Cariogenic Biofilm Research Articles

Anti-biofilm and anti-glucosyltransferase effects of nano liposomal plant extracts against Streptococcus mutans

The role of Streptococcus mutans in the initiation of caries is related to its acidogenicity, aciduricity, and polysaccharides extracellular layer production by glucosyltransferases in dental biofilms. Therefore, inhibition of glucosyltransferase activity impairs the virulence of cariogenic biofilms, which can be used to prevent dental caries. We evaluated the anti-bacterial, anti-biofilm, and anti-glucosyltransferases effects of nanoliposomal herbal aqueous extracts of Liquorice (Glycyrrhiza glabra; G. glabra), Ginger (Zingiber officinale; Z. officinale), Pomegranate (Punica granatum; P. granatum), and Rose (Rosa damascene; R. damascene) via minimum bactericidal concentration and minimum inhibitory concentration against Streptococcus mutans strain ATCC 35,668. An anti-biofilm assay was performed using a minimum biofilm inhibitory concentration test. Among herbs, only P. granatum showed an antibacterial effect. Therefore, a nanoliposomal formulation of P. granatum was developed and characterized. Its effect on S.mutans glucosyltransferases was assessed by measuring glucan amount. The nanoliposomal formulation of P.granatum showed a significantly higher anti-biofilm effect than P. granatum aqueous extract. Their similar potential in blocking glucosyltransferases showed that the nanoliposomal formulation of P.granatum blocked other pathways rather than blocking glucosyltransferases for its anti-biofilm effect. Collectively, the nanoliposomal formulation of P.granatum, due to its anti- Streptococcus mutans characteristics, would be a production which open a new horizon for the oral pharmaceutical industry.

Illumina Sequencing in Conjunction with Propidium Monoazide to Identify Live Bacteria After Antiseptic Treatment in a Complex Oral Biofilm: A Study Using an Ex Vivo Supragingival Biofilm Model

Background/Objectives: The evaluation of the efficacy of antibacterial treatments in complex oral ecosystems is limited by the inability to differentiate live from dead bacteria using omic techniques. The objective of this study was therefore to assess the ability of the combination of the 16S rRNA Illumina sequencing methodology and the action of propidium monoazide (PMA) to study viable bacterial profiles in oral biofilms after exposure to an antiseptic compound. Methods: Cariogenic supragingival biofilms were developed in an ex vivo model for 96 h, using saliva from healthy volunteers. The biofilms were treated with 0.12% chlorhexidine (CHX) combined with 0.05% cetylpyridinium chloride (CPC), for 60 s, using phosphate buffered saline as a control. After exposure, each biofilm was treated or not with PMA to then extract the bacterial DNA, quantify it by Qubit, quantify the bacterial population using qPCR, and perform the metataxonomic study of the samples using Illumina 16S rRNA sequencing. Results: A significantly lower DNA concentration in the PMA-treated biofilms (p < 0.05 compared with those not exposed to PMA) was observed. The viable bacterial count obtained by qPCR differed significantly from the total bacterial count in the biofilm samples exposed to the antiseptic (p < 0.05). The viable microbiome differed significantly from the total bacterial profile of the samples treated with CHX/CPC after exposure to PMA (p < 0.05 at the α- and β-diversity levels). Conclusions: The combination of Illumina 16S rRNA sequencing and PMA helps solve the inability to evaluate the efficacy of antibacterial treatments in the bacterial profile of complex ecosystems such as oral biofilms.

