奥替溴铵抑制革兰阳性菌生长与生物被膜形成的活性及机制研究

Antibacterial and anti-biofilm activities of otilonium bromide and its antibacterial mechanism against Gram-positive bacteria

目的探究奥替溴铵抑制革兰阳性菌生长及抑制生物被膜活性和作用机制。方法采用肉汤稀释法测定奥替溴铵对金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、屎肠球菌和无乳链球菌的最小抑菌浓度(minimum inhibitory concentration,MIC),并通过生长曲线分析奥替溴铵对浮游菌生长的影响,杀菌曲线评估奥替溴铵杀菌活性;结晶紫染色生物被膜含量测定和激光共聚焦显微镜检测奥替溴铵的抗生物被膜活性;定量蛋白质组学研究奥替溴铵对金黄色葡萄球菌的抗菌机制。结果奥替溴铵对革兰阳性菌表现出广谱抗菌活性和杀菌活性,MIC_(90)≤12.5μmol/L,奥替溴铵在1/2×MIC浓度时对细菌生长有显著的抑制作用;此外,奥替溴铵能够以剂量依赖形式抑制金黄色葡萄球菌生物被膜的形成,同时可灭杀成熟生物被膜中的细菌;蛋白质组分析显示奥替溴铵处理能够显著抑制细菌初级代谢过程,诱导过氧化应激。结论奥替溴铵对革兰阳性菌具有较佳的抗菌活性和抗生物被膜活性,通过抑制细菌初级代谢和引发过氧化应激达到抑菌效果。

Objective This study aims to explore antibacterial and anti-biofilm activities of otilonium bromide and its antibacterial mechanism against Gram-positive bacteria.Methods The MIC of otilonium bromide against Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis,Enterococcus faecium,and Streptococcus agalactiae was determined by the broth dilution method,and the inhibitory effect of otilonium bromide on the growth of planktonic cells was assessed by growth curve.Crystal violet staining and confocal laser scan microscopy were performed to evaluate the anti-biofilm activity of otilonium bromide.The antibacterial mechanism of otilonium bromide against S.aureus was studied by quantitative proteomics.Results The MIC_(90) of otilonium bromide against all Gram-positive bacteria tested were all≤12.5μmol/L,otilonium bromide significantly inhibited the growth of planktonic cells at subinhibitory concentration of 1/2×MIC concentration.In addition,otilonium bromide could inhibited the biofilm formation of S.aureus in a dose-dependent manner as well as killed bacteria embedded in mature biofilm.Proteomic analysis showed that otilonium bromide significantly inhibited the primary metabolic process and induced oxidative stress response.Conclusion Otilonium bromide exhibited antibacterial and anti-biofilm activities against Gram-positive bacteria,and the antibacterial mechanisms of otilonium bromide against otilonium bromide may be related to disrupting the primary metabolic process and inducing oxidative stress.