巴西苏木素对金黄色葡萄球菌生物膜形成的抑制作用及其机制  

Inhibitory effect and mechanism of Brazilian hematoxylin on formation of Staphylococcus aureus biofilm

作　　者：晏嘉 徐子善 陈旋 简永欢 周飘雁 温伟洪[1] 徐令清[1] YAN Jia;XU Zi-shan;CHEN Xuan;JIAN Yong-huan;ZHOU Piao-yan;WEN Wei-hong;XU Ling-qing(The Sixth Affiliated Hospital of Guangzhou Medical University/Qingyuan People's Hospital,Qingyuan,Guangdong 500518,China)

机构地区：[1]广州医科大学附属第六医院/清远市人民医院检验科,广东清远500518

出　　处：《中华医院感染学杂志》2023年第8期1121-1126,共6页Chinese Journal of Nosocomiology

基　　金：广东省中医药局基金资助项目(20201407);广东省科技创新战略专项基金资助项目(DZXQY002);广东省医学科学技术研究基金(A2021490);清远市人民医院医学科研基金支持项目(20190209,202301-201);清远市科技计划项目(200808114560452);广州医科大学大学生科技创新项目(2021A098,2020ALY07)。

摘　　要：目的探究巴西苏木素对金黄色葡萄球菌生物膜的影响、抑制生物膜的机制。方法收集耐甲氧西林金黄色葡萄球菌(MRSA)40株,刚果红平板法和96孔板结晶紫染色法鉴定其产膜能力;采用二倍稀释法测定药物对强产膜菌最小抑菌浓度(MIC)和最小杀菌浓度(MBC);绘制生长曲线观察药物对生物膜生长的影响;油镜下观察药物对生物膜生长的影响;观察在药物处理后的生物膜内存活菌的数量反映对生物膜的作用效果;紫外分光光度计检测DNA及RNA等大分子物质的外渗水平;聚丙烯酰胺凝胶电泳(SDS-PAGE)测定对可溶性蛋白的影响;检测溶血环大小反映对分泌α-溶血素的影响。结果筛选出19株生物膜阳性菌,其中强产膜菌9株、弱产膜菌10株;巴西苏木素对强产膜菌的MIC为64μg/ml,MBC为128μg/ml;生长曲线提示对强产膜菌的抑制效果与药物浓度呈正相关;镜下观察到药物浓度越高,菌体数量越少、聚集越松散;药物浓度增加,生物膜内的存活菌量减少;巴西苏木素对强产膜菌的DNA、RNA的外渗随药物浓度增加,外渗量增大;SDS-PAGE结果表明药物浓度增加,对强产膜株可溶性蛋白形成的抑制作用增强;药物浓度增加,溶血环直径减小。结论巴西苏木素对强产膜菌生物膜具有良好的抑制作用且具有浓度依赖性,可能通过减少其可溶性蛋白的形成、破坏生物膜、减少生物膜胞外DNA和RNA、减少溶血素来发挥作用。OBJECTIVE To investigate the effect of Brazilian hematoxylin on Staphylococcus aureus biofilm formation and the mechanism of inhibiting biofilm.METHODS Forty strains of methicillin-resistant S.aureus(MRSA)were collected and their film-producing capacity was detected by Congo red plate method and 96-well plate crystal violet staining method.The minimum inhibitory concentration(MIC)and the minimum bactericidal concentration(MBC)of the drug against strong membrane-producing bacteria were determined by two-fold dilution method.The growth curve was drawn to observe the effect of drugs on the growth of biofilm,which was observed under oil microscope.The number of viable bacteria in the biofilm treated with the drug was counted to evaluate its effect on the biofilm.The exosmosis levels of macromolecular substances such as DNA and RNA were detected by ultraviolet spectrophotometer.The influence on soluble proteins were determined by polyacrylamide gel electrophoresis(SDS-PAGE).The hemolytic ring size was detected to show the effect onα-hemolysin secretion.RESULTS Nineteen strains of biofilm-positive bacteria were screened,including 9 strains of strong membrane-producing bacteria and 10 strains of weak membrane-producing bacteria.The MIC and MBC of Brazilian hematoxylin against strong membrane-producing bacteria were 64μg/ml and 128μg/ml,respectively.The growth curve indicated that the inhibitory effect on strong membrane-producing bacteria was positively correlated with drug concentrations.Microscopically,with the higher the concentration of drug,the number of bacteria was less and the aggregation was looser.With the increase of drug concentration,the amount of viable bacteria in the biofilm decreased;the exosmosis of Brazilian hematoxylin to DNA and RNA in strong membrane producing bacteria increased with the increase of drug concentration.SDS-PAGE showed that with the increase of drug concentration,the expression of soluble protein was inhibited and the diameter of hemolytic ring was decreased.CONCLUSION Brazilian h

关 键 词：耐甲氧西林金黄色葡萄球菌 生物膜 产膜能力 巴西苏木素 抑制作用 机制 

分 类 号：R285.5[医药卫生—中药学]