白马雪山云南黄芪与灰毛康定黄芪根际微生物物种多样性及抗生物膜活性菌株筛选

Rhizosphere microbial diversity of Astragalus yunnanensis and A.tatsienensis var.incanus in Baima Snow Mountain and screening of microorganisms with anti-biofilm activity

【背景】细菌生物膜是造成病原菌耐药性增强和持续感染的主要因素,但目前尚无针对抗菌膜的特效药物。特境植物根际微生物可产生大量具有提高宿主免疫功能的活性成分,极具抗生物膜药源开发潜力。【目的】了解滇西北高寒特境白马雪山分布的云南黄芪与灰毛康定黄芪植物根际微生物的物种多样性,并对可培养菌株进行抑菌与抗生物膜活性筛选。【方法】采用宏基因组技术结合传统微生物培养方法,对采自我国云南迪庆藏族自治州德钦县白马雪山的云南黄芪与灰毛康定黄芪的根际微生物进行物种多样性研究,并通过“孔板法”测定其可培养菌株发酵液乙酸乙酯粗浸膏的抗菌、抗生物膜活性。【结果】宏基因组测序结果显示,云南黄芪根际土壤样本中的微生物来自6门7纲8目8科9属10种,其中栖热菌属为优势菌群;灰毛康定黄芪根际土壤样本中的微生物来自6门8纲10目11科14属15种,其中慢生根瘤菌属为优势菌群。通过纯培养共获得145株可培养菌株,包括112株细菌和33株真菌。其中,云南黄芪根际细菌59株,共计16属35种,优势属为假单胞菌属和链霉菌属;根际真菌19株,共计4属5种,优势属为曲霉属;灰毛康定黄芪根际细菌53株,归属于16属29种,优势属为芽孢杆菌属与寡养单胞菌属;根际真菌14株,归属于3属4种,优势属为曲霉属。从不同种水平上选择51株细菌和7株真菌为代表菌株进行抗生素药源评估,发现5株细菌及1株真菌发酵液的乙酸乙酯粗浸膏具有中等至较强的抗革兰阳性菌活性,而且其中4株具有抗MRSA生物膜活性,最终确定了链霉属放线菌Streptomyces fulvissimus KTA1和曲霉属真菌Aspergillus fumigatus YNF5为潜力活性菌株。【结论】首次报道了滇西北地区高寒特境黄芪属植物根际微生物具有较好的物种多样性,而且具有一定的抗生素药用资源开发潜力。本研究对滇西北高寒特境特色植物来源的微生物资源开发利用与保护具有重要的借鉴意义。

[Background]Bacterial biofilm is a major factor causing bacterial resistance and recurrent nosocomial infections.But up to now there have been no effective anti-biofilm drugs.Rhizosphere microorganisms of some plants in high-cold,special environment can produce large quantities of active components which can improve the host immunity,and are thought to be a medicinal resource with great anti-biofilm potential.[Objective]To understand the rhizosphere microbial diversity of Astragalus yunnanensis and A.tatsienensis var.incanus growing in Baima Snow Mountain,and to screen cultivable strains with antibacterial and anti-biofilm activities.[Methods]In this study,metagenomics and traditional culture-dependent methods were employed to explore the rhizosphere microbial diversity of A.yunnanensis and A.tatsienensis var.incanus from Baima Snow Mountain in Deqin County,Diqing Tibetan Autonomous Prefecture,Yunnan Province.The microplate assay was conducted for determining the antibacterial and anti-biofilm activity of these cultivable microbes.[Results]Metagenomic sequencing indicated that the rhizosphere microorganisms of A.yunnanensis samples belonged to 6 phyla,7 classes,8 orders,8 families,9 genera and 10 species,and Thermus was the dominant microflora.The rhizosphere microbes of A.tatsienensis var.incanus samples belonged to 6 phyla,8 classes,10 orders,11 families,14 genera and 15 species,and Bradyrhizobium was the dominant microflora.A total of 145 cultivable strains were obtained by pure cultivation,including 112 bacteria and 33 fungi.Among them,59 bacterial strains of 16 genera and 35 species and 19 fungal strains of 4 genera and 5 species were isolated from the rhizosphere soil of A.yunnanensis,with Streptomyces,Pseudomonas and Aspergillus being predominant.The other 53 bacterial isolates of 16 genera and 29 species and 14 fungal isolates of 3 genera and 4 species were derived from the rhizosphere soil of A.tatsienensis var.incanus,among which Bacillus,Stenotrophomonas and Aspergillus were the most abundant genera.For the screened microbes with antibacterial and anti-biofilm activity,51 bacterial strains and 7 fungal strains were chosen at different species levels as representatives to explore their potential as medicinal resources of natural antibiotics.The crude ethyl acetate extract of the fermentation broth of 5 bacteria and 1 fungus were demonstrated to have moderate to strong activity against Gram-positive pathogens,and 4 strains of them showed anti-biofilm activity against methicillin-resistant Staphylococcus aureus(MRSA).Finally,two microbial strains Streptomyces fulvissimus KTA1 and Aspergillus fumigatus YNF5 were considered as the promising bioactive strains.[Conclusion]It is reported for the first time that there is abundant species community composition in the rhizosphere microorganisms of Astragalus plants distributed in northwestern Yunnan province.These cultivable microorganisms could be regarded as a great medicinal source for the exploitation of natural antibiotics.This study was significant for utilization and protection of characteristic plants-associated microorganisms in high-cold,special environment of northwestern Yunnan.