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目的:探讨染色体和质粒介导铜绿假单胞菌(PA)对喹诺酮类(QNs)的耐药机制。方法:采用微量肉汤稀释法测定环丙沙星对PA的最低抑菌浓度,用PCR方法扩增染色体基因gyrA、gyrB、parC、parE与质粒介导喹诺酮类耐药(PMQR)基因qnrA、qnrB、qnrC、qnrD、qnrS、aac(6’)-Ib-cr、qepA,并对PCR阳性产物进行测序以确定染色体基因喹诺酮类耐药决定区(QRDR)突变情况和PMQR基因亚型。结果:256株PA中有1株检测到qnrA1基因,环丙沙星对该菌株的MIC为2μg/ml,未检测到qnrB、qnrC、qnrD、qnrS、aac(6’)-Ib-cr和qepA基因。65株耐环丙沙星PA中gyrA基因错义突变49株(75.4%),均为ACC-83→ATC;parC基因错义突变15株(23.1%),为TCG-87→TTG;gyrB基因错义突变2株(3.1%),分别为TCC-467→TTC和CAG-468→CAT;parE基因未发现错义突变。结论:国内首次从PA中检测到介导环丙沙星低水平耐药的qnrA1基因。QRDR突变为染色体介导PA对QNs耐药的主要机制之一。
Objective: To investigate the mechanism of chromosome and plasmid-mediated resistance to quinolones (QNs) by Pseudomonas aeruginosa (PA). Methods: The minimal inhibitory concentrations of ciprofloxacin against PA were determined by micro broth dilution method. The chromosomal genes gyrA, gyrB, parC, parE and plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB , QnrC, qnrD, qnrS, aac (6 ’) - Ib-cr, qepA, and PCR positive products were sequenced to determine the mutation of quinolone resistance determinant (QRDR) and PMQR gene subtypes in chromosomal genes. RESULTS: qnrA1 gene was detected in one of 256 PA strains. MIC of ciprofloxacin to this strain was 2μg / ml. No qnrB, qnrC, qnrD, qnrS, aac (6 ’) - Ib-cr and qepA gene. There were 49 (75.4%) gyrA gene missense mutations in 65 resistant Ciprofloxacin PA groups, all of which were ACC-83 → ATC; 15 (23.1%) parC gene missense mutations were TCG-87 → TTG; gyrB gene Two missense mutations (3.1%) were TCC-467 → TTC and CAG-468 → CAT respectively. No missense mutation was found in parE gene. Conclusion: qnrA1 gene, which mediates low level of ciprofloxacin resistance, was detected in PA for the first time in China. QRDR mutation is one of the major mechanisms of chromosome-mediated PA resistance to QNs.