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.     

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.     

Novel pit and fissure sealant containing nano-CaF2 and dimethylaminohexadecyl methacrylate with double benefits of fluoride release and antibacterial function

ObjectivePit and fissure sealants with antibacterial and remineralization properties have broad application prospects in caries prevention. The objectives of this study were to: (1) develop a novel pit and fissure sealant containing CaF₂ nanoparticles (nCaF₂) and dimethylaminohexadecyl methacrylate (DMAHDM); and (2) investigate the effects of nCaF₂ and DMAHDM on biofilm response and fluoride (F) ion release for the first time.MethodsHelioseal F was used as a control. Bioactive sealants were formulated with DMAHDM and nCaF₂. Flow properties, enamel shear bond strength, hardness and F ion releases were measured. Streptococcus mutans (S. mutans) biofilms were grown on sealants. Biofilm metabolic activity, lactic acid production, colony-forming units (CFU), and pH of biofilm culture medium were measured.ResultsAdding 5% DMAHDM and 20% nCaF₂ did not reduce the paste flow and enamel bond strength, compared to control (p < 0.05). Hardness of sealants with 20% nCaF₂ and DMAHDM was higher than control (p < 0.05). The F ion release from 20% nCaF₂ was much higher than that of commercial control (p < 0.05). The sealant with DMAHDM reduced the S. mutans biofilm CFU by 4 logs. The pH in biofilm medium of the new bioactive sealant was much higher (pH 6.8) than that of commercial sealant (pH 4.66) (p < 0.05).SignificanceThe new bioactive pit and fissure sealant with nCaF₂ and DMAHDM achieved high fluoride release and strong antibacterial performance. This novel fluoride-releasing and antibacterial sealant is promising to inhibit caries and promote the remineralizaton of enamel and dentin.     

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.     

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.     

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.     

In vitro comparison of biofilm formation and acidogenicity between human breast milk and other milk formulas

ObjectiveTo evaluate the ability and acidogenicity of human breast milk (HBM) biofilm formation relative to other milk formulas.Materials and methodsPooled HBM from six donor mothers, plain milk-based formula, milk-based formula supplemented with sucrose, soy-based formula, and brain heart infusion (BHI) containing 10% sucrose were utilized for the evaluation of biofilm formation and its acidogenicity. Biofilms formed by these tested groups, with and without 10⁵ CFU/ml of Streptococcus mutans, were stained with crystal violet that was quantified by measuring the absorbance value (OD) at 595 nm. The acidogenicity of the biofilms was assessed after a 24-hour incubation period.Statistical analysisMultivariate analysis, the Kruskal Wallis test, and the Bonferroni test were performed. A P-value less than 0.05 was considered to be statistically significant.ResultsHBM containing S. mutans had significantly less potential to form biofilms as compared to other milk formulas. The overall biofilm OD value was significantly lower in the HBM group as compared to the other groups (P < 0.05). Additionally, the pH of HBM biofilm formation did not drop under critical pH after 24 h of incubation.ConclusionsHBM tends to be non-cariogenic due to its low acidogenicity and limited biofilm formation.     

Effect of arginine on the growth and biofilm formation of oral bacteria

BackgroundAlkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries.ObjectiveTo investigate the effect of arginine on the growth and biofilm formation of oral bacteria.Methods and resultsPolymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2 h and 8 h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P = 0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P < 0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P = 0.32, 0.06, 0.44 respectively).ConclusionsArg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries.     

Effect of bovine lactoferrin on enamel demineralization and acid fermentation by Streptococcus mutans

The purpose of this study was to evaluate the effects of bovine lactoferrin on acid fermentation and enamel demineralization using Streptococcus mutans in a culture system and an artificial mouth model system. The antibacterial activity of bovine lactoferrin (bLF) against S. mutans was analyzed by a radial diffusion assay. In the culture system, the effect of bLF on the synthesis and adherence of water insoluble glucan (WIG) and the adherence of S. mutans to a glass surface was examined by a batch culture. In the artificial mouth model system, cell suspension of S. mutans, heart infusion broth supplemented with sucrose, and PBS or lactoferrin solution were supplied separately and constantly for 21 hours. The following parameters were determined for evaluation: the amount of artificial biofilm, the changes in pH underneath the biofilm; and the changes in enamel microhardness measured by a Vicker's hardness tester. The antibacterial activity of bLF against S. mutans was observed. The amounts of bacterial cells in the total adherent fractions were inhibited by bLF in a dose dependent manner. The amounts of WIG in a firm-adherent fraction were significantly inhibited by 0.1–1.0% bLF. The changes in microhardness on enamel slabs in the bLF group (2.4 ± 0.8) showed significantly less hardness reduction than those in the control group (22.3 ± 2.5) (P < 0.001). The artificial biofilm accumulation was not reduced by bLF. The results of this study suggest that bLF might have inhibitory effects against acid fermentation and demineralization of enamel by S. mutans.     

