Dental Caries Induction in Experimental Animals by Clinical Strains of Streptococcus mutans Isolated from Japanese Children

Oral implantation and the cariogenic activity of clinical strains of Streptococcus mutans which had been isolated from Japanese children and labeled with streptomycin-resistance were examined in specific pathogen-free Sprague-Dawley rats. All the seven strains tested were easily implanted and persisted during the experimental period. Extensive carious lesions were produced in rats inoculated with clinical strains of S. mutans belonging to serotypes c, d, e, and f, and maintained on caries-inducing diet #2000. Noninfected rats did not develop dental caries when fed diet #2000. Type d S. mutans preferentially induced smooth surface caries in the rats. Strains of other serotypes primarily developed caries of pit and fissure origin. Caries also developed in rats inoculated with reference S. mutans strains BH-TR and FAIR (type b) that had been maintained in the laboratories for many years. However, the cariogenicity of the laboratory strains was found to have decreased markedly. All three S. sanguis strains could be implanted, but only one strain induced definite fissure caries. Two S. salivarius strains could not be implanted well in the rats and therefore they were not cariogenic. Four different species of lactobacilli also failed to induce dental caries in rats subjected to similar caries test regimen on diet #2000. S. mutans strain MT6R (type c) also induce caries in golden hamsters and ICR mice, but of variable degrees.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

Inhibition of Rat Dental Caries by Dextranase from a Strain of Spicaria violacea

Dextranase AD17 obtained from a culture liquor of a strain of Spicaria violacea was assessed for its ability to inhibit the development of dental caries in conventional Sprague-Dawley rats which had been infected with one of the Streptococcus mutans strains, MT6R (serotype c), OMZ 176R (d), or MT-703R (e). These experiments showed that caries was significantly inhibited when rats were given cariogenic diet # 2000 and drinking water containing AD17 at a concentration of 10 units/g, as compared to control rats not given dextranase. The inhibitory effects of AD17 were more prominent in smooth surface caries than in total caries. AD17 had a tendency to retard both the establishment of inoculated S. mutans and plaque deposition on tooth surfaces. However, S. mutans could be implanted in the rat oral cavity after repeated inoculation of the bacteria, even in the presence of AD17. These results suggest that the anticaries activity of AD17 is due to not only inhibition of adherence of S. mutans cells on tooth surfaces but also to physicochemical changes of dental plaque formed under the enzymatic action of AD17. Preliminary histopathological examination showed that AD17 had no significant toxicity in rats.     

Sulfated vizantin inhibits biofilm maturation by Streptococcus mutans

Streptococcus mutans is the main pathogen of dental caries and adheres to the tooth surface via soluble and insoluble glucans produced by the bacterial glucosyltransferase enzyme. Thus, the S. mutans glucosyltransferase is an important virulence factor for this cariogenic bacterium. Sulfated vizantin effectively inhibits biofilm formation by S. mutans without affecting its growth. In this study, less S. mutans biofilm formation occurred on hydroxyapatite discs coated with sulfated vizantin than on noncoated discs. Sulfated vizantin showed no cytotoxicity against the human gingival cell line Ca9-22. Sulfated vizantin dose-dependently inhibited the extracellular release of cell-free glucosyltransferase from S. mutans and enhanced the accumulation of cell-associated glucosyltransferase, compared with that observed with untreated bacteria. Sulfated vizantin disrupted the localization balance between cell-associated glucosyltransferase and cell-free glucosyltransferase, resulting in inhibited biofilm maturation. These results indicate that sulfated vizantin can potentially serve as a novel agent for preventing dental caries.     

Clinically acceptable restorations may be a hotbed for cariogenic microbes

doi: 10.1111/j.1741‐2358.2011.00571.x Clinically acceptable restorations may be a hotbed for cariogenic microbes Objective: The aim of this study was to investigate the cross‐sectional association of dental restorations with salivary cariogenic pathogens among the elderly to establish effective parameters of caries risk for this population. Materials and methods: Stimulated whole saliva was collected from 289 community‐dwelling older adults (66.2 ± 3.9 years old) who had 20 or more teeth. Salivary levels of three cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus and lactobacilli) were estimated using quantitative polymerase chain reaction (real‐time PCR) method. Results: The mean number of residual teeth was 26.4, and restored teeth with crowns, inlays and composite resin were 7.35, 3.88 and 0.68, respectively. The number of crowns correlated positively with salivary S. mutans, S. sobrinus and lactobacilli. Multiple linear regression analyses showed that the number of restored teeth with crowns was independently associated with salivary S. mutans, S. sobrinus and lactobacilli after controlling for age, gender, number of residual teeth and salivary flow rate. Salivary flow rate was independently associated with salivary S. mutans and lactobacilli. Conclusion: The number of crowns had an association with salivary levels of cariogenic bacteria, suggesting that this parameter may be a caries risk indicator for the elderly population.     

