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. 2025 Feb 6;40(2):104–115. doi: 10.1111/omi.12492

The Effect of Environmental Factors on the Nitrate and Nitrite Metabolism of Oral Actinomyces and Schaalia Species

Tomona Otake 1,2, Jumpei Washio 1,✉, Kazuko Ezoe 1, Satoko Sato 1, Yuki Abiko 1, Kaoru Igarashi 2, Nobuhiro Takahashi 1
PMCID: PMC11904265  PMID: 39912537

ABSTRACT

Actinomyces naeslundii and Schaalia odontolytica belong to the most predominant nitrite‐producing bacteria in the oral microbiome. Nitrite has antibacterial and vasodilatory effects that may contribute to maintaining oral and systemic health. We have previously elucidated the metabolic characteristics of the nitrite‐producing activity of oral Veillonella species and the effects of oral environmental factors. However, this is still unknown for Actinomyces and Schaalia species. Furthermore, these bacteria are thought to degrade nitrite. Therefore, this study aimed to comprehensively elucidate the effects of environmental factors (pH, oxygen concentration, glucose, lactate, and the presence of nitrate/nitrite during growth) on nitrate and nitrite metabolism of these bacterial species using the type strains. Nitrite was quantified by Griess reagent, and final metabolites were analyzed by high‐performance liquid chromatography (HPLC). The nitrite‐producing activity of A. naeslundii and S. odontolytica was affected variously by environmental factors. Especially in A. naeslundii, under anaerobic conditions, the activity increased in a concentration‐dependent manner with the addition of glucose or lactate and was higher at lower pH when lactate was added. The nitrite‐degrading activity of both bacteria was lower than the nitrite‐producing activity and was less affected by environmental factors. Metabolites from glucose by A. naeslundii were different with and without nitrate, suggesting that nitrate altered metabolic pathways. The growth was inhibited under anaerobic conditions but promoted under aerobic conditions. These results indicate that the nitrite‐producing capacity of the oral microflora must take into account not only the composition and abundance of bacteria but also the variation in metabolic activity due to various environmental factors.

Keywords: Actinomyces, metabolism, nitrate, nitrite, oral microbiome, Schaalia

1. Introduction

Recently, the reduction of nitrate (NO3 −) into nitrite (NO2 −) by the oral microbiome has garnered increasing interest. It is well known that nitrate is abundant in green leafy vegetables such as spinach and lettuce (Doel et al. 2004; Noda et al. 1981), a component that is regularly ingested from our daily diet. This dietary nitrate is reduced to nitrite by bacteria in the oral microbiome, subsequently ingested, absorbed through the gastrointestinal tract, and finally transferred into the bloodstream, where it is gradually re‐oxidized to nitrate, a part of which is secreted back into the oral cavity as saliva. Consequently, the environment in oral cavity is thought to be abundant with nitrate derived from diet and saliva. In general, the concentrations of nitrate and nitrite in human saliva have been reported to be 0.8–4 and 0.2–2 mM, respectively (Sánchez et al. 2014; Silva‐Mendez et al. 1999).

Nitrite has antibacterial and vasodilatory effects. In fact, the metabolism and growth of bacteria associated with dental caries and periodontal disease were inhibited by nitrite in vitro (Xia et al. 2006) and also inhibited sugar metabolism and acid production in real human plaque in vivo (Yamamoto et al. 2017, 2024). This increase in oral pH due to the antimicrobial activity of nitrite has attracted attention as a possible anti‐caries prebiotic due to nitrates. It has also been suggested that nitrite contributes to improve systemic blood flow, potentially reducing cardiovascular disease (Lundberg and Weitzberg 2010). Thus, nitrite may play an important role in maintaining and enhancing both oral and systemic health.

Furthermore, saliva collected directly from the parotid gland lacked nitrite (Xia and Wang 2003), and the use of antimicrobial mouthwashes reduced blood nitrite levels (Kapil et al. 2013), suggesting that the oral microbiome may play an indispensable role in nitrite production in our bodies. Therefore, understanding the nitrite‐producing capacity of nitrate‐reducing bacteria in the oral cavity is very important from the viewpoint of their contribution not only to oral but also systemic health.

