Cariogenic Biofilm Research Articles

Novel Bioactive Nanocomposites Containing Calcium Fluoride and Calcium Phosphate with Antibacterial and Low-Shrinkage-Stress Capabilities to Inhibit Dental Caries.

Composites are commonly used for tooth restorations, but recurrent caries often lead to restoration failures due to polymerization shrinkage-stress-induced marginal leakage. The aims of this research were to: (1) develop novel low-shrinkage-stress (L.S.S.) nanocomposites containing dimethylaminododecyl methacrylate (DMADDM) with nanoparticles of calcium fluoride (nCaF2) or amorphous calcium phosphate (NACP) for remineralization; (2) investigate antibacterial and cytocompatibility properties. Nanocomposites were made by mixing triethylene glycol divinylbenzyl ether with urethane dimethacrylate containing 3% DMADDM, 20% nCaF2, and 20% NACP. Flexural strength, elastic modulus, antibacterial properties against Streptococcus mutans biofilms, and cytotoxicity against human gingival fibroblasts and dental pulp stem cells were tested. Nanocomposites with DMADDM and nCaF2 or NACP had flexural strengths matching commercial composite control without bioactivity. The new nanocomposite provided potent antibacterial properties, reducing biofilm CFU by 6 logs, and reducing lactic acid synthesis and metabolic function of biofilms by 90%, compared to controls (p < 0.05). The new nanocomposites produced excellent cell viability matching commercial control (p > 0.05). Bioactive L.S.S. antibacterial nanocomposites with nCaF2 and NACP had excellent bioactivity without compromising mechanical and cytocompatible properties. The new nanocomposites are promising for a wide range of dental restorations by improving marginal integrity by reducing shrinkage stress, defending tooth structures, and minimizing cariogenic biofilms.

Effects of Tea Polyphenols and Theaflavins on Three Oral Cariogenic Bacteria.

In order to investigate the antibacterial mechanism of tea polyphenols and theaflavins against oral cariogenic bacteria, the pH value of the culture medium, the number of bacteria adhering to the smooth glass tube wall, and the electrical conductivity value within 10 h were measured, respectively. The effects of four concentrations of tea polyphenols and theaflavins below the MIC value were studied on acid production, adhesion, and electrical conductivity of oral cariogenic bacteria. The live/dead staining method was used to observe the effects of four concentrations of tea polyphenols and theaflavins below the MIC value on the biofilm formation of oral cariogenic bacteria under a laser scanning confocal microscope. With the increase in concentrations of tea polyphenols and theaflavins, the acid production and adhesion of the cariogenic bacteria gradually decreased, and the conductivity gradually increased. However, the conductivity increase was not significant (p < 0.05). Compared with the control group, the 1/2MIC and 1/4MIC tea polyphenols and theaflavins treatments significantly reduced the biomass of the cariogenic biofilm (p < 0.05). The confocal laser scanning microscope showed that the integrated optical density of green fluorescence of the cariogenic biofilm gradually decreased with the increase in agent concentration after the action of tea polyphenols and theaflavins.

Mutanase from Trichoderma harzianum inductively Produced by Mutan: Short-Term Treatment to Degrade Mature Streptococcus mutans Biofilm.

This study aimed to evaluate the disruptive effect of fungal mutanase against cariogenic biofilm after short-term treatment. For that, mature Streptococcus mutans biofilms (n = 9) were exposed to active or inactivated enzymes produced by Trichoderma harzianum for 1 min, two times per day. Biofilms were analyzed by amount of matrix water-insoluble polysaccharides, bacterial viability, acidogenicity, and morphology by scanning electron microscopy (SEM). The group treated with active enzymes (AE) had a significantly lower amount of insoluble polysaccharides (893.30 ± 293.69) when compared to the negative control group (NaCl, 2192.59 ± 361.96), yet no significant difference was found when comparing to the positive control group (CHX, 436.82 ± 151.07). Also, there was no significant effect on bacteria metabolism and viability (P-value < 0.05). Data generated by the quantitative analysis were confirmed through scanning electron microscopy images. Thus, fungal mutanase degraded the biofilm after a short-term treatment without interfering with bacterial viability and metabolism. Such findings offer insight to the development of routine oral care products containing this input.

