藤黄微球菌拮抗菌株筛选及抑菌活性物质分离鉴定

Screening of an antagonistic strain against Micrococcus luteus and isolation and identification of an antibacterial compound

摘要:

【背景】藤黄微球菌(Micrococcus luteus)是一种条件致病菌，能引起肺炎、心内膜炎、败血症等疾病。近年来，从沙漠环境中分离具有抑菌作用的放线菌及其抑菌活性物质成为研究热点。【目的】筛选藤黄微球菌拮抗放线菌菌株，分离纯化抑菌活性物质，并检测其细胞毒性。【方法】利用平板对峙法，从沙漠土壤中筛选藤黄微球菌拮抗菌株，以具有较强抑制作用的菌株为研究对象，结合形态学观察和16S rRNA基因序列进行菌种鉴定。采用硅胶柱和Sephadex LH-20凝胶层析等方法从目标菌株中分离抑菌活性物质。通过微量孔法测定其最小抑菌浓度(minimum inhibitory concentration,MIC)和最小杀菌浓度(minimum bactericidal concentration,MBC)，并使用噻唑蓝(methylthiazolyldiphenyl tetrazolium bromide,MTT)法检测该物质对RAW264.7巨噬细胞的细胞毒性。【结果】从塔克拉玛干沙漠土壤中分离到一株具有显著抑制作用的放线菌TRM 58446-WSY，鉴定为灰肉色链霉菌(Streptomyces griseoincarnatus)。从其发酵菌体中分离纯化到一种具有藤黄微球菌拮抗活性的化合物——3,4-二叔丁基苯酚(3,4-di-tert-butylphenol,3,4-DTBP)。该化合物对藤黄微球菌的MIC为55.34 μg/mL，MBC为57.62 μg/mL，并且对RAW264.7巨噬细胞没有毒性。【结论】拮抗菌株TRM 58446-WSY及其代谢产物3,4-二叔丁基苯酚具有良好的藤黄微球菌拮抗活性且没有细胞毒性，为开发天然抑菌药物提供菌株支持和理论基础。

Abstract:

[Background] Micrococcus luteus is an opportunistic pathogen that can cause a variety of diseases such as pneumonia, endocarditis, and sepsis. The isolation of actinomycetes from desert environments and identification of their antibacterial compounds have become research hotspots in recent years. [Objective] To screen antagonistic actinomycete strains against M. luteus, isolate and purify antibacterial compounds, and evaluate their cytotoxicity. [Methods] The antagonistic strains of M. luteus were screened from desert soil by the dilution plate method. The strains with strong inhibitory effects were identified by 16S rRNA sequencing and morphological observation. The antibacterial compounds were isolated by gel column chromatography and Sephadex LH-20 chromatography after fermentation in the optimum fermentation media. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the microtiter-well method, and the cytotoxicity of the compounds to RAW264.7 macrophages was evaluated by the methylthiazolyldiphenyl tetrazolium bromide (MTT) method.[Results] An actinomycete strain TRM 58446-WSY exhibiting significant inhibitory activity was isolated from the soil of the Taklimakan Desert and identified as Streptomyces griseoincarnatus. A compound, 3,4-di-tert-butylphenol (3,4-DTBP), antagonistic to M. luteus, was isolated and purified from the fermentation broth of the strain. This compound showed the MIC of 55.34 μg/mL, MBC of 57.62 μg/mL, and no toxicity to RAW264.7 macrophages. [Conclusion] The antagonistic strain S. griseoincarnatus TRM 58446-WSY and its metabolite 3,4-DTBP had good antagonistic activity against M. luteus and no cytotoxicity, serving as a strain and a compound supporting the development of natural antibacterial drugs.