铜绿假单胞菌环丙沙星耐药株Ⅱ类拓扑异构酶基因突变的研究

Study of type Ⅱ topoisomerase mutation in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa

用PCR方法 ,扩增 2 6株临床分离铜绿假单胞菌环丙沙星耐药株、4株环丙沙星敏感菌株和PA0 1菌株的Ⅱ类拓扑异构酶 gyrA、gyrB、parC和 parE基因的喹诺酮类药物耐药决定区 (QRDR)基因片断 ;并对铜绿假单胞菌II类拓扑异构酶基因突变与耐药性关系进行研究。结果表明 90 %以上的铜绿假单胞菌耐药株表现出Ⅱ类拓扑异构酶基因突变 (2 6株中有 2 4株 )。突变主要发生在 gyrA基因 (2 2株 )和 parC基因 (14株 )上 ,其中 gyrA基因的第 83位氨基酸密码子表现出高频突变 (2 2株中有 2 1株 ,ACC→ATC ,占 90 % ) ,parC基因第 80位氨基酸密码子也表现出较高的突变率 (14株中有 12株 ,TCG→TTG ,占 60 % ) ,4株耐药株的 gyrB和 3株耐药株的 parE基因发生单点突变 ,2株耐药株II类拓扑异构酶基因未检测到突变。用二倍稀释法 ,测定部分喹诺酮和 β 内酰胺类药物对耐药突变株的体外抗菌活性。表明铜绿假单胞菌Ⅱ类拓扑异构酶基因突变株对诺氟沙星、左氧氟沙星、哌拉西林、头孢他啶和亚胺培南表现出不同的敏感性。试验所用 2 4株突变株对诺氟沙星呈现 10 0 %的耐药 ;对左氧氟沙星 65 %的耐药 ;40 %的菌株对哌拉西林表现出耐药 ;3 0 %的菌株对头孢他啶耐药 ;75

The quinolone resistance determining regions (QRDR) of gyrA, gyrB,parC and parE genes encoding type Ⅱ topoisomerase from 26 clinical isolates of ciprofloxacin resistant Pseudomonas aeruginosa were analyzed with PCR amplification and DNA sequencing. The correlation of typeⅡtopoisomerase mutation to drug resistance in ciprofloxacin resistant clinical isolates of Pseudomonas aeruginosa was investigated. The mutations in type Ⅱ topoisomerase genes were detected for more than 90% ciprofloxacin resistant isolates (24 of 26). The mutations were found mainly in the gyrA (22 strains) and parC (14 strains). 21 isolates possessed mutations at codon 83 (ACC→ATC) in gyrA . 12 isolates had mutations at codon 80 (TCG→TTG) in parC . The gyrA and parE mutation only occured in 4 and 3 resistant isolates. Two resistant isolates did not have any mutations in type Ⅱ topoisomerase genes. The antibacterial activities of norfloxacin, levofloxacin, piperacillin, ceftazidime and imipenem against type Ⅱ topoisomerase gene mutants of P.aeruginosa were investigated. The susceptibilities of the mutants with typeⅡ topoisomerase gene to norfloxacin, levofloxacin, Piperacillin, ceftazidime and imipenem were different in this study. All of mutants disclosed the high level resistance to norfloxacin. 65% of mutants showed the resistance to levofloxacin; 40% of mutants were resistant to piperacillin and 30% of them were resistant to ceftazidime; 75% of them were susceptibility to imipenem.