Quantitative analysis of biofilm bacteria according to different stages of early childhood caries

ObjectiveMonitoring selected key species related to health or disease may facilitate caries risk assessment and discovery of novel ecological preventive and therapeutic approaches. This study aimed at quantifying Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Lactobacillus casei group, Streptococcus gordonii, Mitis group and Streptococcus mutans by quantitative polymerase chain reaction (qPCR) in dental biofilm from Brazilian children with different stages of early childhood caries (ECC).DesignSeventy-five preschool children were clinically evaluated by ICDAS criteria and divided into groups: caries-free (CF; n = 20), enamel caries lesions (ECL; n = 17) and dentine caries lesions (DCL; n = 38). Plaque samples from all children were collected for detection and quantification of the selected bacteria.ResultsL. acidophilus and L. casei group were absent in almost all plaque samples. No differences in relative proportions of A. naeslundii, Mitis group and S. gordonii were observed in any stage of caries. However, S. mutans and Bifidobacterium spp. were present at higher concentrations in the biofilm of children with DCL (p < 0.001). Multivariate analysis showed that S. mutans and Bifidobacterium spp. were strongly associated with biofilm in children with DCL.ConclusionDifferences were observed in the proportion of acidogenic and aciduric bacteria with dental caries progression. The data indicate that S. mutans and Bifidobacterium spp. in dental biofilm may be involved in some progression processes for ECC.     

Hydroalcoholic extracts of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves on Streptococcus mutans biofilm and tooth demineralization

ObjectivesThis study evaluated the effect of the hydroalcoholic extracts of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves (alone or combined) on the viability of Streptococcus mutans biofilm and on the prevention of enamel demineralization.MethodsStrain of S. mutans (ATCC 21175) was reactivated in BHI broth. Minimum inhibitory concentration, minimum bactericidal concentration, minimum inhibition biofilm concentration and minimum eradication biofilm concentration were determined in order to choose the concentrations to be tested under biofilm model. S. mutans biofilm (5 × 10⁵ CFU/ml) was produced on bovine enamel, using McBain saliva under 0.2% sucrose exposure, for 3 days. The biofilm was daily treated with the extracts for 1 min. The biofilm viability was tested by fluorescence and the enamel demineralization was measured using TMR.ResultsMyracrodruon urundeuva All. (Isolated or combined) at the concentrationsc ≥0.625 mg/ml was able to reduce bacteria viability, while Qualea Grandflora Mart. alone had antimicrobial effect at 5 mg/ml only (p < 0.05). On the other hand, none of the extracts were able to reduce enamel demineralization.ConclusionsThe hydroalcoholic extracts of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves (isolated or combined) have antimicrobial action; however, they do not prevent enamel caries under S. mutans biofilm model.     

Effect of the association of maltodextrin and sucrose on the acidogenicity and adherence of cariogenic bacteria

ObjectiveThe aim was to investigate the effect of maltodextrin and sucrose association on the acidogenic and adherence profiles of cariogenic bacteria.DesignStreptococcus mutans (S. mutans) and Lactobacillus casei (L. casei) were cultivated in culture medium containing maltodextrin, sucrose, maltodextrin–sucrose mixture or glucose. Analyses of the acidogenicity and microbial adherence were conducted in triplicate for each microorganism and tested carbohydrate.ResultsFor L. casei, maltodextrin, sucrose and maltodextrin–sucrose mixture showed lower acidogenic potential compared to glucose. When the microorganism was S. mutans, sucrose and maltodextrin–sucrose mixture presented higher acidogenic potential compared to maltodextrin and glucose. Microbial adherence analysis revealed higher adherence for S. mutans in presence of sucrose and maltodextrin–sucrose mixture compared to maltodextrin and glucose. For L. casei, all the carbohydrates showed similar adherence percentages.ConclusionThe addition of maltodextrin to sucrose does not increase the cariogenicity of sucrose in terms of acidogenicity and adherence of the cariogenic bacteria.     