New Preventive Strategy against Oral Biofilm Formation in Caries-Active Children: An In Vitro Study.

Quorum quenching (QQ) is the inhibition of bacterial communication, i.e., quorum sensing (QS). QS is a key mechanism in regulating biofilm formation and phenotype in complex bacterial communities, such as those found within cariogenic biofilms. Whereas QQ approaches were shown to effectively reduce biomass, knowledge of their impact on the taxonomic composition of oral polymicrobial biofilms remains scarce. Here, we investigate the effect of the QQ lactonase Aii20J on biomass production and taxonomical composition of biofilms. We collected supragingival plaque samples from 10 caries-free and 10 caries-active children and cultured them to generate in vitro biofilms. We describe significant biomass reductions upon Aii20J exposure, as assessed by crystal violet assays. Taxonomical profiling using 16S rRNA gene amplicon sequencing revealed no significant changes in bacterial composition at the genus level. Interestingly, at the species level Aii20J-treatment increased the abundance of Streptococcus cristatus and Streptococcus salivarius. Both S. cristatus and S. salivarius express pH-buffering enzymes (arginine deiminase and urease, respectively) that catalyze ammonia production, thereby potentially raising local pH and counteracting the biofilm's cariogenic potential. Within the limitations of the study, our findings provide evidence of the biofilm-modulating ability of QQ and offer novel insights into alternative strategies to restore homeostasis within dysbiotic ecosystems.

Antimicrobial activity of probiotic Streptococcus salivarius LAB813 on in vitro cariogenic biofilms

ObjectiveTo investigate the antimicrobial activity of a novel commensal strain of Streptococcus salivarius, LAB813, against Streptococcus mutans biofilms. MethodsThe inhibitory activity of LAB813 towards S. mutans was tested using mono-, dual-, and multi-species cariogenic biofilms formed on three types of orthodontic appliances (metal, ceramic, aligner). The activity of the commercially available probiotic, BLIS M18™ was used as control. ResultsLAB813 significantly inhibited S. mutans biofilms with cell killing approximating 99% for all materials. LAB813 showed effectiveness at inhibiting S. mutans in more complex multi-species biofilms with cell killing approximating 90% for all three materials. When comparing the killing kinetics of the probiotics, LAB813 had a faster rate of killing biofilms than M18. Experiments conducted with cell-free culture supernatant confirmed the presence of an inhibitory substance of proteinaceous nature. The addition of xylitol, a common sugar substitute used for human consumption, potentiated the inhibitory effects of LAB813 against S. mutans embedded in a more complex fungal-bacterial biofilm. ConclusionsLAB813 possesses strong antimicrobial activity, potent anti-biofilm properties, and enhanced antimicrobial activity in the presence of xylitol. The identification and characterization of strain LAB813 exhibiting antimicrobial activity towards S. mutans hold exciting promise for this novel strain to be developed as an oral probiotic for use in the prevention of dental caries.

Anticaries properties of natural berries: systematic literature review.