Clotrimazole and econazole inhibit Streptococcus mutans biofilm and virulence in vitro

Objective: The aim of this study was to determine the inhibitory effect of eight antifungal drugs on S. mutans growth, biofilm formation and virulence factors.MethodsThe actions of antifungal drugs on S. mutans were determined by recovery plates and survival kinetic curves. Biofilms were observed by scanning electron microscopy and the viable cells were recovered on BHI plates, meanwhile biofilms were stained by BacLight live/dead kit to investigate the biofilm viability. Bacteria/extracellular polysaccharides staining assays were performed to determine the EPS production of S. mutans biofilms. Acidogenicity and acidurity of S. mutans were determined using pH drop and acid tolerance assays, and the expression of ldh gene was evaluated using qPCR.ResultsWe found that clotrimazole (CTR) and econazole (ECO) showed antibacterial activities on S. mutans UA159 and S. mutans clinical isolates at 12.5 and 25 mg/L, respectively. CTR and ECO could also inhibit S. mutans biofilm formation and reduce the viability of preformed biofilm. CTR and ECO affected the live/dead ratio and the EPS/bacteria ratio of S. mutans biofilms. CTR and ECO also inhibited the pH drop, lactate acid production, and acid tolerance. The abilities of CTR and ECO to inhibit S. mutans ldh expression were also confirmed.ConclusionsWe found that two antifungal azoles, CTR and ECO, had the abilities to inhibit the growth and biofilm formation of S. mutans and more importantly, they could also inhibit the virulence factors of S. mutans.     

The impact of glass ionomer cement and composite resin on microscale pH in cariogenic biofilms and demineralization of dental tissues

ObjectiveSecondary caries is among the most frequent reasons for the failure of dental restorations. Glass ionomer cement (GIC) restorations have been proposed to protect the surrounding dental tissues from demineralization through the release of fluoride and by buffering the acid attack from dental biofilms. In contrast, the lack of buffering by composite resin (CR) restorations has been suggested as a promoting factor for the development of secondary caries.MethodsThe present study employed transversal microradiography and confocal microscopy based pH ratiometry to quantify mineral loss and map microscale pH gradients inside Streptococcus mutans biofilms grown on compound specimens consisting of enamel, dentin and either GIC or CR.ResultsMineral loss in dentin was significantly lower next to GIC than next to CR, but no significant differences in local biofilm pH were observed between the two restorative materials.SignificanceThe cariostatic effect of GIC relies predominantly on the provision of fluoride and not on a direct buffering action. The lack of buffering by CR did not affect local biofilm pH and may therefore be of minor importance for secondary caries development.     

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.     

Effects of S. mutans gene-modification and antibacterial monomer dimethylaminohexadecyl methacrylate on biofilm growth and acid production

ObjectivesAntibacterial quaternary ammonium monomers (QAMs) are used in resins. The rnc gene in Streptococcus mutans (S. mutans) plays a key role in resisting antibiotics. The objectives of this study were to investigate for the first time: (1) the effects of rnc deletion on S. mutans biofilms and acid production; (2) the combined effects of rnc deletion with dimethylaminohexadecyl methacrylate (DMAHDM) on biofilm-inhibition efficacy.MethodsParent S. mutans strain UA159 (ATCC 700610) and the rnc-deleted S. mutans were used. Bacterial growth, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) were measured to analyze the bacterial susceptibility of the parent and rnc-deleted S. mutans against DMAHDM, with the gold-standard chlorhexidine (CHX) as control. Biofilm biomass, polysaccharide and lactic acid production were measured.ResultsThe drug-susceptibility of the rnc-deleted S. mutans to DMAHDM or CHX was 2-fold higher than parent S. mutans. The drug-susceptibility did not increase after 10 passages (p < 0.05). Deleting the rnc gene increased the biofilm susceptibility to DMAHDM or CHX by 2-fold. The rnc-deletion in S. mutans reduced biofilm biomass, polysaccharide and lactic acid production, even at no drugs. DMAHDM was nearly 40 % more potent than the gold-standard CHX. The combination of rnc deletion + DMAHDM treatment achieved the greatest reduction in biofilm biomass, polysaccharide synthesis, and lactic acid production.SignificanceGene modification by deleting the rnc in S. mutans reduced the biofilm growth and acid production, and the rnc deletion + DMAHDM method showed the greatest biofilm-inhibition efficacy, for the first time. The dual strategy of antibacterial monomer + bacterial gene modification shows great potential to control biofilms and inhibit caries.     

