临床分离耐喹诺酮类铜绿假单胞菌gyrA基因单点突变研究

Study of the resistance against quinolones and its mechanism of clinical isolated Pseudomonas aeruginosa

目的 研究临床分离铜绿假单胞菌耐喹诺酮类药物的分子机制 ,对PCR RFLP SSCP分析铜绿假单胞菌gyrA基因突变的可行性评价。方法 以铜绿假单胞菌gyrA基因序列为靶序列 ,用PCR、PCR SSCP、PCR RFLP、DNA测序、OMIGA软件分析等方法 ,对铜绿假单胞菌gyrA基因突变进行研究。结果 在铜绿假单胞菌 10株耐药突变株中 ,有 8株的gyrA基因的 83位表现出高频的单点突变 ,其突变方式全为ACC→ATC。gyrA的PCR扩增产物SacⅡ酶切片段与测序结果一致。SSCP带谱与测序结果比较 ,除 1株 (PSA2 )其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。结论 临床分离的铜绿假单胞菌耐喹诺酮类药物分子机制主要表现为gyrA基因 83位氨基酸密码子突变 (Thr 83→Ile) ,利用PCR SSCP RFLP系统 ,可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中至少 1个碱基的差异。

Objective To study the mechanism of resistance to quinolones in Pseudomonas aeruginosa and evaluate the application of PCR, PCR-SSCP-RFLP in analysis of gyrA mutation. Methods The gyrA gene sequence in P.aeruginosa was analyzed with PCR, PCR-SSCP, PCR-RFLP and DNA sequencing. Results gyrA mutation (ACC→ATC) was observed in 8 of 10 quinolone-resistant strains isolated from patients in hospitals and care centers in Chengdu, China. Analyzing with the aid of computer, a variety of the secondary structure of the PCR products of gyrA were found. This may increase the resistance to quinolone of P.aeruginosa. Conclusion Compare the result of PCR-RFLP-SSCP analysis with DNA sequencing, the PCR-RFLP-SSCP analysis is simple, fast and sensitive. It should be applied for epidemiological surveillance.