丁香酚对压疮致病菌金黄色葡萄球菌及其耐药菌的抑菌作用及机制  

Study on inhibitory effect and mechanism of eugenol against Staphylococcus aureus and its drug-resistant bacteria that causes pressure sores

作　　者：于杰[1,2] 郑淑媚 张文海 周瑞刚 赵钢 李金贵[3] YU Jie;ZHENG Shu-mei;ZHANG Wen-hai;ZHOU Rui-gang;ZHAO Gang;LI Jin-gui(Heilongjiang University of Chinese Medicine,Harbin 150040,China;The Affiliated Suqian First People's Hospital of Nanjing Medical University,Suqian 223800,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]黑龙江中医药大学,黑龙江哈尔滨150040 [2]南京医科大学附属宿迁第一人民医院,江苏宿迁223800 [3]扬州大学兽医学院,江苏扬州225009

出　　处：《现代药物与临床》2023年第9期2184-2194,共11页Drugs & Clinic

基　　金：国家自然科学基金资助项目(82004375);中国博士后科学基金资助项目(2021MD703836);宿迁市“西楚英才”雄英计划双创人才资助项目(卫生创新)[(2022)-0051]。

摘　　要：目的明确丁香酚对压疮致病菌金黄色葡萄球菌Staphylococcus aureus的体外抑菌作用及机制。方法采用微量肉汤稀释法和和生长曲线确定丁香酚对金黄色葡萄球菌的抑菌活性;通过碱性磷酸酶(AKP)活性、细菌核酸蛋白泄漏的检测,细菌超微结构的扫描和透射电镜观察以及菌体对碘化丙啶(PI)和N-菲酰-次巯基-L-丙氨酸(NPN)的摄取来探讨丁香酚处理后金黄色葡萄球菌细胞膜的通透性和完整性;以结晶紫染色法和实时荧光定量PCR法研究丁香酚对菌体生物被膜(BF)生成、成熟以及菌体黏附、侵袭相关毒力因子表达水平的影响。结果丁香酚对金黄色葡萄球菌标准菌和耐甲氧西林金葡菌(MRSA)的最低抑菌浓度(MIC)分别为0.25、0.125 mg/mL。丁香酚处理后可以提高细菌培养液中核酸、蛋白浓度及AKP的活性;菌体出现皱缩和破裂,菌体内膜和外膜中分别出现PI和NPN荧光,菌体结构破坏和荧光强度与丁香酚浓度呈正相关。丁香酚能够抑制金黄色葡萄球菌菌株BF的生成,且对成熟BF具有明显清除作用,显著降低BF生成相关基因agr A、sar A、cid A和icaA及黏附因子clf A、clf B、fnb A和fnbB的基因表达水平;与标准菌株相比,丁香酚对MRSA的作用更明显。结论丁香酚对金黄色葡萄球菌标准菌株和耐药菌株均具有显著抗菌作用,可破坏菌体细胞结构,改变细胞膜通透性和完整性,抑制BF的生成,并对成熟BF具有清除作用,抑制细菌BF及黏附侵袭相关毒力因子的转录和表达,可能是艾灸促进压疮创面修复的机制之一。Objective To identify the antibacterial mechanism of eugenol against Staphylococcus aureus that causes pressure sores.Methods The minimum inhibitory concentration(MIC)of eugenol on S.aureus was determined by microbroth dilution method and growth curve.Subsequently,AKP activity,bacterial nucleic acid protein leakage,bacterial ultrastructure observed by scanning electron microscopy and transmission electron microscopy,as well as PI and NPN uptake of eugenol on S.aureus were detected to determine its permeability and integrity of cell membrane of S.aureus treated with eugenol.The effect of eugenol on the formation and maturation of S.aureus ATCC29213 and MRSA biofilms was studied by crystal violet staining.The expression levels of eugenol on biofilms and adhesion and invasion-related virulence factors were detected by real-time fluorescent quantitative PCR after exposure.Results The results showed that the MIC of eugenol against S.aureus ATCC29213 and MRSA were respectively 0.25 and 0.125 mg/mL.After eugenol intervention,the concentration of nucleic acid and protein as well as the activity of AKP can be increased in bacterial culture medium.The bacteria were crumpled and ruptured,and PI and NPN fluorescence appeared in the inner membrane and outer membrane,respectively.The damage of cell structure and fluorescence intensity were positively correlated with eugenol concentration.Eugenol can inhibit the BF formation of S.aureus strain,and has obvious scavenging effect on mature BF.Eugenol significantly reduced the expression levels of biofilm related genes including agrA,sarA,cidA and icaA and significantly inhibited the expression levels of adhesion factors such as clfA,clfB,fnbA and fnbB.It was also found that eugenol had a more significant inhibitory effect on MRSA.Conclusion Eugenol has significant antibacterial effect on both standard and drug-resistant strains of S.aureus.Eugenol can damage the cell structure of S.aureus,change both the permeability of cell membrane and the integrity of cell wall.Eugenol can inhi

关 键 词：丁香酚 压疮 金黄色葡萄球菌 抑菌 艾烟 创面修复 艾灸 

分 类 号：R965[医药卫生—药理学]