临床分离铜绿假单胞菌喹诺酮耐药株gyrA基因突变及其对β-内酰胺类药物敏感性研究

Study on the gyrA mutation and susceptibility to β-lactams in quinolone resistant clinical isolates of Pseudomonas aeruginosa

利用 PCR、限制性片段多态性分析 (RFL P)、DNA单链构像多态性分析 (SSCP)和 DNA测序等方法 ,对 10株成都地区临床分离耐喹诺酮类药物铜绿假单胞菌 gyr A基因进行研究。结果表明 ,成都地区耐喹诺酮类药物铜绿假单胞菌 gyr A的喹诺酮耐药决定区编码第 83位氨基酸密码子表现出高频单点突变 (10株中有 8株 ) ,其突变方式为 ACC→ATC。gyr A的 PCR扩增产物 Sac II酶切片段与测序结果一致。SSCP的带谱与测序结果比较 ,除 1株 (PSA2 )的 SSCP带谱与标准株相同 ,但测序结果有点突变外 ,其余菌株与测序结果一致。因此 ,利用 PCR- SSCP- RFL P系统 ,可快速、准确的检测耐喹诺酮类药物的铜绿假单胞菌 gyr A中至少一个碱基的差异。用二倍稀释法测定喹诺酮和 β-内酰胺类药物对耐药突变株体外抗菌活性 ,结果表明 ,铜绿假单胞菌 gyr A基因突变株对诺氟沙星、环丙沙星、左氧氟沙星、头孢他啶、亚胺培南和哌拉西林表现出不同的敏感性。试验所用 8株突变株对诺氟沙星和环丙沙星表现出高水平抗性 ;3株对左氧氟沙星敏感 ;3株对头孢他啶和哌拉西林表现出耐药 ,2株对亚胺培南耐药。

The gyrA mutations of 10 fluoroquinolone resistant Pseudomonas aeruginosa clinical isolates were analyzed with PCR, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism analysis (SSCP) and DNA sequencing. The results of DNA sequence analysis showed that a point mutation of the Thr 83→Ile alteration (ACC→ATC) was the most frequent in gyrA gene of fluoroquinoloneresistant clinical isolates of P.aeruginosa (8 of the 10 isolates) in our hospital in Chengdu. Results of PCR RFLP SSCP analysis were almost consistant with DNA sequence except one strain (PSA2) which showed no change in SSCP analysis but harbored a point mutation, PCR RFLP SSCP analysis is simple, rapid, and sensitive. It could be suitable for epidemiological surveillance for at least one base change. The antibacterial activities of norfloxacin, ciprofloxacin, levofloxacin, ceftazidime, imipenem and piperacillin against gyrA mutants of P.aeruginosa was measeured by double dilution method. The susceptibilities of the mutants with a Thr 83→Ile alteration to norfloxacin, ciprofloxacin, levofloxacin, ceftazidime, imipenem and piperacillin were different. All of the gyrA mutants disclosed the high level resistance to norfloxacin and ciprofloxacin. Three mutants showed the susceptibility to levofloxacin, three were resistant to ceftazidime and piperacillin, and two were resistant to imipenem.