Cariogenic Biofilm Research Articles

In vitro assessments of antimicrobial potential and cytotoxicity activity of an orthodontic adhesive doped with nano-graphene oxide.

Formation of white spots and initial carious lesions are the most important complications of fixed orthodontic treatment. Preparation of orthodontic adhesives containing antimicrobial agents might be a practical solution for the prevention of the mentioned defects.

Metataxonomic and metabolomic evidence of biofilm homeostasis disruption related to caries: An in vitro study.

The ecological dysbiosis of a biofilm includes not only bacterial changes but also changes in their metabolism. Related to oral biofilms, changes in metabolic activity are crucial endpoint, linked directly to the pathogenicity of oral diseases. Despite the advances in caries research, detailed microbial and metabolomic etiology is yet to be fully clarified. To advance this knowledge, a meta‐taxonomic approach based on 16S rRNA gene sequencing and an untargeted metabolomic approach based on an ultra‐high performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry analysis (UHPLC/Q‐TOF‐MS) were conducted. To this end, an in vitro biofilm model derived from the saliva of healthy participants were developed, under commensal and cariogenic conditions by adding sucrose as the disease trigger. The cariogenic biofilms showed a significant increase of Firmicutes phyla (p = 0.019), due to the significant increase in the genus Streptococcus (p = 0.010), and Fusobacter (p < 0.001), by increase Fusobacterium (p < 0.001) and Sphingomonas (p = 0.024), while suffered a decrease in Actinobacteria (p < 0.001). As a consequence of the shift in microbiota composition, significant extracellular metabolomics changes were detected, showed 59 metabolites of the 120 identified significantly different in terms of relative abundance between the cariogenic/commensal biofilms (Rate of change > 2 and FDR < 0.05). Forty‐two metabolites were significantly higher in abundance in the cariogenic biofilms, whereas 17 metabolites were associated significantly with the commensal biofilms, principally related protein metabolism, with peptides and amino acids as protagonists, latter represented by histidine, arginine, l‐methionine, glutamic acid, and phenylalanine derivatives.

Effect of pH-sensitive nanoparticles on inhibiting oral biofilms

Dental caries is a biofilm-related preventable infectious disease caused by interactions between the oral bacteria and the host’s dietary sugars. As the microenvironments in cariogenic biofilms are often acidic, pH-sensitive drug delivery systems have become innovative materials for dental caries prevention in recent years. In the present study, poly(DMAEMA-co-HEMA) was used as a pH-sensitive carrier to synthesize a chlorhexidine (CHX)-loaded nanomaterial (p(DH)@CHX). In vitro, p(DH)@CHX exhibited good pH sensitivity and a sustained and high CHX release rate in the acidic environment. It also exhibited lower cytotoxicity against human oral keratinocytes (HOKs) compared to free CHX. Besides, compared with free CHX, p(DH)@CHX showed the same antibacterial effects on S. mutans biofilms. In addition, it had no effect on eradicating healthy saliva-derived biofilm, while free CHX exhibited an inhibitory effect. Furthermore, the 16s rDNA sequencing results showed that p(DH)@CHX had the potential to alter oral microbiota composition and possibly reduce caries risk. In conclusion, the present study presents an alternative option to design an intelligent material to prevent and treat dental caries.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

Dual-sensitive antibacterial peptide nanoparticles prevent dental caries.

