基于细胞膜损伤的蔗糖月桂酸酯对金黄色葡萄球菌的作用机制

Sucrose Laurate Exerts Antibacterial Activity against Staphylococcus aureus by Destroying the Cell Membrane

研究蔗糖月桂酸酯对金黄色葡萄球菌的抑菌活性及其对细胞膜的损伤机制。首先采用二倍稀释法考察了蔗糖月桂酸酯对金黄色葡萄球菌的最小抑菌浓度,用平板计数法绘制了时间-杀菌曲线;通过DiSC_3(5)探针标记荧光分光光度法考察了蔗糖月桂酸酯对菌体细胞膜电势差的影响,使用荧光显微镜结合流式细胞术研究了菌体细胞膜渗透性的影响,使用紫外分光光度法考察了大分子物质的泄漏情况,使用PBFI钾离子探针测定了胞内钾离子泄漏量,最后通过透射电子显微镜观察了蔗糖月桂酸酯处理前后菌体的超微结构。结果表明:蔗糖月桂酸酯对金黄色葡萄球菌的最小抑菌浓度(minimum inhibitory concentration,MIC)为0.312 5 mg/mL;经蔗糖月桂酸酯作用的金黄色葡萄球菌与对照组相比,Di SC_3(5)探针的荧光强度呈现剂量依赖性增大;PI探针对菌体的沾染率增加,MIC处理30 min菌体PI沾染率达到84.7%;260 nm波长处的吸光度随作用时间的延长逐渐增大,但增大量较小;蔗糖月桂酸酯会导致金黄色葡萄球菌细胞钾离子泄漏,且泄漏量与添加的蔗糖月桂酸酯质量浓度呈正相关;经MIC蔗糖月桂酸酯作用1 h后,金黄色葡萄球菌体表面变粗糙,边缘模糊。结论:蔗糖月桂酸酯可以通过破坏细胞膜渗透性消散细胞膜电势,从而导致胞内物质发生轻微泄漏,最终达到抑菌作用,本研究可为多功能性糖酯抑菌产品的开发提供理论依据。

The antibacterial activity of sucrose laurate against Staphylococcus aureus was determined, and the mechanism of cell membrane damage by sucrose laurate was investigated. The minimum inhibitory concentration(MIC) against S. aureus was determined by the two-fold dilution method, and the time-killing curve was assayed by the plate counting method. We studied the effect of sucrose laurate on the membrane potential of S. aureus was studied by fluorescence spectroscopy using DiSC3(5) as the probe, the effect on the membrane permeability by fluorescence microscopy and flow cytometry, the effect on the leakage of macromolecular substances by ultraviolet spectrophotometry, the effect on the leakage of intracellular potassium ions using the fluorescent probe PBFI, and the effect on the ultrastructure by transmission electron microscopy.The results showed that the MIC of sucrose laurate against S. aureus was 0.312 5 mg/mL. Compared with the control group, the fluorescence intensity of S. aureus with the probe DiSC3(5) increased in a concentration-dependent manner after treatment with sucrose laurate;The percentage of S. aureus stained with PI was 84.7% after the treatment of sucrose laurate at MIC for 30 min. The absorbance at 260 nm gradually increased with increasing treatment time at each concentration of sucrose laurate, but the increment was small. Sucrose laurate caused leakage of intracellular potassium ions and this effect was positively correlated with its concentration. After treatment with sucrose laurate at MIC for 1 h, the surface of bacterial cells became rough with blurred edges. Accordingly we concluded that sucrose laurate could dissipate cell membrane potential and destroy membrane permeability, leading to slight leakage of intracellular substances and consequently exerting antibacterial activity. This investigation can provide a theoretical basis for the development of versatile antibacterial products with glycolipids.