Back Cover: Lysine aggregates‐based nanostructured antimicrobial peptides for cariogenic biofilm microenvironment‐activated caries treatment

Back Cover: Lysine aggregates‐based nanostructured antimicrobial peptides for cariogenic biofilm microenvironment‐activated caries treatment

Preventing Dental Caries with Calcium-Based Materials: A Concise Review

This concise review provides an update on the use of calcium-based materials for the prevention of dental caries. Some calcium-based materials promote remineralization and neutralize bacterial acids, disrupting cariogenic biofilms and inhibiting bacterial growth. Medical Subject Headings of [Dental Caries] and [Calcium] were adopted to search publications. Information related to the aim of this review was extracted and summarized. Common calcium-based materials are calcium phosphate, hydroxyapatite, calcium carbonate, calcium fluoride and casein phosphopeptide–amorphous calcium phosphate (CPP-ACP). Calcium phosphate is commonly used in toothpaste. It provides calcium and phosphate ions, enhances the incorporation of fluoride into caries lesions and increases mineral density. Hydroxyapatite is a form of calcium phosphate that is chemically similar to the mineral found in teeth. It can be applied on teeth to prevent caries. Calcium carbonate can be found in toothpastes. It neutralizes bacterial acids and acts as a calcium reservoir during remineralization. Calcium fluoride is found in dental products and promotes remineralization as a source of fluoride, which can be incorporated into tooth enamel, forming fluorapatite and increasing resistance to caries. CPP-ACP is derived from milk proteins. It contains calcium and phosphate, which help to remineralize tooth enamel. CPP-ACP inhibits cariogenic bacteria. It also interacts with bacterial biofilms and disrupts their formation. These calcium-based materials can be used to boost the preventive effect of fluorides or, alternatively, as a therapy for caries prevention.

Inhibitory effect of helium cold atmospheric plasma on cariogenic biofilms

ABSTRACT This study aimed to determine the effects of low-temperature plasma jet produced in gas helium (LTP-helium) on cariogenic biofilms composedby Streptococcusmutans, Streptococcus sanguinis and Streptococcus gordonii, and also by the combination of Candida albicans, Lactobacillus acidophilus and S. mutans. Biofilms were treated for 1, 3, 5, and 7 minutes. A 0.12% chlorhexidine solution was used as the positive control and sterile physiologic solution was the negative control. Biofilm viability was analyzed by viable cell recovery, scanning electron microscopy, and confocal laser scanning microscopy. All assays were performed intriplicate in three independent experiments. Multispecies biofilms exposed to LTP-helium had a significant reduction in viability when compared to the negative control (p < 0.0001). For biofilm formedby S. mutans, S. sanguinis, and S. gordonii, LTP treatments for 5 and 7 minutes caused similar reduction of morethan 2 log10. Also, a significant reduction in the viability of biofilms formedby C. albicans, L. acidophilus, and S. mutans was detected (p < 0.0001). In conclusion, LTP-helium reduced theviability of cariogenic biofilms with different microbial compositions, which indicates that LTP-helium is a potential tool for developing new protocols for dental caries prevention and treatment.

Exploring the efficacy of in-vitro low-temperature plasma treatment on single and multispecies dental cariogenic biofilms

The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms.

Comparative evaluation of cranberry extract and sodium fluoride as mouth rinses on S. mutans counts in children: a double-blind randomized controlled trial.

Recently, research in the field of caries prevention is emphasizing upon identification of edible non-toxic compounds that can interfere with the formation of cariogenic biofilm. This trial aimed to assess the effectiveness of non-dialyzable material (NDM) containing cranberry mouth rinse (C-MR) on the Streptococcus mutans (S. mutans) counts and compare with that of a sodium fluoride (F-MR) mouth rinse. 280 children (8-12years old) were enrolled in this double-blind single center, parallel, one-month, IRB/IEC approved non-inferiority trial. The participants fulfilling the inclusion criteria were randomly allocated into one of the two groups: C-MR and F-MR. All participants were given verbal and written instructions about at-home mouth-rinsing regimen under parental supervision for a month. Dental plaque samples were collected before and after a month of mouth-rinsing and subjected to culture techniques. S. mutans counts were assessed and compared in both groups. Primary outcome was the difference in the S. mutans counts between two interventions. Both intention-to-treat (ITT) and per-protocol analysis were carried out using two-sample t test with equal variance. The mean S. mutans counts (after log transformation) in C-MR group were: 14.66 (90% CI 14.4,14.9) at baseline and 12.85 (90% CI 12.5, 13.2) colony-forming units/ml (CFU/ml) at one-month post intervention (p = 0.001); while these counts were 14.69 (90% CI 14.5, 14.9) at baseline and 12.71 (90% CI 12.3, 13.1) at one-month intervention in F-MR groups (p = 0.001). ITT analysis showed an inter- group difference of 0.14CFU/ml (90% CI -0.32,0.59) in post-SM counts between groups (p = 0.629). The study demonstrated that the cranberry mouth rinse was non-inferior to the fluoride mouth rinse in terms of S. mutans levels change. Cranberry-based mouth rinse can be used effectively to reduce the S. mutans counts in children. Registration number in case of Clinical Trials-CTRI/2019/05/019395.