We have already comprehensively isolated and identified bacteria exhibiting nitrite‐producing activity from the dental and tongue microbiomes of adults and children, most of which were indigenous bacteria such as Actinomyces, Schaalia, Veillonella, Neisseria, and Rothia species (Sato‐Suzuki et al. 2020). Rosier et al. (2020) also conducted similar isolation and identification using a medium with different nutrient composition, and their results also indicated that the Rothia species is predominant as nitrite‐producing bacteria. In general, bacterial metabolism is known to be influenced by various environmental factors such as oxygen concentration and pH. In the oral cavity, environmental factors such as pH, oxygen concentration, and various metabolic substrates such as sugars are constantly fluctuating due to diet, plaque maturation, brushing, salivary cleansing, and fluctuations in these factors may potentially affect nitrite production by the oral microbiome.

We have also shown that the nitrite‐producing activity of the genus Veillonella is enhanced under acidic pH and in the presence of lactate and by cultivation in the presence of nitrate and nitrite (Wicaksono et al. 2020). Therefore, understanding the metabolic characteristics of nitrate‐reducing bacteria under various environments expected in the real oral cavity may provide valuable insights for maintaining both oral and systemic health. It was also shown that the nitrite‐producing activity of Rothia species was increased under acidic conditions (Rosier et al. 2020). However, the details of nitrate metabolism by nitrate‐reducing bacteria other than these bacterial species remain unclear. In the study by Sato‐Suzuki et al. (2020), the genera Actinomyces and Schaalia (reclassified from Actinomyces odontolyticus to Schaalia odontolytica in 2018) were detected most frequently, suggesting that these genera may have a greater impact on nitrite production in the oral cavity. Actinomyces species are known to have the ability to metabolize sugars and proliferate in aerobic environments (Takahashi and Yamada 1996, 1999a, 1999b). Therefore, the effect of environmental factors on the nitrate metabolism of Actinomyces species may be different from that of the other species. Furthermore, genomic data from the KEGG database indicate that Actinomyces naeslundii and S. odontolytica possess not only nitrate‐reducing (nitrite‐producing) enzymes but also enzymes that further degrade nitrite (nitrite‐degrading enzymes). This suggests that these bacteria have the potential not only to produce nitrite from nitrate but also to further degrade the produced nitrite, which may play a role in reducing nitrite in the oral environment. Hence, in order to evaluate the ability to provide nitrite to the oral cavity, it is necessary to evaluate the ability to metabolize both nitrate and nitrite.

Therefore, in this study, we investigated the effect of various environmental conditions on the nitrite production and nitrite reduction by representative species of the genera Actinomyces and Schaalia in vitro.

2. Materials and Methods

2.1. Bacterial Strains, Culture Conditions, and Preparation of Bacterial Suspensions

Type strains of A. naeslundii (ATCC 12104) and Schaalia odontlytica (JCM14871) were used. These bacteria were maintained on the blood agar plate (CDC Anaerobes Blood Agar plate, BD, Tokyo, Japan) at 4°C under anaerobic conditions. The anaerobic condition was provided by an anaerobic chamber (80% nitrogen, 10% hydrogen, and 10% carbon dioxide: Hirasawa Works, Tokyo, Japan), whereas aerobic experiments were performed under atmospheric conditions. When these bacteria were required to grow for experimental use, a single colony of A. naeslundii and S. odontlytica on the agar plate was transferred into the broth containing 1.7% tryptone (Difco Laboratories, Detroit, MI, USA), 0.3% yeast extract (Difco Laboratories, Detroit, MI, USA), 0.5% NaCl (Fujifilm Wako Chemicals, Osaka, Japan), 0.5% glucose (Fujifilm Wako Chemicals, Osaka, Japan), 0.1% ammonium hydrogen carbonate (Fujifilm Wako Chemicals, Osaka, Japan), and 50 mM potassium phosphate buffer (PPB) (pH 7) and grown under both anaerobic and aerobic conditions. Bacterial proliferation was assessed by measuring the optical density (OD) at 660 nm using a spectrophotometer (UV‐1800, Shimadzu Corporation, Kyoto, Japan).

