Jae-Gyu Jeon

Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

BackgroundThe association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.ResultsTwice-daily treatment (one-minute exposure) during biofilm formation affected the gene expression by S. mutans both at early (49-h) and later (97-h) stages of biofilm development. Biofilms treated with combination of agents displayed lower mRNA levels for gtfB and gtfD (associated with exopolysaccharides synthesis) and aguD (associated with S. mutans acid tolerance) than those treated with vehicle-control (p < 0.05). Furthermore, treatment with combination of agents markedly affected the structure-architecture of S. mutans biofilms by reducing the biovolume (biomass) and proportions of both EPS and bacterial cells across the biofilm depth, especially in the middle and outer layers (vs. vehicle-control, p < 0.05). The biofilms treated with combination of agents were also less acidogenic, and had reduced amounts of extracellular insoluble glucans and intracellular polysaccharides than vehicle-treated biofilms (p < 0.05).ConclusionThe data show that the combination of naturally-occurring agents with fluoride effectively disrupted the expression of specific virulence genes, structural organization and accumulation of S. mutans biofilms, which may explain the enhanced cariostatic effect of our chemotherapeutic approach.

Influences of trans‐trans farnesol, a membrane‐targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed‐species oral biofilms

Trans‐trans farnesol (tt‐farnesol) is a bioactive sesquiterpene alcohol commonly found in propolis (a beehive product) and citrus fruits, which disrupts the ability of Streptococcus mutans (S. mutans) to form virulent biofilms. In this study, we investigated whether tt‐farnesol affects cell‐membrane function, acid production and/or acid tolerance by planktonic cells and biofilms of S. mutans UA159. Furthermore, the influence of the agent on S. mutans gene expression and ability to form biofilms in the presence of other oral bacteria (Streptococcus oralis (S. oralis) 35037 and Actinomyces naeslundii (A. naeslundii) 12104) was also examined. In general, tt‐farnesol (1 mmol‐L−1) significantly increased the membrane proton permeability and reduced glycolytic activity of S. mutans in the planktonic state and in biofilms (P<0.05). Moreover, topical applications of 1 mmol‐L−1 tt‐farnesol twice daily (1 min exposure/treatment) reduced biomass accumulation and prevented ecological shifts towards S. mutans dominance within mixed‐species biofilms after introduction of 1% sucrose. S. oralis (a non‐cariogenic organism) became the major species after treatments with tt‐farnesol, whereas vehicle‐treated biofilms contained mostly S. mutans (>90% of total bacterial population). However, the agent did not affect significantly the expression of S. mutans genes involved in acidogenicity, acid tolerance or polysaccharide synthesis in the treated biofilms. Our data indicate that tt‐farnesol may affect the competitiveness of S. mutans in a mixed‐species environment by primarily disrupting the membrane function and physiology of this bacterium. This naturally occurring terpenoid could be a potentially useful adjunctive agent to the current anti‐biofilm/anti‐caries chemotherapeutic strategies.

Relationship between Fluoride Concentration and Activity against Virulence Factors and Viability of a Cariogenic Biofilm: in vitro Study

Despite widespread use of various concentrations of fluoride for the prevention of dental caries, the relationship between fluoride concentration and activity against cariogenic biofilms has not been much studied. Herein we investigated the relationship between fluoride concentration and activity against virulence factors and viability of Streptococcus mutans biofilms. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs. The 70-hour-old biofilms were exposed to 0, 1, 3, 10, 30, 100, 300, 1,000 or 2,000 ppm F-. The changes of virulence factors and viability of the biofilms were analyzed using biochemical methods and laser scanning confocal fluorescence microscopy. At 1-2,000 ppm F-, the activity of fluoride against acid production, acid tolerance, and extracellular polysaccharide formation of S. mutans biofilms accurately followed a sigmoidal pattern of concentration dependence (R2 = 0.94-0.99), with EC50 values ranging from 3.07 to 24.7 ppm F-. Generally, the activity of fluoride against the virulence factors was concentration-dependently augmented in 10-100 ppm F- and did not increase further at concentrations higher than 100 ppm F-. However, fluoride did not alter glucosyltransferase activity and viability of S. mutans biofilm cells in all concentrations tested. These results can provide a basis for the selection of appropriate fluoride concentrations that reduce the physiological ability of cariogenic biofilms.

