Pathways Linking Oral Bacteria, Nitric Oxide Metabolism, and Health.

Nitrate-reducing oral bacteria have gained a lot of interest due to their involvement in nitric oxide (NO) synthesis and its important cardiometabolic outcomes. Consortia of nitrate-metabolizing oral bacteria associated with cardiometabolic health and cognitive function have been recently identified. Longitudinal studies and clinical trials have shown that chronic mouthwash use is associated with increased blood pressure and increased risk for prediabetes/diabetes and hypertension. Concurrently, recent studies are beginning to shed some light on the complexity of nitrate reduction pathways of oral bacteria, such as dissimilatory nitrate reduction to ammonium (DNRA), which converts nitrite into ammonium, and denitrification, which converts nitrite to NO, nitrous oxide, and dinitrogen. These pathways can affect the composition and metabolism of the oral microbiome; consequently, salivary nitrate and nitrite metabolism have been proposed as targets for probiotics and oral health. These pathways could also affect systemic NO levels because NO generated through denitrification can be oxidized back to nitrite in the saliva, thus facilitating flux along the NO3--NO2--NO pathway, while DNRA converts nitrite to ammonium, leading to reduced NO. It is, therefore, important to understand which pathway predominates under different oral environmental conditions, since the clinical consequences could be different for oral and systemic health. Recent studies show that oral hygiene measures such as tongue cleaning and dietary nitrate are likely to favor denitrifying bacteria such as Neisseria, which are linked with better cardiometabolic health. A vast body of literature demonstrates that redox potential, carbon-to-nitrate ratio, and nitrate-to-nitrite ratio are key environmental drivers of the competing denitrification and DNRA pathways in various natural and artificial ecosystems. Based on this information, a novel behavioral and microbial model for nitric oxide metabolism and health is proposed, which links lifestyle factors with oral and systemic health through NO metabolism.

Oral Bacterial Acid-Base Metabolism in Caries Screening: A Proof-Of-Concept Study.

The objective of this cross-sectional study was to clinically validate an array of biochemical tests for oral acid/alkali generation as caries screening instruments. 185 adult subjects (mean 33.6±10.6 years) were examined clinically for dental caries using the ICDAS criteria. Bitewing radiographs were used to confirm interproximal surfaces of posterior teeth. For the purposes of this study, subjects were classified as "caries-active" if they had at least one untreated caries lesion with ICDAS 4 or higher. Pooled supragingival plaque and unstimulated saliva samples were collected and assayed for pH changes from sucrose and urea metabolism using colorimetric tests. The validity of each test to discriminate between "caries-inactive" and "caries-active" subjects was assessed and compared to a commercial bacteriological caries-screening test using roc regression and logistic regression models. The AUCs of the plaque-urea (PU: 0.59 (0.51, 0.67)), plaque-urea-glucose (PUG: 0.59 (0.51, 0.67)) and saliva-urea-glucose (SUG: 0.59 (0.51, 0.67)) tests did not differ significantly from the bacteriological tests (CRT-mutans: 0.62 (0.54, 0.70); CRT-lactobacillus: 0.63 (0.56, 0.71) (P>0.05), but the plaque-glucose (SG), saliva-glucose (SG), saliva-urea (SU) and saliva-plaque-glucose (SPG) tests had significantly smaller AUCs (P<0.05). The AUCs for the PU, PUG, SUG, and the CRT-mutans tests were higher in subjects who had no existing dental restorations (PU: 0.90 (0.77, 1.04); PUG: 0.90 (0.79, 1.01); SUG: 0.89 (0.69, 1.08); CRT-mutans: 0.90 (0.73, 1.08)). The incorporation of the biochemical tests into a multidimensional bacteriological/psychosocial caries screening model significantly increased its diagnostic values (Se+Sp: 160.6, AUC: 0.846). In conclusion, as a proof of concept, the results of this study indicate that measuring the ability of dental plaque and saliva to metabolize urea together with the ability to generate acid from sugars may have a promising role in caries screening either independently, or as part of a multidimensional biological test.

