槲皮素抗多重耐药牛乳房炎金黄色葡萄球菌作用

Inhibitory effects of quercetin against multidrug-resistant Staphylococcus aureus causing bovine mastitis

摘要:

【背景】 牛乳房炎金黄色葡萄球菌(Staphylococcus aureus)具有多重耐药和广泛耐药性。目前已有的抗菌药大多数疗效降低，并且容易残留。天然药物对耐药菌有良好的抑制作用。【目的】 探究槲皮素对牛乳房炎金黄色葡萄球菌抗菌作用及机制。【方法】 采用K-B法测定牛乳房炎金黄色葡萄球菌对15种抗菌药的敏感性。采用微量肉汤稀释法测定槲皮素对牛乳房炎金黄色葡萄球菌的最小抑菌浓度(minimum inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC)。通过生长曲线和杀菌曲线分析槲皮素对牛乳房炎金黄色葡萄球菌抑菌和杀菌特征。采用棋盘法测定槲皮素联合氨苄西林、恩诺沙星和多西环素对牛乳房炎金黄色葡萄球菌的抗菌作用。通过生物膜抑制测定、核酸和蛋白质外泄测定、细菌生存活力观察、菌体内槲皮素蓄积测定、膜蛋白构象变化测定及分子对接等揭示槲皮素抗金黄色葡萄球菌的作用机制。【结果】 槲皮素对牛乳房炎金黄色葡萄球菌的MIC和MBC分别为256 μg/mL和512 μg/mL；槲皮素能够抑制牛乳房炎金黄色葡萄球菌生长，12 h内完全杀灭细菌。槲皮素分别与氨苄西林、恩诺沙星和多西环素联合应用，抗牛乳房炎金黄色葡萄球菌的分级抑制浓度指数(fractional inhibitory concentration index, FICI)分别为0.50、0.75、0.50，呈现协同或相加作用。槲皮素能够显著抑制生物膜的形成(P < 0.05)。核酸和蛋白质外泄以及细菌生存活力分析结果表明槲皮素增加了细胞膜通透性。槲皮素在菌液中的荧光强度显著降低(P < 0.05)，说明其在菌体内的浓度随作用时间逐渐增加。槲皮素能够导致菌体膜蛋白二级和三级构象变化。槲皮素与酪氨酰-tRNA合成酶通过范德华力和氢键结合，结合稳定，从而抑制酶活性。【结论】 槲皮素能够抑制生物膜活性、增强细胞膜通透性、影响膜蛋白构象和抑制酪氨酰-tRNA合成酶活性，从而对牛乳房炎金黄色葡萄球菌产生抗菌作用。

Abstract:

[Background] Staphylococcus aureus, the pathogen of bovine mastitis, has multidrug resistance and extensive drug resistance. Currently, most existing antibacterial drugs demonstrate limited efficacy and are prone to residue. Natural medicines have good inhibitory effects against drug-resistant bacteria. [Objective] To study the inhibitory activity and mechanism of quercetin against S. aureus causing bovine mastitis. [Methods] The K-B method was used to determine the sensitivity of S. aureus causing bovine mastitis to 15 antibiotics. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of quercetin against S. aureus causing bovine mastitis were determined by the broth microdilution method. The growth curve and bactericidal curve were established to analyze the inhibitory and killing effects of quercetin against S. aureus causing bovine mastitis. The chessboard method was adopted to determine the effects of quercetin combined with ampicillin, enrofloxacin, and doxycycline against S. aureus causing bovine mastitis. The mechanism of quercetin against S. aureus causing bovine mastitis was revealed through biofilm inhibition assay, nucleic and protein leakage assay, bacterial viability observation, quercetin accumulation analysis in bacteria, membrane protein conformation change assay, and molecular docking. [Results] The MIC and MBC of quercetin against S. aureus were 256 μg/mL and 512 μg/mL, respectively. Quercetin inhibited the growth of S. aureus and completely killed the bacteria within 12 h. The fractional inhibitory concentration indexes (FICIs) of quercetin with ampicillin, enrofloxacin, and doxycycline against S. aureus were 0.50, 0.75, and 0.50, respectively, which indicated synergistic or additive effects. Quercetin inhibited the biofilm formation of S. aureus (P < 0.05). Nucleic acid and protein leakage and bacterial viability analyses indicated that quercetin increased cell membrane permeability. The fluorescence intensity of quercetin in the bacterial suspension reduced (P < 0.05), which suggested that the concentration in bacterial cells gradually increased over time. Quercetin caused secondary and tertiary conformational changes of bacterial membrane proteins. Quercetin bound to tyrosyl-tRNA synthetase through van der Waals force and hydrogen bonding, which resulted in stable binding, thereby inhibiting enzyme activity. [Conclusion] Quercetin has inhibitory effect against S. aureus causing bovine mastitis by inhibiting the biofilm formation, enhancing the permeability of cell membrane, affecting the conformation of membrane proteins, and suppressing the activity of tyrosyl-tRNA synthetase.