Following a single blind, cross-over and non-randomized design we investigated the effect of 7-day use of chlorhexidine (CHX) mouthwash on the salivary microbiome as well as several saliva and plasma biomarkers in 36 healthy individuals. They rinsed their mouth (for 1 min) twice a day for seven days with a placebo mouthwash and then repeated this protocol with CHX mouthwash for a further seven days. Saliva and blood samples were taken at the end of each treatment to analyse the abundance and diversity of oral bacteria, and pH, lactate, glucose, nitrate and nitrite concentrations. CHX significantly increased the abundance of Firmicutes and Proteobacteria, and reduced the content of Bacteroidetes, TM7, SR1 and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity, accompanied by an increase in saliva lactate and glucose levels. Lower saliva and plasma nitrite concentrations were found after using CHX, followed by a trend of increased systolic blood pressure. Overall, this study demonstrates that mouthwash containing CHX is associated with a major shift in the salivary microbiome, leading to more acidic conditions and lower nitrite availability in healthy individuals.Chlorhexidine (CHX) has been commonly used in dental practice as antiseptic agent since 1970, due to its long-lasting antibacterial activity with a broad-spectrum of action 1 . Since then, many clinical trials have shown effective results of CHX for the clinical management of dental plaque and gingival inflammation and bleeding 2-4 . This is supported by other studies using in vitro methods and reporting positive results of CHX in reducing the proliferation of bacterial species associated with periodontal disease, such as Enterobacteria, Porphyromonas gingivalis, Fusobacterium nucleatum, as well as different species of Actinomyces and Streptococcus, including Streptococcus mutans, which is considered the main etiological agent of dental caries 4,5 . Other studies have also reported that the use of CHX was effective in the treatment of halitosis, especially in reducing the levels of halitosis-related bacteria colonising the dorsal surface of the tongue 6 .The anti-microbial activity of CHX however, has been extensively studied using in vitro culture methods, which limit the identification and cultivation of all microorganisms in the environment 4 . To the best of our knowledge, only one recent study has investigated the effect of CHX mouthwash on mixed bacterial communities (microbiome) of the tongue using new genome sequencing techniques such as 16 S rRNA 7 . The study found differences in over 10 different species colonizing the tongue, and a lower microbial diversity after using CHX for a week, but did not analyse other parameters related to oral health such as pH, lactate production or buffering capacity 7 . Additionally, we and others have recently shown that the use of CHX in healthy subjects can attenuate the nitrate-reducing activity of oral bacteria by at least 80% 8-11 . This in turn leads to lo...

Nitric oxide (NO) can be generated endogenously via NO synthases or via the diet following the action of symbiotic nitrate-reducing bacteria in the oral cavity. Given the important role of NO in smooth muscle control there is an intriguing suggestion that cardiovascular homeostasis may be intertwined with the presence of these bacteria. Here, we measured the abundance of nitrate-reducing bacteria in the oral cavity of 25 healthy humans using 16S rRNA sequencing and observed, for 3.5 h, the physiological responses to dietary nitrate ingestion via measurement of blood pressure, and salivary and plasma NO metabolites. We identified 7 species of bacteria previously known to contribute to nitrate-reduction, the most prevalent of which were Prevotella melaninogenica and Veillonella dispar. Following dietary nitrate supplementation, blood pressure was reduced and salivary and plasma nitrate and nitrite increased substantially. We found that the abundance of nitrate-reducing bacteria was associated with the generation of salivary nitrite but not with any other measured variable. To examine the impact of bacterial abundance on pharmacokinetics we also categorised our participants into two groups; those with a higher abundance of nitrate reducing bacteria (> 50%), and those with a lower abundance (< 50%). Salivary nitrite production was lower in participants with lower abundance of bacteria and these individuals also exhibited slower salivary nitrite pharmacokinetics. We therefore show that the rate of nitrate to nitrite reduction in the oral cavity is associated with the abundance of nitrate-reducing bacteria. Nevertheless, higher abundance of these bacteria did not result in an exaggerated plasma nitrite response, the best known marker of NO bioavailability. These data from healthy young adults suggest that the abundance of oral nitrate-reducing bacteria does not influence the generation of NO through the diet, at least when the host has a functional minimum threshold of these microorganisms.

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There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CV B ), and analytical imprecision (CV A ) in experimental endpoints. However, no study has quantified the CV A and CV B of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CV B , CV A, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CV B of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CV B in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ± 5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (~12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitratereducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CV B for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CV B for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CV B for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), 3Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CV B in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.