Nitrate Reduction in the Oral Cavity - Relevance to Caries

Objectives: In man, the enterosalivary nitrate circulation encourages the growth of bacteria in the clefts of the tongue. Nitrate respiration by these organisms results in nitrite production. Nitrite in contact with acid, such as that produced by Streptococcus mutans and other cariogenic bacteria, results in the production of antimicrobial oxides of nitrogen, including nitric oxide. Thus, nitrite in saliva may control the growth of such acidogenic bacteria. The aim of this study was to identify the major species of bacteria involved in nitrate reduction within the oral cavity. Methods: Sterile toothbrushes were used to sample the posterior dorsal surface of the tongue from ten individuals. These were vortexed in a reduced transport fluid and cultured for 72h at 37^oC, aerobically and anaerobically, on a tryptone-yeast extract-cysteine agar medium with glucose (0.5% w/v), serum (2% v/v) and haemin (5mg/ml). Nitrate-reducing bacteria were isolated using a nitrate agar overlay method and identified using 16S rDNA sequencing. The extent of nitrate reduction was also measured using nitrate-impregnated filter papers at different oral sites and whole mouth nitrate washes. Results: Isolates identified from the tongue comprised Veillonella dispar (37%), Veillonella atypica (28%), Actinomyces odontolyticus (16%), Rothia mucilaginosa (12%), Staphylococcus epidermidis (7%) and Rothia dentocariosa (2%). Measures of nitrate reduction (mean 43.7mM nitrite / 5 min from 1mM potassium nitrate mouth wash) varied widely between individuals, as did the ability to isolate Veillonella spp. from their tongues, the major site of nitrate reduction. Conclusion: The major species responsible for nitrate reduction in the oral cavity were found to be Veillonella atypica and Veillonella dispar. We hypothesise that individuals with a high nitrate reducing flora, which is facilitated by a high nitrate diet, will afford protection against dental caries. This work was supported by the Royal London Hospital Special Trustees (RAC 399).