Identification of anti-biofilm components in Withania somnifera and their effect on virulence of Streptococcus mutans biofilms.

AIMS: The aim of this study was to identify components of the Withania somnifera that could show anti-virulence activity against Streptococcus mutans biofilms. METHODS AND RESULTS: The anti-acidogenic activity of fractions separated from W. somnifera was compared, and then the most active anti-acidogenic fraction was chemically characterized using gas chromatography-mass spectroscopy. The effect of the identified components on the acidogenicity, aciduricity and extracellular polymeric substances (EPS) formation of S. mutans UA159 biofilms was evaluated. The change in accumulation and acidogenicity of S. mutans UA159 biofilms by periodic treatments (10 min per treatment) with the identified components was also investigated. Of the fractions, n-hexane fraction showed the strongest anti-acidogenic activity and was mainly composed of palmitic, linoleic and oleic acids. Of the identified components, linoleic and oleic acids strongly affected the acid production rate, F-ATPase activity and EPS formation of the biofilms. Periodic treatment with linoleic and oleic acids during biofilm formation also inhibited the biofilm accumulation and acid production rate of the biofilms without killing the biofilm bacteria. CONCLUSIONS: These results suggest that linoleic and oleic acids may be effective agents for restraining virulence of S. mutans biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Linoleic and oleic acids may be promising agents for controlling virulence of cariogenic biofilms and subsequent dental caries formation.

Effect of brief cetylpyridinium chloride treatments during early and mature cariogenic biofilm formation.

OBJECTIVES: The aim of this study was to investigate the antibiofilm activity of brief cetylpyridinium chloride (CPC) treatments during early and mature Streptococcus mutans biofilm formation. METHODS: Streptococcus mutans biofilms were formed on saliva-coated hydroxyapatite disks. The biofilms were treated with CPC twice daily (1 min/treatment) from 0 to 50 h or from 48 to 98 h. Acidogenicity, dry weight, viability, and water-insoluble extracellular polysaccharides of the biofilms were analyzed. Confocal laser scanning microscopy (CLSM) images were obtained to confirm the antibiofilm activity during mature biofilm formation and to evaluate the relationship between treatment time and the antibiofilm activity. RESULTS: CPC showed complete antibiofilm activity during early biofilm formation at 0.025% to 0.1%. During mature biofilm formation, CPC inhibited dry weight, viability, and acidogenicity at 0.075% and 0.1%. CLSM images showed an increase in dead cells at 0.075% and 0.1% CPC. The antibiofilm activity during mature biofilm formation increased as the concentration of CPC increased. Images from the CLSM study also showed that antibiofilm activity increased as treatment time increased. CONCLUSION: Our findings suggest that brief CPC treatments have strong anti-S. mutans biofilm activity. The antibiofilm activity was dependent on the stage of biofilm formation, CPC concentration, and treatment time.

Relationships between the antibacterial activity of sodium hypochlorite and treatment time and biofilm age in early Enterococcus faecalis biofilms.

AIM: To determine the relationships between the antibacterial activity of NaOCl and treatment time and biofilm age in early Enterococcus faecalis biofilms using a linear fitting procedure. METHODOLOGY: Enterococcus faecalis biofilms were formed on hydroxyapatite discs. To investigate the relationship between the antibacterial activity of NaOCl and biofilm age, 22-, 46-, 70- and 94-h-old biofilms were exposed to NaOCl (0-3%) for 5 min. To investigate the relationship between the antibacterial activity of NaOCl and treatment time, 70-h-old biofilms were exposed to NaOCl (0-3%) for 1, 3, 5 and 7 min. After treatment, colony-forming units (CFUs) were counted. To determine the relationships between these variables, linear fitting was performed. RESULTS: The change in the minimum biofilm eradication concentration (MBEC) of NaOCl followed a linear pattern of biofilm age (R = 0.941, R(2)  = 0.886) or treatment time dependence (R = -0.948, R(2)  = 0.898). Below the MBEC, the fitting lines for bacterial CFU count versus NaOCl concentration (R ≤ -0.973, R(2)  ≥ 0.948) in the 22-, 46-, 70- and 94-h-old biofilms implied that the antibacterial activity of NaOCl decreased as the biofilm age increased. The fitting lines for bacterial CFU count versus NaOCl concentration (R ≤ -0.970, R(2)  ≥ 0.942) in the 1-, 3-, 5- and 7-min treatments implied that the antibacterial activity of NaOCl increased with treatment time. CONCLUSIONS: These results suggest that the antibacterial activity of NaOCl against early E. faecalis biofilms in root canals may follow a linear pattern depending on biofilm age or treatment time.

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 (R(2) = 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.

Apoptotic effect of Polygonum Cuspidatum in oral cancer cells through the regulation of specificity protein 1.

