唐古特大黄内生细菌多样性及抗菌活性

Diversity and antibacterial activity of endophytic bacteria in Rheum tanguticum

摘要:

【背景】 由于抗生素的过度使用和误用，加速了耐药细菌的出现，对全球卫生与发展构成威胁，因此迫切需要开发新型抗生素。唐古特大黄(Rheum tanguticum)是一种传统中药，具有多种药效活性成分，药理作用主要有抗菌、抗病毒、止血、利胆、抗肿瘤。植物内生菌在与宿主长期进化的过程中会产生与宿主相同或相似的代谢产物，已成为人们寻找新型抗菌药物的重要来源。【目的】 探究唐古特大黄内生细菌的多样性，筛选出具有抗菌活性的细菌，为开发天然抗菌药物提供微生物资源。【方法】 采用稀释涂布法在3种培养基上分离内生细菌，通过16S rRNA基因序列确定内生细菌的分类地位；采用琼脂块法对内生细菌进行抑菌活性初筛；对目标菌株进行抑菌活性检测和全基因组测序分析。【结果】 从唐古特大黄中共分离得到100株内生细菌，分属于4门8纲17目26科43属，其中厚壁菌门(Firmicutes, 35%)为优势菌门，芽孢杆菌属(Bacillus, 9%)为优势菌属。抑菌活性初筛结果显示，有13株菌对不同病原菌表现出了抑制作用，其中1株类芽孢杆菌(Paenibacillus sp.) RTM5-22对5种病原菌有很好的抑制活性；菌株RTM5-22液体发酵正丁醇相粗提物对7种病原菌均有抑制活性；菌株RTM5-22基因组中包含聚酮类、萜烯类、核糖体合成和翻译后修饰肽等次级代谢基因簇。【结论】 唐古特大黄内生细菌物种多样性较为丰富，而且获得多株具有抗菌活性的菌株，为唐古特大黄内生细菌的后续研究提供数据支撑。菌株RTM5-22的代谢产物有广谱抗菌活性，可为寻找新型、高效的天然抑菌活性物质提供新的菌种资源。

Abstract:

[Background] The overuse and misuse of antibiotics accelerate the emergence of drug-resistant bacteria and pose a threat to global health and development. Therefore, it is urgent to develop new antibiotics. Rheum tanguticum, a Chinese medicinal herb, contains a variety of active ingredients and has antimicrobial, antiviral, hemostatic, cholagogic, and antitumor effects. Endophytes of medicinal plants can produce the same or similar metabolites in the long-term evolution with their hosts, becoming an important source for the development of new drugs. [Objective] To explore the diversity of endophytic bacteria in R. tanguticum and screen out the bacteria with antibacterial activity, thus providing microbial resources for the development of natural antibacterial drugs. [Methods] The endophytic bacteria were isolated by dilution coating on three culture media and identified by 16S rRNA gene sequencing. The agar block method was used to screen the endophytic bacteria with antibacterial activity. The antibacterial activities and whole genome sequences of the strains screened out were determined. [Results] A total of 100 strains of endophytic bacteria were isolated from R. tanguticum, belonging to 43 genera, 26 families, 17 orders, 8 classes of 4 phyla, of which Firmicutes was the dominant phylum (35%) and Bacillus (9%) was the dominant genus. The preliminary screening results showed that 13 strains exerted inhibitory effects on different pathogens, and one of them, RTM5-22, had strong inhibitory effects on 5 pathogenic bacteria. The crude n-butanol extract of the fermentation broth of RTM5-22 showed inhibitory activities against 7 pathogens. The genome of RTM5-22 contained the gene clusters for synthesizing secondary metabolites such as polyketones and terpenes, ribosomes, and post-translational modified peptides. [Conclusion] There are rich endophytic bacteria in R. tanguticum. We isolated a number of strains with excellent antibacterial activities, which provides data support for the follow-up study of endophytic bacteria from R. tanguticum. The metabolites of RTM5-22 have broad-spectrum antibacterial activities, which can provide resources for mining new and efficient natural antibacterial substances.