肺炎克雷伯菌gyrA基因与parC基因的突变研究

Study on the gyrA and gyrC gene mutation in Klebsiella pneumoniae

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%(118/231)和45.9%(106/231);gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。

Objective To investigate the antibiotic susceptibility of Klebsiella pneumoniae to ciprofloxacin and levofioxacin, the gyrA and the parC gene mutation in fiuoroquinolone resistant and susceptible isolates. Methods 231 isolates of Klebsiella pneumoniae were collected and their susceptibility to ciprofioxacin and levofioxacin were tested by K-B disk diffuison method. Quinolone resistance determining regions in gyrA gene and parC gene were amplified by PCR in ran- domly selected isolates （ four ciprofloxacin-resistant and three susceptible isolates）. The length of amplified fragments were 625 and 319 bp,respectively. PCR products were purified and sequenced. Results The resistance rates of the isolates to ciprofloxacin and levofloxacin were 45.0% （ 104/231 ） and 44. % （ 103/231 ） ,respectivly. GyrA mutations were found at po- sition 83,87 and 27 amino acids in one isolate, position 83 in two isolates, and position 47 in one;no gyrA gene mutation was found in fiuoroquinolone-susceptible strain. A Serine to Arginine substitution in parC at postion 80 were found in all the four ciprofloxacin-resistance isolates and two ciprofioxacin-susceptible isoaltes as well. Conclusion Klebsiella pneumoniae in Harbin had remarkable resistance to ciprofloxacin ; the gyrA and the parC gene mutation were found in fiuoroquinolone re- sistance isolates;the parC gene mutation had also been found in fiuoroquinolone-susceptible isolates.