Insoluble Extracellular Polysaccharide Research Articles

Characterizations of biogenic selenium nanoparticles and their anti-biofilm potential against Streptococcus mutans ATCC 25175

IntroductionS. mutans has been identified as the primary pathogenic bacterium in biofilm-mediated dental caries. The biogenic selenium nanoparticles (SeNPs) produced by L. plantarum KNF-5 were used in this study against S. mutans ATCC 25175. ObjectivesThe aims of this study were: (1) the biosynthesis of SeNPs by L. plantarum KNF-5, (2) the characterization of SeNPs, (3) the investigation of the inhibitory effect of biogenic SeNPs against S. mutans ATCC 25175, and (4) the determination of the anti-biofilm potential of SeNPS against S. mutans ATCC 25175. Methodology3 mL of the culture was added to 100 mL of MRS medium and incubated. After 4 h, Na2SeO3 solution (concentration 100 μg/mL) was added and incubated at 37 °C for 36 h. The color of the culture solution changed from brownish-yellow to reddish, indicating the formation of SeNPs. The characterization of SeNPs was confirmed by UV-Vis spectrophotometry, FTIR, SEM-EDS and a particle size analyzer. The antibacterial activity was determined by the disk diffusion method, the MIC by the micro-double dilution method, and the biofilm inhibitory potential by the crystal violet method and the MTT assay. The effect of SeNPs on S. mutans ATCC 25175 was determined using SEM and CLSM spectrometry techniques. The sulfate-anthrone method was used to analyze the effect of SeNPs on insoluble extracellular polysaccharides. The expression of genes in S. mutans ATCC 25175 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Preparation of nanoparticlesSeNPs produced by probiotic bacteria are considered a safe method. In this study, L. plantarum KNF-5 (probiotic strain) was used for the production of SeNPs. ResultsThe biogenic SeNPs were spherical and coated with proteins and polysaccharides and had a diameter of about 270 nm. The MIC of the SeNPs against S. mutans ATCC 25175 was 3.125 mg/mL. Biofilm growth was also significantly suppressed at this concentration. The expression of genes responsible for biofilm formation (GtfB, GtfC, BrpA and GbpB,) was reduced when S. mutans ATCC 25175 was treated with SeNPs. ConclusionIt was concluded that the biogenic SeNPs produced by L. plantarum KNF-5 was highly effective to inhibit the growth of S. mutans ATCC 25175. Novelty statementThe application of biogenic SeNPs, a natural anti-biofilm agent against S. mutans ATCC 25175. In the future, this study will provide a new option for the prevention and treatment of dental caries.

Antibacterial Effects of Ramulus mori Oligosaccharides against Streptococcus mutans.

Ramulus mori has been widely used in traditional Chinese medicine because of its physiological activities, including antibacterial, anti-inflammatory, and antioxidant activities. Antimicrobial properties of Ramulus mori extract have been well described. However, no information is available regarding on Ramulus mori oligosaccharides (RMOS). The aim of this study was to investigate the effects of RMOS on the growth and virulence properties of the cariogenic bacterium Streptococcus mutans. The effects of RMOS on the biofilm structure and virulence gene expression of S. mutans were also evaluated, and the results were compared with the effects of commercial prebiotic galactooligosaccharides. RMOS were found to have an antibacterial effect against S. mutans, resulting in significant reductions in acid production, lactate dehydrogenase activity, adhesion, insoluble extracellular polysaccharide production, glucosyltransferase activity, and biofilm formation in a dose-dependent manner. Moreover, the biofilm structure was visibly damaged. A quantitative real-time PCR assay revealed downregulation of virulence gene-regulated acid production, polysaccharide production, adhesion, biofilm formation, and quorum sensing. These findings suggest that RMOS may be a promising natural product for the prevention of dental caries.

