耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变研究

目的　研究临床分离的耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变情况。方法　测定临床分离的 5 5株铜绿假单胞菌MIC值 ,从中筛选出 1株敏感菌和 8株耐药菌 ,以标准敏感菌株ATCC2 785 3作为质控菌株。用聚合酶链反应 (PCR)扩增gyrA及parC基因的喹诺酮耐药决定区 (QR DR) ,扩增产物片段长度分别为 35 1bp、397bp。用限制性内切酶SacⅡ消化gyrAPCR产物 ,同时对上述 10株菌的gyrA及parC基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果　有 8株耐菌株的gyrA基因在 83位 (ACC→ATC)有突变 ,导致氨基酸Thr→Ile的改变 ;有 3株高度耐药菌gyrA基因同时在 87位 (GAC→GGC)有突变 ,导致氨基酸Asp→Gly的改变 ;有 4株耐药菌株的parC基因在 87位有TCG→TTG突变 ,导致氨基酸由Ser→Leu的改变。同时具gy rA和parC突变MIC值是仅具gyrA突变菌株MIC值的 2～ 16倍。未发现parC突变单独存在。另外 ,有 6株耐药菌gyrA的 132位有CAC→CAT的突变 ;所有耐药菌株parC基因 115位有GCT→GCG的突变 ,该突变未引起氨基酸的改变。结论　gyrA83、87位突变及parC基因 87位突变都可引起铜绿假单胞菌对氟喹诺酮类药物产生耐药 ,但以gyrA基因 83位突变为主 ,合并gyrA基因 87位及parC基因 87位突变可增加耐药程度。

gyrA and parC genes mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa

Aim To study gyrA and parC mutations of clinical Pseudomonas aeruginosa strains. Methods MIC values of 55 clinical P.aeruginosa isolates were determined by agar dilution test and 1 sensitive strain and 8 resistant strains were selected with standard sensitive strain ATCC27853 as control, the quinolone determining region (QRDR) of the gyrA and parC genes were amplified by PCR, the lengths of PCR products were 351 bp and 397 bp. The gyrA PCR products(351 bp) were digested with enzyme sacⅡ. The gyrA and parC gene were sequenced. Results In this study, gyrA genes of all resistant strains had an ACC to ATC mutation in codon 83, leading to the amino acid substitution of an isoleucine for a threonine, and three high level resistant strains also showed a GAC to GGC mutation in codon 87, leading to the substitution of a glycine for an aspartic acid. In addition, four resistant strains also had an TCG to TTG mutation in codon 87 of parC gene, leading to the amino acid substitution of a serine for a leucine. The strains with both gyrA and parC mutations were two to sixteen times more resistant than the strains which had only gyrA mutations. At the same time, a silent mutation (CAC to CAT) in codon 132 of gyrA gene and a silent mutation(GCT to GCG) in codon 115 of parC gene occured, which did not lead to amino acid change. Conclusion The mutations of 83 and 87 codons of gyrA and the mutatations of 87 codon of parC gene were related to fluroquinolone resistance, and the mutations of the 83 codon of gyrA gene were more important.