A comparison of oral microbiome composition between highly trained competitive athletes and untrained controls

Abstract

The oral microbiome has a crucial role in nitric oxide (NO) production and contributes to oral and systemic health. This study compared oral microbiome composition and markers of NO production between highlytrained competitive athletes and inactive controls. Competitive athletes and untrained controls (N = 10 per group) were recruited. Saliva, plasma, supragingival plaque and the tongue dorsum microbiome were sampled. The microbiome was examined using long-read 16S rRNA sequencing and ozone-based chemiluminescence used to measure nitrate (NO₃^-) and nitrite (NO₂^-) levels. Weekly training duration was recorded and aerobic fitness capacity (V̇O_2max) assessed via maximal exercise testing.The beta-diversity of the tongue dorsum microbiome differed between groups (Adonis p = 0.046) and athletes had a higher relative abundance of NO₃^--reducing Rothia mucilaginosa and unclassified Gemella species. No significant differences were detected in the supragingival plaque. Positive correlations were detected between R. mucilaginosa and unclassified Gemella species and aerobic fitness. Athletes had higher levels of salivary NO₃^- (p = 0.003) and NO₂^- (p = 0.03). Exercise training may impact the tongue dorsum microbiome more than supragingival plaque, with the relative abundance of specific health-associated bacteria higher in the tongue dorsum microbiome of athletes. The robust methodologies employed in this study highlight a possible link between consistent exercise and the development of an oral microbiome conducive to health. However, further research is needed to explore the mechanisms connecting exercise, the oral microbiome, and overall health.

Keywords: 16 s rRNA; Dental disease; Exercise; Exercise training; Nitrate; Nitrate-reduction; Nitric oxide; Nitrite-production; Oral microbiome; Physical activity.

Fig. 1

Shows a schematic of the study’s data collection procedures. Created in Biorender.com.

Fig. 2

Comparison of training status and aerobic fitness between the trained (TR) and untrained (UTR) participants. (a) Time spent carrying out exercise training in the week before testing^⊗. (b) Relative V̇O_2max ^∅ . Each point represents a participant in the study. N = 10 for each group. ^⊗Wilcoxon test with data shown as median with IQR, ^∅Unpaired t-test with data shown as mean ± SD. **** p < 0.0001.

Fig. 3

Differences in the saliva and plasma between groups and differences in weekly NO₃^- consumption between trained (TR) and untrained (UTR) participants. (a) Plasma NO₃^-⊗. (b) Plasma NO₂^-∅. (c) Salivary NO₃^-⊗. (d) Salivary NO₂^-⊗. (e) Resting salivary pH^∅. (f) NO₂^- produced following administration of a NO₃^- mouth rinse^∅. Each point represents a participant in the study. N = 10 for each group for salivary values, N = 9 for the untrained group for plasma values. and N = 9 for both groups for the dietary NO₃^- consumption estimate. ^⊗Wilcoxon test with data shown as median with IQR, ^∅Unpaired t-test with data shown as mean ± SD. * p < 0.05, ** p < 0.01.

Fig. 4

Assessing the impact of long-term intensive aerobic exercise training on the diversity and composition of the oral microbiome. (a) Rarefaction curves for observed species. Differences in alpha diversity indices (b) dbp. (c) Shannon index. (d) Chao1 index between trained (TR) and untrained (UTR) participants^⊗ and between the microbiome of the tongue and subgingival plaque in participants of the same training status^∅. Data shown as median with IQR. Differences in beta-diversity between the microbiome of trained and untrained participants (e) Tongue dorsum. (f) Subgingival plaque. (g) Combined differences in beta-diversity between microbiome samples of different groups. Most abundant species in the microbiome of trained and untrained participants (h) Tongue dorsum. (i) Supragingival plaque in the trained and untrained groups. All species with relative abundance > 1% are shown, with the remaining species described as ‘other’. ^⊗Wilcoxon test .^∅Wilcoxon signed-rank test. * p < 0.05, ** p < 0.01.

Fig. 4

Assessing the impact of long-term intensive aerobic exercise training on the diversity and composition of the oral microbiome. (a) Rarefaction curves for observed species. Differences in alpha diversity indices (b) dbp. (c) Shannon index. (d) Chao1 index between trained (TR) and untrained (UTR) participants^⊗ and between the microbiome of the tongue and subgingival plaque in participants of the same training status^∅. Data shown as median with IQR. Differences in beta-diversity between the microbiome of trained and untrained participants (e) Tongue dorsum. (f) Subgingival plaque. (g) Combined differences in beta-diversity between microbiome samples of different groups. Most abundant species in the microbiome of trained and untrained participants (h) Tongue dorsum. (i) Supragingival plaque in the trained and untrained groups. All species with relative abundance > 1% are shown, with the remaining species described as ‘other’. ^⊗Wilcoxon test .^∅Wilcoxon signed-rank test. * p < 0.05, ** p < 0.01.

Fig. 4

Assessing the impact of long-term intensive aerobic exercise training on the diversity and composition of the oral microbiome. (a) Rarefaction curves for observed species. Differences in alpha diversity indices (b) dbp. (c) Shannon index. (d) Chao1 index between trained (TR) and untrained (UTR) participants^⊗ and between the microbiome of the tongue and subgingival plaque in participants of the same training status^∅. Data shown as median with IQR. Differences in beta-diversity between the microbiome of trained and untrained participants (e) Tongue dorsum. (f) Subgingival plaque. (g) Combined differences in beta-diversity between microbiome samples of different groups. Most abundant species in the microbiome of trained and untrained participants (h) Tongue dorsum. (i) Supragingival plaque in the trained and untrained groups. All species with relative abundance > 1% are shown, with the remaining species described as ‘other’. ^⊗Wilcoxon test .^∅Wilcoxon signed-rank test. * p < 0.05, ** p < 0.01.

Fig. 5

Shows significant species-level differences between the trained (TR) and untrained (URT) groups. (a) Relative abundance of Rothia mucilaginosa. (b) Relative abundance of unclassified Gemella. N = 10 for each group. Data shown as median with IQR. Each point represents a participant in the study. Groups were compared using a Wilcoxon test, carried out on the ANCOMBC-2 transformed relative abundance values. * p < 0.05.

Fig. 6

Associations between the composition of the tongue dorsum microbiome, NO₃^- and NO₂^- levels and the aerobic training status of all participants. Heatmaps with associations between markers of exercise training status, NO₃^- and NO₂^- levels, salivary pH and oral NO₂^- production levels and bacterial abundance at (a) the genus level and (b) species level. These heatmaps show the association between bacterial abundances after ANCOM-BC2 transformation and other parameters, which were obtained based on their projection onto a correlation circle plot derived from a principal component analysis. The correlation circle plots are shown in Supplementary Fig. 4. Only negative associations below -0.4 and positive associations above 0.4 between species are shown. To complement the association heatmaps, correlations between species and other parameters were determined with Spearman’s rho and marked with asterisks on the heatmap (* p-adjusted < 0.05, ** p-adjusted < 0.01). Scatterplots showing the V̇O_2max and ANCOM-BC2 transformed relative abundance of (c) Rothia. (d) R. mucilaginosa. (e) Unclassified Gemella species.