Bacterial cells for the metabolism experiments were harvested at a late logarithmic phase (OD at 660 nm = 1.0), immediately centrifuged at 4°C, 10,000 rpm, for 7 min (6930, KUBOTA, Osaka, Japan), and washed twice with 2 mM PPB (pH 7) containing 75 mM potassium chloride, 75 mM sodium chloride, 5 mM magnesium chloride, and resuspended in the same buffer to be an OD of 10 at 660 nm. All these operations were carried out under anaerobic or aerobic conditions along each experimental condition. Reagents, deionized water, and buffer solutions required for the experiment under anaerobic conditions remained in the anaerobic chamber for at least 3 days to ensure anaerobicity. Bacterial suspensions were stored in ice‐cold conditions until use.

2.2. Assessment of the Nitrite‐Producing Activity and the Effect of Environmental Factors on Activity

The prepared bacterial suspension (final concentration: OD = 1), PPB (pH 7 or 5) (final concentration: 40 mM), and potassium nitrate solution (final concentration: 1.0 mM) were mixed in a sterilized tube. After the addition of glucose or sodium lactate (final concentration 0.1–10 mM), the reaction mixture was incubated at 37°C for 30 min under either anaerobic or aerobic conditions; the mixture was then centrifuged (4°C, 10,000 rpm, for 3 min), and the nitrite concentration in the supernatant was quantified using the Griess reagent method (Wicaksono et al. 2020). Metabolic experiments under anaerobic conditions were performed in another anaerobic chamber (90% nitrogen and 10% hydrogen gas) (Hirasawa Works Co. Ltd, Tokyo, Japan). The experiments under aerobic conditions were performed under atmospheric conditions.

2.3. Analysis of Organic Acids in the Sample

After measuring the nitrite‐producing activity in Section 2.2, the organic acids (succinate, lactate, formate, and acetate) in the metabolites produced along with nitrite production in each experimental condition were analyzed (Han et al. 2021). The supernatant of the sample was pretreated with a 0.22 µm filter (DISMIC HP020AN, Advantec Toyo Corporation, Tokyo, Japan), and the filtrate was analyzed with high‐performance liquid chromatography (Prominence, Shimadzu Corporation, Kyoto, Japan).

2.4. Assessment of the Nitrite‐Degrading Activity and the Effect of Environmental Factors on the Activity

Following the procedure described in Section 2.2, the prepared bacterial suspension (final concentration: OD = 1), PPB (pH 7 or 5) (final concentration: 40 mM), and potassium nitrite solution (final concentration: 0.1 mM) were mixed in a sterilized tube. After the addition of glucose or sodium lactate (final concentration 1 mM), the reaction mixture was incubated at 37°C for 30 min under either anaerobic or aerobic conditions. The mixture was centrifuged (4°C, 10,000 rpm, for 3 min), and nitrite remaining in the supernatant was quantified using the Griess reagent (Wicaksono et al. 2020).

2.5. Effect of Nitrate and Nitrite on Growth Ability

To investigate whether the growth ability of A. naeslundii and S. odontlytica is affected by nitrite, the growth in the medium with/without potassium nitrate or potassium nitrite (final concentration: 1.0 mM) was evaluated. At the same time, fluctuations in nitrite concentration and pH in the medium were monitored for 48 h.

2.6. Statistical Analysis

A paired t‐test was used for the comparisons between two groups, and Tukey's test was used for the comparisons inter three or more groups. A p value of less than 0.05 was defined as statistically significant. The software used for these analyses was Excel or Stat Flex Ver. 7 (Artec Co. Ltd, Osaka, Japan).

3. Results

3.1. Evaluation of Nitrite‐Producing Activity and Its Effect by Environmental Factors

In A. naeslundii, the nitrite‐producing activity was significantly higher in the anaerobic culture than in the aerobic culture. In both culture conditions, nitrite‐producing activity from nitrate was lower in the absence of glucose or lactate but increased with glucose or lactate supplementation. In anaerobically cultured bacteria, nitrite‐producing activity was higher under anaerobic conditions than under aerobic conditions. Nitrite production in the presence of glucose was similar at pH 7 and 5, whereas the production in the presence of lactate increased, especially at pH 5 and in the presence of more than 1 mM lactate (Figure 1).

FIGURE 1.

FIGURE 1

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Effect of environmental factors (oxygen concentration, pH, glucose, and lactate) on nitrite‐producing activity.

