Drip Flow Biofilm Reactor for Dynamically Profiling Periodontal Multispecies Communities
Objectives: The onset of periodontitis is related to a complex interplay between bacterial interactions in the oral community and its environment, inducing a state of microbial imbalance. Revealing largely unknown mechanisms behind dysbiosis initiation benefits from in vitro model systems which allow in-depth biofilm profiling under dynamically-controlled conditions. In this study, live confocal imaging and metabolic profiling of a 5-species periodontal biofilm in a drip flow reactor are established. Aerobic flow conditions mimic the oral cavity and return spatiotemporal information.
Methods: The drip flow biofilm reactor is inoculated with S. gordonii-GFPmut3* (Sg), S. oralis-GFPmut3* (So), S. sanguinis-pVMCherry (Ss), F. nucleatum (Fn), and P. gingivalis-SNAP26 (Pg) for an anaerobic recirculation start-up phase of 8 hours, followed by an aerobic feeding phase. Bacterial composition derived from confocal images and qPCR, metabolites and structural biofilm features are monitored each day.
Results: Over 72 hours, the biofilm reaches a compositional and metabolic steady state. Quantification based on 28 confocal images after 72 hours shows a mean thickness of 39.0±6.5µm, a substratum coverage of 70.9±6.1% and a roughness coefficient of 0.018±0.006 based on three biological replicates. Depth profiles illustrate the dominance of the GFP-labeled organisms, being Sg and So, over the entire biofilm depth (67.8±2.6%), followed by Ss (23.6±0.8%) and Pg (8.6±0.46%) and this is confirmed by qPCR measurements. Glucose from the medium is fully consumed, pyruvate is partially consumed, and ornithine, lactic acid, acetic acid and formic acid are the observed products.
Conclusions: The drip flow setup shows reproducible growth of a 5-species periodontal biofilm with relative abundancies similar to those found in the oral cavity. This promising model will be used to investigate interactions between key bacteria in multispecies oral communities.