耐氟喹诺酮类铜绿假单胞菌的gyrA基因突变研究

DNA gyrase gyrA mutation in quinolone-resistant clinical isolates of Pseudomonas aeruginosa

目的 研究临床分离的耐氟喹诺酮类药物铜绿假单胞菌 gyrA基因突变情况。方法 测定临床分离的 5 5株铜绿假单胞菌的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基因的喹诺酮耐药决定区 (QRDR) ,扩增产物片段长度为3 5 0bp。用限制性内切酶SacⅡ消化PCR产物 ,同时对上述 10株菌的gyrA基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果 临床分离铜绿假单胞菌敏感菌株的 gyrA基因QRDR经限制性内切酶SacⅡ消化后出现 118bp、2 3 2bp两条片段 ,表明该酶切位点未发生突变 ,此结果经DNA序列分析证实。而耐药菌在酶切后均为一条片段 ,表明该酶切位点消失 ,经DNA序列分析发现 ,8株耐药株在 83位 (ACC→ATC)均有突变 ,该单位点突变引起氨基酸由Thr→Ile的改变 ;其中有 3株高度耐药菌同时发现在 87位 (GAC→GGC)有突变 ,该单位点突变引起氨基酸由Asp→Gly的改变 ,但没有发现 87位点突变单独存在。且双位点突变菌株的MIC值与单一位点突变的MIC值相比 ,有显著的升高 ,MIC增加 4～ 3 2倍。除此之外 ,有 6株耐药菌株在 13 2位有一静止突变 (CAC→CAT) ,该突变未引起氨基酸的改变。结论 gyrA基因突变是铜绿假单胞菌对氟喹诺酮类药物产?

Objective To study the gyrA mutation of clinical Pseudomonas aeruginosa strains. Methods By determing the MIC value, one sensitive strain and eight resistant strains were selected from 55 clinical isolates of P.aeruginosa . Taking standard sensitive strain ATCC27853 as control, the quinolone determining region (QRDR) of the gyrA gene was amplified by PCR, the PCR products (350bp) was digested with enzyme Sac Ⅱ and directly sequenced by PCR DNA. Results The quinolone sensitive clinical isolates of P.aeruginosa had two fragments after digested by Sac Ⅱ, and their length were 118bp and 232bp respectively. Which mean that the Sac Ⅱ site was still exist and this result was verified by DNA sequence. But resistant strains only had a fragment, which showed that the site was absent from the mutant gyrA gene. DNA sequence showed that all resistant strains had an ACC to ATC mutation in codon 83, leading to the amino acid substitution of an isoleucine for a threonine, at the same time, three high level resistant strains also had a GAC to GGC mutation in codon 87, leading to the substitution of a glycine for an aspartic acid. In this study no isolates only had alterations in codon 87. Overall, the strains with a double mutations were four to thirty two times more resistant to the strain with a single mutation. In addition, a silent mutation (CAC to CAT) in codon 132 occured which did not lead to amino acid change. Conclusion The mutation of gyrA gene is one of mechanisms which response for resistance to fluroquinolone, the Thr 83 Ile and Asp 87 Gly mutations were the most frequent.