Background: Dental caries is the most prevalent bacterial biofilm-induced disease. Current clinical prevention and treatment agents often suffer from adverse effects on oral microbiota diversity and normal tissues, predominately arising from the poor biofilm-targeting property of the agents.Methods: To address this concern, we herein report dual-sensitive antibacterial peptide nanoparticles pHly-1 NPs upon acid and lipid-binding for treatment of dental caries. Amino acid substitutions were performed to design the peptide pHly-1. The potential, morphology and secondary structure of pHly-1 were characterized to elucidate the mechanisms of its pH and lipid sensitivity. Bacterial membrane integrity assay and RNA-seq were applied to uncover the antimicrobial mechanism of peptides under acidic condition. The in vitro and ex vivo antibiofilm assays were used to determine the antibiofilm performance of pHly-1 NPs. We also carried out the in vivo anti-caries treatment by pHly-1 NPs on dental caries animal model. Oral microbiome and histopathological analyses were performed to assess the in vivo safety of pHly-1 NPs.Results: The pHly-1 peptide underwent the coil-helix conformational transition upon binding to bacterial membranes in the acidic cariogenic biofilm microenvironment, thereby killing cariogenic bacteria. Under normal physiological conditions, pHly-1 adopted a β-sheet conformation and formed nanofibers, resulting in negligible cytotoxicity towards oral microbes. However, in acidic solution, pHly-1 NPs displayed reliable antibacterial activity against Streptococcus mutans, including standard and clinically isolated strains, mainly via cell membrane disruption, and also suppressed in vitro and human-derived ex vivo biofilm development. Compared to the clinical agent chlorhexidine, in vivo topical treatment with pHly-1 NPs showed an advanced effect on inhibiting rat dental caries development without adverse effects on oral microbiota diversity and normal oral or gastric tissues.Conclusion: Our results demonstrated the high efficacy of dual-sensitive antimicrobial peptides for the selective damage of bacterial biofilms, providing an efficient strategy for preventing and treating dental caries.

Identification of Potential Glucosyltransferase Inhibitors from Cinnamic Acid Derivatives Using Molecular Docking Analysis: A Bioinformatics Study

Background: Dental caries is one of the most common oral chronic diseases. Streptococcus mutans is the main pathogenic bacteria playing a role in degrading the mineral texture of the teeth. Glucosyltransferase (GTFase) of S. mutans is responsible for producing glucan, which is the main exopolysaccharide found in the cariogenic biofilms. Further, previous studies have reported that cinnamic acid diminished biofilm formation of S. mutans. Therefore, we hypothesized that cinnamic acid and its derivatives might act as GTFase inhibitors. Methods: The binding affinity of a total of 12 plant-based compounds including cinnamic acid and its 11 derivatives to the GTFase active site were examined by utilizing the AutoDock tool. The possible interactions between top-ranked cinnamic acid derivatives and the residues within the GTFase catalytic site were also taken into consideration. Results: Five of the cinnamic acid derivatives including rosmarinic acid (RA), cynarine, chlorogenic acid (CGA), caffeic acid 3-glucoside, and N-p-coumaroyltyramine demonstrated inhibitory effects on GTFase at nanomolar concentration. Stabilizing interactions such as π–π stack pairing and pi-charge interactions were detected between top-ranked GTFase inhibitors and residues within the enzyme active site. Conclusions: The present study suggests that RA, cynarine, CGA, caffeic acid 3-glucoside, and N-p-coumaroyltyramine might have protective effects on dental caries, and therefore, may be considered as anti-tooth caries compounds.

VicRK and CovR polymorphisms in Streptococcus mutans strains associated with cardiovascular infections.

Introduction. Streptococcus mutans, a common species of the oral microbiome, expresses virulence genes promoting cariogenic dental biofilms, persistence in the bloodstream and cardiovascular infections.Gap statement. Virulence gene expression is variable among S. mutans strains and controlled by the transcription regulatory systems VicRK and CovR.Aim. This study investigates polymorphisms in the vicRK and covR loci in S. mutans strains isolated from the oral cavity or from the bloodstream, which were shown to differ in expression of covR, vicRK and downstream genes.Methodology. The transcriptional activities of covR, vicR and vicK were compared by RT-qPCR between blood and oral strains after exposure to human serum. PCR-amplified promoter and/or coding regions of covR and vicRK of 18 strains (11 oral and 7 blood) were sequenced and compared to the reference strain UA159.Results. Serum exposure significantly reduced covR and vicR/K transcript levels in most strains (P<0.05), but reductions were higher in oral than in blood strains. Single-nucleotide polymorphisms (SNPs) were detected in covR regulatory and coding regions, but SNPs affecting the CovR effector domain were only present in two blood strains. Although vicR was highly conserved, vicK showed several SNPs, and SNPs affecting VicK regions important for autokinase activity were found in three blood strains.Conclusions. This study reveals transcriptional and structural diversity in covR and vicR/K, and identifies polymorphisms of functional relevance in blood strains, indicating that covR and vicRK might be important loci for S. mutans adaptation to host selective pressures associated with virulence diversity.

