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 investigate the prevalence of plasmid-mediated quinolone resistance(PMQR) among unique clinical strains of K.pneumoniae, and to identify the chromosome-mediated mechanisms of DNA gyrAse and Topoisomerase IV among those PMQR-positive isolates.Methods Identification and antimicrobial susceptibility were performed by Vitek-2 compact system.Screening of genes involved in PMQR, including qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA , was carried out by PCR amplification.Among PMQR-positive strains, MIC of ciprofioxacin was evaluated by E-test.Genes related to chromosome mediated quinolone resistance including gyrA, gyrB, parc and parE were sequenced and analyzed after amplifying by PCR in those PMQR- borne strains.Results The presence of qnrS, qnrB, aac-(6')-Ib-cr and qepA gene was 14.93％(8/67), 2.99％(2/67), 2.99％(2/67) and 16.42％(9/67) among the strains detected respectively.And co-existence of qnr, qepA and aac-(6')-Ib-cr gene were displayed in 2 strains.The results demonstrated that MICs of ciprofloxacin to PMQR-positive strains range from 0.032μg/mL to ≥ 64μg/mL,and 8 strains displayed high ciprofloxacin MIC(≥ 64μg/mL).Sequence analysis showed that most of the mutation located at 83, 87 of gyrA and 80 of parC gene for those strains with ciprofloxacin MIC above 8μg/mL.And no sequence change has been found in 3 strains with ciprofloxacin MIC below 0.5μg/mL.However, no mutation of gyrB and parE was identified in all PMQR-borne strains.Conclusion The results suggested the susceptibility of PMQR-borne isolates to ciprofloxacin may be sensitive or resistance.The presence and/or co-existence of those PMQR genes might contribute to quinolone resitance in clinical K.pneumoniae isolates.And chromosome-mediated target mutation still plays important roles in K.pneumoniae resistant to quinolones.
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