多重耐药大肠埃希菌喹诺酮类外排基因qepA的研究

Quinolone efflux pump gene, qepA, in multidrug-resistant isolates of Escherichia coli

目的对临床分离的多重耐药大肠埃希菌(E.coli)喹诺酮外排泵基因qepA的携带率、接合传递、基因功能等进行研究。方法临床分离的多重耐药E.coli 136株,经PCR筛选qepA基因,阳性菌株作16S rRNA甲基化酶基因rmtB的检测,PCR产物均经测序确认;阳性菌株进行质粒接合实验;扩增qepA基因全长,与pET-12a质粒连接,构建重组质粒,以研究qepA功能。结果 136株多重耐药E.coli qepA基因的检出率为14.0%(19/136),其中16株携带rmtB基因,二者的共存率高达84.2%(16/19)。接合实验的耐药质粒传递率为63.2%(12/19),9株接合子中检出rmtB基因,占75.0%(9/12)。与E.coli BL21(pET-12a)相比,诺氟沙星、环丙沙星和左氧氟沙星对E.coli BL21(pET-12a-qepA)的MIC分别升高64倍、32倍和4倍;而与羰基氰氯苯腙(CCCP)联合应用时三种药物的MIC值则至少降低为原值的1/4。进一步检测菌体内环丙沙星含量,在不含CCCP时,E.coli BL21(pET-12a-qepA)菌体中环丙沙星低于E.coli BL21(pET-12a)或E.coli BL21(P<0.01);加入CCCP后,菌体内环丙沙星含量明显升高(P<0.01)。结论本组多重耐药E.coli的qepA基因检出率较高(14.0%)。qepA基因和rmtB基因具有较密切的相关性和共存率,且容易在菌际间传播。QepA为质子依赖的外排蛋白,可降低某些喹诺酮类药物在细菌体内的含量,使得对药物的敏感性下降。

Objective To investigate plasmid-mediated quinolone effiux pump gene qepA in multidrug- resistant isolates of E. coli. Methods A total of 136 clinical multidrug-resistant isolates of E.coli were collected. The qepA gene screening was carried out by PCR, and 16S rRNA methylase gene, rmtB, was detected among qepA- positive isolates by PCR. DNA sequencing was employed to determine the nucleotide sequence. Transconjugation was performed. The whole qepA gene was cloned to pET-12a, and thus the functional assay of its gene product was performed. Results The qepA gene was present in 19 of the 136 isolates （14.0%）. rmtB was found in 16 of 19 qepA- positive isolates, thus the coexistence rate was 84.2% （16/19）. The efficiency of transformation was 63.2% （12/19）. rmtB was found in 9 of 12 transconjugants （75.0%）. E.coli BL21 （pET-12a-qepA） has 64-, 32- and 4-fold higher MICs, respectively, for norfloxacin, ciprofloxacin and levofloxacin in comparison with E.coli BL21 （pET-12a）. In combination with carbonyl cyanide m-chlorophenylhydrazone （CCCP）, MICs of the three quinolones deceased by at least 4 folds for E.coli BL21 （pET- 12a-qepA）. The accumulation of ciprofloxacin in E.coli BL21 （pET- 12a-qepA） was significantly lower （P〈0.01） than that in E.coli BL21 （pET- 12a） or E. coli BL21, and became significantly higher after the addition of CCCP（P〈0.01）. Conclusion qepA was highly prevalent （14.0%） in multidrug-resistant isolates of E.coli and there was a strong linkage between qepA and rmtB. The efficiency of transformation was 63.2% （12/19）, indicating that qepA could be transmitted among the bacteria with the same genus. QepA, the proton dependent effiux pump, was involved in the decline of susceptibility and resistance to some quinolones.