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.     

Inhibitory effects of tea catechin epigallocatechin-3-gallate against biofilms formed from Streptococcus mutans and a probiotic lactobacillus strain

ObjectiveEffects of tea catechin epigallocatechin-3-gallate (EGCG) against biofilm formation by Streptococcus mutans and probiotic Lactobacillus casei in Yakult^® (LcY) were examined.DesignBiofilms were formed by S. mutans alone (Sm) and co-culture of S. mutans and LcY (Sm + LcY) in the absence or presence of EGCG. The biomass of biofilms, which were sonicated or not, was measured by the crystal violet assay. Biofilm morphology was observed by scanning electron microscopy. Bacterial viability and extracellular polysaccharides were determined by SYTO9/propidium iodide and dextran-conjugated fluorescein staining, respectively, and confocal microscopy. Gene expression of glucosyltransferase was determined by quantitative polymerase chain reaction.ResultsWhile 250 μg/ml EGCG significantly decreased the biomass and acid production of Sm biofilms, 500 μg/ml EGCG was required to inhibit Sm + LcY biofilm formation and acid production. EGCG decreased the amount of live bacteria present in both Sm and Sm + LcY biofilms. The level of dead bacteria in Sm + LcY biofilms was higher than in Sm biofilms when formed in the presence of 250 μg/ml EGCG. EGCG decreased levels of extracellular polysaccharides in Sm and Sm + LcY biofilms. The extent of biofilm removal by sonication was not different between Sm and Sm+LcY biofilms formed in the absence or presence of 62.5 or 125 μg/ml EGCG. The level of Sm gtfB and gtfD expression in Sm + LcY biofilms was higher than those in the Sm biofilms when formed in the presence of EGCG at 250 μg/ml.ConclusionThe results indicated that LcY might interfere the inhibitory effects of EGCG against biofilm formation by S. mutans.     

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea

ObjectiveStreptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified.MethodsEffects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography.ResultsAssam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10 kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10 kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation.ConclusionsThe higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.     

Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans

Objective

The aim of this study was to evaluate the acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans.

Materials and methods

The following strains were tested: Bifidobacterium dentium DSM20436, Parascardovia denticolens DSM10105, and Scardovia inopinata DSM10107. Streptococcus mutans UA159 and Lactobacillus acidophilus ATCC4356 were used as control. Bifidobacteria were studied planktonically as they were not able to form monospecies biofilm, they were grown in biofilms associated with S. mutans. Endogenous polysaccharide reserves of cultures at log phase were depleted. Standardized suspensions of the microorganisms were incubated in growth media supplemented with 10 mM glucose, lactose, raffinose, glucose, or xylitol. S. mutans biofilms were grown on glass cover slips for 24 h to which bifidobacteria were added. After 24 h, the dual-species biofilms were exposed to the same carbon sources, and after 3 h, the pH of spent culture media and concentrations of organic acids were measured. Statistical analyses were carried out using ANOVA and Tukey’s test (α = 0.05).

Results

A higher pH drop was observed when S. mutans was associated with P. denticolens or S. inopinata, in either planktonic or biofilm cultures, than with S. mutans alone. Bifidobacteria showed a higher pH drop in the presence of raffinose than S. mutans or L. acidophilus.

Conclusions

Dual-species biofilms of bifidobacteria and S. mutans produced more acid and greater pH drops than biofilms of S. mutans alone.

Clinical relevance

New insights on the complex process of caries pathogenicity contribute to the establishment of preventive and therapeutic measures, in particular in specific cases, such as in early childhood caries.

