基于BIPES指导的肠道微生物的新培养体系研究

More than 4000-5000 bacterial species have been detected in human gut. They form highly organized microbial communities and are responsible for many diseases, such as obesity, diabetes and Inflammatory Bowel Disease. The prevention and treatment of these diseases require a comprehensive knowledge of gastrointestinal microbial communities, which largely relies on culture-dependent methods to have detailed phenotypic and physiological analysis of these communities. However, most of the currently dilution separation methods can only selectively support the growth of a limited number of bacterial species within these communities, and fail to sustain the original intestinal microbial diversity. Recently, there has been a renewed interest in culture methods for ‘non-cultivable’ species. But simple, high-throughput culture system is still lacking, it's out of great significance for the development of new training methods to uncultured strains stripped from the gut "black box system"..Previous studies of our research group found that DL malic acid and ammonium molybdate have a greater impact on the flora diversity, but the lack of a more well system to facilitate access to targeted high diversity of flora culture.In this study, using Barcoded Illumina Paired End Sequencing (BIPES) technology as an index to systematically survey and analyze the selectivity of commonly used lab media, cultural conditions, we developed a new system which can support different sub-populations within the original gut microbial community derived from faeces. This BIPES-guided method could also be used to develop novel media for other complex microbial communities. It will provide a reference method for the analysis of microbial dark matter, a powerful bank for the development of the intestinal microbial resources.

肠道菌群中寄居着4000-5000种细菌，具有非常重要的生物学功能，可应用于人体疾病和慢性代谢障碍的防治。宏基因组学方法揭示肠道中有大量未养部分菌株-微生物暗物质占70%以上，使得微生物培养技术在组学时代重新成为热点。但简便的高通量培养体系仍然缺乏，开发新的培养方法把未培养菌株从肠道“黑箱系统”中剥离出来具有重要意义。项目组前期研究发现DL苹果酸和钼酸铵对菌群培养的多样性有较大影响，但缺乏更系统的体系便于目标明确的获得多样性高的菌群培养物。本项目以此为基础，拟利用BIPES技术、结合电化学和厌氧工作站，从群体角度筛选肠道细菌多样性培养的培养基和培养条件，并系统优化分离、鉴定方法，解决传统培养中的多样性不足的难题。该方法可大量获得目前尚未培养的新微生物，开发新的培养基；同时促进肠道菌群与疾病的关系解读。为微生物暗物质的解析提供参考方法，为肠道微生物资源的开发、利用提供了强大的菌种资源基础。

人体肠道细菌的数量几乎与细胞数目相等，并在人体健康的预防和疾病治疗中发挥着重要的在作用。但是大部分数据来自于高通量测序的结果，具体哪些菌株在起作用由于纯培养菌种的缺乏，而使得这些功能的注释受到了很大的限制。而现有的肠道细菌的培养结果多数是肠杆菌，肠球菌，大肠杆菌等常见可培养菌种。其中大部分菌种是很难获得培养的暗物质。本项目拟建立的基于BIPES指导的一种高通量培养方法，挖掘微生物暗物质。以健康成人的肠道样品为原材料，设计了多种培养成分和培养条件以及生长因子，利用96孔板首先液体培养，然后稀释涂布在不同的培养基上，同时对液体培养液进行16S rRNA多样性分析，对固体平板上的菌落进行纯化，鉴定，并对纯化的菌株测试短链脂肪酸的能力。结果发现：碳源和氮源以及生长因子对微生物的多样性影响非常大；共获得120个纯培养物，分布在5个科，15个属，42个种，其中一株潜在的新种；一株首次报道有产脂肪酸的肠道细菌，两株能够改善大鼠肠道菌群和功能的潜在应用菌株。主要的研究进展包括BIPES高通量测序技术能够指导肠道细菌的定向培养。通过该方法已经获得了脱硫弧菌，获得了3株脱硫弧菌的纯培养菌株，获得了一种培养脱硫狐菌的特异性培养基。这将为脱硫弧菌的分离、培养提供借鉴。TMAO降解菌对治疗动脉粥样硬化有着重要的潜在作用，能够分离到TMAO降解菌是关键步骤，我们分离到了多株降解菌并在鼠体内进行了验证，这为神经内科治疗该类病人提供了基础数据参考。短链脂肪酸菌调控着肠道内多种营养物质的吸收及激素产生，广泛参与能量代谢。我们获得了60株高效产短链脂肪酸的纯培养菌株，分布在多个属。我们试验了一株高效菌在SD大鼠体内的效果，经过检测灌菌前后的SCFAs的浓度和肠道菌群数量的变化，发现所使用菌株CF11对大鼠肠道菌群和产量都有很明显的改进作用，这为临床治疗方案的制定提供了基础数据支持。