On the other hand, in S. odontolytica, the nitrite‐producing activity in the absence of glucose or lactate was higher than that of A. naeslundii. Glucose enhanced nitrite production slightly, whereas lactate did not show similar effects. Overall, nitrite production tended to be higher under anaerobic than aerobic conditions and at pH 5 than at pH 7. No nitrite was detected when the reaction mixture was incubated without nitrate (Figure 1). Furthermore, the pH did not change before or after the reaction under all conditions (data not shown).

S. odontolytica could not grow enough under aerobic conditions (data not shown); therefore, experiments with aerobically cultured bacteria were not performed with this bacterial species.

3.2. Identification of Acidic End Products Along With Nitrite Production

To elucidate the intricacies of the mechanism in nitrite production in A. naeslundii, which was particularly influenced by environmental factors (glucose, lactate, and pH), supernatant samples of each reaction solution before and after incubation were analyzed for acidic end products together with nitrite (Figure 2). Overall, the total amount of acidic end products produced by anaerobically cultured bacteria was higher under anaerobic conditions than under aerobic conditions. The presence of nitrate decreased the total amount of acidic end products produced in anaerobic metabolism in all cases, except when lactate was added at pH 5. Conversely, no significant changes were observed in any group of aerobically cultured bacteria. Acetate, formate, and succinate were consistently produced in the absence of nitrate under anaerobic conditions. Lactate was produced in addition in the glucose‐added group. When nitrate was added under anaerobic conditions, acetate production increased significantly, formate decreased overall, and succinate and lactate were no longer produced. Conversely, under aerobic conditions, the predominant acidic end product was acetate. Aerobically cultured bacteria produced the same amount of acidic end products as anaerobically cultured bacteria, except that acetate, formate, and succinate were detected only when the bacteria metabolized with glucose in the absence of nitrate at pH 5. Quantification of lactate in the condition added lactate was omitted because of the large amount of lactate added to the lactate group and the difficulty in estimating its utilization after metabolism (Figure 2, upper graph).

FIGURE 2.

FIGURE 2

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Production of acidic end products along with nitrite production in Actinomyces naeslundii.

3.3. Evaluation of Nitrite‐Degrading Activity and Its Effect by Environmental Factors

Nitrite degrading activity was higher in A. naeslundii than S. odontolytica in all experimental conditions (Figure 3). The activity of A. naeslundii was similar under all culture and metabolic conditions, but slightly higher under anaerobic than aerobic metabolic conditions. On the other hand, the activity of S. odontolytica did not differ between anaerobic and aerobic metabolic conditions. In A. naeslundii, the addition of glucose and lactate had little effect on the nitrite degradation, whereas S. odontolytica showed an inhibitory effect, especially when lactate was added under aerobic conditions.

FIGURE 3.

FIGURE 3

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Effect of environmental factors (oxygen concentration, glucose, and lactate) on nitrite‐degrading activity.

3.4. Effects of Nitrate and Nitrite on Bacterial Growth, Changes in Nitrite Concentration, and pH in the Medium

Bacterial growth, changes in pH, and changes in nitrite concentration in the culture medium due to the addition of potassium nitrate and potassium nitrite to the culture medium were monitored over time (Figure 4a,b). In anaerobic culture, the growth of A. naeslundii was inhibited by nitrate and nitrite in a concentration‐dependent manner from 12 to 32 h after the start of culture, but the growth was restored after 44 h. The pH of the medium decreased with the degree of bacterial growth. In addition, the nitrite concentration in the nitrate‐supplemented medium reached its maximum within 12 h after the start of culture, and the concentration was close to the nitrate concentration initially added. Thereafter, the nitrite concentration gradually decreased and reached almost 0 at 32 h when 0.1 mM nitrate was added and at 44 h when 0.5 mM nitrate was added. Even in the medium supplemented with 1 mM nitrate, the concentration eventually decreased to about half.

FIGURE 4.

