Influence of Streptococcus mutans on Enterococcus faecalis Biofilm Formation

IntroductionAn important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of E. faecalis in the presence of other microorganisms.MethodsEight clinical strains of E. faecalis were tested for biofilm formation on hydroxyapatite disks in the presence and absence of a Streptococcus mutans biofilm.ResultsSignificantly more E. faecalis viable cells were found in biofilms in the presence of S. mutans. This phenomenon was, however, strain-dependent. Of the 8 strains tested, biofilm formation of strains AA-OR34, ER5/1, and V583 was not influenced by S. mutans biofilms.ConclusionsThe results from this study, especially the strain difference, underline the importance of studying biofilm formation in a more realistic multispecies setting.     

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.     

Epigallocatechin-3-gallate (EGCG) enhances the therapeutic activity of a dental adhesive

ObjectivesThe purpose of this study was to evaluate the antibacterial potential and physicochemical properties of a dental adhesive incorporated with epigallocatechin-3-gallate (EGCG) in different concentration over time.MethodsEGCG was incorporated at a ratio of 100, 200, and 300 μg/ml into a dental adhesive. The effects of the cured adhesives on the growth of Streptococcus mutans were determined by direct contact test immediately or one month later and by scanning electron microscopy (SEM), respectively. Microtensile bond strength (μTBS) test was used to test the mechanical property of the adhesives immediately or six months later. The degree of conversion (DC) of the adhesives was evaluated by Fourier Transform Infrared Spectroscopy (FTIR).ResultsCompared with negative control, the 200 μg/ml and 300 μg/ml EGCG-incorporated dental adhesive were found to exhibit inhibitory effect on the growth of S. mutans. The μTBS of the EGCG-incorporated dental adhesive was higher than the control. The DC of the adhesive system was not affected by the addition of EGCG.Conclusions200 μg/ml EGCG incorporated dental adhesives could accomplish therapeutic goals that play in antimicrobial function whilst keeping the durability of resin–dentine bond.     

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.     

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.     

Chemical composition of Pistacia vera L. oleoresin and its antibacterial,anti-virulence and anti-biofilm activities against oral streptococci,including Streptococcus mutans

ObjectiveThe aim of this study was to characterize the chemical composition of oleoresin of Pistacia vera L. and to determine its antimicrobial and anti-virulence activity versus selected oral streptococci.DesignA gaschromatografic analysis of the oleoresin was performed. The antimicrobial and anti-virulence activity of the oleoresin and its fractions was evaluated by the Minimum Inhibitory Concentration (MIC) and/or Minimum Bactericidal Concentration (MBC), biofilm production and haemolytic activity inhibition experiments.ResultsThe oleoresin MBCs were ≥1024 μg/mL for all tested strains; the neutral and acidic fraction MBCs ranged from 128 to 2048 μg/mL. Essential oil’s MBCs (from 256 to 2048 μg/mL) were almost identical to MICs, suggesting a bactericidal effect. P. vera oleoresin at sub-lethal concentrations significantly reduced biofilm production by Streptococcus mutans (up to 49.4%) and by Streptococcus sanguinis (up to 71.2%). In addition, the acidic fraction showed a specific anti-biofilm activity against S. mutans (up to 41.3% reduction). A significant dose-dependent reduction in the haemolytic activity of S. mutans (up to 65.9%) and of S. anginosus (up to 78.3%) was observed after growth in the presence of oleoresin at sub-lethal concentrations. The acidic fraction reduced haemolytic activity (up to 54.3% at 64 μg/mL) of S. mutans only.ConclusionsGiven the anti-virulence activity of the P. vera oleoresin and its acidic fraction against S. mutans, our findings suggest their potential use in oral hygiene. These data represent the first step in the exploitation of P. vera L. oleoresin.     

