Small RNA SmsR1 modulates acidogenicity and cariogenic virulence by affecting protein acetylation in Streptococcus mutans.

Post-transcriptional regulation by small RNAs and post-translational modifications (PTM) such as lysine acetylation play fundamental roles in physiological circuits, offering rapid responses to environmental signals with low energy consumption. Yet, the interplay between these regulatory systems remains underexplored. Here, we unveil the cross-talk between sRNAs and lysine acetylation in Streptococcus mutans, a primary cariogenic pathogen known for its potent acidogenic virulence. Through systematic overexpression of sRNAs in S. mutans, we identified sRNA SmsR1 as a critical player in modulating acidogenicity, a key cariogenic virulence feature in S. mutans. Furthermore, combined with the analysis of predicted target mRNA and transcriptome results, potential target genes were identified and experimentally verified. A direct interaction between SmsR1 and 5'-UTR region of pdhC gene was determined by in vitro binding assays. Importantly, we found that overexpression of SmsR1 reduced the expression of pdhC mRNA and increased the intracellular concentration of acetyl-CoA, resulting in global changes in protein acetylation levels. This was verified by acetyl-proteomics in S. mutans, along with an increase in acetylation level and decreased activity of LDH. Our study unravels a novel regulatory paradigm where sRNA bridges post-transcriptional regulation with post-translational modification, underscoring bacterial adeptness in fine-tuning responses to environmental stress.

Avaliação antimicrobiana de uma resina Bulk Fill Fluida com inserção do xilitol / Antimicrobial evaluation of a Fluid Bulk Fill resin with xylitol insertion

Introdução: As resinas Bulk Fill apresentam uma boa procura pelos profissionais, pois o seu uso diminui o tempo clínico, como também a melhora qualidade das restaurações, porém não possuem atividade antibacteriana, sendo um dos fatores que ainda causam impacto negativo na vida das pessoas. A biomodificação com o xilitol tem o sentido de produzir ação microbiana e com isso aperfeiçoar as suas características clínicas. Objetivo: Avaliar a ação antimicrobiana de uma resina Bulk Fill flow após a inserção do xilitol. Metodologia: A resina Tetric® N-Flow Bulk Fill foi misturada às concentrações de xilitol (0% [Controle], 2,5% e 5% p/p). Amostras cilíndricas (n=5 do grupo controle e n=3 dos demais grupos experimentais) foram confeccionadas em moldes acrílicos de diâmetro de 2mm espessura, fotoativadas por 10s e armazenadas a 37ºC por 24h. Os espécimes foram esterilizados por luz ultravioleta por 20 minutos antes de serem acomodadas em uma placa de 48 poços estéril, sendo adicionado em cada poço 0,5mL de caldo Mueller Hinton. Então, adicionou-se 50µL do inóculo de S. mutans nos poços correspondentes. A placa foi incubada a 37 ± 1 ˚C durante 48 horas. Após o período de incubação, os espécimes foram gentilmente removidos e o crescimento microbiano foi indicado pela adição de 100µL da solução aquosa de resazurina (SigmaAldrich) a 0,01% com a posterior incubação a 37 ± 1 ˚C por duas horas. Micro-organismos viáveis reduzem o corante mudando sua coloração azul para rosa e a CIM foi definida como a menor concentração da substância que inibiu a mudança de coloração da resazurina. Em um poço contendo o grupo controle foi acrescentado clorexidina a 0,12% com o intuito de comparar o resultado gerado dos grupos testes. Resultado: Não houve inibição do crescimento bacteriano nos poços com inóculos que continham S. mutans e corpo de prova de resina acrescida de xilitol. Conclusão: Esse estudo mostrou que o acréscimo de 2,5% e 5% de Xilitol à resina Tetric® N-Flow Bulk Fill não apresentou inibição do crescimento bacteriano (AU).

