产KPC型β-内酰胺酶肺炎克雷伯菌对氟喹诺酮类耐药机制研究

Investigation of fluoroquinolone-resistant mechanism of KPCβ-lactamase-producing Klebsiella pneumoniae isolates

目的探讨产KPC型β-内酰胺酶肺炎克雷伯菌对氟喹诺酮类药物的耐药机制,为指导临床合理使用抗菌药物提供依据。方法两所三甲综合性医院临床分离的23株产KPC型β-内酰胺酶肺炎克雷伯菌,采用K-B纸片法药敏试验,聚合酶链反应(PCR)方法对氟喹诺酮类药物作用靶位编码基因gyrA、parC和可移动遗传元件介导的aac(6′)-Ⅰb-cr、qnrA、qnrB、qnrS、qepA基因进行检测,对阳性产物进行测序,测序结果作BLAST对比分析。结果 23株产KPC型β-内酰胺酶肺炎克雷伯菌检出拓普异构酶ⅡA亚单位编码基因,gyrA喹诺酮耐药决定区(QRDR)的第83位密码子TCC→ATC有义突变(氨基酸序列S→I),第87位密码子GAC→GGC有义突变(氨基酸序列D→G),且23株菌株均出现拓普异构酶ⅣC亚单位编码基因parC喹诺酮耐药决定区(QRDR)第80位密码子AGC→ATC有义突变(氨基酸序列S→I);但23株产KPC型β-内酰胺酶肺炎克雷伯菌由可移动遗传元件可介导的氟喹诺酮类耐药相关基因(aac(6′)-Ⅰb-cr、qnrA、qnrB、qnrS、qepA)检测均为阴性。结论 23株产KPC型β-内酰胺酶肺炎克雷伯菌的gyrA与parC基因QRDR区的有义突变是氟喹诺酮类药物耐药的主要机制,加重了其耐药性,给临床抗菌药物选择带来困难,需引起重视。

OBJECTIVE To investigate the fluoroquinolne-resistant mechanism of KPC β-lactamase-producing Klebsiella pneumoniae isolates so as to guide the reasonable clinical use of antibiotics. METHODS Totally 23 strains of KPC β-1actamase-producing Klebsiella pneumoniae isolated from two three A hospitals were collected, then the drug susceptibility testing was performed, and the polymerase-chain-reaction （PCR） was used to detect fluoroquinolones target encoding genes such as gyrA and parC and the mobile genetic elements-mediated genes like aac（6＇）- I trcr, qnrA, qnrB, qnrS, and qepA, the positive PCR products were verified by DNA sequencing, and the results were compared and analyzed with BLAST searching. RESULTS The topoisomerase Ⅱ A subunit genes have been detected in all the 23 strains of KPC β-lactamase-producing Klebsiella pneumoniae, and the sense mutation of the 83th （amino acid sequence S→I） and 87th （amino acid sequence D→G） codon （TCC→ATC, GAC→GGC respectively） on quinolone-resistant determining region（QRDR） of gyrA occurred, and the 80th codon （AGC→ATC） （ amino acid sequence S→I）of topoisomerase IV C subunit genes parC occurred in all the 23 strains in every strains. Meanwhile, the detection of the fluoroquinolne-resistance-associated genes （aac （6＇）-I b-cr, qnrA, qnrB, qnrS, qepA） mediated by mobile genetic elements were all negative. CONCLUSION The fluoroquinolone-resistant mechanism of the 23 strains of KPC β-lactamase-producing Klebsiella pneumoniae is primarily attributed to the sense mutation occuring on QRDR of gyrA and parC, which aggregates the antimicrobial resistance and bring difficulties to the clinical use of antibiotics, it should be paid aattention to.