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

描述(申请人提供)：要全面了解人类口腔健康和疾病，需要对人类口腔微生物群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大障碍是，35%的主要人类微生物区系无法使用标准培养方法进行培养。该提案涉及RFA-DE-14-003的目标：通过多学科的团队科学方法，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，研究口腔微生物区系中未培养的细菌的创新方法和技术。制定具体目标是为了实现最先进技术和创新方法的系统级整合。该建议是假说驱动的；它将提供关于在受试者种群中发现的所有未开垦的口腔分类群不可耕种的性质的具体知识。这一建议可能会建立一种新的范例，通过首先在联合体中培养来培养以前不可培养的口腔微生物区系成员， 迅速降低联合体的复杂性，并将它们以共生或共养关系中的二元对引入共育。含有未培养细菌的临床样本将在新的生长条件下培养，以丰富含有不可培养细菌和提供必要营养的辅助物种的减少的联合体。我们将使用生物信息学和代谢建模方法来确定营养缺陷症的重要性，并确定新的生长因子。这项提议是第一次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。将确定支持以前无法培养的分类群生长的化合物，并允许制定新的培养介质，以支持更广泛的口腔微生物区系的常规培养。未知营养素的身份将通过质谱学方法确定。我们将使用新型微流控设备和光谱成像技术来研究细菌-细菌以及以前无法培养的细菌与其他细胞之间的细菌-宿主细胞相互作用。微流控系统将被用来量化不可培养物种在减少的联合体中的生长，成对的或当补充所需的代谢物或宿主因子时。使用模拟社区进行的牙周病研究将提供一个了解以前不可培养的细菌对共生微生物区系的影响，疾病的进展，宿主对致病联合体的免疫和炎症反应，以及中性粒细胞和单核细胞对以前不可培养的细菌单独或在致病联合体中的反应。将创建一个关于以前未种植的类群的流行率、种植状况、共同栽培伙伴、营养缺陷区和驯化历史的网络可访问数据库，以便随时进入科学界。分离的菌株将可用于体内和体外的生态学研究 互动和与主人的互动。为培养目前无法培养的口腔细菌而开发的高通量方法将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.