Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability

Abstract

The reduction of nitrate to nitrite by the oral microbiota has been proposed to be important for oral health and results in nitric oxide formation that can improve cardiometabolic conditions. Studies of bacterial composition in subgingival plaque suggest that nitrate-reducing bacteria are associated with periodontal health, but the impact of periodontitis on nitrate-reducing capacity (NRC) and, therefore, nitric oxide availability has not been evaluated. The current study aimed to evaluate how periodontitis affects the NRC of the oral microbiota. First, 16S rRNA sequencing data from five different countries were analyzed, revealing that nitrate-reducing bacteria were significantly lower in subgingival plaque of periodontitis patients compared with healthy individuals (P < 0.05 in all five datasets with n = 20-82 samples per dataset). Secondly, subgingival plaque, saliva, and plasma samples were obtained from 42 periodontitis patients before and after periodontal treatment. The oral NRC was determined in vitro by incubating saliva with 8 mmol/L nitrate (a concentration found in saliva after nitrate-rich vegetable intake) and compared with the NRC of 15 healthy individuals. Salivary NRC was found to be diminished in periodontal patients before treatment (P < 0.05) but recovered to healthy levels 90 days post-treatment. Additionally, the subgingival levels of nitrate-reducing bacteria increased after treatment and correlated negatively with periodontitis-associated bacteria (P < 0.01). No significant effect of periodontal treatment on the baseline saliva and plasma nitrate and nitrite levels was found, indicating that differences in the NRC may only be revealed after nitrate intake. Our results suggest that an impaired NRC in periodontitis could limit dietary nitrate-derived nitric oxide levels, and the effect on systemic health should be explored in future studies.

Fig. 1

Study overview. Part A: Bioinformatic analysis comparing health and periodontitis. 16S rRNA sequencing data of five studies from five different countries (Japan, Brazil, Chile, USA and Spain, n = 20–82 samples per study) were analyzed with the Dada2 pipeline to compare nitrate-reducing bacteria in health and periodontitis. Part B) Sample analysis of periodontitis treatment study. Subgingival plaque, saliva, and plasma samples from 42 periodontitis patients located in Glasgow (Scotland, United Kingdom) were collected before and 90 days after periodontal treatment. The subgingival plaque bacterial composition was determined using Illumina sequencing of the 16S rRNA gene, and the quantity of the nitrate-reducing biomarker genus Rothia was determined by qPCR. Measurements of nitrate and nitrite in saliva and plasma were performed and the salivary nitrate reduction capacity (NRC) was determined after three hours of incubation in vitro and compared with the NRC of 15 healthy individuals

Fig. 2

Confirmed nitrate-reducing bacteria in periodontitis and health. Bar graphs show the relative abundance of bacteria in subgingival plaque samples from different countries, as estimated by high-throughput sequencing of the 16 S rRNA gene. Bacteria were grouped in confirmed nitrate-reducing species, periodontitis-associated or red complex according to Rosier et al., Pérez-Chaparro et al., and Socransky et al. respectively (the bacterial species in each group are listed in Supplementary Table 2). Healthy individuals (blue bars) were compared with individuals with periodontitis (red bars). In Supplementary Fig. 1, all known nitrite-producing bacteria (some of which may produce nitrite by pathways other than nitrate reduction) are shown following the same pattern and showing a significant difference between health and periodontitis. The datasets include individuals from Japan, Spain, the USA, Brazil, and Chile (n = 20–82 per dataset, see Supplementary Table 1 for additional information). It should be noted that the levels of groups of bacteria in different studies are affected by the criteria used to describe periodontitis, regional and host factors, as well as the DNA extraction methods or the sequencing techniques used in the original studies. *adjusted P < 0.05 of compositional data standardized by ANCOM-BC and compared with a Wilcoxon test

Fig. 3

Confirmed nitrate-reducing species and disease-associated bacteria before and 90 days after periodontal treatment. a Relative abundances of confirmed nitrate-reducing bacteria, red complex, and periodontitis-associated bacteria before (baseline, BL) and 90 days after treatment (D90) of 42 periodontitis patients. *adjusted P < 0.05, ***P < 0.001 of compositional data standardized by ANCOM-BC and compared with a Wilcoxon test. b, c Rothia cells determined by qPCR before (BL) and 90 days after (D90) treatment per sample (absolute amount, n = 37) (b) or normalized per ng of DNA (c). *P < 0.05 determined by a Wilcoxon test. d, e Correlations between the abundance of periodontal pathogens and nitrate-reducing bacteria at baseline (BL) and 90 days after treatment (D90). **adjusted P < 0.01 of Spearman’s rank correlation (n.s. = not significant). In Supplementary Fig. 2, the comparisons and correlations of all known nitrite-producing bacteria (some of which may produce nitrite by other pathways than nitrate reduction) are shown

Fig. 4

Nitrate reduction capacity (NRC) in health and periodontitis. a, b Bar graphs represent the concentration of nitrate (NO₃⁻) and nitrite (NO₂⁻) of saliva samples cultured in vitro for 3 h in the presence of 8 mmol/L nitrate at 37 °C before (Baseline, BL) and 90 days after periodontal treatment (n = 42) of periodontal patients, compared to healthy controls (n = 15). c Bars show the pH during this incubation period for the same groups of individuals. Asterisks represent statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) determined by a Wilcoxon test (paired) or a Mann–Whitney U-test (unpaired)

Fig. 5

Plasma nitrate and nitrite before and 90 days after periodontal treatment. Data is shown from 42 periodontitis patients before (BL) and after (D90) treatment. a Plasma nitrite. b Plasma nitrate. c Ratio nitrate/nitrite