(a) Effect of nitrate and nitrite on bacterial growth in Actinomyces naeslundii and changes in pH and nitrite concentration in the medium during growth. (b) Effect of nitrate and nitrite on bacterial growth in Schaalia odontolytica and changes in pH and nitrite concentration during growth in medium.

graphic file with name OMI-40-104-g001.jpg

graphic file with name OMI-40-104-g005.jpg

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In the aerobic culture, the growth of A. naeslundii was promoted by nitrate and nitrite, especially with the addition of nitrite, which was obviously different from the trend in the anaerobic culture. The pH of the medium decreased with the bacterial growth, and the rate of pH decrease was also higher when growth was promoted by nitrate and nitrite. On the other hand, in the control and 1 mM nitrate addition groups, the bacterial growth was slow, and the pH hardly decreased until the end. Furthermore, the nitrite concentration in the 1 mM nitrate‐supplemented medium increased more slowly than in the anaerobic culture, reaching its maximum 40 h after the start of culture, and the concentration was about half of the initial nitrate concentration. After 40 h, the nitrite concentration in the 1 mM nitrate‐supplemented medium gradually decreased but did not reach 0, in contrast to anaerobic culture.

In anaerobic culture, the growth of S. odontolytica was inhibited by nitrate in a concentration‐dependent manner (0.5 and 1 mM nitrate) at the early stage of culture but was eventually restored. With 0.1 mM nitrate, growth was slightly accelerated. On the other hand, nitrite supplementation had little effect on the growth. The change in medium pH corresponded to the degree of growth; the rate of pH decrease was also lower when growth was inhibited by nitrate and nitrite.

4. Discussion

4.1. Effect of Environmental Factors on Nitrite‐Producing Activity

The nitrite‐producing activity of A. naeslundii and S. odontolytica was found to be affected by pH, oxygen concentration, and metabolic substrates (Figure 1). S. odontolytica used in this study was isolated from caries lesions in 1958 and was initially classified in the genus Actinomyces (Batty 1958), but in 2018, genome analysis determined that it is a new genus, Schaalia (Nouioui et al. 2018). Therefore, the two species used in this study are considered to be closely related, but the trends in the effects of environmental factors on nitrite‐producing activity were different.

Anaerobically cultured A. naeslundii showed significantly higher nitrite production under anaerobic than aerobic conditions, and the addition of glucose and lactate increased its activity in a concentration‐dependent manner (Figure 1). In our previous study with Veillonella species (Wicaksono et al. 2020), their nitrite‐producing activity was also increased under acidic and anaerobic conditions in the presence of lactate. It was also shown that the nitrite‐producing activity of Rothia species was increased under acidic conditions (Rosier et al. 2020). A. naeslundii also showed an increase in nitrite production even when glucose was added, a result not seen in the other species. Although the possibility of fructose metabolism has recently been demonstrated (Mashima et al. 2021), Veillonella species basically do not use glucose as an energy substrate, whereas Actinomyces species can use both glucose and lactate as metabolic substrates (Takahashi and Yamada 1996, 1999b), which may result in enhanced nitrite‐producing activity. On the other hand, A. naeslundii cultured aerobically showed significantly lower nitrite production than that cultured anaerobically (Figure 1). The promoting effect by the addition of glucose and lactate was also lower compared to anaerobically cultured cells. These biochemical mechanisms may be related to the redox balance between the respective metabolisms, which will be discussed in the next section.

In S. odontolytica, the nitrite‐producing activity of the control group was high, and its activity was hardly increased by the addition of glucose or lactate (Figure 1). Actinomyces species, including the current genus Schaalia, are known to accumulate intracellular polysaccharides, depending on the species and strain (Komiyama et al. 1983), and it is likely that the S. odontolytica used in this study also accumulated intracellular polysaccharides. Intracellular polysaccharides are polymers of glucose with a structure similar to glycogen (Komiyama et al. 1983), which are converted to glucose 1‐phosphate by hydrolysis and further converted to glucose 6‐phosphate to join glycolysis (Figure 5). Therefore, it is likely that even in the absence of glucose addition, nitrite‐producing activity, as observed in A. naeslundii on glucose addition, was detected. The slight increase in nitrite production observed with glucose addition (Figure 1) suggests the glucose‐induced enhancement of nitrite production as explained before, but the high accumulation of polysaccharides in S. odontolytica used in this experiment may have masked the effect of glucose addition on nitrite production. Further studies on the accumulation and metabolism of polysaccharides in S. odontolytica are required.

FIGURE 5.

FIGURE 5

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Proposed metabolic pathways, metabolic enzymes, and metabolic regulation.

