耐环丙沙星铜绿假单胞菌的耐药性及耐药机制研究

Study on antimicrobial resistance and drug resistance mechanisms of ciprofloxacin-resistant Pseudomonas aeruginosa

目的了解耐环丙沙星铜绿假单胞菌(CRPAE)的药敏谱特征及其对环丙沙星的耐药机制。方法采用微量肉汤稀释法测定环丙沙星对铜绿假单胞菌(PAE)的最低抑菌浓度(MIC),K-B纸片扩散法检测PAE对常用抗菌药物的敏感性,并比较CRPAE与环丙沙星敏感铜绿假单胞菌(CSPAE)的耐药性差异;应用PCR方法扩增CRPAE的gyrA、gyrB、parC、parE基因,并对PCR产物进行测序分析以确定基因的突变。结果 256株无重复PAE中,CRPAE 65株,CSPAE 180株,CRPAE对多数抗菌药物的耐药率明显高于CSPAE(P<0.05);65株CRPAE中gyrA基因第83位密码子(ACC→ATC)错义突变49株占75.4%,parC基因第87位密码子(TCG→TTG)错义突变15株占23.1%,gyrB基因第467位密码子(TCC→TTC)和第468位密码子(CAG→CAT)错义突变各1株占3.1%,未发现parE基因错义突变株;gyrA+gyrB、gyrA+parC双突变株的环丙沙星MICs高于单纯gyrA突变株。结论 CRPAE多药耐药现象严重,gyrA基因第83位密码子(ACC→ATC)突变是CRPAE对环丙沙星耐药的主要机制之一,同时存在gyrB或parC基因突变可进一步升高CRPAE对环丙沙星的耐药性。

OBJECTIVE To explore the antimicrobial resistance and the drug resistance mechanisms of ciprofloxacin- resistant Pseudomonas aeruginosa （CRPAE）. METHODS Minimal inhibitory concentrations （MICs） of eiprofloxacin to P. aeruginosa （PAE） were determined by broth microdilution method, and antimicrobial susceptibility testing was performed by Kirby-Bauer disk diffusion method, and the drug resistance rates of CRPAE were compared with ciprofloxacin-suseeptible P. aeruginosa（CSPAE）. Chromosome genes gyrA, gyrB, parC, pare of CRPAE were amplified by PCR, then the PCR products were sequenced to identify the mutations in these genes. RESULTS There were 65 strains of CRPAE and 180 strains of CSPAE in 256 non-repetitive PAE isolates. The resistant rates of CRPAE to most antimicrobial agents were significantly higher than that of CSPAE （P^O. 05）. Among the 65 CRPAE isolates, missense mutations of codon 83 （ACC-~ATC） of gyrA gene were found in 49 strains（75.4%） , and codon 87（TCG-~TTG） of parC gene in 15 strains（23.1~）, while codon 467 （TCC--~TTC） or codon 468（CAG--~CAT） of gyrB gene in 1 strain（3. 1 ~） respectively. Missense mutation of pare gene was not detected in all CRPAE isolates. MICs of ciprofloxacin to the strains with double mutations of both gyrA and gyrB, or gyrA and parC gene, were significantly higher than that of the strains with gyrA gene mutation only. CONCLUSION The CRPAE strains are highly drug resistant;mutation of 83 codon （ACC--ATC） of gyrA gene is one of the main resistant mechanisms of CRPAE to ciprofloxacin, and this resistance will be enhanced by gyrB or parC gene mutations at the same time.