Cultivation, Nature, Ecology and Pathogenicity of the Uncultivable Oral Microbiom

描述（由申请人提供）：全面了解人类口腔健康和疾病需要对人类口腔微生物组及其与人类宿主的相互作用进行全面分析。实现这一目标的一个重要障碍是，35%的主要人类微生物群无法使用标准培养方法进行培养。本提案通过多学科、团队科学的方法，汇集了微生物学、基因组学、代谢组学、信息学、微流体学和成像方面的专家，解决了RFA-DE-14-003的目标：检测口腔微生物群中未培养细菌的创新方法和技术。制定的具体目标是实现最先进技术和创新办法在系统一级的综合。这个提议是由假设驱动的；它将为在一个主题群体中确定的所有未开垦口腔分类群的不可耕性性质提供具体的知识。这一建议可能会建立一种新的模式，通过先在群体中培养，培养以前无法培养的口腔微生物群成员。

DESCRIPTION (provided by applicant): A full understanding of human oral health and disease requires a comprehensive analysis of the human oral microbiome and its interactions with the human host. A significant barrier to this goal is that 35% of the predominant human microbiota are uncultivable using standard cultivation methods. This proposal addresses the goals of RFA-DE-14-003: Innovative Approaches and Technologies for Examining the Uncultivated Bacteria of the Oral Microbiota through a multidisciplinary, team science approach, bringing together experts in microbiology, genomics, metabolomics, informatics, microfluidics and imaging. The specific aims have been constructed to achieve a systems-level integration of state-of-the-art technologies and innovative approaches. The proposal is hypothesis driven; it will provide specific knowledge on the nature of uncultivability for all uncultivated oral taxa identified in a subject population. This proposal will likely establish a new paradigm for cultivating previously uncultivable members of the oral microbiota by culturing first in consortia, rapidly reducing the complexity of the consortia, and bringing them into co-cultivation as binary pairs in commensal or syntrophic relationships. Clinical samples containing uncultivated bacteria will be incubated under novel growth conditions to enrich for reduced consortia containing uncultivables and helper species that supply essential nutrients. We will use bioinformatic and metabolic modeling approaches to determine the importance of auxotrophy and to identify novel growth factors. The proposal is the first to track genome evolution in studies to determine the genetic changes underlying bacterial domestication. Compounds that support the growth of previously uncultivable taxa will be identified and allow the formulation of novel culture media to support routine cultivation of a broader range of the oral microbiota. The identity of unknown nutrients will be determined by mass spectrometric methods. We will use novel micro-fluidic devices and spectral imaging technology to examine bacteria-bacteria and bacteria-host cell interactions of previously-uncultivable bacteria with other cells. Microfluidic systems will be used to quantify growth of uncultivable species in reduced consortia, in binary pairs, or when supplemented with a required metabolite or host factor. Periodontal disease studies using mock communities will provide an under-standing of the impact of previously-uncultivable bacteria on the commensal microbiota, the progression of disease, the host immune and inflammatory responses to pathogenic consortia, and the response of neutrophils and monocytes to previously-uncultivable bacteria alone or in pathogenic consortia. A web-accessible database on the prevalence, cultivation status, co-cultivation partners, auxotrophies, and domestication histories of previously-uncultivated taxa will be created for ready access to the scientific community. Isolates will be available for in vivo and in vitro studies of ecological interactions and interactions with the host. The high-throughput methods developed to cultivate currently- uncultivable oral bacteria will be generalizable to other body sites and to environmental microbiology.