Influences of Dryopteris crassirhizoma Extract on the Viability, Growth and Virulence Properties of Streptococcus mutans

Dryopteris crassirhizoma is traditionally used as an herbal remedy for various diseases, and has been identified in a previous study as a potential anti-caries agent. In this study, the effect of a methanol extract of D. crassirhizoma on the viability, growth and virulence properties of Streptococcus mutans, a cariogenic dental pathogen, was investigated. In addition, the phytochemical composition of the extract was analyzed. The extract showed bactericidal and bacteriostatic activity against oral bacteria (MIC and MBC of S. mutans: 62.5 and 250 μg/mL, respectively). At two times the MBC, the extract significantly eliminated S. mutans up to 99.9% after 1 h incubation. The extract also dose-dependently reduced growth rates of S. mutans at sub-MIC levels. Furthermore, at sub-MIC levels, virulence properties (acid production, acid tolerance, glucosyltransferase activity and sucrose-dependent adherence) of S. mutans were also inhibited in a dose-dependent manner. GC-MS analysis revealed the presence of mono and disaccharides (44.9%), fatty acids (12.3%) and sugar alcohols (6.8%) in the extract. These data indicate that the extract might be useful for the control of dental caries.

Effect of 1-Minute Fluoride Treatment on Potential Virulence and Viability of a Cariogenic Biofilm.

Fluoride is a well-studied and widely used agent for the prevention of dental caries. Although dental caries is strongly related to cariogenic biofilms, the effect of brief fluoride treatment on the virulence properties of biofilms has not been well studied. This study evaluated the effect of a 1-min fluoride treatment on the virulence properties and viability of cariogenic biofilms, using a Streptococcus mutans biofilm model. For this study, 46-hour-old S. mutans biofilms were formed on saliva-coated hydroxyapatite discs and were treated with fluoride (0, 30, 300, 1,000, and 2,000 ppm F-) for 1 min. Viability and changes in acidogenicity, aciduricity and extracellular polysaccharide (EPS) formation of the biofilms were analyzed using biochemical and microbiological methods (pH drop, H+ permeability, acid killing, and bacterial colony-forming unit assays). Laser scanning confocal fluorescence microscopy study was also performed. After the 1-min fluoride treatment, acidogenicity, aciduricity, and EPS formation of 46-hour-old S. mutans biofilms were significantly reduced when treated with concentrations ≥300 ppm F-. The antivirulence activities of the 1-min fluoride treatment increased in a concentration-dependent pattern. However, the 1-min fluoride treatments did not affect viability, biovolume, and microcolony appearance of biofilm bacteria, even at high concentrations. These results suggest that the brief treatment with fluoride at concentrations ≥300 ppm F- is an effective measure for controlling cariogenic biofilms.

Recovery of Acid Production in Streptococcus mutans Biofilms after Short-Term Fluoride Treatment

Fluoride is commonly used as an ingredient of topical oral hygiene measures. Despite the anti-acidogenic activities of fluoride against cariogenic biofilms, the recovery of the biofilms from fluoride damage is unclear. Herein, we investigated the recovery of acid production in Streptococcus mutans biofilms after short-term or during periodic 1-min fluoride treatments. For this study, 46-hour-old S. mutans biofilms were treated with fluoride (0-2,000 ppm F-) for 1-8 min and then incubated in saliva for 0-100 min. The 74-hour-old biofilms were also periodically treated with the fluoride concentration during biofilm formation (1 min/treatment). Changes in acidogenicity and viability were determined via pH drop and colony-forming unit assays, respectively. In this study, acid production after a 1-min fluoride treatment was recovered as saliva incubation time increased, which followed a linear pattern of concentration dependence (R = 0.99, R2 = 0.98). The recovery pattern was in a biphasic pattern, with an initial rapid rate followed by a second slow recovery. Furthermore, recovery from fluoride damage was retarded in a concentration-dependent manner as treatment time increased. In periodic 1-min fluoride treatments, acid production in the biofilms was not diminished during the non-fluoride treatment period; however, it was reduced in a concentration-dependent manner during the fluoride treatment period. The viability of the biofilm cells did not change, even at high fluoride concentrations. Collectively, our results suggest that brief fluoride treatment does not sustain anti-acidogenic activity against S. mutans in biofilms since the damage is recoverable with time.

