松萝酸对耐甲氧西林金黄色葡萄球菌的抑菌作用机制研究

Antibacterial mechanism of usnic acid on methicillin-resistant Staphylococcus aureus

【目的】通过探究松萝酸对耐甲氧西林金黄色葡萄球菌(MRSA)细胞膜、细胞壁、蛋白质、乳酸脱氢酶(LDH)和生物被膜的影响,探讨松萝酸对MRSA的抑菌作用机制,为开发新型抗MRSA药物提供理论依据.【方法】采用微量肉汤稀释法、琼脂平板菌落记数法和比浊法分别测定松萝酸对USA300的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和生长曲线;通过测定外渗DNA量确定松萝酸对细胞膜和细胞壁的影响;通过SDS-PAGE和ELISA法分别检测松萝酸对可溶性蛋白代谢和乳酸脱氢酶(LDH)的影响;利用透射电镜观察经松萝酸处理后菌株的超微结构;通过结晶紫染色法测定松萝酸对生物被膜的影响.【结果】松萝酸对USA300菌株具有一定的抑制作用,其MIC为128μg/mL,MBC为512μg/mL.与对照组相比,2MIC松萝酸作用菌体1 h后,DNA外渗量增加(46.73±3.32) ng/μL,作用6 h后,菌体可溶性蛋白的含量降低(13.36±3.11)%.松萝酸能改变USA300菌体的固有形态.经64μg/mL松萝酸处理后,USA300生物被膜总量比对照组减少(85.85±2.19)%.【结论】松萝酸通过改变菌体细胞膜通透性,干扰其蛋白质代谢来抑制细菌的生长,破坏其固有形态,在亚抑菌浓度能够抑制生物被膜的形成.

【Objective】 To provide a theoretical basis for the development of new anti-MRSA drugs,the bacteriostatic mechanism of usnic acid on MRSA was explored by investigating the effects of usnic acid on the cell membrane,cell wall,protein,lactate dehydrogenase(LDH),ultrastructure and biofilm of MRSA.【Method】 The minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC) and growth curve of usnic acid to USA300 were determined via microbroth dilution method,colony counting method and turbidimetry,respectively. DNA exosmosis amount was detected to investigate the effect of usnic acid on cell membrane and cell wall. SDS-PAGE and ELISA were used to detect the effect of usnic acid on soluble protein synthesis and LDH respectively. The microscopic structure of the bacteria strain treated by usnic acid was observed through transmission electron microscope. The effect of usnic acid on biofilms was investigated via crystal violet staining method.【Result】 Usnic acid had definite inhibitory activity on USA300,the MIC of usnic acid against USA300 was 128 μg/mL and MBC was 512 μg/mL. Compared with the control group,after treating the strain with 2 MIC usnic acid for 1 h,the DNA exosmosis amount improved(46.73±3.32) ng/μL. After treating for 6 h,the content of soluble protein reduced(13.36±3.11)%. Usnic acid changed the intrinsic morphology of USA300. The 64 μg/mL usnic acid inhibited(85.85±2.19)% of biofilms formation,compare with control group.【Conclusion】 Usnic acid inhibits the growth of USA300 by changing its cell membrane and cell wall permeability,disturbing protein synthesis and destroying normal form. In addition,usnic acid inhibit biofilm formation at subinhibitory concentrations.