Abstract:

To obtain the small colony variants （SCVs） of Staphylococcus aureus （S. aureus） is important biological materials for the research of anti-SCVs drugs. In this study， S. aureus ATCC43300 was used as the starting strain， the stable SCVs were obtained by secondary induction technique of low mass concentration （1.25~5.00 μg·mL^-1） gentamicin，and their characteristics were analyzed. The results showed that the stable SCVs could be obtained by secondary induction at 1/2×minimum inhibitory concentration（MIC）gentamicin 6 hours and 2×MIC gentamicin 12 hours. SCVs grew slowly， small colonies could be seen after incubated for 24 hours， even took 7 hours to reach the exponential growth phase；the sensitivity to gentamicin and the ability to produce membrane were increased 3 and 1 times；the recovery mutation rate was 0% after successive 20 passages； anti-stress， drug sensitivity and anti-coagulation were all enhanced. The SigB regulatory systems and the biofilm-related genes were up-regulated， the Agr regulatory system and the virulence factors were down-regulated. This method proposed successful new screening strategy for screening SCVs， and also provided stable test strains for pathogenesis of SCVs and the development of novel anti-SCVs drugs.

Key words: Staphylococcus aureus, small colony variants（SCVs）, gentamicin

摘要：

稳定型金黄色葡萄球菌（Staphylococcus aureus）小菌落突变体（small colony variants， SCVs）的获得，为抗SCVs药物的研究提供了重要的生物学材料。以S. aureus ATCC43300为出发菌株，利用低质量浓度（1.25~5.00 μg·mL^-1）庆大霉素二次诱导技术获得稳定型SCVs，并对其特性进行分析。结果表明，1/2×最小抑菌浓度（minimum inhibitory concentration， MIC）及2×MIC庆大霉素分别诱导6 和12 h，可获得稳定型SCVs。SCVs生长缓慢，培养24 h可见较小菌落，培养7 h至生长对数期；对庆大霉素敏感性及产生物膜能力分别提高3和1倍；传代20次后恢复突变率为0%，抗逆性、药物敏感性、抗凝血性均增强，SigB调控系统及生物膜相关基因呈上调趋势，Agr调控系统及毒力因子下调。该方法的成功建立为SCVs的获得提出新的筛选策略，并为开展SCVs致病机理研究及新型抗SCVs药物研发提供稳定型受试菌株。

关键词: 金黄色葡萄球菌, 小菌落突变体, 庆大霉素