Bioresponsive nanotherapy for preventing dental caries by inhibiting multispecies cariogenic biofilms

Early childhood caries (ECC) is a public healthcare concern that greatly reduces the quality of life of young children. As a leading factor of ECC, cariogenic biofilms are composed of acidogenic/aciduric pathogens and extracellular polysaccharides (EPSs), creating an acidic and protected microenvironment. Antimicrobial photodynamic therapy (aPDT) is a noninvasive, painless, and efficient therapeutic approach that is suitable for treating ECC. However, due to the hyperfine structure of cariogenic biofilms, most photosensitizers (PSs) could not access and penetrate deeply in biofilms, which dramatically hamper their efficiency in the clinic. Herein, bioresponsive nanoparticle loaded with chlorin e6 (MPP-Ce6) is developed, which largely increases the penetration depth (by over 75%) and retention (by over 100%) of PS in the biofilm compared with free Ce6. Furthermore, MPP-Ce6-mediated aPDT not only kills the bacteria in preformed biofilms but also inhibits multispecies biofilm formation. A rampant caries model is established to mimic ECC in vivo, where the population of cariogenic bacteria is decreased to 10% after MPP-Ce6-mediated aPDT. Importantly, the number and severity of carious lesions are efficiently reduced via Keyes’ scoring and micro-CT analysis. This simple but effective strategy can serve as a promising approach for daily oral hygiene in preventing ECC.

Applications of Silver Diamine Fluoride in Dentistry

Various studies published in the literature around the world indicate a significant prevalence of early childhood caries (ECC). Treatment of early childhood caries (ECC) using silver diamine fluoride (SDF) has become a viable alternative, particularly for young and anxious children, because traditional restorative treatments is unlikely to combat this prevalent condition. Arrested caries is identified by the hardening of teeth and it changes its color from dark brown to black. More restorative treatments can be performed if necessary. Traditional ECC restorative treatments are not always inexpensive or feasible, as they involve patient cooperation to achieve a satisfactory result. Japan was the first country to promote silver diamine fluoride in 1960. SDF has been used to prevent the spread of caries in various Asian nations, including Japan and China. It was enacted as a fluoride to treat sensitive teeth in the United States by Food and Drug Administration ( FDA). For young children or individuals with specific needs, it is painless and simple to use .The fluoride in SDF stimulates remineralization while the silver ions act as an antimicrobial agent when applied to carious lesions and stop cariogenic biofilms from growing. SDF also prevents future degradation of the dentin's collagen. With a 38 percent use rate, SDF has been utilized in a number of nations. According to a review, SDF is a harmless, economical caries control agent with a wide utilisation in dentistry, and it may meet the WHO Millennium Development Goal (MDG) as well as the United States Institute of Medicine's criteria. The applications of SDF in dental treatment are discussed in this article.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 μg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R2 = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes.

Nanoparticle carrier co-delivery of complementary antibiofilm drugs abrogates dual species cariogenic biofilm formation in vitro

ABSTRACT Background Dental caries is a multifactorial disease caused by pathogenic biofilm. In particular, Streptococcus mutans synthesizes biofilm exopolysaccharides, while Candida albicans is associated with the development of severe carious lesions. Aim This study aimed to prevent the formation of S. mutans and C. albicans biofilms by exploiting pH-sensitive nanoparticle carriers (NPCs) with high affinity to exopolysaccharides to increase the substantivity of multi-targeted antibiofilm drugs introduced topically in vitro. Methods Dual-species biofilms were grown on saliva-coated hydroxyapatite discs with sucrose. Twice-daily, 1.5 min topical treatment regimens of unloaded and drug-loaded NPC were used. Drugs included combinations of two or three compounds with distinct, complementary antibiofilm targets: tt-farnesol (terpenoid; bacterial acid tolerance, fungal quorum sensing), myricetin (flavonoid; exopolysaccharides inhibitor), and 1771 (lipoteichoic acid inhibitor; bacterial adhesion and co-aggregation). Biofilms were evaluated for biomass, microbial population, and architecture. Results NPC delivering tt-farnesol and 1771 with or without myricetin completely prevented biofilm formation by impeding biomass accumulation, bacterial and fungal population growth, and exopolysaccharide matrix deposition (vs. control unloaded NPC). Both formulations hindered acid production, maintaining the pH of spent media above the threshold for enamel demineralization. However, treatments had no effect on pre-established dual-species biofilms. Conclusion Complementary antibiofilm drug-NPC treatments prevented biofilm formation by targeting critical virulence factors of acidogenicity and exopolysaccharides synthesis.

