肺炎克雷伯菌质粒介导的喹诺酮类抗生素耐药机制的研究  

Research of plasmid-mediated quinolone resistance among clinical isolates of Klebsiella pneumoniae

作　　者：马晓波[1] 林粼[2] 张加勤[1] 赵元勋[1] 郑港森[1] 郑燕青[1] 房丽丽[1] 宋秀宇[1] 吴维生[1] 

机构地区：[1]厦门大学附属第一医院检验科,厦门361003 [2]厦门大学附属第一医院中心实验室,厦门361003

出　　处：《中国抗生素杂志》2011年第8期625-629,共5页Chinese Journal of Antibiotics

基　　金：厦门市科技局(3502Z20084003;3502Z20089003);福建省卫生厅青年课题2010-2-90

摘　　要：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－2compact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，（69－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qn，和卵p4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（≥64μgg／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gyrA83位、87位~[1parC80位上。所有PMQR阳性的肺炎克雷伯菌的∥膪和pa旭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 ciprofloxacin was evaluated by E-test. Genes related to chromosome mediated quinolone resistance including gyrA, gyrB, parC andparE 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 ciprofloxaein to PMQR-positive strains range from 0.032gg/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 ofgyrA and 80 ofparC 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 ofgyrB 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.

关 键 词：肺炎克雷伯菌 质粒介导喹诺酮类耐药 QNR aac-(6')-Ib-cr QEPA GYRA 

分 类 号：R378.2[医药卫生—病原生物学]