The potential use of glycosyl-transferase inhibitors for targeted reduction of S. mutans biofilms in dental materials

Streptococcus mutans is the primary oral caries-forming bacteria, adept at producing “sticky” biofilms via the synthesis of insoluble extracellular polysaccharides (EPS), catalyzed by glucosyltransferases (GTFs). To circumvent the use of broad-spectrum antibiotics to combat these bacteria, this study sought to modify existing EPS-targeting small molecules with the ultimate goal of producing anti-biofilm polymer surfaces specifically targeting S. mutans. To achieve this, a known GTF inhibitor (G43) was modified with methoxy or tetraethyleneglycol substitutions in different positions (nine derivatives, tested at 50-µM) to pinpoint potential sites for future methacrylate functionalization, and then assessed against single-species S. mutans biofilms. As expected, the compounds did not diminish the bacterial viability. In general, the compounds with methoxy substitution were not effective in reducing EPS formation, whereas the tetraethyleneglycol substitution (G43-C3-TEG) led to a decrease in the concentration of insoluble EPS, although the effect is less pronounced than for the parent G43. This aligns with the reduced GTF-C activity observed at different concentrations of G43-C3-TEG, as well as the consequent decrease in EPS formation, and notable structural changes. In summary, this study determined that G43-C3-TEG is non-bactericidal and can selectively reduce the biofilm formation, by decreasing the production of EPS. This molecule will serve to functionalize surfaces of materials to be tested in future research.

Triclosan to Improve the Antimicrobial Performance of Universal Adhesives.

To solve the proble ms of composite restoration failure caused by secondary caries, this study reports a light curable antibacterial triclosan derivative (TCS-IH), which was synthesized and added to the existing commercial universal adhesive to achieve a long-term antibacterial effect The effect of mixing different mass percentages of TCS-IH on the bond strength of dentin was also investigated.TCS-IH was synthesized by solution polymerization and characterized by nuclear magnetic resonance hydrogen spectroscopy (1H NMR) and Fourier transform infrared (FTIR) spectroscopy. Two commercial universal adhesives, Single Bond Universal and All Bond Universal, were selected and used as the control group, and universal adhesives with different mass percentages (1 wt%, 3 wt%, 5 wt% and 7 wt%) of TCS-IH were used as the experimental group. The antibacterial properties were analysed by means of colony count experiments, biofilm formation detection, plotting of growth curves, biofilm metabolic activity detection, insoluble extracellular polysaccharide measurements and observations by confocal laser scanning microscopy and scanning electron microscopy (SEM). The effect of adhesives on biofilm formation, metabolism, extracellular matrix production, distribution of live and dead bacteria, and bacterial morphology of Streptococcus mutans (S. mutans) was analysed. The mechanical properties were evaluated by the degree of conversion and microtensile bonding strength under different conditions. Its biosafety was tested. We found that the addition of TCS-IH significantly improved the antibacterial performance of the universal adhesive, with the 5 wt% and 7 wt% groups showing the best antibacterial effect and effectively inhibiting the formation of biofilm. In addition, the adhesive strength test results showed that there was no statistical difference (p < 0.05) in the microtensile bond strength measured under various factors in all experimental groups except for the 7 wt% group in the self-etch bonding mode, and all of them had good biosafety. In summary, the 5 wt% group of antibacterial monomer TCS-IH was selected as the optimum addition to the universal adhesive to ensure the antimicrobial properties of the universal adhesive and the stability and durability of the adhesive interface. This study provides a reference for the clinical application of adhesives with antimicrobial activity to improve the stability and durability of adhesive restorations.

Effect of glaze and chlorhexidine on physical and mechanical properties of bis-acryl resin: An in situ study.