Photoinactivation of multispecies cariogenic biofilm mediated by aluminum phthalocyanine chloride encapsulated in chitosan nanoparticles.

This study aimed to characterize the aluminum phthalocyanine chloride (AlClPc) encapsulated in chitosan nanoparticles (CN) and apply it in antimicrobial photodynamic therapy (aPDT) on multispecies biofilm composed of Streptococcus mutans, Lactobacillus casei, and Candida albicans to analyze the antimicrobial activity and lactate production after treatment. Biofilms were formed in 24-well polystyrene plates at 37°C for 48h under microaerophilia. The following groups were evaluated (n = 9): as a positive control, 0.12% chlorhexidine gluconate (CHX); phosphate-buffered saline (PBS) as a negative control; 2.5% CN as release vehicle control; the dark toxicity control of the formulations used (AlClPc and AlClPc + CN) was verified in the absence of light; for aPDT, after 30min incubation time, the photosensitizers at a final concentration of 5.8 × 10-3mg/mL were photoirradiated for 1min by visible light using a LED device (AlClPc + L and AlClPc + CN + L) with 660nm at the energy density of 100J/cm2. An in vitro kit was used to measure lactate. The biofilm composition and morphology were observed by scanning electron microscopy (SEM). The antimicrobial activity was analyzed by quantifying colony forming units per mL (CFU/mL) of each microorganism. Bacterial load between groups was analyzed by ANOVA and Tukey HSD tests (α = 0.05). A lower lactate dosage was observed in the aPDT AlClPc + CN + L and CHX groups compared to the CN and AlClPc groups. The aPDT mediated by the nanoconjugate AlClPc + CN + L showed a significant reduction in the viability of S. mutans (3.18 log10 CFU/mL), L. casei (4.91 log10 CFU/mL), and C. albicans (2.09 log10 CFU/mL) compared to the negative control PBS (p < 0.05). aPDT using isolated AlClPc was similar to PBS to the three microorganisms (p > 0.05). The aPDT mediated by the nanoconjugate AlClPc + CN+ L was efficient against the biofilm of S. mutans, L. casei, and C. albicans.

Anti-Biofilm Formation of an Adhesive Containing Calcium Salts of Acidic Monomers against Oral Candida Related to Root Caries

The proportion of older people within the general population is expanding due to the decline of birth rate and the increase of life expectancy. Increasing elderly populations, which is retaining its teeth longer, leads to oral health problems, for example, root caries. The main etiological factor for the initiation and development of root caries is the appearance of cariogenic biofilm. The acid-producing and acid-tolerant bacteria and oral yeast, such as Candida albicans are the major contributors in root caries formation. A new generation of adhesives is developed with several modifications for the prevention of dental caries by incorporating antimicrobial components. The aim of this study was to investigate the antimicrobial effect of adhesive containing calcium salt of acidic monomers on the biofilm formation of oral Candida related to root caries. In the experiments, the flat-bottom surfaces of 96-well plate were painted with an adhesive containing calcium salts of 4-methacryloxyethyl trimellitic acid (CMET) and 10-methacryloyloxydecyl dihydrogen calcium phosphate (MDCP) (Bio-Coat, CA, Sun Medical, Japan). Then they were LED light-cured and coated with sterile saliva at 37 °C for 60 min. The biofilm formation was made by adding of Candida albicans (ATCC 10238 and two clinical strains) suspensions (107 colony forming unit/mL) and incubated at 37°C for 24 h. The amount of vital biofilm was determined by WST-8 Microbial Cell Counting Kit (Dojindo Molecular Technologies, USA). All experiments were done in triplicate and repeated three times. Statistical analysis was performed using Student’s t-test. The results clearly showed that adhesive could significantly inhibit biofilm formation of all tested Candida compared with a control. This suppressive effect was not different among the strains of Candida. The percentages of vital biofilm reduction were 25% to 40%. The ability of this adhesive to suppress biofilm of oral yeast may be its antimicrobial property of acidic monomer or the effect of calcium ion within the adhesive, which can alter Candida cell morphology, and influence their structures or process of biofilm formation. In conclusion, an adhesive containing calcium salts of acidic monomers could significantly inhibit biofilm formation of C. albicans. This adhesive could be effectively applied to exposed root surfaces to prevent or inhibit the progression of root caries. Further studies are necessary to clarify the effect on multispecies biofilm, on long-term activity, and in vivo conditions.

