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面临许多挑战，其中包括解决实际问题，如获得未扭曲和具有代表性的样品，以及确定如何将先进技术用于发现未表征的、看似不可培养的、代表性差的微生物，以应用于小样本量。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着稀释和扭曲肠道微生物群。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肠道样本的分析，这些样本可能不足以代表更多的居住和地理特异性粘膜相关细菌群落。在这方面，新的和代表性不足的物种，具有特殊的条件或公共属性，促进其接近宿主可能会被错过。这些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，它们的组成和群落组织的区域差异也必须考虑在内。在本提案中，这些问题将在开发新的和改进的非培养技术时加以考虑，这些技术将最终促进对大量以前未被鉴定的人类肠道微生物组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自然状态下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方法，旨在获得高质量的微生物群落复合DNA或富集/纯化的代表性不足的未分类微生物物种样本。第一种方法是对人类结肠不同区域的粘膜相关细菌进行激光捕获显微解剖，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16S rDNA和宏基因组测定的独特核糖探针结合荧光活化细胞分选（FACS）。产量，富集和纯度将优化，以发现和分离新的，未分类的和罕见的微生物从人类结肠微生物组进行全基因组测序的高通量测序中心。我们相信这些研究将产生非基于培养的技术，这将推动对大量以前未表征的人类肠道微生物组成员的基因组测序和宏基因组分析。公共卫生相关性：该应用旨在开发新的和改进的非基于培养的技术，最终将促进基因组测序和宏基因组分析大量以前未被鉴定的人类肠道微生物组成员。这将通过激光捕获显微解剖和FISH-FACS方法的发展来实现，这些方法来自于人类肠道微生物群中完整的粘膜相关微生物群落。

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.