Temporary prosthesis protects the oral tissues, in addition to providing aesthetic look and masticatory function until a definitive prosthesis is manufactured. To evaluate the effect of glaze and 0.12% chlorhexidine (CHX) on the physical and mechanical properties of bis-acryl, and to evaluate the antimicrobial efficacy of CHX. Eighty specimens of bis-acryl resin were made. Over 40 of them the glaze was applied. One specimen with and 1 specimen without glaze were placed in niches of an appliance manufactured for each patient. Each of the 20 volunteers received 2 devices. Initially, the volunteers used one device and treated it with sucrose for 7 days (control), and later they used the other device and treated it with sucrose and CHX for 7 days (test). Color, microhardness, roughness, surface energy, and insoluble extracellular polysaccharides (EPS) tests were performed. All results were submitted to the Tukey's test, with the exception of the EPS results, which were submitted to the Student's t test. The ΔE00 of the unglazed control group was significantly higher than that of the unglazed test group. In all groups, a significant decrease in microhardness occurred over time. At both times, the glaze significantly increased the microhardness of the specimens (in all the glazed groups). At the final time, the test glaze group showed significantly higher microhardness compared with the control glaze group. Roughness in the groups without glaze increased significantly with CHX treatment over time. At both times, the glaze generated a significant reduction in roughness in the control and test groups. There was a significant reduction in surface energy over time in all groups. In most comparisons, the glazed groups showed significantly higher surface energy values compared with the unglazed control group. At the final time point, the unglazed test group showed a significantly higher surface energy value than the unglazed control group; and the glazed test group showed a significantly higher surface energy value compared with the glazed control group. The resins that received CHX had a significantly lower amount of biofilm. Color values were clinically acceptable in all tested groups. At both time points, the roughness values were clinically acceptable only in the glazed groups. Glaze increased the microhardness of the specimens. Microhardness and surface energy were reduced over time in all groups. Chlorhexidine can help prevent microhardness degradation. Glaze and CHX can increase surface energy. Chlorhexidine reduced the amount of bacterial biofilm.

Mucoadhesive controlled-release formulations containing morin for the control of oral biofilms

This study aimed to evaluate the antimicrobial and anti-biofilm activity of morin on polymicrobial biofilms and its cytotoxicity in controlled-release films and tablets based on gellan gum. Polymicrobial biofilms were formed from saliva for 48 h under an intermittent exposure regime to 1% sucrose and in contact with films or tablets of gellan gum containing 2 mg of morin each. Acidogenicity, bacterial viability, dry weight and insoluble extracellular polysaccharides from biofilms were evaluated. The cytotoxicity of morin was evaluated in oral keratinocytes. Morin released from the systems reduced the viability of all the microbial groups evaluated, as well as the dry weight and insoluble polysaccharide concentration in the matrix and promoted the control of acidogenicity when compared with the control group without the substance. Morin was cytotoxic only at the highest concentration evaluated. In conclusion, morin is an effective agent and shows antimicrobial and anti-biofilm activity against polymicrobial biofilms.

Effect of different salivary glucose concentrations on dual-species biofilms of Candida albicans and Streptococcus mutans

Diabetes mellitus is one of the most prevalent diseases worldwide. The glucose levels found in the blood of diabetic patients can be reflected in the saliva, which can favor biofilm growth and predispose denture wearers to oral candidiasis. This study aimed to evaluate the effect of different salivary glucose concentrations on dual-species biofilms of Candida albicans and Streptococcus mutans. A 96-h biofilm was developed on acrylic resin specimens exposed to ‘feast’ (10% sucrose) and ‘famine’ periods. Biofilms were constantly exposed to salivary glucose concentrations equivalent to 0, 20, 60 and 100 mM. Higher salivary glucose concentrations resulted in increased counts of C. albicans and a higher quantity of insoluble extracellular polysaccharides. All biofilms presented high phospholipase activity. The biofilms were characterized by the predominance of yeast cells and microcolonies in all the groups analyzed with co-localization of both species. Higher salivary glucose concentrations formed more robust and potentially virulent biofilms.

