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.
The proposed research is relevant of public health because, despite increasing knowledge of the development of periodontal diseases, these infections affect more than 64 million US adults and are more prevalent among minorities. These diseases have been linked to systemic conditions and their treatment costs billions annually. This project is relevant to NIDCR's mission because understanding of the role of uncultured/unrecognized subgingival bacteria can provide new targets for prevention, diagnosis and treatment of oral infections.
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