In vitro evaluation of composite containing DMAHDM and calcium phosphate nanoparticles on recurrent caries inhibition at bovine enamel-restoration margins

ObjectiveRecurrent caries is a primary reason for restoration failure caused by biofilm acids. The objectives of this study were to: (1) develop a novel multifunctional composite with antibacterial function and calcium (Ca) and phosphate (P) ion release, and (2) investigate the effects on enamel demineralization and hardness at the margins under biofilms.MethodsDimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) were incorporated into composite. Four groups were tested: (1) Commercial control (Heliomolar), (2) Experimental control (0% DMAHDM + 0% NACP), (3) antibacterial group (3% DMAHDM + 0% NACP), (D) antibacterial and remineralizing group (3% DMAHDM + 30% NACP). Mechanical properties and Ca and P ion release were measured. Colony-forming units (CFU), lactic acid and polysaccharide of Streptococcus mutans (S. mutans) biofilms were evaluated. Demineralization of bovine enamel with restorations was induced via S. mutans, and enamel hardness was measured. Data were analyzed via one-way and two-way analyses of variance and Tukey’s multiple comparison tests.ResultsAdding DMAHDM and NACP into composite did not compromise the mechanical properties (P > 0.05). Ca and P ion release of 3% DMAHDM + 30% NACP was increased at cariogenic low pH. Biofilm lactic acid and polysaccharides were greatly decreased via DMAHDM, and CFU was reduced by 4 logs (P < 0.05). Under biofilm acids, enamel hardness at the margins was decreased to about 0.5 GPa for control; it was about 1 GPa for antibacterial group, and 1.3 GPa for antibacterial and remineralizing group (P < 0.05).ConclusionsThe novel 3% DMAHDM + 30% NACP composite had strong antibacterial effects. It substantially reduced enamel demineralization adjacent to restorations under biofilm acid attacks, yielding enamel hardness that was 2-fold greater than that of control composites. The novel multifunctional composite is promising to inhibit recurrent caries.     

Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms

IntroductionNanoemulsions (NE) are a unique class of disinfectants produced by mixing a water immiscible liquid phase into an aqueous phase under high shear forces. NE have antimicrobial properties and are also effective anti-biofilm agents.Materials and methodsThe effectiveness of nanoemulsion and its components was determined against Streptococcus mutans and Lactobacillus casei by live/dead staining. In vitro antimicrobial effectiveness of nanoemulsion against planktonic S. mutans, L. casei, Actinomyces viscosus, Candida albicans and mixed culture was determined by a serial dilution technique to obtain minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). In addition, efficacy was investigated by kinetics of killing, adherence and biofilm assays.ResultsCompared to its components, nanoemulsion showed notable antimicrobial activity against biofilm organisms, up to 83.0% kill within 1 min. NE dilutions ranging from 243 to 19683 were effective against planktonic S. mutans, L. casei, A. viscosus, C. albicans and mixed culture of these four strains as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. The level of adhesion on glass surface was reduced by 94.2–99.5% in nanoemulsion treated groups (p < 0.001). 4-Day-old S. mutans, L. casei, A. viscosus, C. albicans and mixed cultures biofilms treated with NE showed reductions of bacterial counts with decreasing dilutions (p < 0.001).ConclusionThese results suggest that nanoemulsion has effective anti-cariogenic activity against cariogenic microorganisms and may be a useful medication in the prevention of caries.     

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar‐overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.     