Oral spray containing plant-derived compounds is effective against common oral pathogens

ObjectivesPlant-derived compounds are a good source of therapeutic agents and inhibitors of inflammatory process. Dental caries, periodontal diseases and candidiasis are common oral infections caused by virulent biofilms. The objectives of this study were to develop oral spray containing plant-derived compounds; α-mangostin (α-MG) and/or lawsone methyl ether (2-methoxy-1,4-naphthoquinone) (LME) and determine its antimicrobial, anti-biofilm, and anti-inflammatory activities.DesignOral spray formulations were prepared containing α-MG (5 mg/ml) and/or LME (250 μg/ml). Antimicrobial activity against Candida albicans, Streptococcus mutans, and Porphyromonas gingivalis and anti-biofilm formation activities were determined as well as cytotoxicity and anti-inflammatory effects.ResultsThe oral spray demonstrated antimicrobial activity against all three of the oral pathogens tested with stronger effects on C. albicans and S. mutans than P. gingivalis. The formulation containing α-MG (2.5 mg/ml) and LME (125 ug/ml) reduced growth of the microorganisms about 1–2 Log CFU/ml at 1–3 h and the killing effects were complete at 24 h. Based on biofilm assay, the oral spray containing both α-MG and LME showed greater inhibitory effects than those with α-MG or LME. In addition, the oral spray containing both α-MG and LME demonstrated more inhibition of nitric oxide production than α-MG alone. All the formulations were safe and demonstrated greater anti-inflammatory activity at lower concentration (<6.25 μg/ml) than at a higher concentration.ConclusionOral spray containing α-MG and/or LME is effective against common oral pathogens without significant cytotoxicity. Thus, it has the potential to prevent the infections and may serve as adjunctive treatment to conventional therapy.     

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.     

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.     

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.     

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.     

New perspective to improve dentin–adhesive interface stability by using dimethyl sulfoxide wet-bonding and epigallocatechin-3-gallate

ObjectivesTo determine whether dentin–adhesive interface stability would be improved by dimethyl sulfoxide (DMSO) wet-bonding and epigallocatechin-3-gallate (EGCG).MethodsEtched dentin surfaces from sound third molars were randomly assigned to five groups according to different pretreatments: group 1, water wet-bonding (WWB); group 2, 50% (v/v) DMSO wet-bonding (DWB); groups 3–5, 0.01, 0.1, and 1 wt% EGCG-incorporated 50% (v/v) DMSO wet-bonding (0.01%, 0.1%, and 1%EGCG/DWB). Singlebond universal adhesive was applied to the pretreated dentin surfaces, and composite buildups were constructed. Microtensile bond strength (μTBS) and interfacial nanoleakage were respectively examined after 24 h water storage or 1-month collagenase ageing. In situ zymography andStreptococcus mutans (S. mutans) biofilm formation were also investigated.ResultsAfter collagenase ageing, μTBS of groups 4 (0.1%EGCG/DWB) and 5 (1%EGCG/DWB) did not decrease (p > 0.05) and was higher than that of the other three groups (p < 0.05). Nanoleakage expression of groups 4 and 5 was less than that of the other three groups (p < 0.05), regardless of collagenase ageing. Metalloproteinase activities within the hybrid layer in groups 4 and 5 were suppressed. Furthermore, pretreatment with 1%EGCG/DWB (group 5) efficiently inhibited S. mutans biofilm formation along the dentin–adhesive interface.SignificanceThis study suggested that the synergistic action of DMSO wet-bonding and EGCG can effectively improve dentin–adhesive interface stability. This strategy provides clinicians with promising benefits to achieve desirable dentin bonding performance and to prevent secondary caries, thereby extending the longevity of adhesive restorations.     