Prevention of secondary caries using fluoride-loaded chitosan nanoparticle-modified glass-ionomer cement.

To study the effect of incorporating chitosan and fluoride-loaded chitosan nanoparticles into a glass-ionomer cement (GIC) to prevent secondary caries. A standard cervical cavity (mesio-distal width 6mm, cervico-occlusal width 2mm, and depth 2mm) was prepared on 30 molars for the following restoration groups: group 1, conventional GIC restoration; group 2, chitosan (10%) modified GIC restoration; group 3, fluoride loaded chitosan nanoparticles (10%) modified GIC restoration. The restored teeth were subjected to 1,500 thermal cycles before undergoing a multi-species cariogenic biofilm challenge. The restored teeth were examined by micro-computed tomography (micro-CT), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX). Data were analyzed by the one-way ANOVA, Tukey HDS, Kruskal Wallis, and Dunn's test. Micro-CT determined outer lesion depths for groups 1-3 were: 614 ± 20μm, 589 ± 17μm, and 560 ± 19μm respectively. Both modifications with chitosan and fluoride-loaded chitosan nanoparticles significantly affected outer lesion depth (p < 0.05). The modification with fluoride-loaded chitosan nanoparticles statistically significantly decreased the outer lesion depth compared to all other groups (p < 0.05). SEM/EDX showed an increase of calcium, phosphorus, and fluoride at the root dentine adjacent to the restoration in groups 2 and 3 (modified GIC). This increase was statistically significantly higher in the group modified with fluorine-loaded nano chitosan particles compared to the other groups (p < 0.05). Incorporation of 10% chitosan and 10% fluoride-loaded chitosan nanoparticles into GIC restorative material can prevent secondary root caries development. 10% fluoride-loaded chitosan nanoparticles were more effective. Glass ionomer cement modified with fluoride-loaded chitosan nanoparticles may be a promising restorative material in pediatric and preventive dentistry due to their controlled release properties.

Preventing proximal enamel caries in neighboring tooth with glass ionomer cement restoration and silver diamine fluoride pretreatment

