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目的了解临床分离的携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌对喹诺酮类药物的耐药机制,为临床治疗提供参考依据。方法在2008年11月-2009年7月从住院患者中分离19株携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌,采用聚合酶链反应(PCR)及序列分析的方法分析两种喹诺酮类药物作用靶位编码基因(gyrA、parC)和5种质粒介导的喹诺酮类耐药相关基因。结果 19株携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌gyrA和parC基因PCR扩增均阳性,1株(5.3%)aac(6′)-Ⅰb-cr基因阳性,qnrA、qnrB、qnrS和qepA基因均阴性;序列分析结果表明,19株gyrA和parC基因均发生突变,分别导致gyrA基因喹诺酮耐药决定区(QRDR)出现两个位点错义突变,导致第83位丝氨酸(Ser)被异亮氨酸(Ile)取代、第87位天冬氨酸(Asp)被甘氨酸(Gly)取代,parC基因QRDR出现1个位点错义突变,导致第80位丝氨酸被异亮氨酸取代。结论染色体介导的耐药机制仍是临床分离的携带blaKPC-2型碳青霉烯酶基因泛耐药肺炎克雷伯菌对喹诺酮类药物耐药主要机制。
Objective To understand the mechanism of resistance to quinolones in clinical isolates of Klebsiella pneumoniae carrying blaKPC-2 carbapenemase gene and to provide a reference for clinical treatment. Methods Nineteen strains of pan-resistant Klebsiella pneumoniae carrying blaKPC-2 carbapenemase gene were isolated from hospitalized patients from November 2008 to July 2009. PCR-based polymerase chain reaction (PCR) and sequence analysis Methods The genes encoding gyrA, parC and quinolone resistance genes of two quinolones were analyzed. Results A total of 19 strains of gyrA and parC gene of Klebsiella pneumoniae carrying blaKPC-2 carbapenemase gene were positive for PCR amplification and 1 (5.3%) for aac (6 ’) -Ib-cr gene was positive, qnrA, qnrB, qnrS and qepA genes were negative. The results of sequence analysis showed that 19 gyrA and parC genes were mutated, leading to missense mutations in the gyrA gene quinolone resistance determining region (QRDR) Serine is replaced by isoleucine, the 87th aspartic acid is replaced by glycine, the parC gene QRDR has a 1-site missense mutation, resulting in serine 80 Isoleucine substitution. Conclusion Chromosome-mediated mechanism of drug resistance is still the main mechanism of clinical isolates of quinolones resistant to pan-resistant Klebsiella pneumoniae carrying the blaKPC-2 carbapenemase gene.