大黄素抑制MRSA生物膜活性及其作用机制研究

Inhibitory effect of emodin on MRSA biofilm and its mechanism

目的探究大黄素抗耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)生物膜作用及其机制。方法微量肉汤稀释法测定大黄素对MRSA标准株及5种不同地区临床分离株的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC),绘制抑菌曲线评估其动态抑菌效果;结晶紫染色测定大黄素抑制MRSA生物膜的作用,扫描电镜和激光共聚焦显微镜观察其对MRSA生物膜形态的影响;荧光定量PCR检测ica操纵子基因的表达。结果大黄素对不同MRSA菌株的MIC为2~8μg/mL,MBC为4~16μg/mL;大黄素可显著抑制MRSA生物膜的形成,并呈剂量依赖关系;大黄素对生物膜结构具有破坏作用,且显著增加了生物膜内死细菌的比例;荧光定量PCR结果提示大黄素显著下调了ica操纵子基因的表达。结论大黄素具有抑制MRSA生物膜的作用,并可能通过下调ica操纵子基因的表达抑制细菌生物膜的形成。

Objective To investigate anti-biofilm activity and mechanisms of emodin against methicillin-resistant Staphylococcus aureus(MRSA)in vitro.Methods Microbroth dilution was explored to determine minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of emodin against MRSA252 strain and clinical isolations.Inhibitory curve were draw to estimate dynamic bacteriostatic effect.Crystal violet staining was used to investigate the inhibitory effect of emodin against MRSA biofilm.The effect of emodin on the morphology of MRSA biofilm was observed by scanning electron microscopy(SEM)and laser scanning confocal microscopy(LSCM).The expression of intercellular adhesion(ica)operon was detected by fluorescence quantitative PCR.Results The MICs of emodin against different strains of MRSA ranged from 2 to 8μg/mL,while MBCs were from 4 to 16μg/mL.Emodin obviously inhibited the formation of biofilm in a dose-dependent manner.Besides,it destroyed biofilm structure and increased the ratio of dead bacteria under biofilm.Fluorescence quantitative PCR showed that emodin significantly down-regulated the expression of ica operon.Conclusion Emodin can inhibit MRSA biofilm in vitro,and may restrain the formation of bacterial biofilm by down-regulating the expression of ica operon.