Technologies for the discovery of novel human colonic mucosal-associated microbes

描述（由申请人提供）：人类肠道微生物组的研究存在许多挑战，其中包括解决实际问题，例如获得未失真和代表性样品，以及确定如何将用于发现未表征、看似不可培养和代表性差的微生物的先进技术应用于小样本量。然而，迄今为止的研究也未能认识到，用于为标准结肠镜检查准备结肠的结肠灌洗会显著稀释和扭曲肠道微生物组。此外，我们对人类肠道微生物组的了解在很大程度上是基于对粪便和管腔样品的分析，这些样品可能不足以代表粘膜相关细菌的更具居住性和地理特异性的群落。在这方面，新的和代表性不足的物种，有特殊的条件或公共属性，使他们接近主机可能会被错过。这些微生物可能对人类健康和疾病有直接影响。在研究人类肠道微生物组时，还必须考虑其组成和社区组织的区域差异。在这项提案中，这些问题将在开发新的和改进的非培养技术时得到考虑，这些技术将最终促进人类肠道微生物组大量先前未表征成员的基因组测序和宏基因组分析。我们建议在未准备的人类结肠内获得处于自然状态的粘膜相关微生物的区域特异性样品。然后，我们将开发和完善两种非培养为基础的方法，旨在获得高品位，复合DNA的微生物群落或富集/纯化样品的代表性不足，未分类的微生物物种。第一个涉及激光捕获显微切割来自人类结肠不同区域的粘膜相关细菌，这将主要用于生成用于宏基因组分析的高质量DNA。第二种方法涉及荧光原位杂交（FISH），使用16 S rDNA和宏基因组学确定的独特的核糖核酸探针与荧光激活细胞分选（FACS）相结合。将优化产率、富集和纯度，以从人类结肠微生物组中发现和分离新型、未分类和罕见的微生物，用于高通量测序中心的全基因组测序。我们相信这些研究将产生非培养技术，这将推进人类肠道微生物组中大量先前未表征的成员的基因组测序和宏基因组分析。公共卫生相关性：该申请旨在开发新的和改进的非基于培养的技术，这些技术将最终促进人类肠道微生物组的大量先前未表征的成员的基因组测序和宏基因组分析。这将通过对来自人类肠道微生物组的完整粘膜相关微生物群落的样品开发激光捕获显微切割和FISH-FACS方法来实现。

DESCRIPTION (provided by applicant): There are many challenges to the study of the human enteric microbiome, among them solving practical issues such as obtaining undistorted and representative samples and determining how advanced technologies for discovery of uncharacterized, seemingly uncultivable, and poorly represented microorganisms can be applied to small sample sizes. However, studies to date have also failed to recognize that colonic lavage used to prepare the colon for standard colonoscopy significantly dilutes and distorts the enteric microbiome. Moreover, our knowledge of the human enteric microbiome is heavily based on analyses of stool and luminal samples which may not be sufficiently representative of the more residential and geographically-specific communities of mucosal- associated bacteria. In this regard, novel and underrepresented species that have special conditional or communal properties that facilitate their close proximity to the host are likely to be missed. These organisms are likely to have direct bearing on human health and disease. Regional differences in their composition and community organization must also be factored in when studying the human enteric microbiome. In this proposal, these issues will be taken into consideration in developing new and improved non-cultivation-based technologies that will ultimately facilitate genomic sequencing and metagenomic analysis of substantial numbers of previously uncharacterized members of the human enteric microbiome. We propose to obtain region-specific samples of mucosal associated microbes in their natural state within the unprepped human colon. We will then develop and refine two non-cultivation- based approaches aimed at obtaining high grade, composite DNA of microbial communities or enriched/purified samples of underrepresented, unclassified microbial species. The first involves laser capture microdissection of mucosal-associated bacteria from different regions of the human colon, which will be developed primarily for generating high quality DNA for metagenomic analyses. The second approach involves fluorescence in situ hybridization (FISH) using 16S rDNA and metagenomically-determined unique riboprobes coupled with fluorescence-activated cell sorting (FACS). Yield, enrichment, and purity will be optimized to discover and isolate novel, unclassified, and rare microbes from the human colonic microbiome for whole genome sequencing by high throughput sequencing centers. We believe these studies will produce non-cultivation-based technologies that will advance genomic sequencing and metagenomic analysis of substantial numbers of previously uncharacterized members of the human enteric microbiome. PUBLIC HEALTH RELEVANCE: This application is aimed at developing new and improved non-cultivation-based technologies that will ultimately facilitate genomic sequencing and metagenomic analysis of substantial numbers of previously uncharacterized members of the human enteric microbiome. This will be achieved through development of laser capture microdissection and FISH-FACS approaches on samples derived from intact, mucosal-associated microbial communities of the human enteric microbiome.