Introduction: Bulk Fill resins are in good demand among professionals, as their use reduces clinical time and improves the quality of restorations, but they do not have antibacterial activity, which is one of the factors that still hurt people's lives. Biomodification with xylitol aims to improve its clinical characteristics. Objective: To evaluate the antimicrobial action of a Bulk Fill flow resin after inserting xylitol. Methodology: Tetric® N-Flow Bulk Fill resin was mixed with xylitol concentrations (0% [Control], 2.5% and 5% w/w). Cylindrical samples (n=5 from the control group and n=3 from the other groups) were made in acrylic molds with a diameter of 2 mm thick, light-cured for 10 s and stored at 37ºC for 24h. The specimens were sterilized by ultraviolet light for 20 minutes before being placed in a sterile 48-well plate, with 0.5 mL of Mueller Hinton broth added to each well. The plate was incubated at 37 ± 1 ˚C for 48 hours. After the incubation period, the specimens were gently removed, and microbial growth was indicated by adding 100 µL of 0.01% resazurin aqueous solution with subsequent incubation at 37 ± 1 ˚C for two hours. Viable microorganisms reduce the dye, changing its color from blue to pink. The MIC was defined as the lowest concentration of the substance that inhibited the color change of resazurin. In 0.12%, chlorhexidine was added to a well containing the control group to compare the results generated from the test groups. Result: There was no inhibition of bacterial growth in the wells with inocula containing S. mutans and the resin specimen with xylitol added. Conclusion: This study showed that adding 2.5% and 5% Xylitol to the Tetric® N-Flow Bulk Fill resin did not inhibit bacterial growth (AU).

Evaluation of the collagen-binding properties and virulence of killed Streptococcus mutans in a silkworm model.

Streptococcus mutans, a major pathogen of dental caries, is also known as a causative agent of cardiovascular disease. A 120 kDa collagen-binding protein (Cnm) of S. mutans is an important contributor to the pathogenicity of cardiovascular disease. Although dead bacteria have been detected in cardiovascular specimens by molecular biological methods, the pathogenicity of the bacteria remains unknown. Here, we analyzed the pathogenicity of killed S. mutans by focusing on collagen-binding ability and the effects on silkworms. In live S. mutans, Cnm-positive S. mutans had high collagen-binding activity, while Cnm-negative S. mutans had no such activity. After treatment with killed Cnm-positive S. mutans, amoxicillin-treated bacteria still had collagen-binding ability, while lysozyme-treated bacteria lost this ability. When live and amoxicillin-treated S. mutans strains were administered to silkworms, the survival rates of the silkworms were reduced; this reduction was more pronounced in Cnm-positive S. mutans infection than in Cnm-negative S. mutans infection. However, the administration of any of the lysozyme-treated bacteria did not reduce the survival rate of the silkworms. These results suggest that amoxicillin-killed Cnm-positive S. mutans strains maintain collagen-binding properties and pathogenicity in the silkworm model, and are possibly associated with pathogenicity in cardiovascular diseases.

Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence.

Lysine acetylation is a frequently occurring post-translational modification (PTM), emerging as an important metabolic regulatory mechanism in prokaryotes. This process is achieved enzymatically by the protein acetyltransferase (KAT) to specifically transfer the acetyl group, or non-enzymatically by direct intermediates (acetyl phosphate or acetyl-CoA). Although lysine acetylation modification of glucosyltransferases (Gtfs), the important virulence factor in Streptococcus mutans, was reported in our previous study, the KAT has not been identified. Here, we believe that the KAT ActG can acetylate Gtfs in the enzymatic mechanism. By overexpressing 15 KATs in S. mutans, the synthesized water-insoluble extracellular polysaccharides (EPS) and biofilm biomass were measured, and KAT (actG) was identified. The in-frame deletion mutant of actG was constructed to validate the function of actG. The results showed that actG could negatively regulate the water-insoluble EPS synthesis and biofilm formation. We used mass spectrometry (MS) to identify GtfB and GtfC as the possible substrates of ActG. This was also demonstrated by in vitro acetylation assays, indicating that ActG could increase the acetylation levels of GtfB and GtfC enzymatically and decrease their activities. We further found that the expression level of actG in part explained the virulence differences in clinically isolated strains. Moreover, overexpression of actG in S. mutans attenuated its cariogenicity in the rat caries model. Taken together, our study demonstrated that the KAT ActG could induce the acetylation of GtfB and GtfC enzymatically in S. mutans, providing insights into the function of lysine acetylation in bacterial virulence and pathogenicity.

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.

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.

Inhibition of Streptococcus mutans biofilms with bacterial-derived outer membrane vesicles.

BACKGROUND: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. RESULTS: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced. CONCLUSION: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms.

Inhibition of Streptococcus mutans biofilm formation by strategies targeting the metabolism of exopolysaccharides.

