肠道微生物资源库的构建：进展、方法和展望

Construction of gut microbial culture banks: advances, methods and perspectives

摘要:

基于肠道微生物组的活体药物(live biotherapeutics, LBPs)开发、菌株与宿主互作的分子机制研究及新型抗菌肽、酶、代谢途径的挖掘使得肠道微生物资源库的构建成为必然。本文对近年来国内外不同研究团队肠道微生物资源库构建进行系统比较，分析了不同构建方法间的差异，以期为国内外不同研究者在构建和丰富现有肠道微生物资源库方面提供帮助。目前，肠道微生物资源库共有1 000多种肠道细菌，分属于12个门、22个纲、39个目、96个科和358个属，厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidota)、放线菌门(Actinomycetota)菌株最多。测序结果显示人肠道细菌物种丰富度在2 000左右，因此目前分离到的菌株远未达到饱和。在构建方法上，一般对粪便样本进行或不进行乙醇处理，使用非选择性培养基(以Gifu厌氧培养基为代表)进行涂布分离培养，最后进行纯化培养。使用较为简单的培养方法即可培养得到多数常见的重要肠道微生物类目，如双歧-乳杆菌属(Lactobacillus-bifidobacteria)菌株、阿克曼氏菌(Akkermansia muciniphila)、普拉梭菌(Faecalibacterium prausnitzii)、普雷沃氏菌属(Prevotella)及S24-7科菌株等。为满足功能研究和产品开发的需要，肠道微生物菌种资源库的样本来源应该进一步覆盖更多地域和生活习惯、疾病及健康状态具有显著差异的人群，从而进一步丰富肠道关键物种的菌株多样性。

Abstract:

Recently, the gut microbiota-based live biotherapeutics (LBPs) development, the interaction between gut microbial species and the host, and the mining of new antimicrobial peptides, enzymes and metabolic pathway have received increasing attention. Culturing gut microbial species is therefore of great importance. This review systemically compared the construction advances of gut microbial culture banks and also analyzed the differences of methods used by research groups to give insight into the construction and enrichment of gut microbial resources. Presently, the gut microbial culture banks have included more than 1 000 bacterial species, belonging to 12 phyla, 22 classes, 39 orders, 96 families, and 358 genera. Among these, Firmicutes, Proteobacteria, Bacteroidota, and Actinomycetota exhibited the greatest diversities at the species level. The sequencing data showed that there are more than 2 000 species inhibited in the human gut. Therefore, the cultured gut microbial species are far from saturation. In terms of the construction method, the stool samples were pre-treated with ethanol or directly spread and cultured in the non-selective nutritional rich medium (represented by Gifu anaerobic medium) to obtain single colony. Then single colony was further purified. Generally, a simplified isolation and culture method is sufficient to obtain the most common and important intestinal bacterial species, such as Bifidobacteria-Lactobacillus, Akkermansia muciniphila, Faecalibacterium prausnitzii, Prevotella and S24-7 family strains. Finally, microbial resources with great diversities at the strain level are required for further functional research and product development. Samples covering hosts with distinct physiological status, diets or regions are necessary.