Anticariogenic properties have been ascribed to polyphenolic compounds present in high concentrations in numerous fruits. Berries, in particular, have been reported as potentially having an inhibitory effect on the dental biofilm and subsequently on caries, but the evidence is unclear. The objective of this review was to explore the literature and summarize the evidence for berries having an inhibitory effect on the dental biofilm and an anticariogenic effect. Following Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines, the PubMed, Web of Science, and SCOPUS databases were scanned using predefined and accessible terms, with a search strategy based on a structured PICO question. After article selection, 23 studies met the inclusion criteria, most of them being in vitro studies. A risk assessment was performed, and data were extracted and presented in a table for qualitative analysis. Meta-analyses were conducted using standardized mean differences (SMDs) with a 95% confidence interval (CI) by Review manager 5.4. Only 3 types of berries were found to have a reported anticaries effect: grape seed extract (GSE), cranberry, and sour cherry. Nine studies that fulfilled the eligibility criteria were subjected to quantitative analysis. Meta-analyses showed GSE was associated with enhanced remineralization of dental enamel (SMD = .96 95% CI [.45, 1.46], P < .0002) and of dentin (SMD = .65 95% CI [.13, 1.17], P = .01). Cranberry extracts positively influenced the cariogenic dental biofilm by decreasing the biofilm biomass (SMD = -2.23 95% CI [-4.40, -.05], P = .04), and biovolume (SMD = -2.86 95% CI [-4.34, -1.37], P = .0002), and increasing the biofilm pH (SMD = 7.9 95% CI [3.49, 12.31], P < .0004). Within the limitations of this systematic review and metaanalysis, GSE and cranberries or their active compounds could represent an alternative for caries management. Further clinical trials are needed to verify this effect in a clinical setting. PROSPERO registration no. CRD42020223579.

Effects of caffeic acid phenethyl ester against multi-species cariogenic biofilms.

Dental caries is a biofilm-related disease, widely perceived to be caused by oral ecological imbalance when cariogenic/aciduric bacteria obtain an ecological advantage. Compared with planktonic bacteria, dental plaques are difficult to remove under extracellular polymeric substance protection. In this study, the effect of caffeic acid phenethyl ester (CAPE) on a preformed cariogenic multi-species biofilm was evaluated, which was comprised of cariogenic bacteria (Streptococcus mutans), commensal bacteria (Streptococcus gordonii), and a pioneer colonizer (Actinomyces naeslundii). Our result revealed that treatment with 0.08mg/mL CAPE reduced live S. mutans in the preformed multi-species biofilm while not significantly changing the quantification of live S. gordonii. CAPE significantly reduced the production of lactic acid, extracellular polysaccharide, and extracellular DNA and made the biofilm looser. Moreover, CAPE could promote the H2O2 production of S. gordonii and inhibit the expression of SMU.150 encoding mutacin to modulate the interaction among species in biofilms. Overall, our results suggested that CAPE could inhibit the cariogenic properties and change the microbial composition of the multi-species biofilms, indicating its application potential in dental caries prevention and management.

Epigallocatechin-3-gallate/mineralization precursors co-delivery hollow mesoporous nanosystem for synergistic manipulation of dentin exposure.

As a global public health focus, oral health plays a vital role in facilitating overall health. Defected teeth characterized by exposure of dentin generally increase the risk of aggravating oral diseases. The exposed dentinal tubules provide channels for irritants and bacterial invasion, leading to dentin hypersensitivity and even pulp inflammation. Cariogenic bacterial adhesion and biofilm formation on dentin are responsible for tooth demineralization and caries. It remains a clinical challenge to achieve the integration of tubule occlusion, collagen mineralization, and antibiofilm functions for managing exposed dentin. To address this issue, an epigallocatechin-3-gallate (EGCG) and poly(allylamine)-stabilized amorphous calcium phosphate (PAH-ACP) co-delivery hollow mesoporous silica (HMS) nanosystem (E/PA@HMS) was herein developed. The application of E/PA@HMS effectively occluded the dentinal tubules with acid- and abrasion-resistant stability and inhibited the biofilm formation of Streptococcus mutans. Intrafibrillar mineralization of collagen fibrils and remineralization of demineralized dentin were induced by E/PA@HMS. The odontogenic differentiation and mineralization of dental pulp cells with high biocompatibility were also promoted. Animal experiments showed that E/PA@HMS durably sealed the tubules and inhibited biofilm growth up to 14 days. Thus, the development of the E/PA@HMS nanosystem provides promising benefits for protecting exposed dentin through the coordinated manipulation of dentin caries and hypersensitivity.