Dental caries is one of the most prevalent and costly biofilm-associated infectious diseases affecting most of the world's population. In particular, dental caries is driven by dysbiosis of the dental biofilm adherent to the enamel surface. Specific types of acid-producing bacteria, especially Streptococcus mutans, colonize the dental surface and cause damage to the hard tooth structure in the presence of fermentable carbohydrates. Streptococcus mutans has been established as the major cariogenic pathogen responsible for human dental caries, with a high ability to form biofilms. The exopolysaccharide (EPS) matrix, mainly contributed by S. mutans, has been considered as a virulence determinant of cariogenic biofilm. As EPS is an important virulence factor, targeting EPS metabolism could be useful in preventing cariogenic biofilm formation. This review summarizes plausible strategies targeting S. mutans biofilms by degrading EPS structure, inhibiting EPS production, and disturbing the EPS metabolism-related gene expression and regulatory systems.

Association of Candida albicans and Cbp+ Streptococcus mutans with early childhood caries recurrence.

Early childhood caries (ECC) recurrence occurs in approximately 40% of treated cases within one year. The association of Streptococcus mutans and Candida albicans with the onset of ECC is well known. Also, S. mutans strains harboring collagen-binding proteins (Cbps) avidly bind to collagen-rich dentin and are linked to increased caries risk. Here, we investigated the presence of Cbp+ S. mutans and C. albicans in saliva and dental plaque of children with varying caries statuses, and their salivary microbiome. In this cross-sectional study, 143 children who were caries-free (n = 73), treated for ECC with no signs of recurrence after 6 months (n = 45), or treated for ECC and experiencing recurrence within 6 months following treatment (n = 25) were enrolled. Co-infection with C. albicans and S. mutans, especially Cbp+ S. mutans, was strongly associated with caries recurrence. Subjects of the recurrence group infected with Cbp+ S. mutans showed a greater burden of Candida spp. and of Mutans streptococci in dentin than those infected with Cbp- strains. Salivary microbiome analysis revealed that Streptococcus parasanguinis was overrepresented in the caries recurrence group. Our findings indicate that Cbp+ S. mutans and C. albicans are intimately associated with caries recurrence, contributing to the establishment of recalcitrant biofilms.

Mucin O-glycans suppress quorum-sensing pathways and genetic transformation in Streptococcus mutans.

Mucus barriers accommodate trillions of microorganisms throughout the human body while preventing pathogenic colonization1. In the oral cavity, saliva containing the mucins MUC5B and MUC7 forms a pellicle that coats the soft tissue and teeth to prevent infection by oral pathogens, such as Streptococcus mutans2. Salivary mucin can interact directly with microorganisms through selective agglutinin activity and bacterial binding2-4, but the extent and basis of the protective functions of saliva are not well understood. Here, using an ex vivo saliva model, we identify that MUC5B is an inhibitor of microbial virulence. Specifically, we find that natively purified MUC5B downregulates the expression of quorum-sensing pathways activated by the competence stimulating peptide and the sigX-inducing peptide5. Furthermore, MUC5B prevents the acquisition of antimicrobial resistance through natural genetic transformation, a process that is activated through quorum sensing. Our data reveal that the effect of MUC5B is mediated by its associated O-linked glycans, which are potent suppressors of quorum sensing and genetic transformation, even when removed from the mucin backbone. Together, these results present mucin O-glycans as a host strategy for domesticating potentially pathogenic microorganisms without killing them.

Mutanofactin promotes adhesion and biofilm formation of cariogenic Streptococcus mutans.

Cariogenic Streptococcus mutans is known as a predominant etiological agent of dental caries due to its exceptional capacity to form biofilms. From strains of S. mutans isolated from dental plaque, we discovered, in the present study, a polyketide/nonribosomal peptide biosynthetic gene cluster, muf, which directly correlates with a strong biofilm-forming capability. We then identified the muf-associated bioactive product, mutanofactin-697, which contains a new molecular scaffold, along with its biosynthetic logic. Further mode-of-action studies revealed that mutanofactin-697 binds to S. mutans cells and also extracellular DNA, increases bacterial hydrophobicity, and promotes bacterial adhesion and subsequent biofilm formation. Our findings provided an example of a microbial secondary metabolite promoting biofilm formation via a physicochemical approach, highlighting the importance of secondary metabolism in mediating critical processes related to the development of dental caries.