ObjectiveTo investigate caries preventive effects of 38 % silver diamine fluoride (SDF) pretreatment on neighboring tooth proximal to glass ionomer cement (GIC), including conventional GIC (CGIC) and resin-modified GIC (RMGIC) restorations in an in vitro model. Methods: Human tooth blocks were restored withSDF+CGIC (Group 1), CGIC (Group 2), SDF+RMGIC (Group 3) or RMGIC (Group 4). Enamel specimen simulating proximal surface of neighboring tooth was placed in proximity to the restorations. The specimen underwent cariogenic challenge with cross-kingdom biofilm of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans. After cariogenic challenge, the biofilm's growth kinetics, viability, and morphology were evaluated by propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), respectively. The enamel lesion depth, surface morphology and crystal characteristics were determined by micro-computed tomography (micro-CT), SEM and X-ray diffraction (XRD), respectively. ResultsPMA-qPCR demonstrated lower microbial growth in Group 1 and 3 compared with Group 2 and 4 (p < 0.05). CLSM showed the dead−to-live ratio in Groups 1–4 were 1.15±0.12, 0.53±0.13, 1.10±0.24 and 0.63±0.10, respectively (Group 1,3 > 2,4, p < 0.05). SEM revealed Groups 1 and 3 had scattered biofilm whereas Group 2 and 4 had confluent biofilm. Micro-CT showed the enamel lesion depths (µm) were 98±9, 126±7, 103±6 and 128±7 for Group 1 to 4, respectively (Group 1,3 < 2,4, p < 0.05). SEM revealed oriented and ordered enamel prismatic patterns in Group 1 and 3, not in Group 2 and 4. XRD showed the reflections of hydroxyapatite in Groups 1 and 3 were sharper than Groups 2 and 4. ConclusionSDF pretreatment enhances the preventive effect of GIC on proximal enamel surface on neighboring tooth through inhibiting cariogenic biofilm, reducing enamel demineralization and promoting enamel remineralization. Clinical significanceSDF pretreatment of GIC restorations can help prevent caries on neighboring teeth, particular for patients with high caries risk.

Lacticaseibacillus rhamnosus inhibits the development of dental caries in rat caries model and in vitro

ObjectivesDental caries result from a microbial imbalance in the oral cavity. Probiotics ecologically modulate the oral microflora to prevent caries. This study evaluated the anti-cariogenic effects of two Lacticaseibacillus rhamnosus strains in vitro and in vivo to provide a more theoretical basis for its clinical applications in caries prevention. MethodsIn the study, cariogenic biofilms were grown with L. rhamnosus (LGG) or L. rhamnosus ATCC 7469 and analyzed. Quantitative real-time PCR (qPCR), Scanning Electron Microscope (SEM), and Confocal laser scanning microscope (CLSM) were used to detect the changes in the composition and architectures; cariogenic activity was measured by the lactic acid production and Transverse Microradiography (TMR). The effects of LGG on the 12 Sprague-Dawley rat caries model were assessed using Keyes scores and micro-CT analysis. Oral microbiome changes were evaluated through 16S rRNA gene high-throughput sequencing. ResultsL. rhamnosus can reduce cariogenic bacteria in biofilm by 14.7 % to 48.9 %, with LGG exhibiting more potent inhibitory effects. Both strains of L. rhamnosus can adhere to the surface of biofilms, reduce the extracellular polysaccharides (EPS) matrix, and loosen the biofilm structure. L. rhamnosus inhibited cariogenic activity by reducing the lactic acid production in biofilms. The bovine enamel blocks presented lower mineral loss values and lesion depth values in the group Core+L.rh and Core+LGG. LGG-ingested rats had significantly lower levels of moderate dentin lesions and higher mineral density than the control group. The 16 s rRNA gene sequencing revealed that LGG regulated the beta diversity of the oral microbial community in the rat dental caries model. ConclusionsThis study revealed the promising potential of L. rhamnosus, especially the LGG strain, in the ecological prevention of dental caries. Clinical SignificanceProbiotics may provide a strategy for preventing caries by regulating the oral microecological balance. The study revealed the promising anti-caries potential of the LGG probiotic strain in vivo and in vitro. It is expected that LGG could be used as an oral probiotic for the clinical prevention and treatment of caries.

Engineering a dysbiotic biofilm model for testing root caries interventions through microbial modulation

