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

描述(申请人提供)：人类肠道微生物组的研究面临许多挑战，其中包括解决实际问题，如获得未扭曲和有代表性的样本，以及确定如何将发现未表征、似乎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，到目前为止的研究也没有认识到，用于为标准结肠镜检查准备结肠的结肠灌洗显著稀释和扭曲肠道微生物群。此外，我们对人类肠道微生物群的了解在很大程度上是基于对粪便和腔样本的分析，而这些样本可能不足以代表更多居住和地理上特定的粘膜相关细菌群落。在这方面，可能会错过具有特殊条件或公共属性的新的和代表性不足的物种，这些属性有助于它们与宿主更接近。这些生物很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群时，还必须考虑其组成和群落组织的地区差异。在这项提案中，将在开发新的和改进的非培养技术时考虑到这些问题，这些技术最终将促进对大量以前未描述的人类肠道微生物组成员的基因组测序和元基因组分析。我们建议在未准备好的人类结肠内获得自然状态下的粘膜相关微生物的区域特定样本。然后，我们将开发和提炼两种基于非培养的方法，旨在获得高等级的微生物群落复合DNA，或浓缩/纯化的未被充分代表的未分类微生物物种的样本。第一项涉及激光捕获显微解剖人类结肠不同区域的粘膜相关细菌，这将主要用于产生高质量的DNA，用于元基因组分析。第二种方法包括使用16S rDNA的荧光原位杂交(FISH)和结合荧光激活细胞分选(FACS)的后基因组确定的独特核探针。产量、浓缩和纯度将得到优化，以从人类结肠微生物组中发现和分离新的、未分类的和稀有的微生物，用于高通量测序中心的全基因组测序。我们相信，这些研究将产生非基于培养的技术，这些技术将促进对大量以前未描述的人类肠道微生物组成员的基因组测序和元基因组分析。公共卫生相关性：这项应用旨在开发新的和改进的非培养为基础的技术，最终将促进对大量以前未描述的人类肠道微生物组成员的基因组测序和元基因组分析。这将通过开发激光捕获显微解剖和FISH-FACS方法来实现，这些方法来自完整的、与粘膜相关的人类肠道微生物群落的样本。

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.