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%的主要人体微生物群无法使用标准培养方法培养。该提案解决了RFA-DE-14-003的目标：通过多学科，团队科学方法，汇集微生物学，基因组学，代谢组学，信息学，微流体学和成像方面的专家，检查口腔微生物群中未培养细菌的创新方法和技术。具体目标是实现最先进技术和创新方法的系统级整合。该建议是假设驱动的，它将提供具体的知识，在一个受试者群体中确定的所有未开垦的口腔分类群的性质uncultivability。这项提议可能会建立一种新的模式，通过首先在聚生体中培养， 快速降低聚生体的复杂性，并将它们作为共生或互养关系中的二元对进行共培养。含有未培养细菌的临床样本将在新的生长条件下孵育，以富集含有不可培养物和辅助物种的减少的聚生体，这些物种提供必需营养素。我们将使用生物信息学和代谢建模方法来确定营养缺陷型的重要性，并确定新的生长因子。该提案是第一个在研究中跟踪基因组进化以确定细菌驯化背后的遗传变化的提案。将鉴定支持先前不可培养的分类群生长的化合物，并允许配制新的培养基以支持更广泛的口腔微生物群的常规培养。未知营养素的身份将通过质谱法确定。我们将使用新的微流控装置和光谱成像技术来研究以前无法培养的细菌与其他细胞的细菌-细菌和细菌-宿主细胞相互作用。微流控系统将用于定量减少聚生体中不可培养物种的生长，以二元对或补充所需的代谢物或宿主因子。使用模拟社区的牙周病研究将提供以前无法培养的细菌对肠道微生物群的影响，疾病的进展，宿主对致病性聚生体的免疫和炎症反应，以及中性粒细胞和单核细胞对以前无法培养的细菌单独或致病性聚生体的反应。将建立一个关于以前未种植的类群的流行率、种植状况、共同种植伙伴、营养缺陷型和驯化历史的网络可访问数据库，供科学界随时访问。分离株将可用于体内和体外生态学研究 与宿主的相互作用。为培养目前无法培养的口腔细菌而开发的高通量方法将可推广到其他身体部位和环境微生物学。

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.