枯草芽孢杆菌中bacilosarcin B对美人鱼发光杆菌的抑菌机理

Inhibitory mechanism of bacilosarcin B from Bacillus subtilis against Photobacterium damselae

摘要:

【背景】美人鱼发光杆菌（Photobacterium damselae）是一种海洋条件致病菌，能够引起多种海洋生物和人类疾病。因此，探究美人鱼发光杆菌的生物防治技术具有重要意义。【目的】探究枯草芽孢杆菌（Bacillus subtilis）中bacilosarcin B对美人鱼发光杆菌的抑菌活性及其可能的抑菌机理。【方法】利用高效液相色谱法从枯草芽孢杆菌fmb60发酵液中制备bacilosarcin B，采用分光光度法测定bacilosarcin B对多种致病菌的最小抑菌浓度及其对美人鱼发光杆菌的时间-抑菌曲线。测定bacilosarcin B对美人鱼发光杆菌生物被膜、胞外核酸、蛋白质和胞内碱性磷酸酶含量的影响，结合荧光显微镜、扫描电镜和透射电镜检测美人鱼发光杆菌细胞膜通透性和细胞壁完整性，并研究bacilosarcin B对细菌运动能力和胞内DNA的作用。【结果】Bacilosarcin B对美人鱼发光杆菌最小抑菌浓度为8 μg/mL。抑菌机理研究表明bacilosarcin B通过破坏细菌细胞壁和细胞膜的完整性使细胞膜通透性增强，造成细胞内成分渗出。此外，bacilosarcin B还可与胞内DNA结合，且结合方式与溴化乙锭（ethidium bromide，EB）插入DNA结构类似。【结论】bacilosarcin B对美人鱼发光杆菌具有显著的抑菌效果，具有研发成新型抑菌剂的潜力，对海洋养殖业的健康发展具有一定的意义。

Abstract:

[Background] The marine pathogenic bacterium, Photobacterium damselae, causes various diseases in marine organisms and humans. Therefore, it is of great significance to investigate the approaches for the biocontrol of P. damselae. [Objective] To investigate the activity and mechanism of bacilosarcin B from Bacillus subtilis in inhibiting P. damselae.[Methods] Bacilosarcin B was prepared from the fermentation broth of B. subtilis fmb60 by high-performance liquid chromatography. The minimum inhibitory concentration of bacilosarcin B against various pathogenic bacteria and the time-kill curve against P. damselae were determined using a spectrophotometer. The impacts of bacilosarcin B on the biofilm, extracellular nucleic acids, proteins, and intracellular alkaline phosphatase content of P. damselae were assessed. Fluorescence microscope, scanning electron microscope, and transmission electron microscope were employed to examine the cell membrane permeability and cell wall integrity of P. damselae. Finally, the effects of bacilosarcin B on bacterial motility and intracellular DNA were studied. [Results] The minimum inhibitory concentration of bacilosarcin B against P. damselae was 8 μg/mL. Bacilosarcin B disrupted the integrity of bacterial cell walls and cell membranes and increased membrane permeability to cause the leakage of cellular components. Additionally, it bound to intracellular DNA by the manner similar to the insertion of ethidium bromide into DNA. [Conclusion] Bacilosarcin B exhibits a significant inhibitory effect on P. damselae and has the potential to be developed as a novel antibacterial agent, which is of significance for the healthy development of marine aquaculture.