BackgroundThis study aimed to engineer and optimise a dysbiotic biofilm model to develop in vitro root caries for investigating microbial modulation strategies. The model involved growing complex biofilms from a saliva inoculum collected from four volunteers using two strategies. In the first strategy (“pre-treatment strategy”), bovine root slabs were used, and two natural compounds were incorporated at time 0 of the 10-day biofilm experiment, which included sucrose cycles mimicking the cariogenic environment. In the second strategy (“post-treatment strategy”), mature biofilms were grown in a modified Calgary biofilm device coated with collagen and hydroxyapatite for 7 days and then were exposed to the same natural compounds. The metatranscriptome of each biofilm was then determined and analysed. Collagenase activity was examined, and the biofilms and dentine were imaged using confocal and scanning electron microscopy (SEM). Mineral loss and lesion formation were confirmed through micro-computed tomography (μ-CT).ResultsThe pH confirmed the cariogenic condition. In the metatranscriptome, we achieved a biofilm compositional complexity, showing a great diversity of the metabolically active microbiome in both pre- and post-treatment strategies, including reads mapped to microorganisms other than bacteria, such as archaea and viruses. Carbohydrate esterases had increased expression in the post-treated biofilms and in samples without sugar cycles, while glucosyltransferases were highly expressed in the presence of sucrose cycles. Enrichment for functions related to nitrogen compound metabolism and organic cyclic component metabolism in groups without sucrose compared to the sucrose-treated group. Pre-treatment of the roots with cranberry reduced microbial viability and gelatinase (but not collagenase) activity (p < 0.05). SEM images showed the complexity of biofilms was maintained, with a thick extracellular polysaccharides layer.ConclusionsThis root caries model was optimized to produce complex cariogenic biofilms and root caries-like lesions, and could be used to test microbial modulation in vitro. Pre-treatments before biofilm development and cariogenic challenges were more effective than post-treatments. The clinical significance lies in the potential to apply the findings to develop varnish products for post-professional tooth prophylaxis, aiming at implementing a strategy for dysbiosis reversal in translational research.2cRgxuoMbmgt6TZPkgExXQVideo

Destruction of cariogenic biofilms by antimicrobial photodynamic therapy mediated by phycocyanin and toluidine blue along with sodium fluoride varnish or titanium tetrafluoride

ObjectiveEvaluation of the effect of phycocyanin (PC) and toluidine blue (TBO) along with sodium fluoride varnish (FV) or titanium tetrafluoride (TiF4) under the conditions of antimicrobial photodynamic therapy (PDT) on a dual-species cariogenic biofilm and on remineralization process. DesignAfter the development of Streptococcus mutans and Lactobacillus acidophilus dual-species biofilms on the human enamel disks, they were divided into 11 groups (n = 9): Control (0.9 % saline), PC, TBO, FV, and TiF4 alone, PC and TBO in combination with a 635 nm diode laser (PDT treatment), PC-PDT+ (PC + FV or TiF4 + 635 nm diode laser), and TBO-PDT+ (TBO + FV or TiF4 + 635 nm diode laser). After the treatment, crystal violet assay was performed to determine the reduction of cariogenic biofilms. Enamel remineralization changes were analyzed using energy dispersive X-ray spectroscopy (EDX) and field emission scanning electron microscopy (FESEM) for the calcium and phosphorus (Ca/P) ratio. ResultsOnly TBO-PDT+ showed superior antibiofilm activity when TiF4 was applied. Furthermore, the highest Ca/P ratio was found after treatment of enamel surfaces with TiF4-TBO-PDT+. The FESEM images showed that the enamel disks treated with TiF4 plus TBO-mediated PDT exhibited surface coating. However, TiF4 plus PC-mediated PDT cannot repair demineralized enamel. ConclusionsThese data suggest that TBO-PDT along with TiF4 can effectively reduce cariogenic biofilms and significantly remineralize enamel disks, opening new avenues in caries prevention.

Efficiency of the photodynamic therapy on viability of Streptococcus mutans in the oral cavity using chitosan nanoparticles: an in vitro study.

