Various ingredients are utilized to inhibit the growth of harmful bacteria associated with cavities, gum disease, and bad breath. However, the precise mechanisms by which these ingredients affect the oral microbiome have not been fully understood at the molecular level. To elucidate the molecular mechanisms, a high-throughput bacterial transcriptomics study was conducted, and the gene expression profiles of six common oral bacteria, including two Gram-positive bacteria (Actinomyces viscosus, Streptococcus mutans) and four Gram-negative bacteria (Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, and Prevotella pallens), were analyzed. The bacteria were exposed to nine common ingredients in toothpaste and mouthwash at different concentrations (stannous fluoride, stannous chloride, arginine bicarbonate, cetylpyridinium chloride, sodium monofluorophosphate, sodium fluoride, potassium nitrate, zinc phosphate, and hydrogen peroxide). Across 78 ingredient–microorganism pairs with 360 treatment–control combinations, significant and reproducible ingredient-based transcriptional response profiles were observed, providing valuable insights into the effects of these ingredients on the oral microbiome at the molecular level. This research shows that oral care product ingredients applied at biologically relevant concentrations manifest differential effects on the transcriptomics of bacterial genes in a variety of oral periodontal pathogenic bacteria. Stannous fluoride, stannous chloride, and cetylpyridinium chloride showed the most robust efficacy in inhibiting the growth or gene expression of various bacteria and pathogenic pathways. Combining multiple ingredients targeting different mechanisms might be more efficient than single ingredients in complex oral microbiomes.

This research examined the effects of daily application of an oral hygiene regimen on the subgingival microbiome over 24 months. Generally healthy adults (107 enrolled, 87 completed) with early periodontitis used a home-care regimen (stannous fluoride paste, cetylpyridinium chloride rinse, power toothbrush, and floss) or usual care (control). Subgingival plaque samples were analyzed enzymatically for bacterial toxins. TLR ligands were measured using TLR-SEAP and TLR-ATP assays. Proinflammatory cytokines and metalloproteinases were quantified via immunoassays. Subgingival DNA was sequenced using a shotgun approach to assess microbial diversity. Increasing levels of bacteria, toxins, TLR activation, inflammatory cytokines, and MMPs were observed for periodontitis versus gingivitis and gingivitis versus healthy sites. The regimen significantly reduced levels of the critical proinflammatory cytokine IL-1β, as well as MMP-1 and MMP-9, at 24 months. By month 6, TLR ligands within subgingival plaques decreased. The abundance of pathogenic bacteria correlated with levels of virulence factors, proinflammatory cytokines, MMPs, and severity of clinical measures. Two distinct constellations of pathogenic bacteria were identified. Gingival sites were categorized into responders and non-responders per clinical symptoms and biomarkers. The regimen yielded more responder sites (70%) versus the control (47%), p = 0.0002914. The regimen reduced pathogenic bacteria, IL-1β, MMP1, and MMP-9, paralleling clinical reductions in periodontal disease.

Poor oral health is a neglected epidemic, potentially contributing to systemic health issues. We employed a multi-omics approach to investigate the biological changes associated with gingivitis and the effects of stannous fluoride (SnF₂) dentifrice on microbial composition and salivary proteomics in an eight-week clinical trial involving 39 participants categorized as high (n = 20) and low bleeders (n = 19). Baseline assessments revealed significant microbial dysbiosis in high bleeders, characterized by a higher abundance of Porphyromonas and Fusobacterium, alongside compromised epithelial barriers and increased inflammation. Following SnF₂ treatment, a substantial reduction in these bacteria, and an increase in Rothia and Haemophulis, were observed, correlating with improved clinical measures, including reduced bleeding and inflammation indices. In total, 80 proteins (including pro-inflammatory cytokines, alarmin keratins, and matrix metalloproteinases) showed a significant reduction in high bleeders after treatment, with 29 overlapping the disease biomarkers in the plasma atlas, supporting the role of SnF₂ in mitigating oxidative stress and enhancing epithelial integrity. Furthermore, SnF₂ treatment significantly reduced collagen degradation, suggesting the preservation of tissue integrity. These findings highlight that SnF₂ not only improves local oral health but may also benefit systemic health, showcasing the value of a multi-omics approach in understanding the interconnections among oral microbiota, inflammatory responses, and systemic health outcomes.

This research analyzed metabolomic and proteomic differences between participants with gingivitis (>20 bleeding sites) and generally healthy participants (≤3 bleeding sites) at baseline and 4 weeks post stannous fluoride (SnF₂) dentifrice treatment. Sixty-two metabolites were different (p < 0.05) between groups at baseline. Forty cytokines were analyzed using immunoassays and a group of proinflammatory cytokines (IL-1α, IL-1β, TNF-α, SAA, ICAM-1, VCAM-1) was elevated in participants with gingivitis (p < 0.1) versus healthy gingiva at baseline, with C-reactive protein (p < 0.05) being significantly elevated. Proteomic analysis carried out in baseline oral lavage revealed four of the top hits (p < 0.0004) were central-metabolism-related: aldolase A, triosephosphate isomerase, lactate dehydrogenase, and malate dehydrogenase. Enzymatic assays confirmed the proteomic finding that malate dehydrogenase and triosephosphate isomerase activities were elevated in gingivitis samples; SnF₂ dentifrice treatment reduced their activity. Collectively, 20 proteins with the lowest p-values in oral lavage appeared to be indicative of periodontal health, potentially forming the basis to cluster samples into healthy and unhealthy groups. A TLR-ATP biosensor model was established and demonstrated that microbial virulence factors induced the observed changes in oral lavage. Combined findings suggest gingivitis involves upregulation of host cell bioenergetic processes involving enzymatic activity in the glycolysis and citric acid cycle pathways.

This research examined host metabolic processes associated with gingivitis and potential impacts of interventions. First, gingival brush samples from a previously reported clinical study in which participants used an intervention (stannous fluoride (SnF₂) toothpaste/cetylpyridinium chloride rinse/oscillating-rotating electric toothbrush) or control (standard fluoride toothpaste/manual toothbrush) were analyzed with Metabolon metabolomic assessment. Next, hypotheses were generated about mechanisms of metabolic observations and tested in two in vitro models. Human primary blood cells were treated with endotoxins and outer membrane vesicles (OMVs) in the presence of SnF₂. In clinical brush samples, 46 metabolites decreased with intervention at Week 3, including 9- and 13-hydroxyoctadecadienoic acid, citrulline, adenine, and hypoxanthine. Seven metabolites related to nitrogen metabolism and citrulline production were selected for targeted quantification. They were low in health and increased in gingivitis. Targeted quantification demonstrated reduced citrulline, succinate, ornithine and deoxycarnitine (p ≤ 0.001 for all) between baseline and Week 6 with intervention; reductions were greater for intervention vs. control for citrulline, succinate and deoxycarnitine at Week 6 (p ≤ 0.019). In vitro, endotoxin and OMVs increased citrulline and nitric oxide (NO). SnF₂ inhibited elevation of both. Citrulline, deoxycarnitine, succinate, and ornithine were identified as potential novel markers of gingivitis. Elevated NO production is a potential driver of oxidative stress, reduced with intervention.