耐甲氧西林金黄色葡萄球菌MRSA252辅酶A二硫化物还原酶缺失延缓苯唑西林的次级损伤效应

Deficiency of coenzyme A disulfide reductase in methicillin-resistant Staphylococcus aureus MRSA252 delays the secondary damage of oxacillin

摘要:

[目的] 耐甲氧西林金黄色葡萄球菌在苯唑西林作用下，其辅酶A二硫化物还原酶表达上调2.3倍。本文研究苯唑西林对该酶缺失的金黄色葡萄球菌的杀菌效应。[方法] 利用同源重组双交换技术对金黄色葡萄球菌进行基因敲除，并用质粒pOS1构建回补株；采用分光光度法检测菌株体外增殖能力；以时间-杀菌法考察苯唑西林对菌株杀菌效应；以2'，7'-二氯荧光黄双乙酸盐为探针检测胞内活性氧水平。[结果] 辅酶A二硫化物还原酶基因敲除株较亲株生长缓慢（P<0.05）；20倍MIC浓度苯唑西林下敲除株的时间-杀菌曲线及胞内活性氧水平与亲株无显著性差异，5倍MIC浓度下敲除株的致死速率及胞内活性氧水平均较亲株下降。[结论] 在较低浓度苯唑西林作用下，辅酶A二硫化物还原酶基因缺失降低胞内活性氧水平，减小杀菌速率，延缓次级损伤效应。

Abstract:

[Objective] The expression of coenzyme A disulfide reductase in methicillin-resistant Staphylococcus aureus was up-regulated by 2.3 folds when the strain was cultured with oxacillin. Here, the bactericidal effect of oxacillin on coenzyme A disulfide reductase deficient Staphylococcus aureus was studied. [Methods] Homologous recombination mediated double-exchange techniques were used to delete genes in Staphylococcus aureus, whereas pOS1 plasmid was used to construct the complemented strain. Spectrophotometry was used to detect the proliferation ability of the strains in vitro. The bactericidal effect of oxacillin on the strains was studied by time-kill method, and 2',7'-dichlorodihydrofluorescein diacetate was used as a probe to detect intracellular reactive oxygen species levels. [Results] Coenzyme A disulfide reductase gene deleted strain grew more slowly than the parental strain (P<0.05). Under the action of oxacillin, the time-kill curve and intracellular reactive oxygen species level of the knockout strains were slightly different from that of the parental strains at 20 times MIC concentration, and the lethal rate and intracellular reactive oxygen species level of coenzyme A disulfide reductase gene deleted strains were lower than that of the parental strains at 5 times MIC concentrations. [Conclusion] At lower concentrations of oxacillin, the deletion of coenzyme A disulfide reductase gene reduced intracellular ROS levels, and the bactericidal rate, meanwhile the secondary damage was delayed.