This study aimed to investigate the efficiency of antimicrobial photodynamic therapy (aPDT) on Streptococcus mutans biofilm in the oral cavity using the photosensitizer chloroaluminum phthalocyanine encapsulated in chitosan nanoparticles (ClAlPc/Ch) at three preirradiation times. Biofilms of Streptococcus mutans strains (ATCC 25,175) were cultivated on bovine tooth blocks and exposed to a 10% sucrose solution three times a day for 1min over three consecutive days. The samples were randomly distributed into five treatment groups (n = 5): (I) aPDT with ClAlPc/Ch with a preirradiation time of 5min (F5), (II) aPDT with ClAlPc/Ch with a preirradiation time of 15min (F15), (III) aPDT with ClAlPc/Ch with a preirradiation time of 30min (F30), (IV) 0.12% chlorhexidine digluconate (CHX), and (V) 0.9% saline solution (NaCl). After treatment, the S. mutans biofilms formed on each specimen were collected to determine the number of viable bacteria (colony-forming units (CFU)/mL). Data were analyzed for normality using the Shapiro-Wilk test and the analysis of variance (ANOVA) and Tukey HSD tests to analyze the number of viable bacteria (α = 0.05). The one-way ANOVA showed a difference between the groups (p = 0.0003), and the Tukey HSD posttest showed that CHX had the highest microbial reduction of S. mutans, not statistically different from the F5 and F15 groups, whereas the NaCl group had the lowest microbial reduction statistically similar to the F30 group. The results demonstrate that aPDT mediated by ClAlPc/Ch when used at preirradiation times of 5-15min can be an effective approach in controlling cariogenic biofilm of S. mutans, being an alternative to 0.12% CHX.

Limosilactobacillus reuteri and Its Probiotic Potential against Cariogenic Bacteria

Bacteriotherapy is a promising option in addressing dental caries, a persistent global public health challenge with multifactorial origin, including dysbiosis. Despite the exploration of various probiotics, outcomes remain inconclusive. Objective: This study aimed to assess the inhibitory potential of L. reuteri and other potential probiotics like S. salivarius and S. oralis on the growth, adhesion, colonization, and viability of major cariogenic pathogens, comparing their probiotic efficacy. Methods: An in vitro experimental study was conducted, encompassing direct competition assays in solid and liquid co-culture tests and the characterization of adhesion to dental enamel and cell viability by life or death assay. Results: L. reuteri exhibited the significant inhibition of S. sobrinus and S. mutans growth in both solid and liquid cultures, with statistically notable differences. Scanning electron microscopy and confocal microscopy demonstrated reduced cariogenic biofilm formation when combined with L. reuteri, corroborated by diminished bacterial viability and decreased dental enamel coverage. These findings underscore L. reuteri’s potential as an effective agent in caries prevention. Conclusion: The study suggests L. reuteri could serve as an effective probiotic in bacteriotherapy against dental caries. It displayed substantial inhibitory activity in vitro against cariogenic bacteria, impeding biofilm formation and adhesion, thereby impacting cell viability.

Flowable resin-based composites modified with chlorhexidine-loaded mesoporous silica nanoparticles induce superior antibiofilm properties

Clinical failure of dental resin-composite restorations is mainly due to bacterial-mediated secondary caries formation. Therefore, the development of a flowable resin-composite material having inherent antibacterial properties is crucial to enhance the durability of dental restorations. Herein, dental flowable resin-composite material was modified with chlorhexidine-loaded mesoporous silica nanoparticles (CHX-MSN) to induce in situ antibacterial properties against S. mutans. Mesoporous silica nanoparticles loaded with chlorhexidine (CHX-MSN) were formulated and characterized for drug-loading/encapsulation efficiency, morphology by electron microscopy, and infrared spectral analysis. CHX-MSN were incorporated into the flowable composite material at different concentrations of 1, 5, and 10% (w/w) and examined at two time points (baseline and 3 months in artificial saliva). The CHX-MSN modified composites exhibited an initial CHX release burst followed by a steady release up to 30 days. The antimicrobial efficacy of the modified composites was evaluated by crystal violet assay, MTT assay, and confocal laser scanning microscopy. In addition to measuring the degree of conversion and cytotoxicity, the mechanical properties were characterized by surface microhardness and flexural strength. The modified composites demonstrated a significant increase in antimicrobial properties compared to the unmodified control (p < 0.05) which is dependent on the concentration of the CHX-MSN nanoparticles. In addition, the modified composites possessed acceptable biocompatibility without adversely affecting mechanical properties and degree of conversion up to 5% addition of CHX-MSN nanoparticles. This study introduced a protocol to develop resin-based flowable dental composite material having superior antibacterial property against cariogenic biofilms aiming for enhancing clinical longevity of dental restorations.Graphical