Cariogenic Potential of Human and Bovine Milk on Enamel Demineralization

The higher cariogenicity of human milk when compared with bovine milk is still a debatable subject. Therefore, we evaluated the effect of human or bovine milk exposure on biofilm composition and enamel demineralization using a validated cariogenic biofilm model. Streptococcus mutans UA159 biofilms (n = 8) were grown on human saliva-coated bovine enamel slabs of known surface hardness. The biofilms were exposed 8×/day to 0.9% NaCl (negative control), human milk, bovine milk, 7.0% lactose (active human milk control), 4.5% lactose (active bovine milk control), or 10% sucrose (positive control). The culture medium was changed twice daily, and the pH was analyzed as an indicator of biofilm acidogenicity. After 120 h of growth, biofilms were harvested to evaluate viable cells, and soluble and insoluble extracellular polysaccharides (EPS). Enamel demineralization was assessed by the percentage of surface hardness loss (%SHL). Data were analyzed by one-way ANOVA/Tukey’s test (α = 5%). In terms of %SHL, negative control (7.7 ± 3.1), human milk control (13.3 ± 7.5), bovine milk control (15.3 ± 8.2), human milk (7.5 ± 5.0), and bovine milk (8.7 ± 6.3) did not differ among them (p > 0.05) but differed (p < 0.05) from sucrose (55.1 ± 5.4). The findings of enamel demineralization (%SHL) were statistically supported by the data of biofilm acidogenicity, bacterial counts and EPS biofilm composition. This experimental study suggests that human and bovine milk have low cariogenic potential to provoke caries lesions in enamel.

In situ evaluation of 200 ppm fluoride toothpaste content trimetaphosphate, xylitol and erythritol on enamel demineralization and dental biofilm

ObjectiveTo evaluate the effect of low-fluoride (F−) toothpaste and sodium trimetaphosphate (TMP) associated with xylitol and erythritol (XE) on enamel demineralization and biofilm composition. MethodsThis crossover double-blind in situ study consisted of five phases (seven days each), in which 14 volunteers wore oral appliances containing four enamel bovine blocks. The cariogenic challenge was performed by exposure to a 30% sucrose solution (6x/day). The toothpaste treatments (3x/day) were as follows: placebo (no F−/TMP/XE); 200 ppm F− (NaF) (200F); 1,100 ppm F− (1100F); 16% Xylitol and 4% Erythritol (XE); and 200 ppm F−, 0.2% TMP, 16% xylitol, and 4% erythritol (200F-TMP-XE). Percentage of surface hardness loss (%SH) and integrated loss of subsurface hardness (ΔKHN), and calcium (Ca2+), phosphate (PO43−), and F− on enamel and biofilm were determined; as well as insoluble extracellular polysaccharide (EPS). ResultsXE and 1100F groups showed no significant difference for %SH and ΔKHN values (p = 0.220 and p = 0.886), and the 200F-TMP-XE group had the lowest mineral loss (p < 0.001). Ca2+ and PO43− in the enamel showed the highest values (p < 0.001) for the 200F-TMP-XE group. Higher values of F− in the enamel and biofilm were observed for the 1100F group (p < 0.001). There was no difference for Ca2+ (p = 1.00) and EPS (p =0.918) values between XE and 200-TMP-XE groups in the biofilm, but their values were higher and lower than the 1100F (p = 0.002 and p = 0.029), respectively. Conclusions200F-TMP-XE promoted a greater protective effect against enamel demineralization and significantly affected the composition of biofilm formed in situ compared to 1100F toothpaste. Clinical SignificanceLow-F− toothpaste containing TMP and polyols can be considered an effective and safe measure to improve the oral health of individuals, especially patients with high caries activity.

Effect of S. mutans combinations with bifidobacteria/lactobacilli on biofilm and enamel demineralization.