4.2. Inference of Metabolic Regulation Mechanisms From Metabolic End Products

The nitrite‐producing activity of anaerobically cultured A. naeslundii was promoted by the addition of glucose (Figure 1). In the process of glycolysis, reducing power such as NADH is produced (Figure 5). However, for metabolism to run smoothly, the reducing power produced must be consumed immediately by other metabolic reactions, and a balance between reduction and oxidation must be maintained at all times. Under anaerobic conditions, A. naeslundii produced succinate, lactate, formate, and acetate from glucose, and nitrite in the presence of nitrate, but only formate and acetate were produced (Figure 2). Under anaerobic conditions, the reducing power produced by glycolysis is consumed in the metabolic process of succinate production and lactate production by lactate dehydrogenase (LDH) to achieve redox balance and smooth metabolism (Figure 5) (Takahashi et al. 1994, 1999b). In the presence of nitrate, the reducing power produced by glycolysis can be used to reduce nitrate catalyzed by nitrate reductase (NAR) to produce nitrite, and glucose cannot produce succinate and lactate, which require reducing power, and can subsequently be catalyzed by pyruvate formate lyase (PFL) (Figure 5). At pH 5, the production of formate is reduced, suggesting that pyruvate dehydrogenase (PDH) (Figure 5) may have replaced the acid‐sensitive PFL, but the details are unknown. Under aerobic conditions, acetate was produced mainly from glucose, probably because most of the reducing power produced by glycolysis and subsequent oxidation of pyruvate by PDH was consumed by the reaction with oxygen catalyzed by NADH oxidase (NADH OD), and metabolic pathways requiring reducing power, such as succinate and lactate production, failed to function (Figure 5). Formate production also decreased, probably because the oxygen‐sensitive PFL was inactivated by oxygen and lost its function (Takahashi et al. 1982; Abbe et al. 1982). The detection of a trace amount of nitrite suggests that some of the reducing power produced by glycolysis and PDH was used by NAR to reduce nitrate, resulting in the production of nitrite (Figures 2 and 5).

Aerobically cultured A. naeslundii produced mainly acetate (Figure 2), similar to the results of anaerobically cultured cells under aerobic conditions, except that nitrite production was lower than in anaerobically cultured cells (Figures 1 and 2). Metabolism may be similarly affected by oxygen as mentioned above, but it is thought that because oxygen metabolism–related enzymes can be more induced under aerobic conditions, aerobically cultured A. naeslundii can utilize oxygen more efficiently than anaerobically cultured cells. Therefore, because reducing power is more efficiently utilized by oxygen than by nitrite, the reducing power required to produce nitrite from nitrate may be insufficient, resulting in a decrease in nitrite production from nitrate.

Without the addition of glucose, A. naeslundii cultured anaerobically produced succinate, formate, and acetate under anaerobic conditions and acetate under aerobic conditions, and these metabolites are thought to be derived from intracellular polysaccharides, as already discussed for S. odontolytica (Figure 5). Basically, the metabolic pathway is similar to that of glucose metabolism except for lactate (Figure 2). This could be because polysaccharide metabolism is slower than glucose metabolism, and the fructose 1,6‐bisphosphate, a glycolytic metabolic intermediate that activates LDH in A. naeslundii (Takahashi and Yamada 1992, 1999b), is not sufficiently high, resulting in an inefficient conversion of pyruvate to lactate. The presence of nitrate would have resulted in the production of mainly acetate, as in the case of glucose addition, because the reducing power produced during polysaccharide metabolism in the bacteria would have been used by NAR to reduce nitrate to nitrite. The anaerobic metabolism of aerobically cultured cells without glucose is thought to be similar to that of anaerobically cultured cells.

When lactate was added in the absence of nitrate, the acidic end products of anaerobically cultured A. naeslundii did not change significantly compared to the control (Figure 2), suggesting that anaerobically cultured A. naeslundii metabolizes little lactate. However, when lactate was added under aerobic conditions, the acetate production increased, especially in pH 5, suggesting that lactate was metabolized and converted to acetate in the presence of oxygen. Takahashi and Yamada (1996, 1999a, 1999b) reported that aerobically cultured Actinomyces can metabolize lactate and that the enzymes necessary for lactate metabolism are induced by aerobic culture, which supported our results.

