环丙沙星体外诱导肺炎链球菌耐药机制研究

Molecular mechanism of ciprofloxacin drug resistance in Streptococcus pneumoniae induced in vitro

目的探讨环丙沙星体外诱导肺炎链球菌耐药的分子机制。方法10株临床分离的肺炎链球菌体外用不同浓度梯度环丙沙星逐级诱导成为耐药株,检测诱导前后菌株对环丙沙星的MICs,PCR扩增诱导前后菌株parC/parE和gyrA/gyrB基因喹诺酮耐药决定区(QRDR)、测序并与GenBank公布序列比对。结果8株临床菌株成功诱导为耐药株,诱导后MICs是诱导前16倍以上,诱导后2株只发现parC/parE基因的QRDR有突变其耐药水平较低MICs分别为8μg/ml和16μg/ml,6株parC/parE和gyrA/gyrB基因的QRDR同时存在点突变耐药水平较高,MICs分别为64μg/ml和128μg/ml。结论逐步增加药物浓度可以诱导肺炎链球菌parC/parE和/或gyrA/gyrB基因的QRDR点突变导致对环丙沙星耐药。

Objective To investigate the molecular mechanisms of ciprofloxacin(Cip)resistance in S.pneumoniae induced in vitro.Methods A total of 10 S.pneumoniae isolates was induced to become drug-resistant strains with different concentrations of gradient ciprofloxacin in vitro.The MICs of ciprofloxacin of isolates before and after induction was detected.The quinolone resistance determining regions(QRDR)of the parC/parE and gyr A/gyr B genes of the strains before and after induction of PCR amplification were sequenced and compared with the sequences published in Gen Bank.Results MIC values of 8 isolates after induction were 16 times higher than induced before,which were induced to drug resistant strains successfully and also resistant to Cip.Mutations of QRDR of only parC/parE genes were detected by sequencing in 2 isolates,and the MICs with low drug resistance level were 8μg/ml and 16μg/ml respectively.The QRDR of parC/parE and gyr A/gyrB genes simultaneously had point mutation in 6 isolates and the MICs with high resistance level were 64μg/ml and 128μg/ml.Conclusion Gradual increase in drug concentration can induce QRDR point mutations in the parC/parE and/or gyr A/gyrB genes of S.pneumoniae,resulting in resistance to ciprofloxacin.