稳定的左氧氟沙星耐药株,且对CIP、SPX和GAT产生不同程度的交叉耐药。7株诱导成功菌株主动外排表型阳性。PCR扩增mex B、opr M和mexR基因5株外排表型阴性菌株均阴性,7株外排表型阳性菌株中5株mex B、opr M和mexR均阳性、1株仅opr M基因阳性,mexR基因PCR产物测序结果与Gen Bank(NC002516)公布mexR基因比对,4株出现基因缺失、插入或替换突变,1株同源性为100%。结论左氧氟沙星可诱导铜绿假单胞菌产生获得性耐药,其耐药机制与主动外排相关。Objective To investigate the induced resistance and cross-drug resistance by levofloxacin in vitro and the resistant mechanism of efflux pumps in P. aeruginosa. Methods Induced resistance to Levofloxacin was performed by multiple-step method in 18 strains of P. aeruginosa. The susceptibility of the induced strains resistant to LVX and ciprofloxacin( CIP),spxacin( SPX),gatifloxacin( GAT) were measured before and after induction by detecting the MICs using microdilution method.Positive strains of active effluent phenotype were screened by effluent pump inhibitor( PaβN). The efflux pump genes of mex B,opr M and mexR were amplified by PCR,and then the nucleotide sequence of mexR gene was analyzed. Results Twelve strains of Pseudomonas aeruginosa induced stable drug resistance to levofloxacin,and showed different degree of cross resistance to CIP,SPX and GAT. 7 strains induced resistance were positive in phenotype tests. PCR-amplified mex B,opr M and mexR genes were negative in all 5 strains with negative pump phenotype,and in all 7 with positive pump phenotype strains,5 strains were positive in mex B,opr M and mexR,and 1 strain was only opr M gene positive. mexR sequencing comparing with Gen Bank accession number NC002516 showed 4 strains of gene deletion,insertion or replacement mutations,and 1 strain had 100%homology. Conclusion The acquired resistance of P. aeruginosa to Levofloxacin could be induced in vitro,and the efflux pump may play a role in some resistant strains.

关 键 词：左氧氟沙星 绿假单胞菌 体外诱导耐药 主动外排 耐药 交叉耐药 

分 类 号：R446.5[医药卫生—诊断学]