Protein Interactomes of Streptococcus mutans YidC1 and YidC2 Membrane Protein Insertases Suggest SRP Pathway-Independent- and -Dependent Functions, Respectively.

Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial cotranslational protein trafficking involves the signal recognition particle (SRP) pathway components Ffh and FtsY, the SecYEG translocon, and YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway and has two yidC paralogs. This study characterized YidC1 and YidC2 interactomes to clarify respective functions alone and in concert with the SRP and/or Sec translocon. Western blots of formaldehyde cross-linked or untreated S. mutans lysates were reacted with anti-Ffh, anti-FtsY, anti-YidC1, or anti-YidC2 antibodies followed by mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates of wild-type and ΔyidC2 strains were reacted with anti-YidC2-coupled Dynabeads, and cocaptured proteins were identified by MS. Last, YidC1 and YidC2 C-terminal tail-captured proteins were subjected to two-dimensional (2D) difference gel electrophoresis and MS analysis. Direct interactions of putative YidC1 and YidC2 binding partners were confirmed by bacterial two-hybrid assay. Our results suggest YidC2 works preferentially with the SRP pathway, while YidC1 is preferred for SRP-independent Sec translocon-mediated translocation. YidC1 and YidC2 autonomous pathways were also apparent. Two-hybrid assay identified interactions between holotranslocon components SecYEG/YajC and YidC1. Both YidC1 and YidC2 interacted with Ffh, FtsY, and chaperones DnaK and RopA. Putative membrane-localized substrates HlyX, LemA, and SMU_591c interacted with both YidC1 and YidC2. Identification of several Rgp proteins in the YidC1 interactome suggested its involvement in bacitracin resistance, which was decreased in ΔyidC1 and SRP-deficient mutants. Collectively, YidC1 and YidC2 interactome analyses has further distinguished these paralogs in the Gram-positive bacterium S. mutansIMPORTANCEStreptococcus mutans is a prevalent oral pathogen and major causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche and cause disease are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms, an important difference being the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein's interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results demonstrated substantial overlap between their interactomes but also revealed several differences in their direct protein binding partners. Membrane transport machinery components were identified in the context of a large network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general and informs our understanding of S. mutans pathogenesis.

Combinatorial therapy of chitosan hydrogel-based zinc oxide nanocomposite attenuates the virulence of Streptococcus mutans.

BACKGROUND: Biofilm formation is an important causative factor in the expansion of the carious lesions in the enamel. Hence, new approaches to efficient antibacterial agents are highly demanded. This study was conducted to evaluate the antimicrobial-biofilm activity of chitosan hydrogel (CS gel), zinc oxide/ zeolite nanocomposite (ZnONC) either separately or combined together [ZnONC / CS gel (ZnONC-CS)] against Streptococcus mutans biofilm. RESULTS: MTT assay demonstrated that the ZnONC-CS exhibits a non-cytotoxic effect (> 90% cell viability) toward human gingival fibroblast cells at different dosages (78.1-625 µg/mL) within 72 h. In comparison with CS gel and ZnONC, ZnONC-CS was superior at biofilm formation and metabolic activity reduction by 33 and 45%, respectively; (P < 0.05). The field emission scanning electron microscopy micrographs of the biofilms grown on the enamel slabs were largely in concordance with the quantitative biofilm assay results. Consistent with the reducing effect of ZnONC-CS on biofilm formation, the expression levels of gtfB, gtfC, and ftf significantly decreased. CONCLUSIONS: Taken together, excellent compatibility coupled with an enhanced antimicrobial effect against S. mutans biofilm has equipped ZnONC-CS as a promising candidate for dental biofilm control.

Probiotic Effects of Lactobacillus paracasei 28.4 to Inhibit Streptococcus mutans in a Gellan-Based Formulation.