     

Curcumin reduces Streptococcus mutans biofilm formation by inhibiting sortase A activity

BackgroundSortase A is an enzyme responsible for the covalent attachment of Pac proteins to the cell wall in Streptococcus mutans. It has been shown to play a role in modulating the surface properties and the biofilm formation and influence the cariogenicity of S. mutans. Curcumin, an active ingredient of turmeric, was reported to be an inhibitor for Staphylococcus aureus sortase A. The aim of this study was to investigate the inhibitory ability of curcumin against S. mutans sortase A and the effect of curcumin for biofilm formation.MethodsThe antimicrobial activity of the curcumin to the S. mutans and inhibitory ability of the curcumin against the purified sortase A in vitro were detected. Western-blot and real-time PCR were used to analysis the sortase A mediated Pac protein changes when the S. mutans was cultured with curcumin. The curcumin on the S. mutans biofilm formation was determined by biofilm formation analysis.ResultsCurcumin can inhibit purified S. mutans sortase A with a half-maximal inhibitory concentration (IC₅₀) of (10.2 ± 0.7) μmol/l, which is lower than minimum inhibitory concentration (MIC) of 175 μmol/l. Curcumin (15 μmol/l) was found to release the Pac protein to the supernatant and reduce S. mutans biofilm formation.ConclusionsThese results indicated that curcumin is an S. mutans sortase A inhibitor and has promising anti-caries characteristics through an anti-adhesion-mediated mechanism.     

Impact of combined CO2 laser irradiation and fluoride on enamel and dentin biofilm-induced mineral loss

Objectives

The caries-protective effects of CO₂ laser irradiation on dental enamel have been demonstrated using chemical demineralization models. We compared the effect of CO₂ laser irradiation, sodium fluoride, or both on biofilm-induced mineral loss (∆Z) and Streptococcus mutans adhesion to enamel and dentin in vitro.

Materials and methods

Ground, polished bovine enamel, and dentin samples were allocated to four groups (n = 12/group): no treatment (C); single 22,600-ppm fluoride (F) varnish (5 % NaF) application; single CO₂ laser treatment (L) with short pulses (5 μs/λ = 10.6 μm); and laser and subsequent fluoride treatment (LF). Samples were sterilized and submitted to an automated mono-species S. mutans biofilm model. Brain heart infusion plus 5 % sucrose medium was provided eight times daily, followed by rinses with artificial saliva. After 10 days, bacterial numbers in biofilms were enumerated as colony-forming units/ml (CFU/ml) (n = 7/group). ∆Z was assessed using transversal microradiography (n = 12/group). Univariate ANOVA with post hoc Tukey honestly-significant-difference test was used for statistical analysis.

Results

Bacterial numbers were significantly higher on dentin than enamel (p < 0.01/ANOVA). On dentin, LF yielded significantly lower CFUs than other groups (p = 0.03/Tukey), while no differences between groups were found for enamel. The lowest ∆Z in enamel was observed for L (mean/SD 2036/1353 vol%×μm), which was not only significantly lower than C (9642/2452 vol%×μm) and F (7713/1489 vol%×μm) (p < 0.05) but also not significantly different from LF (3135/2628 vol%×μm) (p > 0.05). In dentin, only LF (163/227) significantly reduced ∆Z (p < 0.05).

Conclusion/clinical relevance

CO₂ laser irradiation did not increase adhesion of S. mutans in vitro. Laser treatment alone protected enamel against biofilm-induced demineralization, while a combined laser-fluoride application was required to protect dentin.

     

Caries Preventive Effects of Theobromine Containing Toothpaste on Early Childhood Caries: Preliminary Results

ObjectiveEnamel remineralizing effects of theobromine have received much attention from the clinicians. The aim of this study was to investigate the caries preventive effect of theobromine containing toothpaste on children with early childhood caries (ECC).Material and MethodsSalivary pH, buffering capacity and frequency of Streptococcus mutans (SM) levels were measured. Each child was assigned either fluoridated or theobromine containing toothpaste. The changes were analyzed using Laser Fluorescence system. Statistical analyses were performed.ResultsWe included 13 children (mean age 4.25) with 145 teeth in the fluoridated toothpaste (Colgate Kids toothpaste) group; 13 children (mean age 4.46) with 115 teeth in theobromine containing toothpaste (Theodent^TMKids toothpaste) group. Both toothpastes demonstrated enamel remineralization and were effective in increasing the buffering capacity and pH (p < 0.05). A statistically significant decrease in S. mutans levels was found in both toothpaste groups (p<0.05).ConclusionBoth toothpaste group showed a statistically significant amount of enamel remineralization. Since theobromine had the added benefits of increasing the salivary pH and decreasing the S.mutans levels, theobromine containing toothpastes can be considered effective agents in remineralizing white spot lesions and can be used in prevention of early enamel lesions.Key words: Toothpastes, Theobromine, Tooth Remineralization, Preventive Dentistry, Preschool Child