UC Santa Barbara

This dissertation delineates the development of a simplified, reproducible in vitro microbiome model of the human dental plaque, with the specific aims of minimizing procedural complexity and investigating the effects of preservation on a complex laboratory microbiome model. This model is based on a 24-well-plate anaerobic model that uses an enriched, undefined culture media, with complex carbohydrate sources and supplemented by nutritional components required by certain fastidious organisms. Preliminary work with the plaque inoculum from a single host served as a proof-of-concept and foundation for the model. In this stage of the project, the viability, membership and relative abundances of the preliminary model were assessed by fluorescence microscopy and 16S rRNA amplicon sequencing on the Illumina MiSeq platform. Processing and analysis of the sequencing data were performed using the mothur pipeline and phyloseq package in R. Preliminary work validated the cultivation, sequencing, and analytical procedures by showing members and abundances expected of early-stage human oral bacterial communities. These procedures were then adopted in the development of a temporal model with a maximum incubation time of 168 hours. The temporal model was fed on a defined schedules and minimal nutrients. Analysis of this model showed a temporal succession of bacteria that closely follows the order of succession in host oral cavities, as well as developmental differences across different hosts. These results show promise that the temporal model may be modified with periodic re-inoculation and lengthened to capture a broader temporal range and thus a more complex colonization succession in the dental plaque bacterial communities.

The combined results from the preliminary and temporal models formed the basis of the preservation experiments, which investigated the feasibility of refrigerating and cryopreserving 72-hour in vitro communities from three hosts for subsequent propagation. The results indicated that preservation of microbial communities did not lead to substantial changes in membership or relative abundance, though propagating the preserved communities led to relative abundance shifts in favor of early colonizers. What is worth noting is that differences from the host plaque inoculum dominated the observed dissimilarities among preserved and propagated communities. Examination of these communities also implicated potential synergy between members of the Streptococcus and the Veillonella genera, and potential antagonism between members of the Streptococcus and the Prevotella genera. These findings warrant further investigations of organismal relationships in a reduced microcosm, while offer promising potential applications of preserving in vitro dental microbiome models for therapeutic purposes.