Multi-Omics Reveals the Inhibition of Lactiplantibacillus plantarum CCFM8724 in Streptococcus mutans-Candida albicans Mixed-Species Biofilms.

Lactiplantibacillus plantarum CCFM8724 is a probiotic with the potential to prevent dental caries in vitro and in vivo. To explore the effects of this probiotic at inhibiting Streptococcus mutans-Candida albicans mixed-species biofilm and preventing dental caries, multi-omics, including metabolomics and transcriptomics, was used to investigate the regulation of small-molecule metabolism during biofilm formation and the gene expression in the mixed-species biofilm. Metabolomic analysis revealed that some carbohydrates related to biofilm formation, such as sucrose, was detected at lower levels due to the treatment with the L. plantarum supernatant. Some sugar alcohols, such as xylitol and sorbitol, were detected at higher levels, which may have inhibited the growth of S. mutans. In transcriptomic analysis, the expression of the virulence genes of C. albicans, such as those that code agglutinin-like sequence (Als) proteins, was affected. In addition, metabolomics coupled with a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and RNA-seq revealed that the L. plantarum supernatant had an active role in sugar metabolism during the formation of the S. mutans-C. albicans mixed-species biofilm, and the L. plantarum supernatant was also related to carbohydrate utilization, glucan biosynthesis, and mycelium formation. Hence, L. plantarum CCFM8724 decreased the mixed-species biofilm mass from the perspective of gene expression and metabolic reprogramming. Our results provide a rationale for evaluating L. plantarum CCFM8724 as a potential oral probiotic for inhibiting cariogenic pathogen biofilm formation and improving dental caries.

Effects of cigarette smoking on the growth of Streptococcus mutans biofilms: An in vitro study.

The increased incidence of dental caries by cigarette smoking (CS) has been widely reported in epidemiological studies, but the relationship between CS and cariogenic biofilm growth has been rarely studied. This study aims to investigate the effects of CS exposure on the growth and virulence of Streptococcus mutans biofilms (S. mutans). Briefly, S. mutans biofilms were formed on saliva-coated hydroxyapatite disks, which were exposed to CS 1, 3, and 6 times per day, respectively. In addition, S. mutans biofilms without CS exposure were considered as the control group. Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides (IPSs) were analyzed and confocal laser scanning microscopy (CLSM) images of 74-h-old S. mutans biofilms were obtained. The lowest accumulation of biofilms and EPSs were detected in the 6 times/day CS exposure group compared with those of the control group and other CS exposure groups in 74-h-old S. mutans biofilms. CLSM also revealed the lowest bacterial count (live and dead cells) and EPSs biovolume in the six times/day CS exposure group in 74-h-old S. mutans biofilms. CS exposure inhibited the growth of S. mutans biofilm in vitro study, the anti-cariogenic biofilm formation was enhanced with a dose (frequency)-dependent at which frequency has more influence in the present findings.

Combination Effect of Diurnal Exposure to Sucrose and Nocturnal Exposure to Lactose on Enamel Demineralization

We have hypothesized that the association between human milk and caries in breastfeeding children could be explained by the combination of a diurnal cariogenic diet with the nocturnal lactose fermentation, conditions simulated in this experimental study. Cariogenic biofilm was formed on bovine enamel slabs, which were exposed 8x/day for 3 min to a 10% sucrose solution, simulating a highly cariogenic diurnal diet, or 50 mM NaCl solution (control). Simulating the nocturnal retention of milk in mouth, biofilms were transferred to culture medium containing 0.7% lactose for 2 h, or only to culture medium (control). Four groups were designed (n = 12): Ctrl, no exposure to diurnal sucrose or nocturnal lactose; Lac, only nocturnal exposure to lactose (2 h); Suc, only diurnal exposure to sucrose (8x/day); and Suc→Lac, diurnal exposure to sucrose (8x/day) followed by nocturnal exposure to lactose (2 h). The medium was changed 3x/day, at the beginning of the day and after diurnal and nocturnal exposures. Calcium in the medium was determined as a chemical indicator of partial demineralizations occurred during the diurnal and the nocturnal treatments; the medium pH was also determined. After 96 h of growth, biofilms were harvested to evaluate CFU, biomass, and extracellular polysaccharides, soluble and insoluble. The percentage of enamel surface hardness loss (%SHL) was evaluated as cumulative demineralization. Data were analyzed by one-way ANOVA and Tukey’s test (α = 5%). Highest %SHL (p < 0.05) was found for the Suc→Lac (40.6%) group when compared to Suc (32.1%), Lac (6.6%), and Ctrl (2.4%) groups. Calcium released during the diurnal and nocturnal treatments was, respectively, Suc→Lac = Suc > Lac = Ctrl and Suc→Lac > Lac > Suc = Ctrl (p < 0.05). Regarding the Ctrl group, calcium released from nocturnal lactose fermentation by the Suc→Lac group was 4-fold greater than that provoked by the Lac group. The findings were supported by the pH of the media. The data suggest that the biofilm formed under diurnal exposure to sucrose enhances the cariogenicity of nocturnal exposure to lactose.