Candida tropicalis biofilm inhibition by ZnO nanoparticles and EDTA

ObjectiveBiofilm of Candida tropicalis denote as a complex cellular congregation with major implication in pathogenesis. This lifestyle of fungus as a biofilm can inhibit immune system and antifungal therapy in treatment of infectious disease especially medical device associated chronic disease. In this study effects of Zinc Oxide (ZnO) nanoparticles and EDTA were evaluated on C. tropicalis biofilm by using different techniques. ZnO nanoparticles were synthesized from Egg albumin.DesignTo assay the formation of biofilm of yeast cells like Fluconazole-susceptible C. tropicalis (ATCC 13,803) and fluconazole-resistant standard strains of C. tropicalis (ATCC 750) were grown in 24 well plates and antifungal effect of ZnO and EDTA were evaluated on C. tropicalis biofilm using ATP bioluminescence and tetrasodium salt (XTT) reduction assays.ResultsSynthesized ZnO NPs and EDTA had effective antifungal properties at the concentration of 5.2, 8.6 μg/ml for Fluconazole susceptible strain and 5.42, 10.8 μg/ml Fluconazole resistant strains of C. tropicalis biofilms compared to fluconazole drug.ConclusionIn present study we conclude, ZnO considered as a new agent in field of prevention C. tropicalis biofilms especially biofilms formed surface of medical device.     

Inhibitory effects of the phenolic fraction from the pomace of Vitis coignetiae on biofilm formation by Streptococcus mutans

ObjectivesThe anti-cariogenic properties of the phenolic fraction from the pomace of Vitis coignetiae (VcPP) were evaluated by in vitro assays and compared with fruit juices from V. coignetiae and common grapes and with other phenolic fractions. The effects of VcPP against the biofilm of Streptococcus mutans were investigated.DesignSucrose-dependent biofilm formation by S. mutans cultured in the presence of VcPP was measured by crystal violet dye uptake. Inhibition of adhesion to the saliva-coated hydroxyapatite (sHA) beads was quantified using fluorescent-labelled cells. The MIC for S. mutans was determined by colony counting on agar plates containing VcPP. The ability of VcPP to inhibit glucan synthesis by three distinct recombinant glucosyltransferases (Gtfs) was assessed by quantifying the production of water-soluble and -insoluble polysaccharides in bacterial cultures. In addition, the buffering effect of VcPP in cultures of S. mutans was evaluated.ResultsVcPP reduced adhesion of S. mutans to sHA and biofilm formation in a dose-dependent manner. The MIC of VcPP was 7.50 mg/ml. VcPP inhibited GtfB activity associated with the synthesis of water-insoluble glucans. It also inhibited GtfD activity associated with the synthesis of water-soluble glucans at a concentration which was lower than that used for inhibition of GtfB. VcPP had no effect on acidification associated with glucose utilization by S. mutans.ConclusionsThe current study supports the potential of VcPP as a food additive for reducing caries by inhibiting adhesion to the tooth surface and GtfD-mediated soluble glucan synthesis.     

Anti-biofilm activity of silver nanoparticle-containing glass ionomer cements

ObjectiveTo develop a silver nanoparticle (AgNP) formulation for incorporation into glass ionomer cements (GICs) which minimises biofilm growth on restoration surfaces.MethodsGICs, Fuji IX, Ketac Molar, and Riva Selfcure were modified with 6, 10 and 24 μg per GIC capsule of α-lipoic acid-capped AgNPs. Monoculture biofilms of Streptococcus mutans were cultured (72 h) on GIC specimens (n = 3) and biofilm accumulation was quantified using a viability stain with confocal laser scanning microscopy. Compression strength and flexural strength (CS & FS) were measured according to ISO 9917-1:2007 (n = 8, n = 25). GIC colour was measured at 0, 1, and 14 days following AgNP incorporation using a digital spectrophotometer. Silver release from AgNP-modified GIC specimens was monitored at 1, 3, 7 and 14 days using inductively coupled plasma-mass spectrometry.ResultsAgNP-modified Fuji IX demonstrated the greatest reduction in biofilm accumulation, with 10 μg Ag/capsule inhibiting biofilm formation by 99%. Ketac Molar and Riva Selfcure required 24 μg Ag/capsule to achieve 78% biofilm reduction. AgNP-modified GICs demonstrated significantly higher CS and FS than sintered silver-containing GICs, and possessed equivalent or higher strength values when compared to unmodified GICs. The colour shades of AgNP-modified GICs were more comparable to VITA shades of non-modified GICs than were sintered silver-containing GICs. The silver (≥99.6%) remained within the GIC for at least two weeks following incorporation.SignificanceAgNP-modified GICs exhibited significant antibiofilm activity and retained mechanical properties equivalent or superior to non-modified GICs. AgNP-modified GICs could reduce bacterial colonisation on and around restorations thereby reducing restoration failure caused by secondary caries.     

