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目的研究质粒介导的喹诺酮类耐药机制(PMQR)在肺炎克雷伯菌临床分离株上的分布情况,并对阳性菌株上染色体介导的喹诺酮类耐药机制进行分析。方法细菌的鉴定和药敏采用Vitek-2 compact系统;采用PCR法检测质粒介导的喹诺酮类耐药基因qnrA、qnrB、qnrS、aac-(6’)-Ib-cr和qepA的分布情况。对包含PMQR的细菌,采用E-试验测定环丙沙星的MIC大小,同时扩增测序分析染色体基因gyrA、gyrB、parC、parE的突变情况。结果临床分离的67株肺炎克雷伯菌中,qnrS、qnrB、aac-(6’)-Ib-cr、qepA的检出率分别为14.93%、2.99%、2.99%和16.42%。8株细菌同时包含qnr和qepA基因,其中2株qnr、qepA和aac-(6’)-Ib-cr同时阳性。PMQR阳性菌株对环丙沙星的MIC值不定(0.032~≥64μg/mL),其中8株(占61.54%)对环丙沙星高水平耐药(≥64μg/mL)。比对结果显示,环丙沙星MIC≤0.5μg/mL的3株细菌几乎未见染色体的氨基酸序列改变;而环丙沙星MIC≥8μg/mL的菌株全部存在gyrA和parC编码氨基酸序列改变,且突变主要集中在gyrA 83位、87位和parC 80位上。所有PMQR阳性的肺炎克雷伯菌的gyrB和parE均未发现任何氨基酸序列突变。结论临床分离的肺炎克雷伯菌上检测到qnr、aac-(6’)-Ib-cr、qepA的分布与共存。PMQR阳性菌株对环丙沙星的MIC值不定,但染色体机制仍是肺炎克雷伯菌对喹诺酮类抗生素耐药的主要机制,突变主要见于gyrA的83位、87位及parC的80位上。
Objective To study the distribution of plasmid-mediated quinolone resistance mechanism (PMQR) in clinical isolates of Klebsiella pneumoniae and to analyze the mechanism of chromosome-mediated quinolone resistance in positive strains. Methods The bacterial identification and drug susceptibility were determined by Vitek-2 compact system. The distribution of quinolone resistance genes qnrA, qnrB, qnrS, aac- (6 ’) - Ib-cr and qepA were detected by PCR. For PMQR-containing bacteria, the MIC size of ciprofloxacin was determined by the E-test and the chromosomal gene gyrA, gyrB, parC and parE mutations were amplified by sequencing. Results The positive rates of qnrS, qnrB, aac- (6 ’) - Ib-cr and qepA in 67 strains of Klebsiella pneumoniae were 14.93%, 2.99%, 2.99% and 16.42%, respectively. Eight strains contained both qnr and qepA genes, of which two were positive for qnr, qepA and aac- (6 ’) - Ib-cr. MIC values of PMQR-positive strains were variable (0.032 ~ ≥64μg / mL) for ciprofloxacin, of which 8 strains (61.54%) were highly resistant to ciprofloxacin (≥64μg / mL). Alignment of the results showed that almost no chromosomal amino acid sequence was found in the three strains with MIC≤0.5μg / mL. However, all strains with ciprofloxacin MIC≥8μg / mL had gyrA and parC encoded amino acid sequence changes, The mutations were mainly located in gyrA 83, 87 and parC 80. No amino acid sequence mutations were found in gyrB and parE of all PMQR positive Klebsiella pneumoniae. Conclusion The distribution and coexistence of qnr, aac- (6 ’) - Ib-cr and qepA were detected in Klebsiella pneumoniae isolated clinically. The MIC value of PMQR-positive strains was variable for ciprofloxacin, but the mechanism of chromosome was still the main mechanism of quinolone antibiotic resistance in Klebsiella pneumoniae. The mutations were mainly found in gyrA 83, 87 and parC 80.