Urease activity as a risk factor for caries development in children during a three-year study period: a survival analysis approach.

UNLABELLED: Recent cross-sectional studies suggest that reduced ability to generate alkali via the urease pathway in dental plaque may be an important caries risk factor, but it has not been assessed prospectively. OBJECTIVE: To evaluate the effect of plaque and saliva urease activity on the risk for developing new caries over a three-year period in children. METHODS: A panel of 80 children, three to six years of age at recruitment, was followed prospectively for three years. Plaque urease activity, saliva urease activity and dental caries were measured every six months. Survival analysis methodology was used to evaluate the effect of urease on caries development during the study period adjusted for gender, age, baseline caries levels, sugar consumption, amount of plaque, and mutans streptococci levels. RESULTS: The risk for developing new caries increased in a dose-responsive manner with increasing levels of urease activity in saliva (adjusted HR(Q4 vs. Q1): 4.98; 95% CI: 1.33, 18.69) and with decreasing urease activity in plaque (adjusted HR(Q4 vs. Q1): 0.29; 95% CI: 0.11, 0.76). Multiple measurements of urease activity were conducted to overcome the variability of urease activity in this study. Baseline caries and mutans streptococci in saliva were also important predictors of caries risk. CONCLUSIONS: Increased urease activity in saliva can be an indicator of increased caries risk in children, whilst increased urease activity in plaque may be associated with reduced caries risk. The reproducibility of urease measurements must be improved before these findings can be further tested and clinically applied.

Urease activity in dental plaque and saliva of children during a three-year study period and its relationship with other caries risk factors.

UNLABELLED: Bacterial urease activity in dental plaque and in saliva generates ammonia, which can increase the plaque pH and can protect acid-sensitive oral bacteria. Recent cross-sectional studies suggest that reduced ability to generate ammonia from urea in dental plaque can be an important caries risk factor. In spite of this proposed important clinical role, there is currently no information available regarding important clinical aspects of oral ureolysis in children. OBJECTIVE: The objective of this study was to evaluate the distribution and pattern of urease activity in the dental plaque and in the saliva of children during a three-year period, and to examine the relationship of urease with some important caries risk factors. METHODS: A longitudinal study was conducted with repeated measures over a three-year period on a panel of 80 children, aged 3-6 years at recruitment. The dynamics of change in urease activity were described and associated with clinical, biological, and behavioural caries risk factors. RESULTS: Urease activity in plaque showed a trend to remain stable during the study period and was negatively associated with sugar consumption (P<0.05). Urease activity in unstimulated saliva increased with age, and it was positively associated with the levels of mutans streptococci in saliva and with the educational level of the parents (P<0.05). CONCLUSIONS: The results of this study reveal interesting and complex interactions between oral urease activity and some important caries risk factors. Urease activity in saliva could be an indicator of mutans infection in children.

The effect of sucrose on plaque and saliva urease levels in vivo.

OBJECTIVE: Dietary sugar exposures induce an immediate drop of the plaque pH. Based on in vitro observations, it was hypothesized that oral bacteria may rapidly respond to this environmental change by increasing the activity or expression of alkali-generating pathways, such as the urease pathway. The objective of this exploratory in vivo study was to determine the short-term effect of a brief sucrose exposure on plaque and saliva urease activity and expression, and to relate this effect to caries experience. METHODS: Urease activity levels were measured in plaque and saliva samples collected from 20 children during fasting conditions and 30 min after rinsing with a sucrose solution. Streptococcus salivarius ureC-specific mRNA in saliva was quantified using real-time RT-PCR. The impact of host-related factors, such as age, gender, sugar consumption, salivary mutans streptococci levels and caries status on urease activity was evaluated. RESULTS: Plaque urease activity under fasting conditions was higher in subjects with low caries and mutans streptococci levels. This difference was not observed after the sucrose exposure. The response of urease to sucrose in vivo did not depend on caries experience or salivary mutans levels. Significant increase in urease activity of plaque and saliva after exposure to sucrose was observed only in the subjects who had low urease levels at baseline. CONCLUSIONS: The findings of this exploratory study suggest that plaque urease activity may have an important long-term influence in caries development but not during a cariogenic challenge.