Optimization of a real-time high-throughput assay for assessment of Streptococcus mutans metabolism and screening of antibacterial dental adhesives

ObjectiveThe present work shows the optimization of a high-throughput bioluminescence assay to assess the metabolism of intact Streptococcus mutans biofilms and its utility as a screening method for nanofilled antibacterial dental materials.MethodsThe assay was optimized by monitoring changes in bioluminescence mediated by variation of the experimental parameters investigated (growth media and sucrose concentration, inoculum:D-Luciferin ratio, dilution factor, inoculum volume, luminescence wavelength, replicate and luciferase metabolic activity). Confocal microscopy was then used to demonstrate the impact of biofilm growth conditions on the 3-D distribution of extracellular polymeric substance (EPS) within Streptococcus mutans biofilms and its implications as confounding factors in high-throughput studies (HTS).ResultsRelative Luminescence Unit (RLU) values from the HTS optimization were analyzed by multivariate ANOVA (α = 0.05) and coefficients of variation, whereas data from 3-D structural parameters and RLU values of biofilms grown on experimental antibacterial dental adhesive resins were analyzed using General Linear Models and Student–Newman–Keuls post hoc tests (α = 0.05). Confocal microscopy demonstrated that biofilm growth conditions significantly influenced the quantity and distribution of EPS within the 3-D structures of the biofilms. An optimized HTS bioluminescence assay was developed and its applicability as a screening method in dentistry was demonstrated using nanofilled experimental antibacterial dental adhesive resins.SignificanceThe present study is anticipated to positively impact the direction of future biofilm research in dentistry, because it offers fundamental information for the design of metabolic-based assays, increases the current levels of standardization and reproducibility while offering a tool to decrease intra-study variability.     

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.     

Comparative effect of a stannous fluoride toothpaste and a sodium fluoride toothpaste on a multispecies biofilm

ObjectivesThis paper aimed to compare the mode of action of a stannous fluoride-containing toothpaste with a conventional sodium fluoride-containing toothpaste on anti-biofilm properties.MethodsA three-species biofilm model that consists of Streptococcus mutans, Streptococcus sanguinis and Porphyromonas gingivalis was established to compare the anti-biofilm properties of a stannous fluoride-containing toothpaste (CPH), a conventional sodium fluoride-containing toothpaste (CCP) and a negative control (PBS). The 48 h biofilms were subjected to two-minute episodes of treatment with test agents twice a day for 5 consecutive days. Crystal violet staining and XTT assays were used to evaluate the biomass and viability of the treated biofilm. Live/dead staining and bacteria/extracellular polysaccharides (EPS) double-staining were used to visualize the biofilm structure and to quantify microbial/extracellular components of the treated biofilms. Species-specific fluorescent in situ hybridization and quantitative polymerase chain reaction (qPCR) were used to analyze microbial composition of the biofilms after treatment.ResultsThe biomass and viability of the biofilms were significantly reduced after CPH toothpaste treatment. The inhibitory effect was further confirmed by the live/dead staining. The EPS amounts of the three-species biofilm were significantly reduced by CCP and CPH treatments, and CPH toothpaste demonstrated significant inhibition on EPS production. More importantly, CPH toothpaste significantly suppressed S. mutans and P. gingvalis, and enriched S. sanguinis in the three-species biofilm. In all experiments CPH had a significantly greater effect than CCP (p < 0.05) and CCP had a greater effect than PBS (p < 0.05).ConclusionsStannous fluoride-containing toothpaste not only showed better inhibitory effect against oral microbial biofilm, but was also able to modulate microbial composition within multi-species biofilm compared with conventional sodium fluoride-containing toothpaste.     

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.