OBJECTIVES: The aim of this study was to evaluate the growth inhibitory and apoptosis-inducing effects and mechanisms of Polygonum cuspidatum root in oral cancer cells. MATERIALS AND METHODS: The testing materials were separated by normal-phase silica gel liquid chromatography. The effect of P. cuspidatum root on apoptotsis and its mechanism were performed using 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (MTS) assay, western blot analysis, RT-PCR, promoter assay, and (4'-6-Diamidino-2-phenylindole) (DAPI) staining. RESULTS: The methanol extract of P. cuspidatum (MEPC) inhibited the proliferation of oral cancer cells by inducing caspase-dependent apoptosis. Protein and mRNA expression levels and the transactivation of Specificity protein 1 (Sp1) were markedly decreased in KB cells treated with MEPC. Ethyl acetate fraction (EA) from MEPC was more potent than aqueous fraction (AQ) from MEPC to induce apoptosis. F2, F3, and F4 from EA differentially inhibited the growth of KB cells, and it depends on the amount of Emodin in F2, F3, and F4. Moreover, Emodin inhibited oral cancer cell growth and induced caspase-dependent apoptosis by decreasing Sp1. MEPC also decreased an apoptosis-related downstream target of Sp1 protein, survivin. CONCLUSION: The results from this study strongly suggest that MEPC, its fraction, and Emodin may be potential bioactive materials to cause apoptosis mechanism via the down-regulation of Sp1 in oral cancer cells.

Natural products in caries research: current (limited) knowledge, challenges and future perspective.

Dental caries is the most prevalent and costly oral infectious disease worldwide. Virulent biofilms firmly attached to tooth surfaces are prime biological factors associated with this disease. The formation of an exopolysaccharide-rich biofilm matrix, acidification of the milieu and persistent low pH at the tooth-biofilm interface are major controlling virulence factors that modulate dental caries pathogenesis. Each one offers a selective therapeutic target for prevention. Although fluoride, delivered in various modalities, remains the mainstay for the prevention of caries, additional approaches are required to enhance its effectiveness. Available antiplaque approaches are based on the use of broad-spectrum microbicidal agents, e.g. chlorhexidine. Natural products offer a rich source of structurally diverse substances with a wide range of biological activities, which could be useful for the development of alternative or adjunctive anticaries therapies. However, it is a challenging approach owing to complex chemistry and isolation procedures to derive active compounds from natural products. Furthermore, most of the studies have been focused on the general inhibitory effects on glucan synthesis as well as on bacterial metabolism and growth, often employing methods that do not address the pathophysiological aspects of the disease (e.g. bacteria in biofilms) and the length of exposure/retention in the mouth. Thus, the true value of natural products in caries prevention and/or their exact mechanisms of action remain largely unknown. Nevertheless, natural substances potentially active against virulent properties of cariogenic organisms have been identified. This review focuses on gaps in the current knowledge and presents a model for investigating the use of natural products in anticaries chemotherapy.

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.

Bioactivity-guided separation of anti-acidogenic substances against Streptococcus mutans UA 159 from Polygonum cuspidatum.

OBJECTIVES: The aim of this study was to separate the anti-acidogenic substances against Streptococcus mutans UA 159 from Polygonum cuspidatum. MATERIAL AND METHODS: The anti-acidogenic substances were separated by a series of liquid-liquid fractionations followed by normal-phase silica gel liquid chromatography, based on high-performance liquid chromatography and glycolytic pH-drop assay. The effectiveness of the separated substances on the acidogenicity of Streptococcus mutans UA 159 was examined using sodium fluoride as a positive control. The chemical composition and quantities of the components of the substances was also assessed by qualitative-quantitative chromatographic analysis. RESULTS: Among the substances separated from P. cuspidatum, F3 showed the strongest inhibitory effect on the acidogenicity of S. mutans UA 159 in a dose-dependent manner without displaying any bactericidal activity. F3 decreased the acidogenicity of S. mutans even at 12.5 microg ml(-1) (P < 0.05). F3 consisted mainly of resveratrol and emodin (C(14)H(12)O(3) and C(14)H(4)O(2)(OH)(3)CH(3), respectively), which made up approximately 60% of the weight of F3. CONCLUSION: F3 can be considered as a promising agent for controlling the acidogenicity of S. mutans and subsequent dental caries formation.

Public Sense of Water Fluoridation as Reflected on Twitter 2009-2017.

