摘要
目的体外构建喹诺酮耐药肺炎克雷伯菌模型并研究其gyrA、parC基因突变与喹诺酮耐药的关系。方法运用亚抑菌浓度的左旋氧氟沙星与肺炎克雷伯菌在培养基中共培养，并倍比增加诱导药物的浓度，直至肺炎克雷伯菌能够在含高浓度的左旋氧氟沙星培养基上良好生长。收集耐药菌株，用PCR扩增和DNA测序法检测gyrA、parC基因喹诺酮耐药决定区域（QRDR）的突变情况。结果体外成功构建喹诺酮高浓度耐药肺炎克雷伯菌模型，所有耐药菌株QRDR均发生了变异，绝大部分为gyrA和parC双重突变。gyrA基因以Ser83、Asp87突变为主，Ser83→Ile，Asp87→Arg、Gly，也有菌株86位氨基酸发生了突变，由Tyr86→Ser。几乎所有菌株parC基因80位氨基酸的突变均为Ser80→Ile。结论长期低浓度用药可以诱导喹诺酮耐药性的产生。QRDR突变与喹诺酮耐药性有关，gyrA及parC的双重突变可以导致高水平耐药，同时可能存在其他耐药机制。
Objective To construct quinolone resistant model of Klebsiella pneumoniae in vitro and investigate the association of mutations of gyrA and parC gene with the quinolone resistance. Methods Klebsialla pneumoniae were isolated and cultured with levofloxacin at subinhibitory concentration in media first, then the induced drug was increased at double concentration until klebsialla pneumoniae grew well on the plate containing high concentration of levofloxacin. Resistant strains were harvested and detected for mutations of quinolone resistance determining regions （QRDR） in gyrA gene and parC gene by PCR amplification and DNA sequence. Results Quinolone resistant model of Klebsiella pneumoniae in vitro was constructed successfully at high concentration. All the strains had mutations at the QRDR, most of which had double mutations of both gyrA and parC. Mutations of gyrA related to quinolone resistance primarily included Ser83 and Asp87 （ Ser83→Ile, Asp87→Arg ） , some of the strains mutated at 86 position amino acid（ Tyr86→Ser）. Most of the strains parC mutated from Ser to Ile at 80 position amino acid. Conclusions Long-term use of quinolone at low concentration can contribute to resistance of Klebsiella pneumoniae. The mutation of QRDR is closely associated to the quinolone resistance. The double mutations of gyrA and parC can result in high resistance, but other unknown mechanism of drug resistance can not be excluded.
出处
《中华生物医学工程杂志》
CAS
2008年第5期341-345,共5页
Chinese Journal of Biomedical Engineering
基金
广东省医学科学技术研究基金（A2008279）