A graphene/zinc oxide nanocomposite film protects dental implant surfaces against cariogenic Streptococcus mutans

Oral biofilms play a crucial role in the development of dental caries and other periodontal diseases. Streptococcus mutans is one of the primary etiological agents in dental caries. Implant systems are regularly employed to replace missing teeth. Oral biofilms accumulate on these implants and are the chief cause of dental implant failure. In the present study, the potential of graphene/zinc oxide nanocomposite (GZNC) against the cariogenic properties of Streptococcus mutans was explored and the anti-biofilm behaviour of artificial acrylic teeth surfaces coated with GZNC was examined. Acrylic teeth are a good choice for implants as they are low cost, have low density and can resist fracture. Microscopic studies and anti-biofilm assays showed a significant reduction in biofilm in the presence GZNC. GZNC was also found to be nontoxic against HEK-293 (human embryonic kidney cell line). The results indicate the potential of GZNC as an effective coating agent for dental implants by efficiently inhibiting S. mutans biofilms.     

Effect of neovestitol–vestitol containing Brazilian red propolis on accumulation of biofilm in vitro and development of dental caries in vivo

The present study examined the influences of the neovestitol–vestitol (NV) containing fraction isolated from Brazilian red propolis on the development of biofilm and expression of virulence factors by Streptococcus mutans using saliva-coated surfaces of hydroxyapatite. In addition, NV was tested in a rodent model of dental caries to assess its potential effectiveness in vivo. Topical applications of NV (800 μg ml^?1) significantly impaired the accumulation of biofilms of S. mutans by largely disrupting the synthesis of glucosyltransferase-derived exopolysaccharides and the expression of genes associated with the adaptive stress response, such as copYAZ and sloA. Of even greater impact, NV was as effective as fluoride (positive control) in reducing the development of carious lesions in vivo. NV is a promising natural anti-biofilm agent that targets essential virulence traits in S. mutans, which are associated with the formation of cariogenic biofilm and the subsequent onset of dental caries disease.     

Anti‐biofilm and bactericidal effects of magnolia bark‐derived magnolol and honokiol on Streptococcus mutans

Dental caries affects people of all ages and is a worldwide health concern. Streptococcus mutans is a major cariogenic bacterium because of its ability to form biofilm and induce an acidic environment. In this study, the antibacterial activities of magnolol and honokiol, the main constituents of the bark of magnolia plants, toward planktonic cell and biofilm of S. mutans were examined and compared with those of chlorhexidine. The minimal inhibitory concentrations of magnolol, honokiol and chlorhexidine for S. mutans were 10, 10 and 0.25 µg/mL, respectively. In addition, each agent showed bactericidal activity against S. mutans planktonic cells and inhibited biofilm formation in a dose‐ and time‐dependent manner. Magnolol (50 µg/mL) had greater bactericidal activity against S. mutans biofilm than honokiol (50 µg/mL) and chlorhexidine (500 µg/mL) at 5 min after exposure, while all showed scant activity against biofilm at 30 s. Furthermore; chlorhexidine (0.5–500 µg/mL) exhibited high cellular toxicity for the gingival epithelial cell line Ca9‐22 at 1 hr, whereas magnolol (50 µg/mL) and honokiol (50 µg/mL) did not. Thus; it was found that magnolol has antimicrobial activities against planktonic and biofilm cells of S. mutans. Magnolol may be a candidate for prevention and management of dental caries.     

Inhibitory effect of zingiber officinale towards Streptococcus mutans virulence and caries development: in vitro and in vivo studies

Background

Streptococcus mutans is known as a key causative agent of dental caries. It metabolizes dietary carbohydrate to produce acids which reduce the environmental pH leading to tooth demineralization. The ability of this bacterium to tolerate acids coupled with acid production, allows its effective colonization in the oral cavity leading to the establishment of highly cariogenic plaque. For this reason, S. mutans is the only bacterium found in significantly higher numbers than other bacteria in the dental plaque. The aim of this study was to evaluate the effect of crude extract and methanolic fraction of Z. officinale against S. mutans virulence properties.