Analysis of cariogenic biofilms by using a swept-source optical coherence tomography in vitro

ObjectiveThe objective was to measure the thickness of Streptococcus mutans (S. mutans) biofilms forming in an oral biofilm reactor (OBR) by using a noninvasive swept-source optical coherence tomography (SS-OCT) system at every 4 h time interval until 20 h and analyze the correlations with the amounts of biofilms. MethodsS. mutans biofilms were formed on square-shaped bovine enamel blocks inside an OBR. Biofilms were analyzed at every 4 h stage (4 h, 8 h, 12 h, 16 h and 20 h) using a SS-OCT system and a laser scanning confocal microscope (LSCM). The amounts of biofilms were measured at each stage by separating the water insoluble glucan (WIG) and bacterial cells. Co-relationships between the SS-OCT measured biofilm thickness and the amounts of adhered biofilms were analyzed. ResultsThe thickness of biofilms detected on SS-OCT images at 4 h stage was 0.059 ± 0.029 (Av ± SD) mm which increased time-dependently in a linear fashion after 8 h stage and reached to 0.435 ± 0.159 mm at 20 h stage and the correlation coefficient was about 0.89. The amounts of biofilms; bacterial optical density (OD) and WIG concentration increased time-dependently were 0.035 ± 0.008 / mm2 and 10.328 ± 2.492 µg/ mm2 respectively at 20 h stage. Correlation coefficients of 0.66 between ‘the amounts of bacteria’ and ‘biofilm thickness on OCT’ and 0.67 between ‘the amounts of WIG’ and ‘biofilm thickness on OCT’ were obtained, suggesting that there was a relatively positive correlation between them. ConclusionThe SS-OCT can be a useful tool to measure time-dependent growth of biofilms. Further studies are needed in order to assess biofilms using SS-OCT more accurately.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Epigallocatechin gallate reduces the virulence of cariogenic Streptococcus mutans biofilm by affecting the synthesis of biofilm matrix components

IntroductionThere have been reports on the effects of epigallocatechin gallate (EGCG) against Streptococcus mutans viability and acidogenesis. However, the effects of EGCG on the virulence of S. mutans biofilm development have yet to be fully investigated using validated cariogenic biofilm models. ObjectiveThus, this study aimed to evaluate the effects of EGCG on S. mutans biofilm virulence using a validated cariogenic model and clinically relevant treatment regimens, twice a day for 1.5 min. MethodsEffects of EGCG on bacterial viability, polyssacharide synthesis and biofilm acidogenesis were evaluated. The morphology and 3D structure of the biofilms were evaluated by scanning electron (SEM) and confocal laser scanning microscopy, respectively. ResultsNo significant change in S. mutans viability or culture medium pH were observed when comparing EGCG-treated and NaCl-treated biofilms. EGCG significantly reduced the accumulation of soluble and insoluble polysaccharides, resulting in the formation of a biofilm with interspaced exopolysaccharide-microcolony complexes unevenly distributed on enamel. The SEM images of the biofilm treated with EGCG depict multilayers of cells arranged in short chains of microorganisms adhered to an unstructured matrix, which is not continuous and does not enmesh or protect the microorganisms entirely. Importantly, confocal images demonstrated that treatment with EGCG affected the 3D structure and organization of S. mutans biofilm, which presented a biofilm matrix more confined to the location of the microcolonies. ConclusionIn conclusion, EGCG lowered the virulence of S. mutans matrix-rich biofilm by reducing the synthesis of biofilm matrix components, altering the biofilm matrix structure, organization, and distribution.