The effect of synbiotic-fluoride therapy on multi-species biofilm

ObjectivesThe study objective was to examine the effect of synbiotic-fluoride (SF) therapy within a multi-species cariogenic biofilm model system comprising of S. mutans, S. sanguinis, and S. gordonii. MethodsThe SF therapy was prepared using 2% L-arginine (Arg), 0.2% NaF and probiotic L. rhamnosus GG (LRG). The 8 treatment groups were: Group 1: No treatment, Group 2: 2% Arg, Group 3: 0.2% NaF, Group 4: LRG, Group 5: 2% Arg+0.2% NaF, Group 6: 2% Arg+LRG, Group 7: 0.2% NaF+LRG, and Group 8: SF therapy (2% Arg+0.2% NaF +LRG). Multi-species biofilm model over 96 h comprising Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii was utilized. The biofilms received cariogenic challenge and SF therapy 2 × /day. The extracellular matrix components were analyzed for carbohydrates, proteins, and extra-cellular DNA (eDNA). The live/dead cells were imaged and quantified using confocal microscopy. The viable/dead bacterial concentrations were estimated using propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR). The gene expressions for gtfB, sagP, arcA, argG, and argH were measured using real-time reverse transcriptase qPCR. ResultsCarbohydrates and protein content with SF therapy were higher than non-LRG containing groups, while eDNA content was lower than other groups (p<0.05). Live bacterial proportions determined using confocal imaging with SF therapy were the lowest (p<0.05). The 2% Arg+LRG and SF therapy showed higher viable L. rhamnosus GG than 0.2% NaF+LRG (p<0.05). The dead S. mutans with SF therapy were higher than the other groups (p<0.05) with no difference from 2% Arg+0.2% NaF and 2% Arg+LRG (p>0.05). The SF therapy significantly downregulates gtfB and upregulates sagP, arcA, argG, argH gene expression (p<0.05). ConclusionSynbiotic-fluoride therapy effectuates multi-fold changes in the multi-species biofilm matrix and cellular components leading to superior ecological homeostasis than its individual contents, prebiotics (arginine), probiotic (L. rhamnosus GG), and fluorides (NaF). Clinical significanceThe ecological-based synbiotic-fluoride caries-preventive therapy aids in maintaining biofilm homeostasis to preempt/restore dysbiosis thereby sustaining dynamic-diverse health-associated microbial stability significant as a preventive regimen for high caries-risk patients.

Main approaches of biofilm formation in composite resins: a concise systematic review

Introduction: Composite resins are widely used in clinical dental practice to restore the structure lost by caries and dental defects, achieving aesthetic and functional results. Failures in the photoactivation process can cause problems in the mechanical resistance to wear in regions of direct occlusal contact on the restorative material. This occurs due to the reduction in the degree of conversion of monomers into polymers, compromising the clinical performance of the composite resin. Polymerization shrinkage leads to microleakage of the restoration, leading to staining and the development of bacterial biofilms. Objective: It was to carry out a systematic review of the main scientific evidence of biofilm formation on composite resin veneers, identifying potential predictors, as well as proposals to mitigate this problem. Methods: The systematic review rules of the PRISMA Platform were followed. The search was carried out from December 2022 to March 2023 in the Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases, using articles from 2015 to 2022. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed accordingly, according to the Cochrane instrument. Results and Conclusion: A total of 125 articles were found, 25 articles were evaluated and 20 were included and developed in this systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 10 studies with a high risk of bias and 70 studies that did not meet GRADE. It was concluded that the surface properties of resin-based composite materials, as well as surface treatments, can strongly affect bacterial adhesion and biofilm formation. In addition, scientific evidence highlights that cariogenic biofilm formation can alter the surface properties of materials, thus stimulating bacterial adhesion and biofilm formation. Photoactivation allows the curing and crosslinking of polymer chains, essential for the clinical longevity of the composite resin. Insufficient photoactivation culminates in marginal microleakage in restorations and biofilm accumulation on surfaces exposed to the oral environment.