Results

We investigated in vitro and in vivo activity of crude extract and methanolic fraction at sub- MIC levels against cariogenic properties of S. mutans. We found that these extracts strongly inhibited a variety of virulence properties which are critical for its pathogenesis. The biofilm formation in S. mutans was found to be reduced during critical growth phases. Furthermore, the glucan synthesis and adherence was also found to be inhibited. Nevertheless, the insoluble glucan synthesis and sucrose dependent adherence were apparently more reduced as compared to soluble glucan synthesis and sucrose- independent adherence. Biofilm architecture inspected with the help of confocal and scanning electron microscopy, showed dispersion of cells in the treated group as compared to the control. The Quantitative Real Time PCR (qRT-PCR) data had shown the down regulation of the virulence genes, which is believed to be one of the major reasons responsible for the observed reduction in the virulence properties. The incredible reduction of caries development was found in treated group of rats as compared to the untreated group which further validate our in vitro data.

Conclusion

The whole study concludes a prospective role of crude extract and methanolic fraction of Z. officinale in targeting complete array of cariogenic properties of S. mutans, thus reducing its pathogenesis. Hence, it may be strongly proposed as a putative anti- cariogenic agent.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0320-5) contains supplementary material, which is available to authorized users.     

Antimicrobial effects of herbal extracts on Streptococcus mutans and normal oral streptococci

Streptococcus mutans is associated with dental caries. A cariogenic biofilm, in particular, has been studied extensively for its role in the formation of dental caries. Herbal extracts such as Cudrania tricuspidata, Sophora flavescens, Ginkgo biloba, and Betula Schmidtii have been used as a folk remedy for treating diseases. The purpose of this study was to evaluate and compare the antibacterial activity of herbal extracts against normal oral streptococci, planktonic and biofilm of S. mutans. Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguinis, and S. mutans were cultivated with brain heart infusion broth and susceptibility assay for the herbal extracts was performed according to the protocol of Clinical and Laboratory Standard Institute. Also, S. mutans biofilm was formed on a polystyrene 12-well plate and 8-well chamber glass slip using BHI broth containing 2% sucrose and 1% mannose after conditioning the plate and the glass slip with unstimulated saliva. The biofilm was treated with the herbal extracts in various concentrations and inoculated on Mitis-Salivarius bacitracin agar plate for enumeration of viable S. mutans by counting colony forming units. Planktonic S. mutans showed susceptibility to all of the extracts and S. mutans biofilm exhibited the highest level of sensitivity for the extracts of S. flavescens. The normal oral streptococci exhibited a weak susceptibility in comparison to S. mutans. S. oralis, however, was resistant to all of the extracts. In conclusion, the extract of S. flavescens may be a potential candidate for prevention and management of dental caries.     

Bacteriolytic Activity of Enzymes Derived from Streptomyces Species

Streptomyces species H–402 and 1829 possessing high lytic activities against cariogenic streptococci which induce dental plaque and caries, were isolated by the screening from soils and sewers. They were identified as Streptomyces griseus and Streptomyces globisporus respectively. The former strain produced lytic enzyme accompanying spore formation during the surface culture, while the latter strain revealed a high activity in the submerged culture. These enzymes had wide substrate specificity against all groups of cariogenic streptococci. The lytic enzymes may be expected as an useful medicament for the prevention of dental caries.     

Effective Immunity to Dental Caries: Protection of Gnotobiotic Rats by Local,Immunization with a Ribosomal Preparation from Streptococcus mutans

A ribosomal preparation from Streptococcus mutans 6715 was characterized for its ability to induce an immune response in gnotobiotic rats which was protective against S. mutans-induced dental caries. Animals injected in the salivary gland region with the S. mutans ribosomal vaccine developed significantly higher (P < 0.01) salivary IgA and serum IgG antibody activities against whole S. mutans cells and ribosomal preparations than nonimmunized rats. Vaccinated animals had significantly lower (67%; P < 0.01) levels of S. mutans adherent to their molar surfaces than the control rats after infection with the homologous, cariogenic S. mutans. The immunized animals had significantly fewer (P < 0.01) carious lesions on their buccal, sulcal, and proximal molar surfaces than the nonimmunized rats following challenge with the virulent organism. Animals injected with the ribosomal preparation developed salivary IgA and serum IgG antibodies with specificities to various cell surface-associated antigens such as lipoteichoic acid and glucosyltransferase, suggesting that the observed protection may be due to antibodies against cell surface contaminants of the ribosomal vaccine. These results are the first demonstration that a ribosomal preparation from S. mutans protected rats from caries formation after challenge with the homologous, virulent S. mutans.     

