目的:据报道Zerumbone具有抗菌作用,但是该化合物发挥作用的机制尚不清楚。因此,本研究旨在探讨zerumbone抗耐甲氧西林金黄色葡萄球菌(MRSA)的作用机制,使用原子力显微镜(AFM),扫描电子显微镜(SEM),和流式细胞术技术。
方法:使用微孔板AlamarBlue测定法测定MRSA(NCTC13277)细胞活力。使用AFM和SEM来确定经零骨处理的MRSA细胞的形态。流式细胞仪分析用于确定零骨对细菌膜通透性和膜电位的影响,采用碘化丙啶(PI)染色法,膜电位敏感荧光探针,和DiBAC4(3)染料。DCFDA染料用于确定MRSA产生的活性氧(ROS)。
结果:Zerumbone显着抑制MRSA生长,最低抑制浓度(MIC)为125µg/ml。AFM分析表明,零骨导致细菌细胞中细胞质内容物的泄漏。超微结构分析显示细菌的小菌落在膜表面上具有孔。零骨处理的MRSAPI和DiBAC4(3)荧光增加,表明细胞膜通透性增加和膜电位降低,最终导致膜结构完整性丧失和细菌死亡。基于DCFDA的染料分析,zerumbone还减少了MRSA产生的ROS。
结论:Zerumbone通过引起膜去极化发挥抗MRSA作用,增加膜的渗透性,最后破坏细胞膜和杀死细菌。
OBJECTIVE: Zerumbone has been reported to exert anti-microbial effects, but the mechanism by which the compound exerts its action is not known. Thus, this study aimed to investigate the mechanism of action of zerumbone against methicillin-resistance Staphylococcus aureus (MRSA), using the atomic force microscopy (AFM), scanning electron microscopy (SEM), and flow cytometry techniques.
METHODS: MRSA (NCTC 13277) cell viability was determined using the microplate AlamarBlue assay. AFM and SEM were used to determine the morphology of zerumbone-treated MRSA cells. Flow cytometric analysis was used to determine the effect of zerumbone on bacterial membrane permeability and membrane potential, using the propidium iodide (PI) staining method, membrane potential-sensitive fluorescence probe, and DiBAC4(3) dye. DCFDA dye was used to determine the generation of reactive oxygen species (ROS) by MRSA.
RESULTS: Zerumbone significantly inhibited MRSA growth with a minimum inhibitory concentration (MIC) of 125 µg/ml. The AFM analysis showed that zerumbone caused leakage of cytoplasmic content from the bacterial cells. Ultrastructure analysis showed small colonies of the bacteria with pores on the membrane surface. There were increases in zerumbone-treated MRSA PI and DiBAC4(3) fluorescence, indicating an increase in cell membrane permeability and a decrease in membrane potential that culminated in the loss of membrane structural integrity and bacterial death. Based on DCFDA dye analysis, zerumbone also reduced ROS production by MRSA.
CONCLUSIONS: Zerumbone exerts anti-MRSA effects by causing membrane depolarization, increasing membrane permeability, and finally disrupting cell membrane and bacterial killing.