The present study evaluated the ability of Bifidobacterium and Lactobacillus species associated with streptococci to increase insoluble extracellular polysaccharide (EPS) production and initial caries lesion progression. Bovine enamel blocks (n = 190; 4 mm x 4 mm) were prepared, selected according to initial surface hardness (SH), and divided into two groups: a) double combinations: S. mutans with Bifidobacterium or Lactobacillus, and b) triple combinations: S. mutans and S. sobrinus with Bifidobacterium or Lactobacillus species. The blocks were exposed to the bacterial associations for 7 days. Subsequently, quantity of EPS from biofilms and caries lesion depth were determined by means of colorimetric and cross-sectional enamel hardness (ΔKHN) analysis. The data were submitted to one-way analysis of variance, followed by the Bonferroni test (p < 0.05). S. mutans with B. animalis or B. dentium produced a higher quantity of EPS; S. mutans + B. animalis led to the highest ∆KHN. S. mutans + S. sobrinus + B. longum induced greater EPS and ∆KHN values. In conclusion, associations of B. animalis and B. longum with streptococci promoted EPS production and caries lesion progression.

The role of sodium alginate and gellan gum in the design of new drug delivery systems intended for antibiofilm activity of morin

The use of controlled drug delivery systems represents an alternative and promising strategy for the use of antimicrobials in the oral cavity. Microparticles, films and oral tablets based on alginate and gellan gum were developed also as a strategy to overcome the low aqueous solubility of morin. The systems were characterized in terms of morphological characteristics, mucoadhesion and in vitro drug release. Antibiofilm activity was analyzed for acidogenicity, microbial viability and the composition of the extracellular matrix of single-species biofilms. Scanning Electron Microscopy demonstrated that the microparticles were spherical, rough and compact. The film and the tablet presented smooth and continuous surface and in the inner of the tablet was porous. These systems were more mucoadhesive compared to the microparticles. The in vitro morin release profiles in artificial saliva demonstrated that the microparticles controlled the release better (39.6%), followed by the film (41.1%) and the tablet (91.4%) after 20 h of testing. The morin released from the systems reduced the acidogenicity, microbial viability, concentration of insoluble extracellular polysaccharides and dry weight of biofilms, when compared to the control group. The findings of this study showed that the morin has antibiofilm activity against cariogenic microorganisms.

Modulatory Effect of Glycated Collagen on Oral Streptococcal Nanoadhesion

Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococci to dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients.

Hindering of Cariogenic Streptococcus mutans Biofilm by Fatty Acid Array Derived from an Endophytic Arthrographis kalrae Strain.

Streptococcus mutans has been considered as the major etiological agent of dental caries, mostly due to its arsenal of virulence factors, including strong biofilm formation, exopolysaccharides production, and high acid production. Here, we present the antivirulence activity of fatty acids derived from the endophytic fungus Arthrographis kalrae isolated from Coriandrum sativum against Streptococcus mutans. The chemical composition of the fatty acids was analyzed by gas chromatography–mass spectrometry GC-MS and revealed nine compounds representing 99.6% of fatty acids, where unsaturated and saturated fatty acids formed 93.8% and 5.8 % respectively. Oleic and linoleic acids were the major unsaturated fatty acids. Noteworthy, the fatty acids at the concentration of 31.3 mg L–1 completely inhibited Streptococcus mutans biofilm, and water insoluble extracellular polysaccharide production in both polystyrene plates, and tooth model assay using saliva-coated hydroxyapatite discs. Inhibition of biofilm correlated significantly and positively with the inhibition of water insoluble extracellular polysaccharide (R = 1, p < 0.0001). Furthermore, Arthrographis kalrae fatty acids at a concentration of 7.8 mg L–1 exhibited acidogenesis-mitigation activity. They did not show bactericidal activity against Streptococcus mutans and cytotoxic activity against human oral fibroblast cells at the concentration used. On the other hand, saliva-coated hydroxyapatite discs treated with sub-minimum biofilm inhibitory concentration of fatty acids showed disturbed biofilm architecture with a few unequally distributed clumped matrices using fluorescence microscopy. Our findings revealed that the intracellular fatty acid arrays derived from endophytic Arthrographis kalrae could contribute to the biofilm-preventing alternatives, specifically Streptococcus mutans biofilms.