Dual-functional adhesive containing amorphous calcium phosphate nanoparticles and dimethylaminohexadecyl methacrylate promoted enamel remineralization in a biofilm-challenged environment

ObjectiveThe cariogenic biofilm on enamel, restoration, and bonding interface is closely related to dental caries and composite restoration failure. Enamel remineralization at adhesive interface is conducive to protecting bonding interface and inhibiting secondary caries. This study intended to assess the remineralization efficiency of adhesive with dimethylaminohexadecyl methacrylate (DMAHDM) and nanoparticles of amorphous calcium phosphate (NACP) on initial caries lesion of biofilm-coated enamel.MethodsArtificial initial carious lesion was created via 72-hour immersion in demineralization solution and cariogenic biofilm was formed after 24-hour culture of Streptococcus mutans (S. mutans). Specimens were then divided into 4 groups: enamel control, enamel treated with NACP, DMAHDM and NACP+DMAHDM respectively. Samples next underwent 7-day cycling, 4 h in BHIS (brain heart infusion broth containing 1 % sucrose) and 20 h in AS (artificial saliva) per day. The pH of BHIS was tested daily. So did the concentration of calcium and phosphate in BHIS and AS. Live/dead staining, colony-forming unit (CFU) count, and lactic acid production of biofilms were measured 7 days later. The enamel remineralization efficiency was evaluated by microhardness testing and transverse microradiography (TMR) quantitatively.ResultsEnamel of NACP+DMAHDM group demonstrated excellent remineralization effectiveness. And the NACP+DMAHDM adhesive released a great number of Ca²⁺ and PO₄^3- ions, increased pH to 5.81 via acid neutralization, decreased production of lactic acid, and reduced CFU count of S. mutans (P < 0.05).SignificanceThe NACP+DMAHDM adhesive would be applicable to preventing secondary caries, strengthening enamel-adhesive interface, and extending the lifespan of composite restoration.     

Antimicrobial and antibiofilm action of Casbane Diterpene from Croton nepetaefolius against oral bacteria

ObjectiveThe antibacterial activity of Casbane Diterpene (CD) was evaluated in vitro against Streptococcus oralis, S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. The viability of planktonic cells was analysed by susceptibility tests (MIC and MBC) and antibiofilm action was assayed.MethodsThe minimal inhibitory and bactericidal concentrations (MIC and MBC) of oral Streptococcus were evaluated through microdilution tests. To assay antibiofilm activity, biofilms were generated on 96-wells polystyrene plates under the presence of CD and quantified by a crystal violet technique and colonies forming units counting.ResultsThe CD isolated from Croton nepetaefolius showed antimicrobial effect on planktonic forms and biofilms of oral pathogens, with MIC values of 62.5 μg/mL against Streptococcus oralis and values between 125 and 500 μg/mL against S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. CD showed an inhibitory effect on S. mutans biofilm formation at 250 μg/mL, and a decrease on viable cell of 94.28% compared to the normal biofilm growth.ConclusionsThe compound CD can be considered as a promising molecule for the treatment against oral pathogens responsible for dental biofilm.     

Evaluation of bacteria-induced enamel demineralization using optical profilometry

ObjectivesStreptococcus mutans is considered a major causative of tooth decay due to its ability to rapidly metabolize carbohydrates such as sucrose. One prominent excreted end product of sucrose metabolism is lactic acid. Lactic acid causes a decrease in the pH of the oral environment with subsequent demineralization of the tooth enamel. Biologically relevant bacteria-induced enamel demineralization was studied.MethodsOptical profiling was used to measure tooth enamel decay with vertical resolution under one nanometer and lateral features with optical resolution as a result of S. mutans biofilm exposure. Comparison measurements were made using AFM.ResultsAfter 72 h of biofilm exposure the enamel displayed an 8-fold increase in the observed roughness average (R_a), as calculated over the entire measured array. Similarly, the average root mean square (RMS) roughness, R_RMS, of the enamel before and after biofilm exposure for 3 days displayed a 7-fold increase. Further, the direct effect of chemically induced enamel demineralization using biologically relevant organic acids was shown. Optical profiles of the enamel surface after addition of a 30% lactic acid solution showed a significant alteration in the surface topography with a corresponding increase in respective surface roughness statistics. Similar measurements with 10% citric acid over seconds and minutes give insight into the demineralization process by providing quantitative measures for erosion rates: comparing surface height and roughness as metrics.SignificanceThe strengths of optical profilometry as an analytical tool for understanding and analyzing biologically relevant processes such as biofilm induced tooth enamel demineralization were demonstrated.     