The relationship between dental caries status and dental plaque urease activity.

INTRODUCTION: Ammonia production from the metabolism of urea by urease enzymes of oral bacteria moderates plaque acidification and may inhibit dental caries, as suggested by in vitro studies and indirect clinical observations. The objective of this study was to examine the relationship of urease activity with dental caries at the clinical level. METHODS: Urease activity was measured in dental plaque and saliva samples from 25 caries-free subjects (CF) and in eight subjects with six or more open caries lesions (CA). Plaque and saliva collection was repeated for each subject 1 week later using identical procedures. RESULTS: Urease-specific activity in the dental plaque of CF subjects was significantly higher compared to that in the subjects with caries. The association of low plaque urease levels with increased caries was further supported by odds ratio analysis using different plaque urease cut-off points. Using a receiver operating characteristic curve it was estimated that there was an approximately 85% probability of correctly classifying the subjects as CA or CF based on the relative ordering of their plaque urease activity levels. No statistically significant differences were observed in salivary urease activity. CONCLUSION: This study suggests that loss of alkali-generating potential of tooth biofilms via the urease pathway has a positive relationship to dental caries.

Analysis of urease expression in Actinomyces naeslundii WVU45.

The hydrolysis of urea by ureases of oral bacteria in dental plaque can cause a considerable increase in plaque pH, which can inhibit the development of dental caries. There is also indirect evidence that urea metabolism may promote the formation of calculus and that ammonia release from urea could exacerbate periodontal diseases. Actinomyces naeslundii, an early colonizer of the oral cavity and a numerically significant plaque constituent, demonstrates comparatively low levels of urease activity on isolation, so this organism has not been considered a major contributor to total oral urease activity. In this study it was observed that urease activity and urease-specific mRNA levels in A. naeslundii WVU45 can increase up to 50-fold during growth under nitrogen-limiting conditions. Using primer extension analysis, a putative, proximal, nitrogen-regulated promoter of the A. naeslundii urease gene cluster was identified. The functionality and nitrogen responsiveness of this promoter were confirmed using reporter gene fusions and 5' deletion analysis. The data indicated that regulation of urease expression by nitrogen availability in A. naeslundii may require a positive transcriptional activator. Plaque bacteria may experience nitrogen limitation when carbohydrates are present in excess. Therefore, based on the results of this study and in contrast to previous beliefs, strains of A. naeslundii may have the potential to be significant contributors to total plaque ureolysis, particularly during periods when there is an increased risk for caries development.

Characterization of the fructosyltransferase gene of Actinomyces naeslundii WVU45.

Oral actinomycetes produce fructosyltransferase (FTF) enzymes which convert sucrose into polymers of D-fructose, known as levans, and these polymers are thought to contribute to the persistence and virulence of the organisms. A gene encoding FTF was isolated from Actinomyces naeslundii WVU45; the deduced amino acid sequence showed significant similarity to known levansucrases of gram-negative environmental isolates but was less similar to FTFs from gram-positive bacteria. A transcriptional start site was mapped by primer extension 70 bp 5' from the putative start codon. Promoter fusions to a chloramphenicol acetyltransferase gene were used to confirm that there was a functional promoter driving ftf expression and to show that sequences located 86 to 218 bp upstream of the transcription initiation site were required for optimal ftf expression. Quantitative slot blot analysis against total RNA from cells grown on different sugars or from different growth phases revealed that ftf was constitutively transcribed. Thus, the A. naeslundii FTF is more similar in primary sequence and the regulation of expression to levansucrases of gram-negative bacteria than gram-positive bacteria.

Genetic and physiologic characterization of urease of Actinomyces naeslundii.

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of Actinomyces naeslundii, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the ureC gene of A. naeslundii WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the A. naeslundii urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the ureABCEFGD genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5' to the start codon of ureA. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the ureC gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by A. naeslundii via a urease-dependent pathway and that ureolysis can protect A. naeslundii against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to A. naeslundii critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.