Streptococcus mutans is considered to be a major bacterium involved in dental caries, and the control of virulence mechanisms is fundamental to prevent disease. Probiotics present a promising preventive method; however, the use of probiotics requires its incorporation into delivery materials to facilitate oral colonization. Thus, we performed a comprehensive study examining preventive effects of Lactobacillus paracasei 28.4-enriched gellan hydrogel materials to inhibit S. mutans in planktonic and biofilm states, addressing its influence in the production of extracellular polysaccharides (EPS) and altered gene expression of several cariogenic virulence factors. L. paracasei 28.4, a strain isolated from the oral cavity of a caries-free individual, was incorporated in three gellan hydrogels (0.5%, 0.75%, and 1% w/v). The pretreatment with probiotic-gellan formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the planktonic growth of S. mutans, independent of the gellan concentrations and pH variations. This pretreatment also had inhibitory activity against S. mutans biofilms, exhibiting a reduction of 0.57 to 1.54 log10 in CFU/mL (p < 0.0001) and a decrease of 68.8 to 71.3% in total biomass (p < 0.0001) compared with the control group. These inhibitory effects were associated with the decreased production of EPS by 80% (p < 0.0001) and the downregulation of luxS, brpA, gbpB, and gtfB genes. The gellan formulation containing L. paracasei 28.4 exhibited probiotic effects for preventing S. mutans growth, biofilm formation, and production of cariogenic factors to suggest possible use in tooth decay prevention.

Molecular mechanisms controlling fructose-specific memory and catabolite repression in lactose metabolism by Streptococcus mutans.

Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long-term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose-phosphate metabolites, F-1-P and F-6-P, could modulate the DNA-binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal-6-P induced the formation of different LacR-DNA complexes. Deletion of lacR resulted in strain-specific growth phenotypes on lactose, but also on a number of mono- and di-saccharides that involve the glucose-PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose-PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.

Light Energy Dose and Photosensitizer Concentration Are Determinants of Effective Photo-Killing against Caries-Related Biofilms.

Caries-related biofilms and associated complications are significant threats in dentistry, especially when biofilms grow over dental restorations. The inhibition of cariogenic biofilm associated with the onset of carious lesions is crucial for preventing disease recurrence after treatment. This in vitro study defined optimized parameters for using a photosensitizer, toluidine blue O (TBO), activated via a red light-emitting diode (LED)-based wireless device to control the growth of cariogenic biofilms. The effect of TBO concentrations (50, 100, 150, and 200 µg/mL) exposed to light or incubated in the dark was investigated in successive cytotoxicity assays. Then, a mature Streptococcus mutans biofilm model under sucrose challenge was treated with different TBO concentrations (50, 100, and 150 µg/mL), different light energy doses (36, 108, and 180 J/cm2), and different incubation times before irradiation (1, 3, and 5 min). The untreated biofilm, irradiation with no TBO, and TBO incubation with no activation represented the controls. After treatments, biofilms were analyzed via S. mutans colony-forming units (CFUs) and live/dead assay. The percentage of cell viability was within the normal range compared to the control when 50 and 100 µg/mL of TBO were used. Increasing the TBO concentration and energy dose was associated with biofilm inhibition (p < 0.001), while increasing incubation time did not contribute to bacterial elimination (p > 0.05). Irradiating the S. mutans biofilm via 100 µg/mL of TBO and ≈180 J/cm2 energy dose resulted in ≈3-log reduction and a higher amount of dead/compromised S. mutans colonies in live/dead assay compared to the control (p < 0.001). The light energy dose and TBO concentration optimized the bacterial elimination of S. mutans biofilms. These results provide a perspective on the determining parameters for highly effective photo-killing of caries-related biofilms and display the limitations imposed by the toxicity of the antibacterial photodynamic therapy's chemical components. Future studies should support investigations on new approaches to improve or overcome the constraints of opportunities offered by photodynamic inactivation of caries-related biofilms.

Optimization of Fungal Dextranase Production and Its Antibiofilm Activity, Encapsulation and Stability in Toothpaste.

Dextranase catalyzes the degradation of the substrate dextran, which is a component of plaque biofilm. This enzyme is involved in antiplaque accumulation, which can prevent dental caries. The activity of crude dextranase from Penicillium roquefortii TISTR 3511 was assessed, and the maximum value (7.61 unit/g) was obtained at 37 °C and pH 6. The Plackett-Burman design was used to obtain significant factors for enhancing fungal dextranase production, and three influencing factors were found: Dextran, yeast extract concentration and inoculum age. Subsequently, the significant factors were optimized with the Box-Behnken design, and the most suitable condition for dextranase activity at 30.24 unit/g was achieved with 80 g/L dextran, 30 g/L yeast extract and five day- old inoculum. The use of 0.85% alginate beads for encapsulation exhibited maximum dextranase activity at 25.18 unit/g beads, and this activity was stable in toothpaste for three months of testing. This study explored the potential production of fungal dextranase under optimal conditions and its encapsulation using alginate for the possibility of applying encapsulated dextranase as an additive in toothpaste products for preventing dental caries.