Inhibitory effects of β-caryophyllene on Streptococcus mutans biofilm

ObjectiveThe biofilm of Streptococcus mutans is associated with induction of dental caries. Also, they produce glucan as an extracellular polysaccharide through glucosyltransferases and help the formation of cariogenic biofilm. β-caryophyllene has been used for therapeutic agent in traditional medicine and has antimicrobial activity. The purpose of this study was to investigate the effect of β-caryophyllene on S. mutans biofilm and the expression of biofilm-related factor.DesignThe susceptibility assay of S. mutans for β-caryophyllene was performed to investigate inhibitory concentration for S. mutans growth. To evaluated the effect of β-caryophyllene on S. mutans biofilm, β-caryophyllene was treated on S. mutans in the various concentrations before or after the biofilm formation. Live S. mutans in the biofilm was counted by inoculating on Mitis-salivarius agar plate, and S. mutans biofilm was analyzed by confocal laser scanning microscope after staining bacterial live/dead staining kit. Finally, the expression of glucosyltransferases of S. mutans was investigated by real-time RT-PCR after treating with β-caryophyllene at the non-killing concentration of S. mutans.ResultsThe growth of S. mutans was inhibited by β-caryophyllene in above concentration of 0.078%, S. mutans biofilm was inhibited by β-caryophyllene in above 0.32%. Also, 2.5% of β-caryophyllene showed anti-biofilm activity for S. mutans biofilm. β-caryophyllene reduced the expression of gtf genes at a non-killing concentration for S. mutans. On the basis on these results, β-caryophyllene may have anti-biofilm activity and the inhibitory effect on biofilm related factor.Conclusionsβ-caryophyllene may inhibit cariogenic biofilm and may be a candidate agent for prevention of dental caries.     

Use of the checkerboard DNA-DNA hybridisation technique for in vivo detection of cariogenic microorganisms on metallic brackets, with or without use of an antimicrobial agent

Objective

Using checkerboard DNA-DNA hybridisation (CDDH) assay, this randomised clinical study evaluated the contamination of metallic brackets by four cariogenic bacterial strains (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei and Lactobacillus acidophilus) and the efficacy of 0.12% chlorhexidine gluconate (CHX) mouthwashes in reducing bacterial contamination.

Methods

Thirty-nine 11-33-year-old patients under treatment with fixed orthodontic appliances were enrolled in the study and had 2 new metallic brackets bonded to premolars. Nineteen patients used a 0.12% CHX mouthwash (Periogard^®) and 20 patients used a placebo mouthwash (control) twice a week. After 30 days, the brackets were removed and samples were obtained for analysis by CDDH. Data were analysed statistically by the Kruskal-Wallis test (α = 0.05) using the SAS software.

Results

S. mutans, S. sobrinus, L. casei and L. acidophilus were detected in 100% of the samples from both groups. However, brackets of the control group were more heavily contaminated by S. mutans and S. sobrinus (P < 0.01). In the experimental group, although all counts decreased after rinsing with the chlorhexidine solution, there was significant difference only for S. mutans (P = 0.03).

Conclusions

The use of 0.12% chlorhexidine gluconate mouthwashes can be useful in clinical practice to reduce the levels of cariogenic microorganisms in patients under treatment with fixed orthodontic appliances.     

Antimicrobial activity of Bifidobacterium spp. isolated from healthy adult Koreans against cariogenic microflora

Objective

Dental caries is the main common infectious disease in the human oral cavity. Streptococcus mutans and Streptococcus sobrinus were reported to be the most important etiological factors in human dental caries. Thus, we examined the inhibitory effects of Bifidobacterium spp. cells and culture supernatants against S. mutans and S. sobrinus, including Streptococcus gordonii, and Aggregatibacter actinomycetemcomitans, which is associated with periodontal disease.

