The antimicrobial effect of different vitamin D compounds on Streptococcus mutans and their impact on glycosyltransferase expression.

Background: Streptococcus mutans is a virulent microorganism associated with dental caries. This in vitro study aimed to investigate the antimicrobial effects of Cholecalciferol (D3) and Doxercalciferol (D2), against S. mutans and on glycosyltransferase gene expression. Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of D3 and D2 for S. mutans were determined according to the Clinical Laboratory Standards Institute guidelines. The effect of the compounds on environmental pH in 1% w/v and 5% w/v sucrose broth cultures after 24 hours were assessed colorimetrically. Additionally, their impact on glycosyltransferases gene expression (GtfB, GtfC, GtfD) in 5% w/v sucrose culture was evaluated using quantitative real-time PCR. Results: The MBCs of D3 and D2 were 83 µg/ml and 166 µg/ml respectively. Both compounds were effective in preventing the local pH drop <5.5 at ≥166 µg/ml in sucrose supplemented cultures. However, the compounds did not inhibit pH drop at MIC values. Notably, D2 upregulated GtfD expression significantly (p < 0.05) and downregulated GtfB and GtfC. Conclusion: Vitamin D2 and D3 inhibited S. mutans mediated pH drop in sucrose supplemented cultures and altered glycosyltransferase expression, suggesting potential therapeutic roles in dental caries prevention. Further research is needed to assess their full impact on S. mutans survival under environmental stresses.

Associations between Host Genetic Variants and Subgingival Microbiota in Patients with the Metabolic Syndrome.

Host genetic variants may affect oral biofilms, playing a role in the periodontitis-systemic disease axis. This is the first study to assess the associations between host genetic variants and subgingival microbiota in patients with metabolic syndrome (MetS); 103 patients with MetS underwent medical and periodontal examinations and had blood and subgingival plaque samples taken. DNA was extracted and processed, assessing a panel of selected single nucleotide polymorphisms (SNPs) first (hypothesis testing) and then expanding to a discovery phase. The subgingival plaque microbiome from these patients was profiled. Analysis of associations between host genetic and microbial factors was performed and stratified for periodontal diagnosis. Specific SNPs within RUNX2, CAMTA1 and VDR genes were associated with diversity metrics with no genome-wide associations detected for periodontitis severity or Mets components at p < 10-7. Severe periodontitis was associated with pathogenic genera and species. Some SNPs correlated with specific bacterial genera as well as with microbial taxa, notably VDR (rs12717991) with Streptococcus mutans and RUNX2 (rs3749863) with Porphyromonas gingivalis. In conclusion, variation in host genotypes may play a role in the dysregulated immune responses characterizing periodontitis and thus the oral microbiome, suggesting that systemic health-associated host traits further interact with oral health and the microbiome.

In vitro effectiveness of pomegranate extract present in pet oral hygiene products against canine oral bacterial species.

Background and Aim: Pomegranate is known to possess antibacterial properties, partly because of its punicalagin content. However, its effect on canine oral bacterial species has not yet been elucidated. In this study, we evaluated the effect of pomegranate extract present in pet dental products on the growth and survival of five canine oral bacterial species in biofilms. Materials and Methods: Five bacterial species, Neisseria shayeganii, Neisseria canis, Porphyromonas gulae, Porphyromonas macacae, and Porphyromonas crevioricanis, were individually cultured for biofilm formation and exposed to pomegranate extract (or control) for 15 min. Cell survival was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was compared between different conditions using a student's t-test. In addition, the individual strains were grown in planktonic suspensions and exposed to serial dilutions of the extract to determine the minimum inhibitory concentration. Results: At a concentration of 0.035% w/v, the extract significantly reduced the survival of P. gulae (-39%, p < 0.001) and N. canis (-28%, p = 0.08) in biofilms. At similar concentrations, the extract also completely or partially inhibited the growth of N. canis and Porphyromonas spp. in planktonic suspensions, respectively. Conclusion: The pomegranate extract found in some pet dental products can limit bacterial growth and survival in the biofilms formed by N. canis and P. gulae in vitro. As P. gulae is involved in periodontal disease progression, limiting its proliferation using products containing pomegranate extract could contribute to disease prevention. Further studies on dogs receiving such products are necessary to confirm these effects.

Analysis of gingival crevicular fluid biomarkers in patients with metabolic syndrome.

