肺炎克雷伯杆菌对氟喹诺酮类抗生素耐药机制的研究

The mechanism of resistance of Klebsiella pneumoniae to Fluoroquinolones

目的研究临床分离的耐氟喹诺酮类药物肺炎克雷伯杆菌的耐药机制。方法临床分离、鉴定的肺炎克雷伯杆菌采用琼脂二倍稀释法测定氟喹诺酮的MIC，从中选取10株耐氟喹诺酮的菌株。通过聚合酶链反应（PCR）扩增gyrA基因和parC基因，扩增产物片段长度分别为625、319bp，经纯化后测序。用琼脂二倍稀释的方法测定5种氟喹诺酮类单独使用及与泵抑制剂CCCP合用后的MIC。结果5株耐氟喹诺酮肺炎克雷伯杆菌的gyrA和parC基因经测序分析，显示gyrA的第83位（TCC→ATC/TTG）均出现了突变，引起了相应氨基酸的改变（Ser-83→Ile／Leu）。其中有一株同时也出现了第87位点（GAC→AAC）的改变，氨基酸也由Asp→Asn。parC有4株出现了第80位点突变（AGC→ATC），引起氨基酸由Ser→Ile的改变。MIC测定结果显示，有一株肺炎克雷伯杆菌在单用5种氟喹诺酮类药物及与CCCP合用后，MIC降低了8～32倍；两株肺炎克雷伯杆菌分别对环丙沙星及左氧氟沙星所测的MIC降低了32倍和4倍；还有一株对左氧氟沙星和司帕沙星在使用CCCP前后的MIC降低了4倍。结论gyrA和parC基因突变是肺炎克雷杆菌对氟喹诺酮类产生耐药机制的主要原因，主动外排机制也是肺炎克雷伯杆菌耐氟喹诺酮类抗生素的因素之一。

OBJECTIVE To study the mechanism of resistance of Klebsiella pneumoniae to Fluoroquinolones. METHODS 10 fluoroquinolone- resistance clinical isolates were collected. Their region of gyrA and parC genes were amplified by polymerase chain reaction （PCR）, the PCR products（625 bp and 319 bp respectively） were purified and directly sequenced. Minimal inhibitory concentrations （MIC） of 5 FQNs with and without CCCP were determined by the agar dilution method. When the MICs of FQNs with CCCP decreased 4 times or less than the MICs of FQNs without CCCP, it indicated existence of efflux mechanism. RESULTS The results by DNA sequencing showed that the all strains had an TCC→ATC or TCC→TTG mutation in codon 83, leading to amino acid change： Ser→Leu or Ser→Ile. At the same. time , a GAC→AAC mutation was found in codon 87 which resulted in amino acid change：Asp→Asn. Furthermore, DNA sequencing analysis of pails also revealed point mutation （AGC→ATC） leading the substitution of a Ile for Set in 4 strains. As for the MICs, there was one strain that the MICs of the 5 Fluoroquinolones with CCCP decreased 8 to 32 times than the MICs without CCCP. The MICs of Ciprofloxacin and Levofloxacin with CCCP was 32 times and 4 times lower than the MICs without CCCP for the other two strains, respectively. MICs of another isolate to Levofloxacin and sparfloxacin with CCCP decreased 4 times than without CCCP. CONCLUSION The mutation of gyrA and parC gene are the primary mechanisms responsing for resistance of Klebsiela pneumoniae to FQNs, and efflux is also an important factor.