Methods

Mutans streptococci or A. actinomycetemcomitans and lactic acid bacteria (LAB) were mixed in 1:1 ratio and then incubated for 90 min at 37 °C. After the incubation, the viability of mutans streptococci or A. actinomycetemcomitans was determined by plate count technique. We also investigated the morphological changes of S. mutans treated with LAB using scanning electron microscopy (SEM).

Results

In vitro viability of S. mutans, S. sobrinus, S. gordonii, and A. actinomycetemcomitans was affected by human intestinal LAB identified as Bifidobacterium adolescentis SPM1005 and Bifidobacterium longum SPM1207. Especially, B. adolescentis SPM1005 cells at 1.0 × 10⁸ CFU had a strong growth-inhibiting effect against S. mutans and induced a 64% loss of its viability (p < 0.05). In addition, swollen and disrupted S. mutans were observed after incubation with B. adolescentis SPM1005. However, the culture supernatant of this strain did not show such inhibitory activity.

Conclusion

B. adolescentis SPM1005 cells decreased the growth of S. mutans, which is a risk factor for dental caries. Therefore, we suggest that this Bifidobacterium strain may be a useful probiotic microorganism for prevention of dental caries that does not have adverse effects.     

In vitro antimicrobial effect of bacteriocin PsVP-10 in combination with chlorhexidine and triclosan against Streptococcus mutans and Streptococcus sobrinus strains

Objectives

To determine the minimal inhibitory concentrations (MICs) of bacteriocin PsVP-10, chlorhexidine and triclosan on S. mutans and S. sobrinus and to study the potential synergistic combination between these antimicrobial and the bacteriocin PsVP-10.

Design

Were determined MICs of bacteriocin PsVP-10, triclosan and chlorhexidine on strains of S. mutans and S. sobrinus, which formed a biofilm or did not form a biofilm. In addition, the synergistic effect was analysed by the determination of respective fractionary inhibitory concentrations (FICs) between bacteriocin PsVP-10 plus chlorhexidine and bacteriocin PsVP-10 plus triclosan.

Results

MICs of three antimicrobials used were higher in those bacterial strains, which formed a biofilm. An interesting synergistic effect on both studied species was observed when bacteriocin and chlorhexidine were combined. A slighter synergy was determined for the combination bacteriocin PsVP-10 and triclosan.

Conclusions

The results showed that the combination of chlorhexidine bacteriocin PsVP-10 could reduce the number of cariogenic bacteria for in vitro studies. In the future this synergistic combination could be an alternative to antimicrobial therapy against S. mutans or S. sobrinus.     

Relationship between caries risk and presence of cariogenic bacteria among Japanese pregnant women

ObjectivesTo analyze the relationship between caries risk and the presence of S. mutans and S. sobrinus from plaques of pregnant women using the Cariostat method and the polymerase chain reaction (PCR) technique.Materials and MethodsThe subjects consisted of 269 pregnant women who were in their 3rd, 4th or 5th month of pregnancy. The presence of S. mutans and S. sobrinus was assessed from their plaques cultured in the Cariostat medium and assessed by PCR.ResultsThe correlation between caries risk scores assessed by the Cariostat method and the presence of S. mutans was statistically significant (P<0.05). The tendency of positive correlation was found between the presence of S. sobrinus and caries activity.ConclusionThis study suggested that pregnant women who have high caries activity should be more aware of the possibility of transmission of cariogenic bacteria to their infants.     

No evidence for the growth-stimulating effect of monomers on cariogenic Streptococci

Background

In spite of contradicting results, the high susceptibility of composites for secondary caries is still often associated with the bacterial growth-stimulating effect of released methacrylate monomers. However, most studies that showed this effect were performed with techniques having inherent limitations (spectrophotometry).

Objectives

Therefore, our objective was to determine the effect of four methacrylate monomers (2-Hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA)) on the growth of two caries-associated bacteria, Streptococcus mutans and sobrinus, and one non-cariogenic species, Streptococcus sanguinis, using TaqMan quantitative polymerase chain reaction (qPCR) to quantify bacterial DNA.

