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agrA、sarA、cidA和icaA及黏附因子clfA、clfB、fnbA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 inhibit the generation of biofilms of S. aureus, promote the removal of mature biofilms as well. It does inhibit the transcription and expression of virulence factors related to bacterial biofilm and adhesion invasion, and it may be one of the mechanisms by which moxibustion can promote the wound repair on pressure ulcer.

R965

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〕