Data supporting the thesis, "Effect of Electron Acceptors on Oral Microbial Community Composition and Function"
Gingivitis is inflammation of the gums and is caused by excessive dental plaque accumulation. The severity of the disease is correlated with both the amount of plaque present and its maturity. As plaque matures its bacterial composition changes from mainly aerobes and facultative anaerobes to one dominated by anaerobic Gram-negative bacteria. Supplementation of the diet with nitrate has been shown to modify salivary bacterial composition with an increase in the proportion of the aerobic and health-associated genera Neisseria and Rothia. It has been suggested that these aerobic bacteria can use nitrate as an alternative electron acceptor to oxygen and thus thrive in the anaerobic conditions of dental plaque. The aim of this work was to investigate the effects of nitrate and other alternative electron acceptors on the growth of health-associated oral bacteria and on the composition of complex biofilms in an in-vitro dental plaque model. In addition, the expression of nitrate-reducing enzymes in biofilms was investigated.
The growth of oral commensal Neisseria and Rothia strains was determined under anaerobic conditions in culture medium supplemented with 0 – 50 mM sodium nitrate, nitrite or sulphate and whole genome sequences were inspected for the presence of relevant enzymes. Sulphate did not enable anaerobic growth for any strain. N. bacilliformis, N. mucosa, N. oralis and all three Rothia spp. showed enhanced growth under anaerobic conditions with nitrate and were shown to encode the respiratory nitrate reductase narG.
Nitrite supplementation resulted in anaerobic growth for the Neisseria species bacilliformis, subflava, sicca and flavescens, as well as R. dentocariosa and R. aeria. Neisseria and Rothia strains encoded the nitrite reductases nirK or nirK and nirBD, respectively.
In-vitro oral biofilms were cultivated in the Calgary-Biofilm Device inoculated with pooled saliva and grown in a mucin-containing complex medium supplemented with 0 - 10 mM sodium nitrate or nitrite. The bacterial community was investigated by 16S rRNA gene sequence analysis. Treatment with 5 and 10 mM nitrate and nitrite resulted in significant changes to the bacterial biofilm community compared to the untreated control. The relative abundance of health-associated Neisseria spp. was significantly higher in nitrate- and nitrite-treated biofilms, while the relative abundance of anaerobic genera Prevotella and Fusobacterium was higher in untreated biofilms. Treatment with nitrate or nitrite did not affect the relative abundance of Rothia spp.
PCR primers specific for narG nitrate reductase genes were designed for a range of oral bacteria and validated at phylum and genus level. NarG gene PCR, cloning and sequencing revealed the diversity of denitrification enzymes to be higher in the saliva inocula than in biofilm samples. Expression of narG from nitrate-reducing genera of interest was successfully measured in biofilm communities using reverse transcription qPCR following a nitrate challenge. However, profiles of expression varied greatly among Neisseria, Rothia, Veillonella and Actinomyces, making the results difficult to interpret.
In conclusion, this work has demonstrated the potential benefits of the use of nitrate and nitrite as prebiotics to beneficially modify dental plaque composition, thereby improving and maintaining oral health.