Oral Streptococci Exhibit Diverse Susceptibility to Human β-Defensin-2: Antimicrobial Effects of hBD-2 on Oral Streptococci

We examined the antimicrobial effects of human -defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human -defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans (S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.     

Anti-biofilm activity of a novel pit and fissure self-adhesive sealant modified with metallic monomers

Abstract

Dental plaque is a biofilm composed of a complex oral microbial community. The accumulation of plaque in the pit and fissures of dental elements often leads to the development of tooth decay (dental caries). Here, potent anti-biofilm materials were developed by incorporating zinc methacrylates or di-n-butyl-dimethacrylate-tin into the light-curable sealant and their physical, mechanical, and biological properties were evaluated. The data revealed that 5% di-n-butyl-dimethacrylate-tin (SnM 5%) incorporated sealant showed strong anti-biofilm efficacy against various single-species (Streptococcus mutans or Streptococcus oralis or Candida albicans) and S. mutans-C. albicans cross-kingdom dual-species biofilms without either impairing the mechanical properties of the sealant or causing cytotoxicities against mouse fibroblasts. The findings indicate that the incorporation of SnM 5% in the experimental pit and fissure self-adhesive sealant may have the potential to be part of current chemotherapeutic strategies to prevent the formation of cariogenic oral biofilms that cause dental caries.     

A three-species biofilm model for the evaluation of enamel and dentin demineralization

Although Streptococcus mutans biofilms have been useful for evaluating the cariogenic potential of dietary carbohydrates and the effects of fluoride on dental demineralization, a more appropriate biofilm should be developed to demonstrate the influence of other oral bacteria on cariogenic biofilms. This study describes the development and validation of a three-species biofilm model comprising Streptococcus mutans, Actinomyces naeslundii, and Streptococcus gordonii for the evaluation of enamel and dentin demineralization after cariogenic challenges and fluoride exposure. Single- or three-species biofilms were developed on dental substrata for 96?h, and biofilms were exposed to feast and famine episodes. The three-species biofilm model produced a large biomass, mostly comprising S. mutans (41%) and S. gordonii (44%), and produced significant demineralization in the dental substrata, although enamel demineralization was decreased by fluoride treatment. The findings indicate that the three-species biofilm model may be useful for evaluating the cariogenic potential of dietary carbohydrates other than sucrose and determining the effects of fluoride on dental substrata.     

Comments on Soltysiak's paper: “Comment: Low dental caries rate in Neandertals: The result of diet or the oral flora compositions?”

A low frequency of dental caries in Neandertal population is still puzzling. Many authors stress that the lower frequency of dental caries was related to a meat diet. However, a recent publication in HOMO – Journal Comparative Human Biology presented a new interpretation of dental caries in Neandertals. In this article, Soltysiak supports the thesis that the lower frequency of caries in the Neandertal population from the Near East could not be related to the low-sugar diet, but rather to the absence of cariogenic bacteria species (S. mutans). Although this hypothesis is interesting, I suspect it to be based on several erroneous assumptions, and a misunderstanding of caries as a disease. Although he stressed that the caries lesion is related to many different factors, in his argument he considers one of two alternatives “a low-sugar diet or a lack of cariogenic bacterial species”.     

Elevated incidence of dental caries in a mouse model of cystic fibrosis

Background

Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model.

Methodology/Principal Findings

We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value<0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16±2 mM vs. 31±2 mM, CF and wildtype mice, respectively; p value<0.01; t test).

Conclusions/Significance

Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.     

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.     

An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans

The objective of this study is to investigate the synergetic action between nisin and sodium fluoride or chlorhexidine against Streptococcus mutans, a primary cariogenic pathogen. In the antibacterial assay, a synergetic effect on S. mutans was found between nisin and sodium fluoride, but there was no interaction between nisin and chlorhexidine by the checkerboard, the fractional inhibitory concentration (FIC) and the fractional bactericidal concentration (FBC) tests. S. mutans survival rates showed a significant decline after treatment with a combination of nisin and sodium fluoride in a time-kill study. Scanning electron microscopy showed that the damage to S. mutans with the combined nisin and sodium fluoride treatment was the most severe among all of the different single and combined antimicrobial treatments. Furthermore, in the antibiofilm test, nisin in combination with sodium fluoride produced a stronger bactericidal effect on a S. mutans biofilm for 4 h and 16 h compared with sodium fluoride alone by confocal laser scanning microscopy. Nisin in combination with sodium fluoride exerted a high bactericidal effect on S. mutans and thereby has the potential to be used as an effective drug combination to prevent dental caries.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.