Targeted Cultivation of New Periodontal Pathogens

描述（由申请人提供）：牙周炎影响6400万美国成年人，导致骨骼和牙齿脱落，并导致全身性疾病。龈下细菌在牙周炎中起主要作用。由于50%的口腔微生物组未被识别或未培养，重要的物种可能被忽视，这代表了知识的空白。 相关口腔未培养类群的培养进展缓慢。现有的少数研究要么是低通量的，要么是开放式的，不太适合培养假定的病原体。因为我们的目标是改善牙周健康，我们的目标是培养与疾病相关的分类群， 而不是任何未经培养的生物。我的R 03的结果显示，许多未识别/未培养的龈下类群是罕见的，因此不太可能是相关的。相比之下，我确定了30个丰富的未培养类群，值得进一步研究，并将成为重点 这一提议。我们的长期目标是确定新的牙周病原体及其致病机制。这项建议的总体目标是恢复和驯化与牙周病有关的未培养的类群。我们的中心假设是，它们的生长结果与社区成员和栖息地，尚未在体外繁殖的相互作用。我们的理由是，有针对性的系统培养相关类群的离体生物膜是一种具有成本效益的方法来分离新的牙周病原体。我们将使用技术和概念创新方法来实现我们的目标。 我们将测试这一假设，并通过追求以下具体目标来实现我们的目标：1）支持体外牙周生物膜中主要未培养类群的生长：我们将使用Calgary生物膜装置来多重培养条件使用16 S rRNA Illumina测序预筛选目标分类群的龈下样品的生长（使用多个生长条件，同时进行）; 2）识别能够维持目标未培养牙周分类群生长的“辅助”物种：我们将使用CLASI-FISH（多重FISH）对生物膜的分析和16 S rRNA数据的相关网络分析，以基于它们与目标未培养分类群的共现和物理接近性，原位和计算机上鉴定最佳“辅助”物种;和3）在辅助物种的小聚生体中驯化“未培养的”牙周分类群：我们将使用上面鉴定的最佳生长条件和辅助物种用于未培养分类群的高通量平行共培养。 这一建议是重要的，因为它将允许新的推定牙周病原体的培养，确定其致病性的关键步骤。我们对这一领域的贡献将是识别允许它们生长的生态关系， 序列以及系统方法的发展，可用于研究其他生物膜疾病。我们的研究结果将支持NIDCR 2014财年的研究计划（不可培养细菌在口腔微生物组中的作用）及其使命，以促进对口腔感染性疾病病因和发病机制的了解。

DESCRIPTION (provided by applicant): Periodontitis affects 64 million US adults, leading to bone and tooth loss and contributing to systemic conditions. Subgingival bacteria play a major role in periodontitis. Because 50% of the oral microbiome is unrecognized or uncultured, important species may have been overlooked, representing a gap in knowledge. The progress in cultivating relevant oral uncultured taxa has been slow. The few studies available are either low-throughput or open-ended, which are less suitable to cultivate putative pathogens. Because our goal is to improve periodontal health, we aim at cultivating taxa that are relevant in disease, and not just any uncultured taxa. Results from my R03 showed that many unrecognized/uncultured subgingival taxa were uncommon, thus unlikely to be relevant. In contrast, I identified 30 abundant uncultured taxa that merit further pursuit and will be the focus of this proposal. Our long term goal is to identify new periodontal pathogens and their pathogenic mechanisms. The overall objective of this proposal is to recover and domesticate uncultured taxa associated with periodontal disease. Our central hypothesis is that their growth results from interactions with community members and the habitat, which have not yet been reproduced in vitro. Our rationale is that targeted systematic cultivation of relevant taxa in ex vivo biofilms is a cost-effective approach to isolate new periodontal pathogens. We will use technological and conceptual innovative approaches to achieve our goals. We will test this hypothesis and accomplish our objective by pursuing the following Specific Aims: 1) Support the growth of prominent uncultured taxa in ex vivo periodontal biofilms: we will use the Calgary Biofilm Device to multiplex the cultivation conditions (using multiple growth conditions in paralle) of subgingival samples pre-screened for the target taxa using 16S rRNA lllumina sequencing; 2) Identify "helper" species that can sustain the growth of target uncultured periodontal taxa: we will use CLASI-FISH (a multiplex FISH) on the biofilms and correlation network analysis of 16S rRNA data to identify in situ and in silico the best "helper" species based on their co-occurrence and physical proximity with target uncultured taxa; and 3) Domesticate "uncultured" periodontal taxa in a small consortium of helper species: we will use the best growth conditions and helpers species identified above for the high-throughput parallel co-cultivation of uncultured taxa. This proposal is significant because it will allow the cultivation of novel putative periodontal pathogens, a key step to determine their pathogenicity. Our contribution to the field will be the identification of ecological relationships that allow their growth, the generation of whole genomic sequences as well as the development of a systematic approach that can be useful to study other biofilm diseases. The outcomes of our study will support NIDCR's research initiatives for FY2014 (the role of uncultivable bacteria in the oral microbiome) and their mission to foster the knowledge of the etiology and pathogenesis of oral infectious diseases.