In the presence of nitrate, both anaerobically and aerobically cultured A. naeslundii accelerated the production of nitrite from nitrate, accompanied by an increase in acetate production, and under anaerobic conditions, the production of formate was also observed (Figure 2). This indicates that the oxidation of lactate by LDH produced pyruvate, which was further converted to acetate via acetyl CoA and acetyl phosphate catalyzed by PFL (with formate production under anaerobic conditions) and PDH, and that the reducing power generated by LDH and PDH was used to reduce nitrate by NAR, resulting in the production of nitrite (Figure 5). This suggests that the ability to metabolize lactate is latent in this bacterium and that it can metabolize lactate if there is an appropriate substrate that can receive the reducing power generated by LDH and PDH, as in the case of nitrate. In particular, nitrite production is higher in the presence of lactate at pH 5, similar to Veillonella species (Wicaksono et al. 2020). This may be due to the fact that at acidic pH, weak acids such as lactate have an increased proportion of non‐dissociated forms, which are more easily permeable to the bacterial cell membrane, resulting in increased lactate uptake efficiency (Washio et al. 2014; Wicaksono et al. 2020).

In all experimental systems, nitrite productivity was higher under anaerobic conditions, indicating that NARs of A. naeslundii cultured under anaerobic conditions function more efficiently under anaerobic than aerobic conditions. The reduced function under aerobic conditions may have been caused by the oxygen inactivation of NAR or enhanced utilization of reducing power by NADH OD in the presence of oxygen. In addition, in the presence of nitrate, the reducing power produced by glycolysis, LDH, and PDH is preferentially used by NAR and not for the production of succinate or lactate, with acetate as the major end product (Figure 2), suggesting that these reducing powers have a high enzymatic affinity with NAR. However, the details of the biochemical properties of NARs in A. naeslundii are unknown and require further study.

4.3. Evaluation of Nitrite‐Degrading Activity and Its Effect by Environmental Factors

A. naeslundii had a higher nitrite degradation ability than S. odontolytica (Figure 3). Nitrite‐degrading activity of A. naeslundii was higher under anaerobic conditions, as was its nitrite‐producing activity, but there was little activation by the addition of glucose or lactate, which was similar to or slightly less than that of the control. In contrast, the nitrite‐degrading activity of S. odontolytica did not differ between anaerobic and aerobic conditions but was reduced by the addition of glucose and lactate.

As nitrite degradation is known to be catalyzed by nitrite reductase (NIR) with the supply of reducing power (Figure 5) (Rosier et al. 2022), it is possible that it is controlled by the production of reducing power by the addition of glucose and lactate discussed in the previous section. However, this is not the case for the nitrate‐degrading activity of A. naeslundii and S. odontolytica in this study, and thus further studies are needed.

A. naeslundii and S. odontolytica were shown to simultaneously produce (Figure 1) and degrade (Figure 3) nitrite. However, a comparison of the two activities shows that in A. naeslundii, both activities are close in the absence of glucose or lactate, but in the presence of glucose or lactate, the producing activity is much higher, whereas in S. odontolytica, the producing activity is always higher. These findings suggest that these bacteria function as nitrite‐producing bacteria in the oral cavity.

4.4. Effects of Nitrate and Nitrite on Bacterial Growth, Changes in Nitrite Concentration, and pH in the Medium

In both anaerobically cultured A. naeslundii and S. odontolytica, the addition of nitrate and nitrite to the culture medium inhibited and delayed growth in the early stages of culture (Figure 4a,b). The nitrite concentration increased prior to bacterial growth in the nitrate‐added medium, suggesting that nitrate in the medium did not directly inhibit bacterial growth, but that nitrite produced from the added nitrate inhibited it. The fact that the growth curves for the addition of nitrate and nitrite were similar in A. naeslundii supports the idea that the inhibition of growth is mediated by the production of nitrite from nitrate. However, in S. odontolytica, the growth curves for the addition of nitrate and nitrite were somewhat different, especially when higher concentrations of nitrate and nitrite were added. It might be considered that the reactivity to higher concentrations of nitrite is different between A. naeslundii and S. odontolytica, although further investigation is required.

As shown in the previous section, the nitrite‐degrading activity was lower than the nitrite‐producing activity, and the fact that the nitrite concentration increased to almost the same level as the nitrate concentration added to the medium in the early stages of growth also supports that nitrite productivity is superior to nitrite degradation. The nitrite concentration in the medium increased once and then decreased, which was considered to be due to the fact that the nitrate in the medium was completely consumed, resulting in no new nitrite production and continuous nitrite degradation.