Comparative Evaluation of Antimicrobial Efficacy of Fluoride-Based and Self-Assembling Peptide P11-4-based Tooth Remineralization Agents on Streptococcus mutans: A Microbiological Study.

Dental caries is a biofilm-related oral disease that continues to afflict the majority of the world's population. The disease results from an interaction between specific bacteria and dietary constituents within a biofilm known as dental plaque. Among the cariogenic microorganisms, Streptococcus mutans (S. mutans) plays pivotal role in caries-inducing processes. Evaluate and compare the antimicrobial efficacy of self-assembling peptide P11-4-based tooth remineralization agents on S. mutans. An in vitro microbiological study. The antibacterial efficacy of self-assembling peptide P11-4 gel (Group 1), fluoride enhanced hydroxyapatite gel (Group 2), acidulated phosphate fluoride gel (Group 3), chlorhexidine gluconate gel 1.0% w/w (Group 4; positive control), and normal saline (Group 5; negative control) was performed using time-kill assay over a period of 24 h and the number of S. mutans colony-forming units (CFUs) were calculated. Statistical analysis was done using Kruskal-Wallis test and Mann-Whitney post hoc Test. The level of significance was set at P < 0.05. Group 1 showed mean CFUs (× 103) of 841.33 ± 3.51, Group 2 showed 10035.33 ± 60.68, Group 3 showed 1058.00 ± 56.96, Group 4 showed 0.00 ± 0.00, and Group 5 showed mean CFUs with 15226.67 ± 96.67. The difference in the mean CFUs (× 103) between different groups was statistically significant at P < 0.001. The self-assembling peptide P11-4-based tooth remineralization agent exhibited an inhibitory influence on S. mutans and hence formation of cariogenic bacteria dominant biofilm can thus be affected by its application.

Detection of Streptococcus mutans and Streptococcus sobrinus and Their Association with Oral Microbiome Stressors in 6-18-month-old Infants.

To determine the presence of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) and their association with extrinsic and intrinsic variables in 6-18-month-old infants. This was an analytical, cross-sectional study of 65 6-18-month-old infants who visited the Centers for Early Childhood in Buenos Aires City. Three groups were established according to the presence of teeth-group I (GI)-edentulous infants, group II (GII)-infants with 1-8 teeth, and group III (GIII)-infants with 9-16 teeth. Data on the variables, diet, use of artificial teats, and oral hygiene were gathered using a self-administered questionnaire. An oral examination was performed according to the International Caries Detection and Assessment System (ICDAS II) criterion. A saliva sample was taken by aspiration with a sterile plastic syringe. Cariogenic Streptococci (CS) were counted using the adherence test in modified gold broth (AT-MGB). Molecular detection and quantification were performed by quantitative polymerase chain reaction (qPCR) (gtfB, gtfT, and tuf). A total of 12% of infants received oral hygiene, 38% used bottles, 30% used pacifiers, and 55% had sugar intake. S. sobrinus and S. mutans were detected in 57.1 and 28.6% of the children with caries, respectively. Groups I, II, and III had CS counts of log 2, 3.4, and 3.7, respectively. S. sobrinus was detected in 26.7% of GI, 52.9% of GII, and 85.7% of GIII, while S. mutans was detected in 13.3%, 35.3%, and 57.7%, respectively. The prevalence of S. sobrinus was higher than S. mutans in all groups. The presence of CS was significantly associated with sugar intake. No association was found between S. mutans and S. sobrinus and the presence of caries, hygiene habits, or use of artificial teats. This study supports the role of diet in developing a cariogenic biofilm in children under 2 years of age. Cornejo CF, Soken LJ, Salgado PA, et al. Detection of Streptococcus mutans and Streptococcus sobrinus and Their Association with Oral Microbiome Stressors in 6-18-month-old Infants. Int J Clin Pediatr Dent 2023;16(1):68-73.