The effects of bimodal action of photodynamic and photothermal therapy on antimicrobial and shear bond strength properties of orthodontic composite containing nano-graphene oxide

Background: White spot lesions are a common adverse effect of fixed orthodontic treatment and represent the main challenge to achieving esthetic appearance. The purpose of the current study was to evaluate physico-mechanical and antimicrobial potency of orthodontic composite (OC; Transbond XT) containing nano-structured graphene oxide (nGO) (OC-nGO) as a novel composite following photodynamic therapy (PDT) and photothermal therapy (PTT) against Streptococcus mutans. Materials and methods: Following preparation of OC-nGO, shear bond strength (SBS) and adhesive remnant index (ARI) of the test OC-nGO (containing 1, 2, 5, and 10% wt. nGO) were measured using a universal testing machine and stereomicroscope, respectively. The antimicrobial activities of test OC contained different concentrations of nGO were determined by disk agar diffusion (DAD), biofilm formation inhibition, and eluted components assays. After continuously rinsed in the aging process (up to 180 days), the antimicrobial activity of OC-nGO containing the highest concentration of nGO which had simultaneously the highest antimicrobial activity and SBS value were determined by DAD, biofilm formation, metabolic activity, and gtfB gene expression assays following photo-activation using diode laser irradiation against S. mutans. Data were analyzed using One-way Analysis of Variance (ANOVA). The Bonferroni post hoc test was used for comparison between the experimental groups. The significant difference was considered at P values < 0.05. Results: OC with 5% wt. nGO showed simultaneously the highest SBS value (10.64 ± 2.76 MPa, P < 0.05) an antimicrobial and anti-biofilm activities. The OC-nGO in all test concentrations of nGO had ARI scores as same as control group (Transbond XT without the nGO) (P < 0.05). In microbial biofilm formation and gene expression assays, the reduction of photothermal disinfection and anti-virulence activities of the 5% wt. OC-nGO against test bacterium was associated with the time of aging process, so they were reduced significantly up to day 150. Diode laser irradiated 5% wt. OC-nGO suppressed 15.6 and 8.1-fold gtfB mRNA expressions in the biofilm growth of the S. mutans at days 120 and 150 of rinsing (P < 0.05). Microbial biofilm formation and gtfB gene expression in S. mutans at day 180 following PAD had a high level of similarity with OOC as the control group. 5% wt. OC-nGO following photo-activation was not colonized by the S. mutans at day 90 and significant suppressed 91.98% and 76.37% of S. mutans biofilm formation at day 120 and 150, respectively (both P < 0.05). From day 120 onwards, metabolic activity was progressively increased on laser-irradiated 5% wt. OC-nGO discs compared to the control group (OC alone). Photo-activated OC-nGO containing 5% wt. nGO suppressed 86.94% and 46.82% metabolic activity of the S. mutans at days 120 and 150 of rinsing (both P < 0.05). Conclusions: Our data support that the photo-activated 5% wt. OC-nGO can serve as an orthodontic composite/adhesive additive to control cariogenic bacterial biofilms.

Ursolic Acid Targets Glucosyltransferase and Inhibits Its Activity to Prevent Streptococcus mutans Biofilm Formation.

