Metagenomic Analysis of Nitrate-Reducing Bacteria in the Oral Cavity: Implications for Nitric Oxide Homeostasis

Abstract

The microbiota of the human lower intestinal tract helps maintain healthy host physiology, for example through nutrient acquisition and bile acid recycling, but specific positive contributions of the oral microbiota to host health are not well established. Nitric oxide (NO) homeostasis is crucial to mammalian physiology. The recently described entero-salivary nitrate-nitrite-nitric oxide pathway has been shown to provide bioactive NO from dietary nitrate sources. Interestingly, this pathway is dependent upon oral nitrate-reducing bacteria, since humans lack this enzyme activity. This pathway appears to represent a newly recognized symbiosis between oral nitrate-reducing bacteria and their human hosts in which the bacteria provide nitrite and nitric oxide from nitrate reduction. Here we measure the nitrate-reducing capacity of tongue-scraping samples from six healthy human volunteers, and analyze metagenomes of the bacterial communities to identify bacteria contributing to nitrate reduction. We identified 14 candidate species, seven of which were not previously believed to contribute to nitrate reduction. We cultivated isolates of four candidate species in single- and mixed-species biofilms, revealing that they have substantial nitrate- and nitrite-reduction capabilities. Colonization by specific oral bacteria may thus contribute to host NO homeostasis by providing nitrite and nitric oxide. Conversely, the lack of specific nitrate-reducing communities may disrupt the nitrate-nitrite-nitric oxide pathway and lead to a state of NO insufficiency. These findings may also provide mechanistic evidence for the oral systemic link. Our results provide a possible new therapeutic target and paradigm for NO restoration in humans by specific oral bacteria.

Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis

“…Although little is known about how nitrogenous intermediates or denitrifying mechanisms influence competition between diverse micro-organisms within a polymicrobial environment, our study suggests denitrification processes may promote bacterial competition and could potentially impact disease outcomes in a manner that may be beneficial to the host. Nitrite is readily available in the human body for conversion to nitrogenous intermediates and has been linked to improved health, particularly in the oral cavity (Hyde et al, 2014). Elevated salivary nitrite concentrations have been associated with a reduction in dental caries, presumably due to the inhibition of the cariogenic pathogen S. mutans by nitric oxide-generating oral commensal bacteria (Doel et al, 2004).…”

“…Elevated salivary nitrite concentrations have been associated with a reduction in dental caries, presumably due to the inhibition of the cariogenic pathogen S. mutans by nitric oxide-generating oral commensal bacteria (Doel et al, 2004). It is hypothesized that nitric oxide (or other RNS) generated by denitrifying oral commensals may modulate microbial homeostasis (Hyde et al, 2014), and thereby function as an infection control strategy. However, it is important to note that polymicrobial interactions involving P. aeruginosa and oral streptococci in the CF lung are complex, and are often controlled by the dynamics of specific streptococcal populations, colonization sequence and environmental conditions.…”

The influence of the human microbiome and probiotics on cardiovascular health

“…77,78 Studies have shown that bacterial nitrate reduction mostly occurs at the dorsal surface of the tongue, 79 with several bacterial taxa identified as key players, including Veillonella and Actinomyces. 80 In vitro experiments have demonstrated the high capacity of Actinomyces odontolyticus to reduce nitrate without subsequent nitrite reduction. 80 This species could be explored as a novel probiotic designed to be taken immediately prior to, or in conjunction with a meal, in order to maximize nitrate utilization from the diet, increase NO generation, and help lower the risk of CVD.…”

“…80 In vitro experiments have demonstrated the high capacity of Actinomyces odontolyticus to reduce nitrate without subsequent nitrite reduction. 80 This species could be explored as a novel probiotic designed to be taken immediately prior to, or in conjunction with a meal, in order to maximize nitrate utilization from the diet, increase NO generation, and help lower the risk of CVD.…”

A ‘green’ diet‐based approach to cardiovascular health? Is inorganic nitrate the answer?

“…In particular, 7 bacterial species never previously implicated in NO 3 − reduction were identified, including Granulicatella adiacens, Haemophilus parainfluenzae, Actinomyces odontolyticus, Actinomyces viscosus, Actinomyces oris, Prevotella melaninogenica, and Prevotella salivae. Studies exploring oral NO 3 − -reducing potential in rodents has demonstrated some similarity with humans with respect to the relevant NO 3 − reducing bacterial strains within the oral cavity, including Haemophilus parainfluenzae [66,67]. These observations suggest that the rat may provide a suitable laboratory model to assess the impact of dietary NO 3 − on the oral microbiome.…”