Effectiveness of a Combined Toothbrushing Technique on Cariogenic Dental Biofilm in Relation to Stainless Steel and Elastomeric Ligatures in Orthodontic Patients: A Randomized Clinical Trial

Increased dental biofilm commonly occurs during orthodontic treatment. The aim of this study was to evaluate the effect of a combined toothbrushing method on dental biofilm cariogenicity in patients with stainless steel (SSL) and elastomeric (EL) ligatures. At baseline (T1), 70 participants were randomized (1:1 ratio) to the SSL or EL group. Dental biofilm maturity was evaluated using a three-color-disclosing dye. The participants were instructed to brush their teeth using a combined horizontal-Charters-modified Bass technique. Dental biofilm maturity was reassessed at the 4-week follow-up (T2). We found that at T1, new dental biofilm was the highest, followed by mature and cariogenic dental biofilm in the SSL group (p < 0.05). In the EL group, cariogenic dental biofilm was highly observed, followed by mature and new dental biofilm (p < 0.05). After intervention, cariogenic dental biofilm significantly decreased in both groups (p < 0.05). Moreover, a marked decrease in cariogenic dental biofilm was observed in the EL group compared with the SSL group (p < 0.05). However, the change in mature dental biofilm in the groups was similar (p > 0.05). Our results demonstrated that the combined toothbrushing method reduced cariogenic dental biofilm in the SSL and EL groups.

The Activity of Calcium Glycerophosphate and Fluoride against Cariogenic Biofilms of Streptococcus mutans and Candida albicans Formed In Vitro.

This study evaluated the effects of calcium glycerophosphate (CaGP), with or without fluoride (F), on dual-species biofilms of Streptococcus mutans and Candida albicans. The biofilms were treated three times with 0.125, 0.25, and 0.5% CaGP solutions, with or without 500 ppm F (NaF). Additionally, 500 and 1100 ppm F-solutions and artificial saliva served as controls. After the final treatment, the microbial viability and biofilm structure, metabolic activity, total biomass production, and the composition of the extracellular matrix composition were analyzed. Regardless of the presence of F, 0.25 and 0.5% CaGP promoted a higher biomass production and metabolic activity increase than the controls (p < 0.05). F-free CaGP solutions reduced bacterial cell population significantly more than the 500 ppm F group or the negative control (p < 0.05). All the groups reduced the proteins, and 0.5% CaGP combined with F led to the highest reduction in the carbohydrate and nucleic acids content of the extracellular matrix (p < 0.05). It can be concluded that CaGP alone affected the number of bacterial cells and, when combined with F, reduced its production of biomass, metabolic activity, and the expression of the extracellular matrix components.

Bovine Milk and Yogurt Affect Oral Microorganisms and Biofilms In-Vitro.

To evaluate the effect of bovine milk and yogurt on selected oral microorganisms and different oral biofilms. Milk was prepared from 0.5% fat (low-fat) and 16% fat (high fat) milk powder. For yogurt preparation, the strains Lactobacillus delbrueckii ssp. bulgarcius and Streptococcus thermophilus were added to the milk. Minimal inhibitory concentrations (MIC) and minimal microbiocidal concentrations (MMC) of the test compounds were measured against various microorganisms by the microbroth dilution technique. Cariogenic periodontal biofilms and one containing Candida were created on plastic surfaces coated with test substances. Further, preformed biofilms were exposed to the test substances at a concentration of 100% for 10 min and thereafter 10% for 50 min. Both colony forming units (cfu) and metabolic activity were quantified in the biofilms. Neither high-fat milk, low-fat milk nor casein inhibited the growth of any species. Yogurt and L. delbrueckii ssp. bulgaricus at low MIC and MMC suppressed the growth of Porphyromonas gingivalis and other bacteria associated with periodontal disease. High-fat yogurt decreased cfu in the forming periodontal biofilm by 90%. Both low- and high-fat yogurts reduced metabolic activity in newly forming and preformed periodontal and Candida biofilms, but not in the cariogenic biofilm. Yogurt and L. delbru eckii ssp. bulgaricus, but not milk, were bactericidal against periodontopathogenic bacteria. Yoghurt reduced the metabolic activity of a Candida biofilm and a periodontal biofilm. Yogurt and L. delbrueckii ssp. bulgaricus may have potential in prevention and therapy of periodontal diseases and Candida infections.