Effects of sub-lethal dose of antimicrobial photodynamic therapy on major virulence traits of Streptococcus mutans.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) as a complementary step of selective removal of dental caries appears as an ideal choice to help manage caries with minimal interventions. Sub-lethal dose of aPDT (sPDT) impressively effects microbial virulence, although it does not kill microorganisms. Therefore, the present study aimed to investigate the influence of sPDT using diode laser plus chlorophyllin-phycocyanin mixture (PhotoActive+) on changes in gtfB gene expression and the subsequent protein expression of GtfB. MATERIALS AND METHODS: sPDT using PhotoActive+ and 635 ± 10 nm diode laser was used to examine the expression of gtfB mRNA and GtfB protein expression of Streptococcus mutans by quantitative real time PCR (qRT-PCR) method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. RESULTS: In this study, sPDT using 2.4 × 10-3 mol/L PhotoActive+ with 3 min irradiation time of diode laser with energy density of 104 J/cm2, significantly reduced the gtfB gene expression with rates of 3.5-fold (P < 0.05). Also, PhotoActive+ mediated sPDT demonstrated a significant reduction in GtfB protein expression of S. mutans up to 54 % (P < 0.05). CONCLUSIONS: This study demonstrated the potential effect of PhotoActive+ mediated sPDT on one of the most important virulence factors of S. mutans.

Propolis nanoparticle enhances the potency of antimicrobial photodynamic therapy against Streptococcus mutans in a synergistic manner.

Less invasive removal approaches have been recommended for deep caries lesions. Antimicrobial photodynamic therapy (aPDT) and propolis nanoparticle (PNP) are highlighted for the caries management plan. Evidence is lacking for an additive effect of combination PNP with photosensitizer (PS) in aPDT. This study aimed to investigate the individual and synergistic effects of chlorophyllin-phycocyanin mixture (PhotoActive+) and toluidine blue O (TBO) as PSs in combination with PNP in the aPDT process (aPDTplus) against major important virulence factors of Streptococcus mutans. Following characterization, biocompatibility of the PSs alone, or in combination with PNP were investigated on human gingival fibroblast cell. The in vitro synergy of PhotoActive+ or TBO and PNP was evaluated by the checkerboard method. The bacteria's virulence properties were surveyed in the presence of the PSs, individually as well as in combination. When the PSs were examined in combination (synergistic effect, FIC Index < 0.5), a stronger growth inhibitory activity was exhibited than the individual PSs. The biofilm formation, as well as genes involved in biofilm formation, showed greater suppression when the PSs were employed in combination. Overall, the results of this study suggest that the combination of PhotoActive+ or TBO with PNP with the least cytotoxicity effects and the highest antimicrobial activites would improve aPDT outcomes, leading to synergistic effects and impairing the virulence of S. mutans.

Effect of bioactive compounds on the regulation of quorum sensing network-associated genes and virulence in Streptococcus mutans-A systematic review.

OBJECTIVE: The aim of this study was to systematically review the literature on the effect of bioactive compounds and their regulation of quorum sensing (QS)-related and/or -regulated-virulence genes expression in Streptococcus mutans. DESIGN: The search strategy was conducted through the electronic databases Pubmed, Scopus, and Web of Science for studies reporting the effects of natural and synthetic bioactive compounds on the regulation of QS-associated and/or -regulated genes of S. mutans. RESULTS: After full-text reading, 19 studies met the inclusion criteria, in most of them, QS-inhibitors from synthetic origin were evaluated, 16 articles described the effect of the compounds on biofilm formation cultivated in vitro and five studies described these effects on adhesion of biofilm-producing cells. Only 2 studies analyzed the potential target-component of the QS. CONCLUSIONS: Mostly, the bioactive compounds affected the expression of QS-associated and/or -regulated genes and virulence traits (e.g. adhesion, biofilm formation, acid stress tolerance) of S. mutans. Further studies are necessary to elucidate the target-specific QS-system constituent used by bioactive compounds to achieve QS inhibition as well as validate the use of these compounds in controlling dental caries.