Enhanced anti-biofilm and anti-caries potential of arginine combined with calcium glycerophosphate and fluoride

ObjectiveThe aim of this work was to evaluate the antibiofilm and anticaries properties of the association of arginine (Arg) with calcium glycerophosphate (CaGP) and fluoride (F). MethodsAn active attachment, polymicrobial biofilm model obtained from saliva and bovine teeth discs were used. After the initial biofilm growth period, the enamel discs were transferred to culture medium. The treatment solutions were added to the culture media to achieve the desired final concentration. The following groups were used: negative control (Control); F (110 ppm F); CaGP (0.05 %); Arg (0.8 %) and their associations (F + CaGP; Arg + F; Arg + CaGP; Arg +F + CaGP). The following analyses were carried out: bacterial viability (total bacteria, aciduric bacteria and mutans streptococci), pH assessment of the spent culture medium, dry weight quantification, evaluation of surface hardness loss (%SH) and subsurface mineral content. Normality and homoscedasticity were tested (Shapiro-Wilk and Levene's test) and the following tests were applied: two-way ANOVA (acidogenicity), Kruskall-Wallis (microbial viability) and one way ANOVA (dry weight, %SH, mineral content). ResultsThe association Arg + F + CaGP resulted in the lowest surface hardness loss in tooth enamel (-10.9 ± 2.3 %; p < 0.05). Arg +F + CaGP exhibited highest values of subsurface mineral content (10.1 ± 2.9 gHAP/cm3) in comparison to Control and F (p < 0.05). In comparison to Control and F, Arg +F + CaGP promoted the highest reduction in aciduric bacteria and mutans streptococci (5.7 ± 0.4; 4.4 ± 0.5 logCFU/mL, p < 0.05). ConclusionsThe Arg-F-Ca association demonstrated to be the most effective combination in protecting the loss of surface hardness and subsurface mineral content, in addition to controlling important virulence factors of the cariogenic biofilm. Clinical significanceOur findings provide evidence that the Arg-F-Ca association showed an additive effect, particularly concerning protection against enamel demineralization. The combination of these compounds may be a strategy for patients at high risk of caries.

Lysine aggregates‐based nanostructured antimicrobial peptides for cariogenic biofilm microenvironment‐activated caries treatment

AbstractDental caries is one of the most prevalent and costly biofilm‐induced oral diseases that causes the deterioration of the mineralized tooth tissue. Traditional antimicrobial agents like antibiotics and antimicrobial peptides (AMPs) struggle to effectively eradicate bacteria in biofilms without eliciting resistance. Herein, we demonstrate the construction of FeOOH@Fe‐Lysine@Au nanostructured AMPs (nAMPs) distinguished by their AMP‐like antibacterial activity and self‐producing reactive oxygen species (ROS) capacity for caries treatment. On the one hand, FeOOH@Fe‐Lysine@Au nAMPs can catalyze glucose oxidation to generate ROS within the cariogenic biofilm microenvironment, resulting in the disintegration of the extracellular polymeric substance matrix and the exposure of bacteria. On the other hand, FeOOH@Fe‐Lysine@Au nAMPs can attach to bacterial surfaces via electrostatic attractions, proceeding to damage membranes, disrupt metabolic pathways, and inhibit protein synthesis through the aggregated lysine and the generated ROS. Based on this antibacterial mechanism, FeOOH@Fe‐Lysine@Au nAMPs can effectively eradicate Streptococcus mutans and its associated biofilm, significantly impeding the progression of dental caries. Given the straightforward and cost‐efficient preparation of FeOOH@Fe‐Lysine@Au nAMPs compared to AMPs that require specific sequences, and their minimal adverse impacts on gingival/palatal tissues, major organs, and oral/gut microbiomes, our research may promote the development of novel therapeutic agents in dental health maintenance.