Effect of Extracts, Fractions, and Isolated Molecules of Casearia sylvestris to Control Streptococcus mutans Cariogenic Biofilm

The effects of extracts, fractions, and molecules of Casearia sylvestris to control the cariogenic biofilm of Streptococcus mutans were evaluated. First, the antimicrobial and antibiofilm (initial and pre-formed biofilms) in prolonged exposure (24 h) models were investigated. Second, formulations (with and without fluoride) were assessed for topical effects (brief exposure) on biofilms. Third, selected treatments were evaluated via bacterium growth inhibition curves associated with gene expression and scanning electron microscopy. In initial biofilms, the ethyl acetate (AcOEt) and ethanolic (EtOH) fractions from Brasília (BRA/DF; 250 µg/mL) and Presidente Venceslau/SP (Water/EtOH 60:40 and Water/EtOH 40:60; 500 µg/mL) reduced ≥6-logs vs. vehicle. Only the molecule Caseargrewiin F (CsF; 125 µg/mL) reduced the viable cell count of pre-formed biofilms (5 logs vs. vehicle). For topical effects, no formulation affected biofilm components. For the growth inhibition assay, CsF yielded a constant recovery of surviving cells (≅3.5 logs) until 24 h (i.e., bacteriostatic), and AcOEt_BRA/DF caused progressive cell death, without cells at 24 h (i.e., bactericidal). CsF and AcOEt_BRA/DF damaged S. mutans cells and influenced the expression of virulence genes. Thus, an effect against biofilms occurred after prolonged exposure due to the bacteriostatic and/or bactericidal capacity of a fraction and a molecule from C. sylvestris.

Safety Assessment of the Modified Lactoperoxidase System-In Vitro Studies on Human Gingival Fibroblasts.

One strategy in caries prevention is to inhibit the formation of cariogenic biofilms. Attempts are being made to develop oral hygiene products enriched with various antimicrobial agents. One of them is lactoperoxidase-an enzyme that can oxidise (pseudo)halide ions to reactive products with antimicrobial activity. Currently, commercially available products utilise thiocyanate as a substrate; however, several alternatives that are oxidised to products with greater antimicrobial potential have been found. In this study, toxicity against human gingival fibroblasts of the lactoperoxidase system was evaluated using four different (pseudo)halide substrate systems-thiocyanate, iodide, selenocyanate, and a mixture of thiocyanate and iodide. For this purpose, cells were treated with the systems and then apoptosis, cell cycle, intracellular glutathione concentration, and mitochondrial superoxide production were assessed. The results showed that each system, after generating 250 µM of the product, inhibited cell divisions, increased apoptosis, and increased the percentage of dead cells. It was concluded that the mechanism of the observed phenomena was not related to increased superoxide production or the depletion of glutathione concentration. These findings emphasised the need for the further in vitro and in vivo toxicity investigation of the modified lactoperoxidase system to assess its safety and the possibility of use in oral hygiene products.

Druggable sites identification in Streptococcus mutans VicRK system evaluated by catechols

Dental caries is a global public health problem, being the most common non-communicable disease. Streptococcus mutans, the causative agent of human cariogenic dental biofilms, produce glycosyltransferases (Gtfs) whose gene expression is modulated by the VicRK system, which makes them a promising target for dental biofilm inhibitor developments. Bioinformatics have playing a significant role in drug discovery programs mainly in novel hit identification. In this study, potential inhibitors against the S. mutans VicK system have been identified through Structure-based Virtual Screening performed between the VicK druggable sites followed byMolecular Dynamic simulations (MD) with binding affinity analysis by MM-PBSA approach. First, VicK protein was downloaded from PDB, and druggability analyses were performed by PockDrug and FTMap servers describing three interaction sites (S1, S2, and S3) that covered the most important domains for stability and activity. Next, a catechol virtual screening (n = 383) was performed on AutoDock4.2, and better-docked catechols showed strong binding affinity interaction through hydrogen bonding, hydrophobic interactions, and π-stacking with VicK auto kinase and phosphatase activity sites. Ligand efficiency indexes were also calculated (LE, LELP, LLE, and BEI) and showed optimal values. Furthermore, a 200 ns MD simulation run showed stability (RMSD and RMSF) and a high number of hydrogen bonds into peltatoside and maritimein, the two best VicK complexes. These results supported that catechols could potentially inhibit exopolysaccharides synthesis and be used in the biofilm management of new anti-cariogenic and antimicrobial agents.