OBJECTIVES: To assess associations between gingival crevicular fluid (GCF) markers in patients with metabolic syndrome, with or without concomitant periodontitis. METHODS: A total of 95 patients with Metabolic Syndrome (MetS) had a periodontal examination and gingival crevicular fluid samples taken. Proteomic analysis of gingival crevicular fluid (GCF) was carried out by Human XL Cytokine protein arrays in 12 selected patients, followed by multiplex ELISA of 11 analytes in 95 participants. RESULTS: Increased levels of Aggrecan, IL-6 and IL-8 were found in patients with periodontal health compared with moderate and severe periodontitis. The inverse stepwise association between severity of periodontitis and reduced Aggrecan levels was also observed at adjusted linear regression analysis. Diagnosis of diabetes was associated with higher GCF levels of IL-8 and MMP-8. CONCLUSION: Diabetes may affect GCF levels of cytokines, irrespective of periodontal status. Periodontal status may be associated with Aggrecan levels in the GCF of patients affected by metabolic syndrome. CLINICAL SIGNIFICANCE: Investigation of GCF biomarkers may potentially help have diagnostic potential in patients with MetS.

Interdental and subgingival microbiota may affect the tongue microbial ecology and oral malodour in health, gingivitis and periodontitis.

BACKGROUND AND OBJECTIVE: Oral malodour is often observed in gingivitis and chronic periodontitis patients, and the tongue microbiota is thought to play a major role in malodorous gas production, including volatile sulphur compounds (VSCs) such as hydrogen sulphide (H2 S) and methanethiol (CH3 SH). This study aimed to examine the link between the presence of VSCs in mouth air (as a marker of oral malodour) and the oral bacterial ecology in the tongue and periodontal niches of healthy, gingivitis and periodontitis patients. METHODS: Participants were clinically assessed using plaque index, bleeding on probing (BOP) and periodontal probing depths, and VSC concentrations in their oral cavity measured using a portable gas chromatograph. Tongue scrapings, subgingival and interdental plaque were collected from healthy individuals (n = 22), and those with gingivitis (n = 14) or chronic periodontitis (n = 15). The bacterial 16S rRNA gene region V3-V4 in these samples was sequenced, and the sequences were analysed using the minimum entropy decomposition pipeline. RESULTS: Elevated VSC concentrations and CH3 SH:H2 S were observed in periodontitis compared with health. Significant ecological differences were observed in the tongue microbiota of healthy subjects with high plaque scores compared to low plaque scores, suggesting a possible connection between the microbiota of the tongue and the periodontium and that key dysbiotic changes may be initiated in the clinically healthy individuals who have higher dental plaque accumulation. Greater subgingival bacterial diversity was positively associated with H2 S in mouth air. Periodontopathic bacteria known to be prolific VSC producers increased in abundance on the tongue associated with increased bleeding on probing (BOP) and total percentage of periodontal pockets >6 mm, supporting the suggestion that the tongue may become a reservoir for periodontopathogens. CONCLUSION: This study highlights the importance of the periodontal microbiota in malodour and has detected dysbiotic changes in the tongue microbiota in periodontitis.

The Extracellular Metabolome Stratifies Low and High Risk Potentially Premalignant Oral Keratinocytes and Identifies Citrate as a Potential Non-Invasive Marker of Tumour Progression.

Premalignant oral lesions (PPOLs) which bypass senescence (IPPOL) have a much greater probability of progressing to malignancy, but pre-cancerous fields also contain mortal PPOL keratinocytes (MPPOL) that possess tumour-promoting properties. To identify metabolites that could potentially separate IPPOL, MPPOL and normal oral keratinocytes non-invasively in vivo, we conducted an unbiased screen of their conditioned medium. MPPOL keratinocytes showed elevated levels of branch-chain amino acid, lipid, prostaglandin, and glutathione metabolites, some of which could potentially be converted into volatile compounds by oral bacteria and detected in breath analysis. Extracellular metabolites were generally depleted in IPPOL, and only six were elevated, but some metabolites distinguishing IPPOL from MPPOL have been associated with progression to oral squamous cell carcinoma (OSCC) in vivo. One of the metabolites elevated in IPPOL relative to the other groups, citrate, was confirmed by targeted metabolomics and, interestingly, has been implicated in cancer growth and metastasis. Although our investigation is preliminary, some of the metabolites described here are detectable in the saliva of oral cancer patients, albeit at a more advanced stage, and could eventually help detect oral cancer development earlier.

Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis.

Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine.

Activity of a nitric oxide-generating wound treatment system against wound pathogen biofilms.

Wound bioburden plays an important role in impaired healing and development of infection-related complications. The objective of this study was to determine the efficacy of an innovative two-layer nitric oxide-generating system (NOx) to prevent and treat biofilms formed by bacterial and fungal pathogens commonly associated with wound infection, and activity against Pseudomonas aeruginosa virulence factors. Single- and mixed-species biofilms were grown for 24 h on nitrocellulose filters placed on agar. Filters were covered with either NOx or placebo, before and after biofilm formation. Populations of bacteria and yeasts were determined using viable counts. Pyocyanin and elastase production from P. aeruginosa were determined in supernatants derived from suspended biofilms. Efficacy of NOx was demonstrated against Staphylococcus aureus, P. aeruginosa, Acinetobacter baumannii, Escherichia coli and Candida spp. Population reductions between 2- and 10-log fold were observed. Pyocyanin and elastase activities from P. aeruginosa were reduced 1.9- and 3.2-fold, respectively. This study demonstrated activity of NOx against formation and treatment of single- and mixed-species biofilms, including multidrug-resistant strains. NOx represents a new generation of antimicrobial agent with potent, broad-spectrum activity, and with no evidence of resistance development.

Use of Probiotics and Oral Health.

PURPOSE OF REVIEW: The purpose of this study is to critically assess recent studies concerning the use of probiotics to control periodontal diseases, dental caries and halitosis (oral malodour). RECENT FINDINGS: Clinical studies have shown that probiotics when allied to conventional periodontal treatment can ameliorate microbial dysbiosis and produce significant improvement in clinical indicators of disease. However, this effect is often not maintained by the host after the end of probiotic use. Current probiotics also show limited effects in treating caries and halitosis. Novel approaches based up on replacement therapy and using highly abundant health-associated oral species, including nitrate-reducing bacteria, have been proposed to improve persistence of probiotic strains and maintain oral health benefits. SUMMARY: Probiotics have potential in the management of multifactorial diseases such as the periodontal diseases and caries, by more effectively addressing the host-microbial interface to restore homeostasis that may not be achieved with conventional treatments.

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

In vitro growth characteristics and volatile sulfur compound production of Solobacterium moorei.

Solobacterium moorei has recently been implicated as a causative agent of halitosis. In vitro experiments to evaluate the role of S. moorei in halitosis have, however, been complicated by a paucity of information on the ideal conditions for culturing this organism. This work aimed to optimize a liquid culture medium for S. moorei, and to determine the growth-curve of the organism. Further, the ability of S. moorei to generate volatile sulfur compounds was investigated and compared quantitatively to other oral anaerobes by an optimized head-space gas chromatography method. Serum-supplementation of standard liquid growth media gave greater growth of S. moorei than non-supplemented broths, with the best medium found to be serum-supplemented tryptone soya broth. S. moorei was able to metabolize cysteine directly to hydrogen sulfide, but was unable to produce methanethiol from methionine. S. moorei produced 2-3 times more hydrogen sulfide (normalized for colony forming units) than Porphyromonas gingivalis and Veillonella dispar, but considerably less than Fusobacterium nucleatum. The study has identified reliable growth conditions for culture of S. moorei, which were employed to show that S. moorei has the requisite biochemistry consistent with a potential role in halitosis.

In vitro effects of novel toothpaste actives on components of oral malodour.

OBJECTIVES: To evaluate the efficacy of a novel toothpaste containing zinc ions and o-cymen-5-ol to reduce volatile sulfur compounds (VSCs) in in vitro models and to elucidate the mode of action for any activity observed. METHODS: Three models were employed, a chemical neutralisation model to evaluate the chemical reactivity of toothpaste slurries to VSCs, a biofilm perfusion model to measure activity in an orally-relevant biofilm and a planktonic bacterial model to measure antimicrobial effects. RESULTS: The models showed that zinc ions were able to react chemically with hydrogen sulfide to remove this odorous component of halitotic breath. This activity was confirmed within a complex biofilm model, with over 90% of hydrogen sulfide removed from perfusate gas by a slurry of the test toothpaste. CONCLUSIONS: This work provides a mode of action for the clinically observed reduction in VSCs seen for up to 12 hours post brushing with this novel toothpaste.