Streptococcus mutans (S. mutans), the prime pathogen of dental caries, can secrete glucosyltransferases (GTFs) to synthesize extracellular polysaccharides (EPSs), which are the virulence determinants of cariogenic biofilms. Ursolic acid, a type of pentacyclic triterpene natural compound, has shown potential antibiofilm effects on S. mutans. To investigate the mechanisms of ursolic acid-mediated inhibition of S. mutans biofilm formation, we first demonstrated that ursolic acid could decrease the viability and structural integrity of biofilms, as evidenced by XTT, crystal violet, and live/dead staining assays. Then, we further revealed that ursolic acid could compete with the inherent substrate to occupy the catalytic center of GTFs to inhibit EPS formation, and this was confirmed by GTF activity assays, computer simulations, site-directed mutagenesis, and capillary electrophoresis (CE). In conclusion, ursolic acid can decrease bacterial viability and prevent S. mutans biofilm formation by binding and inhibiting the activity of GTFs.

Effects of brief sodium fluoride treatments on the growth of early and mature cariogenic biofilms

Although fluoride has been widely used as a preventive agent for dental caries, the effects of fluoride on the activities of biofilms in different stages of cariogenic biofilm formation are less studied. This study was designed to investigate the antibiofilm activity of sodium fluoride during the early and mature stages of Streptococcus mutans (S. mutans) biofilm formation. S. mutans biofilms were formed on saliva-coated hydroxyapatite disks. In the early (0–46 h) and mature (46–94 h) biofilm stages, the biofilms were treated with different concentrations of fluoride (250, 500, 1000, 2000 ppm; 5 times in total, 1 min/treatment). Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides were analysed, and confocal laser scanning microscopy images were obtained of the two stages of biofilms to determine antibiofilm activities of fluoride at varying concentrations during the formation of early and mature biofilms. In the early stages of cariogenic biofilm formation, test groups with all fluoride concentrations significantly inhibited the growth of S. mutans biofilms. The antibiofilm and anti-EPS formation activities of the brief fluoride treatments increased with a concentration-dependent pattern. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited biofilm accumulation, activity, and intracellular/extracellular polysaccharide content compared with those of the control and other fluoride treatment groups. The antimicrobial effect of fluoride treatment on the growth of S. mutans biofilms was linked with the stage of cariogenic biofilm formation. The inhibition of S. mutans biofilm growth by fluoride treatment was easier in the early formation stage than in the mature stage. Fluoride treatment in the early stage of cariogenic biofilm formation may be an effective approach to controlling cariogenic biofilm development and preventing dental caries.

Antibacterial properties in-vitro of Mexican serviceberry extracts against dental biofilm species

BACKGROUND: The Mexican serviceberry, Malacomeles denticulata, have been used as a successful oral therapy by Mexican communities without enough scientific support. OBJECTIVE: To evaluate the M. denticulata extracts with selective antibacterial properties over dental biofilm bacteria. METHODS: Fruit, Leaf, and Stem of M. denticulata extracts were evaluated with micro-broth dilution method using ATCC bacteria. OD600 values had compared against each positive control (T-student-test). Anaerobically viability had confirmed by Colony-Forming-Units. Thin-Layer-Chromatography was used to identify the number of compounds and phytochemicals to identify secondary metabolites of the selected extracts. RESULTS: Streptococcus mutans showed Minimum-Bactericidal-Concentrations_(MBC) at 30 mg/mL to Fruit, Leaf, and Stem extracts. Periodontal-pathogens Aggregatibacter actinomycetemcomitans serotype b_(MBC = 30 mg/mL_NS); Fusobacterium nucleatum subsp. nucleatum_(MBC = 30 mg/mL_p&lt;0.05); Parvimonas micra_(MBC = 15 mg/mL_NS); Porphyromonas gingivalis_(MBC = 30 mg/mL_NS); and Prevotella intermedia_(MBC = 3.75 mg/mL_NS) presented higher sensitivity to Leaf-Methanol, than the primary colonizers. Phytochemicals showed positive results to anthraquinones, coumarins, flavonoids, saponins, saponins steroids/triterpenoids, steroids/triterpenes, and tannins/phenols. CONCLUSION: We suggest the natural extracts of fruit and leaf of the Mexican serviceberry for the preventive use over the oral cariogenic or periodontal biofilm species, by their selective antibacterial properties against pathogenic species evaluated in-vitro, and due to the presence of antibacterial secondary metabolites identified as flavonoids and saponins of M. denticulata leaf extracts.