Exopolysaccharides of lactic acid bacteria isolated from honeybee gut and effects of their antibiofilm activity against Streptococcus mutans.

Streptococcus mutans is one of the main factors in formation of cariogenic biofilms. New strategies need to be developed to reduce the formation of cariogenic biofilms. For this purpose, bacterial exopolysaccharides (EPS) could be considered as new agents against biofilms. Therefore, cell-bound (b-EPS) and released exopolysaccharides (r-EPS) were extracted from the strains Apilactobacillus kunkeei K1.10 and Latilactobacillus curvatus Kar.9b isolated from the microbiota of honeybees, and their antibiofilm effects on S. mutans biofilm formation were determined. The highest reduction in biofilm formation was achieved by r-EPS of L. curvatus Kar.9b and A. kunkeei K1.10. Scanning electron micrographs (SEM) showed that r-EPS inhibited biofilm formation by reducing adhesion of S. mutans. To increase the production of r-EPS from A. kunkeei K1.10, the effects of different incubation conditions were also analyzed. The highest EPS production was obtained during 48 h-incubation at 37ºC in a medium containing 1% fructose. r-EPS can be used as a raw material to inhibit cariogenic biofilms. Further studies revealing the detailed structural analysis of r-EPS and the mechanism of action of its antibiofilm effect could be beneficial. Finally, b-EPS and r-EPS from lactic acid bacteria were found to have very different properties in terms of their antibiofilm properties.

Effect of titanium dioxide on Streptococcus mutans biofilm.

Streptococcus mutans (S. mutans) participates in the dental caries process. Titanium dioxide (TiO2) nanoparticles produce reactive oxygen species capable of disrupting bacterial DNA synthesis by creating pores in cell walls and membranes. The objective of this study was to determine the effect of TiO2 on the disruption of S. mutans biofilm. This study was conducted in four phases involving a TiO2-containing toothbrush and TiO2 nanoparticles. Each phase was completed using 24 h established S. mutans biofilm growth. Phase one data was collected through a bacterial plating study, assessing biofilm viability. Biofilm mass was evaluated in phase two of the study by measuring S. mutans biofilm grown on microtiter plates following crystal violet staining. The third phase of the study involved a generalized oxygen radical assay to determine the relative amount of oxygen radicals released intracellularly. Phase four of the study included the measurement of insoluble glucan/extracellular polysaccharide (EPS) synthesis using a phenol-sulfuric acid assay. Both exposure time and time intervals had a significant effect on bacterial viability counts (p = 0.0323 and p = 0.0014, respectively). Bacterial counts after 6 min of exposure were significantly lower than after 2 min (p = 0.034), compared to the no treatment control (p = 0.0056). As exposure time increased, the amount of remaining biofilm mass was statistically lower than the no treatment control. Exposure time had a significant effect on oxygen radical production. Both the 30 and 100 nm TiO2 nanoparticles had a significant effect on bacterial mass. The silver nanoparticles and the 30 and 100 nm TiO2 nanoparticles significantly inhibited EPS production. The TiO2-containing toothbrush kills, disrupts, and produces oxygen radicals that disrupt established S. mutans biofilm. TiO2 and silver nanoparticles inhibit EPS production and reduce biofilm mass. The addition of TiO2 to dental products may be effective in reducing cariogenic dental biofilm.