Furthermore, as the recovery of growth was observed as the nitrite concentration in the medium decreased, it is expected that when all the nitrite in the medium is consumed, growth will return to the same level as that of the control. The pH of the medium was also delayed in the nitrate‐ and nitrite‐added medium, but the final pH was expected to decrease to the same level as that of the control as the bacterial growth recovered. However, from a different perspective, this delayed pH decrease in the presence of nitrate and nitrite, observed under anaerobic conditions, suggests that nitrate and nitrite can contribute to the inhibition of caries development. The present study suggests that the nitrite‐producing activity of A. naeslundii is higher at acidic conditions and in the presence of lactate, which is consistent with environments that are known to be caries‐prone. Even in such environments, the abundant presence of nitrate may enable indigenous bacteria such as A. naeslundii to produce nitrite, thereby locally inhibiting the development of dental caries. Although we currently only have information on limited bacterial species, it may be important that further research clarifies such positive roles of bacteria in the oral microbiome. Comparing the results of this study with our previous study using Veillonella species (Wicaksono et al. 2020), Veillonella species showed tolerance to nitrite in the medium and a rather faster growth rate when nitrate was added to the medium, whereas A. naeslundii and S. odontolytica were less tolerant to nitrite in the medium and showed very different results.

On the other hand, in aerobically cultured A. naeslundii, the addition of nitrate and nitrite to the culture medium accelerated the growth in the early stages of culture (Figure 4a). The nitrite concentration gradually increased with bacterial growth in the nitrate‐supplemented medium, but the rate was obviously slower than that in the anaerobic culture. It was suggested that nitrate and nitrite may have some beneficial effects in the aerobic growth process of A. naeslundii. But further consideration is needed in the future.

A. naeslundii and S. odontolytica were used in this study. S. odontolytica is a bacterium that was reclassified in 2018 and renamed from A. odontolyticus. Hence, these two bacterial species were originally closely related. However, their behaviors in nitrate and nitrite metabolism were different in many respects. This suggested that we should consider that the characteristic of the metabolism of each bacterial species might be different at the bacterial species level when the metabolism of these individual bacteria was investigated.

Potassium nitrate and potassium nitrite were used in this study. However, in recent years, the potential effect of potassium on bacterial growth and metabolism is suggested (Stautz et al. 2021; Yost et al. 2017). Comparison and confirmation of the effects of potassium in potassium nitrate and potassium nitrite with other salts may be necessary for the conduct of future studies.

5. Conclusion

In our previous study (Sato‐Suzuki et al. 2020), Actinomyces and Schaalia belonged to the most abundant nitrite‐producing bacteria found in the oral cavity, but on the basis of the results of this study, when considering the contribution to nitrite production in the oral cavity, it is thought that not only the number of bacteria but also the modification in metabolic activity due to environmental factors should be taken into consideration. It should also be taken into account that Actinomyces and Schaalia, although less tolerant to nitrite, have nitrite‐degrading activity that Veillonella does not. Further studies, including metabolic characteristics of these bacteria, are needed to elucidate the full extent of nitrite supply by the oral microbiome.

Author Contributions

Tomona Otake and Jumpei Washio contributed equally as the first authors. Tomona Otake contributed to conception, data acquisition, and analysis and drafted the manuscript. Jumpei Washio contributed to conception and design, data analysis and interpretation, drafting, and critically revising the manuscript. Kazuko Ezoe, Satoko Sato, and Yuki Abiko contributed to data acquisition and analysis and critically revised the manuscript. Kaoru Igarashi contributed to the conception and design and critically revised the manuscript. Nobuhiro Takahashi contributed to conception and design, data analysis, and interpretation, drafting and critically revising the manuscript. All authors gave their final approval and agreed to be accountable for all aspects of the work, ensuring integrity and accuracy.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1111/omi.12492.

Acknowledgments

This study was supported in part by Grants‐in‐Aid for Scientific Research B (21H03151), Grants‐in‐Aid for Scientific Research C (20K10241) from Japan Society for the Promotion of Science.

Funding: This study was supported by the Grants‐in‐Aid for Scientific Research B (21H03151), Grants‐in‐Aid for Scientific Research C (20K10241) from Japan Society for the Promotion of Science.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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