伤寒杆菌耐喹诺酮类机理研究

Quinolone resistant mechanisms in Salmonella typhi

以伤寒杆菌临床分离的喹诺酮类敏感株 S2 75与其诱导耐药株 RG1 与 RG2 ,对有关伤寒杆菌耐喹诺酮类机制进行了研究。结果发现与伤寒杆菌 S2 75相比较 ,RG1 与 RG2 对喹诺酮类、四环素、氯霉素敏感性明显降低 ,对头孢唑林 ,亚胺培南敏感性有所增加。喹诺酮类对 DNA旋转酶活性 5 0 %抑制剂量 ,RG1 与RG2 较 S2 75呈 3～ 8倍增加 ,S2 75与 RG1 、RG2 旋转酶亚单位交叉重建及 DNA旋转酶 A亚单位基因 (gyr A)喹诺酮耐药决定区 PCR- RFL P分析表明 A亚单位变异为耐药原因之一。细菌对氧氟沙星聚积测定 ,RG1 与RG2 分别为 S2 75之 1/ 2与 1/ 8,经 CCCP处理后 ,分别上升为 S2 75原水平与 S2 75之 1/ 2水平。外膜蛋白分析示 RG2 5 5 k D蛋白带消失 ,同时伴生化反应改变。表明伤寒杆菌耐喹诺酮类机制存在 DNA旋转酶变异 ,药物主动外运及外膜通透性改变。

To investigate the quinolone resistant mechanisms in Salmonella typhj , the DNA gyrase, drug uptake and membrane proteins of Salmonella typhi S275 (a clinically isolated quinolone susceptible strain) and its spontaneous quinolone resistant mutants RG 1and RG 2(the first and second step mutants) were examined in this study. In comparison with S275, the susceptibilities of RG 1and RG 2to quinolones, tetracycline and chloramphenicol decreased. The MICs of cefotaxime and cefazolin on RG 1and RG 2decreased by 2 and 8 times than that of S275. ID 50 s of quinolones on DNA gyrase to mutants increased by 3~8 times. The results of ID 50 s of quinolones in the cross reconstituted gyrase from S275 and mutants as well as PCR RFLP of quinolone resistant determining region of gyr A suggested that the mutation of gyr A was one of resistant mechanisms. The accumulation of ofloxacin in RG 1and RG 2decreased to 1/2 and 1/8 as that of S275, respectively. After treatment with carbonyl cyanide m chlorophenyl hydrazone (CCCP), the drug uptake increased to the same level in RG 1or 1/2 in RG 2as that in S275. The analysis of outer membrane proteins showed that a protein of 55kD disappeared in RG 2. These results indicated that the alteration of DNA gyrase, outer membrane permeability defect and active drug efflux are the factors contributing to the quinolone resistance of S.typhi .