Investigating the association between nitrate dosing and nitrite generation by the human oral microbiota in continuous culture

Abstract

The generation of nitrite by the oral microbiota is believed to contribute to healthy cardiovascular function, with oral nitrate reduction to nitrite associated with systemic blood pressure regulation. There is the potential to manipulate the composition or activities of the oral microbiota to a higher nitrate-reducing state through nitrate supplementation. The current study examined microbial community composition and enzymatic responses to nitrate supplementation in sessile oral microbiota grown in continuous culture. Nitrate reductase (NaR) activity and nitrite concentrations were not significantly different to tongue-derived inocula in model biofilms. These were generally dominated by Streptococcus spp., initially, and a single nitrate supplementation resulted in the increased relative abundance of the nitrate-reducing genera Veillonella, Neisseria, and Proteus spp. Nitrite concentrations increased concomitantly and continued to increase throughout oral microbiota development. Continuous nitrate supplementation, over a 7-day period, was similarly associated with an elevated abundance of nitrate-reducing taxa and increased nitrite concentration in the perfusate. In experiments in which the models were established in continuous low or high nitrate environments, there was an initial elevation in nitrate reductase, and nitrite concentrations reached a relatively constant concentration over time similar to the acute nitrate challenge with a similar expansion of Veillonella and Neisseria. In summary, we have investigated nitrate metabolism in continuous culture oral biofilms, showing that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of putatively NaR-producing taxa.IMPORTANCEClinical evidence suggests that blood pressure regulation can be promoted by nitrite generated through the reduction of supplemental dietary nitrate by the oral microbiota. We have utilized oral microbiota models to investigate the mechanisms responsible, demonstrating that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of nitrate-reducing taxa.

Keywords: blood pressure; dietary nitrate; hypertension; multiple Sorbarod device; nitric oxide; nitrite; oral microbiota.

Fig 1

Models utilized to explore nitrate metabolism in vitro. Three models were utilized in this study to (i) validate the use of an oral microcosm model to examine time-dependent changes in microbial community composition and nitrate metabolism, (ii) explore how nitrate (15 mM) intervention manipulates biofilm composition, and (iii) compare the effects of nitrate (15 mM) on biofilm composition and nitrate metabolism relative to a PBS control in oral microcosm communities derived from two individuals. Arrows depict time points for sample collection for 16S rRNA sequencing and nitrate metabolism assays.

Fig 2

Detectable levels of nitrite and nitrate reductase in oral biofilm consortia. An MSD model was established and inoculated with tongue material collected from subject 1 inoculum (n = 2) . Nitrate metabolism of biofilm (filter) and perfusate samples was assessed via nitrite secretion (A) and nitrate reductase assays (B). Differential counts of aerobes, anaerobes (Wilkins-Chalgren agar (WCA)), or total streptococci (Mitis Salivarius agar (MSA)) in biofilm samples were surveyed via plate counting at each time point (C). 16S rRNA sequencing was then exploited to evaluate biofilm community composition (D) over 14 days. CFU, colony-forming unit.

Fig 3

Elevated abundances of Neisseria and Proteus spp. following exposure of pre-formed biofilms to nitrate. Oral biofilm microcosms derived from subject 2 were established for 4 days on cellulose filters in a continuous culture flow-through system, and subsequently exposed to nitrate. Changes in biofilm composition (A), nitrite secretion (B), and nitrate reductase activity (C) were assessed over 7 days from both biofilm (filter) and perfusate samples pre- and post-exposure to sodium nitrate (15 mM, day 4).

Fig 4

Nitrate intervention elevates the abundance of Neisseria and Proteus spp. ASVs. Oral microcosm (subject 3) was inoculated into the continuous flow-through system and exposed to either 15 mM nitrate (NO₃⁻) or PBS (control) continuously for 7 days. Changes in control (PBS) or NO₃⁻treated biofilm composition (A) were assessed via 16S rRNA sequencing. Nitrate metabolism from collected biofilms (filter; B) or perfusate (C) was examined via nitrite secretion (i) and nitrate reductase activity (ii) assays.

Fig 5

Effects of nitrate intervention may depend upon initial microcosm composition. An oral microcosm (subject 4) was inoculated into the continuous flow-through system and exposed to either 15 mM nitrate (NO₃⁻) or PBS (untreated) continuously for 7 days. Changes in biofilm composition (A) were assessed via 16S rRNA sequencing. Nitrate metabolism from collected biofilms (filter; B) or perfusate (C) was evaluated via nitrite secretion (i) and nitrate reductase activity (ii) assays.

Fig 6

Shift toward high nitrate-reducing organisms following nitrate dosing. Oral microcosms derived from two individuals (subject 3, A and subject 4, B) were either dosed with 15 mM nitrate or PBS and data pooled following the acquisition of 16S rRNA data on collection days 3 and 7. Figures depict combined changes in ASVs on days 3 and 7 in nitrate-treated samples, relative to PBS control.