Though controversial, water fluoridation has been hailed as one of the top-ten public-health achievements of the 20th century in the United States of America. In this article, we aim to investigate the public sense of water fluoridation as reflected on Twitter, using data from 2009 to 2017. To this end, tweets related to water fluoridation were collected using queries such as "fluoridated water or fluoride water," "water fluoridation or fluoridation of water," and hashtags related to water fluoridation. The collected tweets (n = 218,748) were examined through informetric, linguistic (word sentiment, word frequency, and word network analyses), and issue tweet analyses. We found that Twitter users who tweeted about water fluoridation in English between 2009 and 2017 constituted about <0.01% of all users including non-English users. In their tweets, words such as "poison" and "waste" were the strong negative sentiment words most often used. Of the top 30 words most frequently used, words related to information sources on water fluoridation and the safety of water fluoridation appeared more often than words related to its efficacy. Additionally, the words related to information sources on water fluoridation and the safety of water fluoridation were found to be core terms in the sentences of tweet mentions. Our linguistic analyses indicate that Twitter users responded sensitively to words that emphasize negative aspects of fluoridation. This is clearly shown in our issue tweet analysis, where tweet mentions expressing negative opinions about water fluoridation accounted for at least 59.2% of all mentions. By contrast, <15% of tweet mentions were found to be positive. These findings suggest that professionals need to reevaluate the current state of online information about water fluoridation, and improve it in a way so that the public can easily access reliable information sources.

Chronologic Trends in Studies on Fluoride Mechanisms of Action.

Fluoride has been widely used for the prevention of dental caries since the mid-20th century. The aim of this study was to investigate the chronologic trends in studies on fluoride mechanisms of action against dental caries during the years 1950 to 2015. To this aim, queries such as "fluoride," "fluoride and demineralization," "fluoride and remineralization," "fluoride and (plaque or biofilms)," and "fluoride and (bacteria or microbials)" were submitted to PubMed to collect research article information, including titles, abstracts, publication dates, author affiliations, and publication journals. The article information that PubMed produced was then collected by an automatic web crawler and examined through informetrics and linguistic analyses. We found that the number of articles concerned with fluoride mechanisms of action against dental caries was 6,903 and gradually increased over time during the years 1950 to 2015. They were published by 1,136 journals-most notably, Caries Research and Journal of Dental Research. Of the articles published, those related to bacteria/microbials had a higher percentage (44%) than those dealing with plaque/biofilms, demineralization, and remineralization. With regard to the geographic distribution of authors, Europe and North America accounted for 65% of the articles during the years 1987 to 2015, although the number of authors in Asia sharply increased in recent years. Among the fluoride compounds, NaF was mentioned more frequently than SnF2, Na2PO3F, amine fluoride, and acidulated phosphate fluoride during the years 1986 to 2015. Water fluoridation received the most attention among the various fluoride application methods (toothpastes, mouthwashes, fluoride varnishes, and fluoride gels) during the same period. These results, obtained from employing informetrics and linguistic analyses, suggest that in studies on fluoride mechanisms of action, 1) the unbalanced geographic distribution of articles and 2) the heavy concentration of articles on particular fluoride compounds and application methods should be overcome in future research.

C-myb Regulates Autophagy for Pulp Vitality in Glucose Oxidative Stress.

Diabetes mellitus is closely related to oral-complicated diseases by oxidative stress. This study investigates whether cellular myeloblastosis (c-myb) could protect human dental pulp cells against glucose oxidative stress and regulate autophagy activity for pulp vitality. Diabetes mellitus was induced by streptozotocin in Sprague-Dawley rats, and their pulp tissue in teeth was analyzed in terms of pulp cavity and molecules by hematoxylin and eosin and immunohistochemistry staining. Human dental pulp cells were serially subcultured and treated with glucose oxidase in the presence of elevated glucose to generate glucose oxidative stress. The replication-deficient adenovirus c-myb and small interfering RNA c-myb were introduced for c-myb expression. The pulp tissue from the diabetic rats was structurally different from normal tissue in terms of narrow pulp capacity, reduced c-myb, and dentinogenesis molecules. Glucose oxidase treatment decreased c-myb and dentinogenesis molecules (bone morphogenetic protein 2 and 7, dentin matrix protein 1, and dentin sialophosphoprotein) in human dental pulp cells. However, overexpression of c-myb by adenovirus c-myb increased dentinogenesis, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B-light chain 3, and Beclin-1), and cell survival via p-AMPK/AKT signaling even with glucose oxidative stress. In contrast, the lack of c-myb decreased the above molecules and cell survival by downregulating p-AMPK/AKT signaling. The results indicate that diabetes leads to irreversible damage to dental pulp, which is related to downexpression of autophagy via the p-AMPK/AKT pathway by decline of c-myb. The findings of this study provide a new insight that c-myb could ameliorate autophagy activity and that it is applicable for monitoring complicated diseases of dental pulp. The involvement of c-myb in pulp pathology could serve a therapeutic target in oral-complicated diseases.