目的 明確丁香酚對(duì)壓瘡致病菌金黃色葡萄球菌Staphylococcus aureus的體外抑菌作用及機(jī)制。方法 采用微量肉湯稀釋法和和生長(zhǎng)曲線確定丁香酚對(duì)金黃色葡萄球菌的抑菌活性；通過堿性磷酸酶（AKP）活性、細(xì)菌核酸蛋白泄漏的檢測(cè)，細(xì)菌超微結(jié)構(gòu)的掃描和透射電鏡觀察以及菌體對(duì)碘化丙啶（PI）和N-菲酰-次巰基-L-丙氨酸（NPN）的攝取來探討丁香酚處理后金黃色葡萄球菌細(xì)胞膜的通透性和完整性；以結(jié)晶紫染色法和實(shí)時(shí)熒光定量PCR法研究丁香酚對(duì)菌體生物被膜（BF）生成、成熟以及菌體黏附、侵襲相關(guān)毒力因子表達(dá)水平的影響。結(jié)果 丁香酚對(duì)金黃色葡萄球菌標(biāo)準(zhǔn)菌和耐甲氧西林金葡菌（MRSA）的最低抑菌濃度（MIC）分別為0.25、0.125 mg/mL。丁香酚處理后可以提高細(xì)菌培養(yǎng)液中核酸、蛋白濃度及AKP的活性；菌體出現(xiàn)皺縮和破裂，菌體內(nèi)膜和外膜中分別出現(xiàn)PI和NPN熒光，菌體結(jié)構(gòu)破壞和熒光強(qiáng)度與丁香酚濃度呈正相關(guān)。丁香酚能夠抑制金黃色葡萄球菌菌株BF的生成，且對(duì)成熟BF具有明顯清除作用，顯著降低BF生成相關(guān)基因agrA、sarA、cidA和icaA及黏附因子clfA、clfB、fnbA和fnbB的基因表達(dá)水平；與標(biāo)準(zhǔn)菌株相比，丁香酚對(duì)MRSA的作用更明顯。結(jié)論 丁香酚對(duì)金黃色葡萄球菌標(biāo)準(zhǔn)菌株和耐藥菌株均具有顯著抗菌作用，可破壞菌體細(xì)胞結(jié)構(gòu)，改變細(xì)胞膜通透性和完整性，抑制BF的生成，并對(duì)成熟BF具有清除作用，抑制細(xì)菌BF及黏附侵襲相關(guān)毒力因子的轉(zhuǎn)錄和表達(dá)，可能是艾灸促進(jìn)壓瘡創(chuàng)面修復(fù)的機(jī)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

國(guó)家自然科學(xué)基金資助項(xiàng)目（82004375）；中國(guó)博士后科學(xué)基金資助項(xiàng)目（2021MD703836）；宿遷市“西楚英才”雄英計(jì)劃雙創(chuàng)人才資助項(xiàng)目（衛(wèi)生創(chuàng)新）〔（2022）-0051〕