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目的了解3种氟喹诺酮类(FQS)体外诱导肺炎克雷伯菌(Klebsiella pneumoniae,Kpn)耐药性的差异,研究肺炎克雷伯菌DNA旋转酶A亚单位(GyrA)和拓扑异构酶ⅣC亚基(ParC)的变异与其耐喹诺酮类药物的关系。方法采用环丙沙星(CIP)、左氧氟沙星(LEX)和加替沙星(GAT)对从临床分离8株Kpn进行体外分步诱导,采用琼脂平板二倍稀释法测定CIP、LVF及GAT对Kpn诱导前、后的最低抑菌浓度(MIC),并对诱导成功的17株Kpn的GyrA的基因(gyrA)和ParC的基因(parC)进行PCR扩增,选取其中8株Kpn DNA测序并进行序列分析比较。结果8株耐FQS菌株都存在GyrA变异,同FQS耐药性相关的变异有Ser83(TCC)→Phe(TTC)、Ile(ATC)和Tyr(TAC),Asp87(GAC)→Ala(GCC)、Ala(GCC)和Glu(GAA),5株Kpn同时存在ParC的变异:丝氨酸Ser80(AGC)→Ile(ATC)。结论本研究体外实验证实了Kpn可在长期低剂量的接触抗菌药物后形成耐药菌株。在高度耐FQS的Kpn中同时存在GyrA和ParC变异。
Objective To investigate the differences in drug resistance of Klebsiella pneumoniae (Kpn) induced by three fluoroquinolones (FQS) in vitro and to investigate the relationship between Klebsiella pneumoniae DNA gyrase A subunit (GyrA) and topoisomerase Ⅳ C Variation of subunit (ParC) and its resistance to quinolone drugs. Methods Ciprofloxacin (CIP), levofloxacin (LEX) and gatifloxacin (GAT) were used to induce eight Kpn strains in vitro. The effects of CIP, LVF and GAT on Kpn The minimum inhibitory concentrations (MICs) before and after induction were determined. GyrA gene and parC gene (parC) of 17 Kpn strains were successfully amplified by PCR. Eight Kpn DNAs were sequenced and sequenced analyse and compare. Results There were GyrA mutations in 8 strains of FQS resistant strains. The mutations associated with FQS resistance were Ser83 (TCC) → Phe (TTC), Ile (ATC) and Tyr (TAC), Asp87 Ala (GCC) and Glu (GAA), five strains of Kpn coexist ParC variation: serine Ser80 (AGC) → Ile (ATC). Conclusion The in vitro experiments in this study confirmed that Kpn could form drug-resistant strains after long-term and low-dose exposure to antibiotics. Both GyrA and ParC mutations exist in Kpn highly resistant to FQS.