Materials and methods

Cultures were exposed to monomer solutions selected after spectrophotometric growth measurements. At baseline and predetermined time intervals, bacterial DNA was extracted and quantified with TaqMan qPCR. Biofilms grown in the presence of monomers were analyzed with scanning electron microscopy (SEM).

Results

Spectrophotometry indeed showed increased growth rates of all three strains with 5 mM TEGDMA, EGDMA, and DEGDMA and increased total biomass of S. sanguinis with 5 mM TEGDMA. However, qPCR failed to show any growth-stimulating effect of these monomers on S. mutans and S. sobrinus. In contrast, some monomers exhibited a growth-inhibiting effect on S. sanguinis. SEM revealed extracellular matter in S. sobrinus and S. sanguinis biofilms, which might be attributed to polymer formation.

Conclusions

Techniques which quantify bacterial DNA are more appropriate to evaluate bacterial growth in the presence of monomers than spectrophotometry.

Clinical relevance

Even though methacrylate monomers did not affect the growth of cariogenic species, growth inhibition of S. sanguinis, a non-cariogenic antagonistic species, may lead to ecological shifts towards higher cariogenicity.

     

Influence of orthodontic brackets design and surface properties on the cariogenic Streptococcus mutans adhesion

ObjectiveTo compare the surface properties of self-ligating metallic (SLM), ceramic esthetic, and conventional metallic (CM) brackets, and evaluate the adhesion of Streptococcus mutans biofilms to their surface, attempting to interpret the correlation between bracket type and enamel demineralization from a microbiological perspective.Materials and methodsTwenty-two brackets of each group were used. The brackets’ surface roughness was defined and the bacterial adhesion was performed using the strain S. mutans ATCC25175 with 8 h or 24 h of incubation time. The total bacterial adhesion (TBA) of biofilms was assessed using optical density (OD) methodology. To quantify bacteria viability (BV), the colony forming units (CFU) were counted. A scanning electron microscopy (SEM) observation of biofilms was also performed. Results: Ceramic brackets exhibited significantly higher roughness (0.304) compared to CM (0.090) and SLM (0.067) ones (C > CM = SLM). The data obtained with the TBA and BV tests showed that S. mutans biofilm formed on bracket groups exhibited similar results for both incubation periods. From the SEM images it is possible to observe that biofilm structure formed for 24 h was denser than that for 8 h of incubation with significantly more aggregates and cells for three groups.ConclusionThis in vitro study suggests that despite the higher surface roughness of ceramic brackets, this alone does not influence the adhesion of the S. mutans biofilms.Clinical relevanceFrom a microbiological perspective, the bracket's design may be more relevant than its surface roughness with respect to the adhesion of cariogenic bacteria biofilm with potential risk to dental enamel integrity.     

Efficacy of red propolis hydro-alcoholic extract in controlling Streptococcus mutans biofilm build-up and dental enamel demineralization