Withania somnifera Attenuates Acid Production, Acid Tolerance and Extra-Cellular Polysaccharide Formation of Streptococcus mutans Biofilms

Withania somnifera (Ashwagandha) is a plant of the Solanaceae family. It has been widely used as a remedy for a variety of ailments in India and Nepal. The plant has also been used as a controlling agent for dental diseases. The aim of the present study was to evaluate the activity of the methanol extract of W. somnifera against the physiological ability of cariogenic biofilms and to identify the components of the extract. To determine the activity of the extract, assays for sucrose-dependent bacterial adherence, glycolytic acid production, acid tolerance, and extracellular polysaccharide formation were performed using Streptococcus mutans biofilms. The viability change of S. mutans biofilms cells was also determined. A phytochemical analysis of the extract was performed using TLC and LC/MS/MS. The extract showed inhibitory effects on sucrose-dependent bacterial adherence (≥ 100 μg/ml), glycolytic acid production (≥ 300 μg/ml), acid tolerance (≥ 300 μg/ml), and extracellular polysaccharide formation (≥ 300 μg/ml) of S. mutans biofilms. However, the extract did not alter the viability of S. mutans biofilms cells in all concentrations tested. Based on the phytochemical analysis, the activity of the extract may be related to the presence of alkaloids, anthrones, coumarines, anthraquinones, terpenoids, flavonoids, and steroid lactones (withanolide A, withaferin A, withanolide B, withanoside IV, and 12-deoxy withastramonolide). These data indicate that W. somnifera may be a potential agent for restraining the physiological ability of cariogenic biofilms.

Trend Analysis of Studies on Water Fluoridation Related to Dental Caries in PubMed

Water fluoridation has been cited as one of the top 10 public health achievements of the 20th century. Herein, we analyzed water fluoridation articles related to dental caries published in PubMed between 1950 and 2016 using informetrics and linguistic methods to investigate trends in the studies. To this aim, queries such as “dental caries and (water fluoridation or fluoridated water),” “dental caries and (fluoride or fluoridation),” and “dental caries” were submitted to PubMed to retrieve information about articles on water fluoridation within the area of dental caries and fluoride – their titles, abstracts, publication dates, author affiliations, and publication journals. This article information was then collected by an automatic web crawler and examined through informetrics and linguistic analyses. It was found that the number of articles concerned with water fluoridation and dental caries was 3,381 and declined over time after 1970. The articles were published by 750 journals – most notably, Community Dentistry and Oral Epidemiology and Caries Research. With regard to the geographic distribution of the authors, Europe and North America, especially the USA and UK, accounted for 59.9% of the articles published during the years 1987 to 2015, though there was a sharp increase in the number of authors in Oceania and Asia in recent years. In the titles and abstracts of the articles, “community” and “fluorosis” were mentioned more frequently than the other key terms selected in this study, regardless of the period examined. Our findings may allow one to assess how the research on water fluoridation has evolved over the past several decades.

The Yngve Ericsson Prize in Preventive Odontology

Patent Revenue Fund and ORCA herby solicit nominations for the 2016 Yngve Ericsson Prize. The award will be SEK 300,000 (approx. EUR 32,000) for one person and SEK 200,000 per person if shared by two recipients. The Prize winner(s) will be selected by a Prize Committee of distinguished scientists, three members of which are appointed by the Patent Revenue Fund for Preventive Odontology and two members appointed by ORCA. The Prize is awarded to persons who have performed outstanding laboratory or clinical research that has contributed specifically to the prevention of dental and oral disease. Candidates are judged on the originality, quality and range of their scientific contributions as well as the range and clinical importance of the results. Individuals who are still active in research are preferred candidates. No preference will be given to candidates from any country. A nomination should contain the name of the candidate and must be accompanied by a statement giving reasons why the candidate would be a worthy awardee, including a list of the candidate’s relevant scientific publications and, if possible, a short CV. Nominations of candidates must be received by the Secretary of the Patent Revenue Fund, Prof. Peter Lingström (Institute of Odontology, Box 450, SE–405 30 Göteborg, Sweden; E-Mail peter.lingström @ odontologi. gu.se), no later than February 15, 2016. Call for Nominations