耐氟喹诺酮类肺炎克雷伯菌GyrA和ParC的变异

A study on GyrA and ParC mutant in fluoroquinolone resistant clinical isolates of Klebsiella pneumoniae

目的 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)的变异与其耐氟喹诺酮类 (FQNL)的关系。方法 采用琼脂平板双倍稀释法测定环丙沙星、左氧氟沙星、依诺沙星和诺氟沙星对临床分离的 31株肺炎克雷伯菌及其标准菌株ATCC 10 0 31的最低抑菌浓度 (MIC) ,并对此 32株菌GyrA的基因 (gyrA)和ParC的基因 (parC)进行PCR扩增和DNA序列的分析比较。结果 19株耐FQNL菌都存在GyrA变异 ,同FQNL耐药性相关的变异有Ser83(TCC)→Phe(TTC)、Ile(ATC)、Tyr(TAC)和Lys(TGC) ;Asp87(GAC)→Ala(GCC)。其中Ser83→Ile、Lys的变异是新发现。在 9株高度耐FQNL和 1株低度耐FQNL菌中同时存在ParC的变异 ,同FQNL耐药性相关的变异有丝氨酸Ser80 (AGC)→Ile(ATC)。ATCC 10 0 31的ParC同大肠埃希菌的ParC具有很好的同源性。结论 在耐FQNL肺炎克雷伯菌中普遍存在着GyrA的变异 ,其中以Ser83的变异多见。当细菌有GyrA的变异 ,同时合并有ParC的变异时 ,其耐药性往往会增强。

Objective To study the relation between the alterations in the DNA gyrase subunit A (GyrA), topoisomerase Ⅳ subunit C (ParC) and fluoroquinolones(FQNL) resistance in Klebsiella pneumoniae . Methods The minimal inhibitory concentrations (MIC) of 31 clinically isolated strains and reference strain (ATCC10031) of Klebsiella pneumoniae to ciproxacin, levofloxacin, enoxacin, and norfloxacin were determined by the two fold agar dilution method.The genes of GyrA (gyrA) and ParC (parC) in the 32 strains were amplified by PCR and their DNA sequences were compared. Results The alterations in GyrA existed in all 19 strains of Klebsiella pneumoniae resistant to FQNL. In this study, the alterations related to FQNL resistance included Ser83 (TCC)→ Phe (TTC), Ile (ATC), Tyr (TAC) and Lys (TGC); Asp87 (GAC)→ Ala(GCC). The alterations of Ser83(TCC)→ Ile (ATC) and Lys (TGC) were new discoveries. Alteration in ParC existed in all 9 strains which were highly resistant to FQNL and one strain was moderately resistant to FQNL, and the alteration related to FQNL resistance was Ser80(AGC)→ Ile (ATC). When alteration occurred in GyrA accompanied by that in ParC, the FQNL resistance of bacteria tended to increase. It was discovered in this study that the alteration related to FQNL resistance was Ser80(AGC)→ Ile(ATC). The ParC gene in ATCC10031 was highly homologous to that in Escherichia coli. Conclusions The alterations in GyrA exist commonly in the strains of Klebsiella pneumoniae resistant to FQNL. The alteration at Ser83(TCC) is common. When alteration occurs in GyrA companied by that in ParC, the FQNL resistance of bacteria tends to intensify.