ObjectiveThe efficacy of a red propolis hydro-alcoholic extract (RP) in controlling Streptococcus mutans biofilm colonization was evaluated. The effect of RP on dental demineralization was also investigated.MethodsChemical composition was determined by High Performance Liquid Chromatography (HPLC). Minimum Inhibitory and Bactericidal Concentration (MIC and MBC, respectively) were investigated against Streptococcus mutans (ATCC 25175). The cytotoxic potential of 3% RP in oral fibroblasts was observed after 1 and 3 min. Bovine dental enamel blocks (N = 24) were used for S. mutans biofilm formation (48 h), simulating ‘feast or famine’ episodes. Blocks/biofilms were exposed 2×/day, for 3 days, to a cariogenic challenge with sucrose 10% (5 min) and treated (1 min) with: 0.85% saline solution (negative control), 0.12% Chlorhexidine (CHX, positive control for biofilm colonization), 0.05% Sodium Fluoride (NaF, positive control to avoid demineralization) and 3% RP. Biofilms were assessed for viability (CFU/mL), and to observe the concentration of soluble and insoluble extracellular polysaccharides (SEPS and IEPS). Dental demineralization was assessed by the percentage of surface hardness loss (%SHL) and through polarized light microscopy (PLM).ResultsThe RP presented 4.0 pH and ºBrix = 4.8. The p-coumaric acid (17.2 μg/mL) and luteolin (15.23 μg/mL) were the largest contents of phenolic acids and flavonoids, respectively. MIC and MBC of RP were 293 μg/mL and 1172 μg/mL, respectively. The 3% RP showed 43% of viably cells after 1 min. Lower number (p < 0.05) of viable bacteria (CFU/mL) was observed after CHX (1.8 × 10⁵) followed by RP (1.8 × 10⁷) treatments. The lowest concentration (μg/CFU) of SEPS (12.6) and IEPS (25.9) was observed in CHX (p < 0.05) followed by RP (17.1 and 54.3), and both differed from the negative control (34.4 and 63.9) (p < 0.05). Considering the %SHL, all groups differed statistically (p < 0.05) from the negative control (46.6%); but NaF (13.9%), CHX (20.1%) and RP (20.7%) did not differ among them (p > 0.05). After all treatments, suggestive areas of caries lesions were observed by PLM, which were lower for CHX and NaF.ConclusionThe 3% RP reduced S. mutans colonization, decreased concentration of extracellular polysaccharides and reduced dental enamel demineralization.     

Effect of sodium fluoride on oral biofilm microbiota and enamel demineralization

ObjectiveFluoride is widely used as an anti-caries agent, e.g. in toothpastes and mouth rinses. However, the nature of the anti-caries action is not entirely clear. Mechanisms suspected to explain the cariostatic effect include inhibitory effects on acid formation by bacteria, inhibition of extracellular polysaccharide (EPS) production, inhibition of enamel demineralization and enhancement of remineralizaton or combination thereof. The aim of this study was to examine with the supragingival Zurich in vitro biofilm model the effect of fluoride in NaF formulation, on the microbiota and on demineralization.MethodsBiofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar and Streptococcus sobrinus, were grown anaerobically on sintered hydroxyapatite or bovine enamel disks, exposed to 200, 400, and 1400 ppm of NaF, or 0.1% chlorhexidine (positive control). The biofilms were harvested after 64 h and CFUs were assessed for total bacteria. Demineralization of enamel disks was measured by quantitative light-induced fluorescence.ResultsNaF did not affect the bacterial numbers. No enamel mineral loss was observed at 1400 and 400 ppm of fluoride, whereas the pH of the surrounding medium was increased to 5.5 and 5.0, respectively, compared to the untreated control (pH 4.5 and mineral loss ΔF of −32%). At 1400 ppm NaF the biofilm’s EPS volume was also significantly reduced.ConclusionsAdministration of NaF completely prevented demineralization without affecting biofilm composition and growth. This protective effect may be attributed to the observed decrease in acid production or EPS volume, or to a shift in the de/remineralization balance.     

Characterization of the Streptococcus sobrinus acid-stress response by interspecies microarrays and proteomics

Streptococcus mutans and Streptococcus sobrinus are considered the primary organisms responsible for human dental caries. The ability to generate acids and to adapt to low pH conditions is directly associated with the cariogenic potential of these bacteria. To survive acidic conditions, both species have been shown to mount an acid‐tolerance response (ATR). However, previous characterization of the S. sobrinus ATR identified critical differences in the mechanisms of acid adaptation between S. mutans and S. sobrinus. Here, interspecies microarray and proteomic approaches were used to identify novel, previously unrecognized genes and pathways that participate in the S. sobrinus acid‐stress response. The results revealed that, among other things, metabolic alterations that enhance energy generation and upregulation of the malolactic fermentation enzyme activity constitute important acid‐resistance properties in S. sobrinus. Some of these acid adaptive traits are shared by S. mutans and might be considered optimal targets for therapeutic treatments designed to control dental caries.