Localisation of nitrate-reducing and highly abundant microbial communities in the oral cavity.

The nitrate (NO3-) reducing bacteria resident in the oral cavity have been implicated as key mediators of nitric oxide (NO) homeostasis and human health. NO3--reducing oral bacteria reduce inorganic dietary NO3- to nitrite (NO2-) via the NO3--NO2--NO pathway. Studies of oral NO3--reducing bacteria have typically sampled from either the tongue surface or saliva. The aim of this study was to assess whether other areas in the mouth could contain a physiologically relevant abundance of NO3- reducing bacteria, which may be important for sampling in clinical studies. The bacterial composition of seven oral sample types from 300 individuals were compared using a meta-analysis of the Human Microbiome Project data. This analysis revealed significant differences in the proportions of 20 well-established oral bacteria and highly abundant NO3--reducing bacteria across each oral site. The genera included Actinomyces, Brevibacillus, Campylobacter, Capnocytophaga, Corynebacterium, Eikenella, Fusobacterium, Granulicatella, Haemophilus, Leptotrichia, Microbacterium, Neisseria, Porphyromonas, Prevotella, Propionibacterium, Rothia, Selenomonas, Staphylococcus, Streptococcus and Veillonella. The highest proportion of NO3--reducing bacteria was observed in saliva, where eight of the bacterial genera were found in higher proportion than on the tongue dorsum, whilst the lowest proportions were found in the hard oral surfaces. Saliva also demonstrated higher intra-individual variability and bacterial diversity. This study provides new information on where samples should be taken in the oral cavity to assess the abundance of NO3--reducing bacteria. Taking saliva samples may benefit physiological studies, as saliva contained the highest abundance of NO3- reducing bacteria and is less invasive than other sampling methods. These results inform future studies coupling oral NO3--reducing bacteria research with physiological outcomes affecting human health.

Low Carbohydrate, High Fat Diet Alters the Oral Microbiome without Negating the Nitrite Response to Beetroot Juice Supplementation.

A low carbohydrate, high fat (LCHF) diet in athletes increases fat oxidation but impairs sports performance, potentially due to impaired exercise economy. Dietary nitrate supplementation can improve exercise economy via an increase in nitric oxide production, which is initiated by the reduction of nitrate to nitrite within the oral cavity. This reaction is dependent on the presence of nitrate-reducing oral bacteria, which can potentially be altered by dietary changes, including a LCHF diet. This study explored the effect of a LCHF diet on the oral microbiome and subsequent changes to plasma nitrite concentration following nitrate supplementation. Following five days of LCHF or high carbohydrate (HCHO) control dietary intervention, highly trained male race walkers consumed 140 mL beetroot juice containing 8.4 mmol nitrate; they then provided (a) blood samples for plasma nitrate and nitrite analysis and (b) saliva samples for 16S rRNA sequencing of the oral microbiome. The LCHF diet (n = 13) reduced oral bacterial diversity and changed the relative abundance of the genera Neisseria (+10%), Fusobacteria (+3%), Prevotella (-9%), and Veillonella (-4%), with no significant changes observed following the HCHO diet (n = 11). Following beetroot juice ingestion, plasma nitrite concentrations were higher for the LCHF diet compared to the HCHO diet (p = 0.04). However, the absence of an interaction with the trial (pre-post) (p = 0.71) suggests that this difference was not due to the dietary intervention. In summary, we found an increase in plasma nitrate and nitrite concentrations in response to nitrate supplementation independent of diet. This suggests the oral microbiome is adaptive to dietary changes and can maintain a nitrate reduction capacity despite a decrease in bacterial diversity following the LCHF diet.

Network analysis of nitrate-sensitive oral microbiome reveals interactions with cognitive function and cardiovascular health across dietary interventions.

Many oral bacteria reduce inorganic nitrate, a natural part of a vegetable-rich diet, into nitrite that acts as a precursor to nitric oxide, a regulator of vascular tone and neurotransmission. Aging is hallmarked by reduced nitric oxide production with associated detriments to cardiovascular and cognitive function. This study applied a systems-level bacterial co-occurrence network analysis across 10-day dietary nitrate and placebo interventions to test the stability of relationships between physiological and cognitive traits and clusters of co-occurring oral bacteria in older people. Relative abundances of Proteobacteria increased, while Bacteroidetes, Firmicutes and Fusobacteria decreased after nitrate supplementation. Two distinct microbiome modules of co-occurring bacteria, that were sensitive to nitrate supplementation, showed stable relationships with cardiovascular (Rothia-Streptococcus) and cognitive (Neisseria-Haemophilus) indices of health across both dietary conditions. A microbiome module (Prevotella-Veillonella) that has been associated with pro-inflammatory metabolism was diminished after nitrate supplementation, including a decrease in relative abundance of pathogenic Clostridium difficile. These nitrate-sensitive oral microbiome modules are proposed as potential pre- and probiotic targets to ameliorate age-induced impairments in cardiovascular and cognitive health.

Nitrate-responsive oral microbiome modulates nitric oxide homeostasis and blood pressure in humans.

Imbalances in the oral microbial community have been associated with reduced cardiovascular and metabolic health. A possible mechanism linking the oral microbiota to health is the nitrate (NO3-)-nitrite (NO2-)-nitric oxide (NO) pathway, which relies on oral bacteria to reduce NO3- to NO2-. NO (generated from both NO2- and L-arginine) regulates vascular endothelial function and therefore blood pressure (BP). By sequencing bacterial 16S rRNA genes we examined the relationships between the oral microbiome and physiological indices of NO bioavailability and possible changes in these variables following 10 days of NO3- (12 mmol/d) and placebo supplementation in young (18-22 yrs) and old (70-79 yrs) normotensive humans (n = 18). NO3- supplementation altered the salivary microbiome compared to placebo by increasing the relative abundance of Proteobacteria (+225%) and decreasing the relative abundance of Bacteroidetes (-46%; P < 0.05). After NO3-supplementation the relative abundances of Rothia (+127%) and Neisseria (+351%) were greater, and Prevotella (-60%) and Veillonella (-65%) were lower than in the placebo condition (all P < 0.05). NO3- supplementation increased plasma concentration of NO2- and reduced systemic blood pressure in old (70-79 yrs), but not young (18-22 yrs), participants. High abundances of Rothia and Neisseria and low abundances of Prevotella and Veillonella were correlated with greater increases in plasma [NO2-] in response to NO3- supplementation. The current findings indicate that the oral microbiome is malleable to change with increased dietary intake of inorganic NO3-, and that diet-induced changes in the oral microbial community are related to indices of NO homeostasis and vascular health in vivo.