Glucose supplementation effect on the acidogenicity, viability, and extracellular matrix of Candida single- and dual-species biofilms.

Evidence of glucose supplementation effect on Candida biofilm metabolism has not been demonstrated. Therefore, the aim of the present study was to evaluate the effect of glucose concentration on Candida biofilms. Single- and dual-species biofilms of Candida were grown on saliva-coated poly(methyl-methacrylate) disks for 72hours. Biofilms (N=8/group) were exposed to the following concentrations of glucose: 100mmol/L (G100), 300mmol/L (G300), and no glucose (G0: control). Biofilms were collected to determine the acidogenicity, viability, amount of soluble and insoluble extracellular polysaccharides (IEPS), and surface roughness. Data were analyzed using analysis of variance and Tukey's tests (α<0.05). Single- and dual-species biofilms from G300 were more acidogenic at 48 and 72hours compared to G100 and G0 (P<0.05). The viability of the G100 and G300 groups did not differ (P>0.05), but differed statistically from G0. The amount of IEPS in the G300 group was statistically higher than the G0 and G100 groups (P<0.05). The G300 group also presented a higher IEPS proportion per number of viable cells compared to others. G300 presented greater surface roughness for both single- (mean roughness=1460μm) and dual-species (mean roughness=1990μm) biofilms. Higher glucose concentration (300mmol/L) during biofilm development favors the growth of single- and dual-species biofilms of Candida.

Anticaries effect of toothpaste with nano-sized sodium hexametaphosphate.

To evaluate the effect of a fluoride toothpaste containing nano-sized sodium hexametaphosphate (HMPnano) on enamel demineralization on the biochemical composition and insoluble extracellular polysaccharide (EPS) in biofilm formed in situ. This crossover double-blind study consisted of four phases (7days each), in which 12 volunteers wore intraoral appliances containing four enamel bovine blocks. The cariogenic challenge was performed using 30% sucrose solution (6×/day). Blocks were treated 3×/day with the following toothpastes: no F/HMP/HMPnano (Placebo), conventional fluoride toothpaste, 1100ppmF (1100F), 1100F + 0.5% micrometric HMP (1100F/HMP), and 1100F + 0.5% nano-sized HMP (1100F/HMPnano). The percentage of surface hardness loss (%SH), integrated loss of subsurface hardness (ΔKHN), and enamel calcium (Ca), phosphorus (P), and fluoride (F) were determined. Moreover, biofilms formed on the blocks were analyzed for F, Ca, P, and insoluble extracellular polysaccharide (EPS) concentrations. Data were analyzed using one-way ANOVA, followed by Student-Newman-Keuls' test (p < 0.001). 1100F/HMPnano promoted the lowest %SH and ΔKHN among all groups (p < 0.001). The addition of HMPnano to 1100F significantly increased Ca concentrations (p < 0.001). The 1100F/HMPnano promoted lower values of EPS when compared with 1100F (~ 70%) (p < 0.001) and higher values of fluoride and calcium in the biofilms (p < 0.001). 1100F/HMPnano demonstrated a greater protective effect against enamel demineralization and on the composition of biofilm in situ when compared to 1100F toothpaste. This toothpaste could be a viable alternative to patients at high risk of caries.

Candida albicans Increases Dentine Demineralization Provoked by Streptococcus mutans Biofilm

Streptococcus mutans are considered the most cariogenic bacteria, but it has been suggested that Candida albicans could increase their cariogenicity. However, the effect of this dual-species microorganisms’ combination on dentine caries has not been experimentally evaluated. Biofilms of C. albicans, S. mutans and C. albicans + S. mutans (n = 12/biofilm) were grown in ultra-filtered tryptone yeast extract broth culture medium for 96 h on root dentine slabs of known surface hardness and exposed 8 times per day for 3 min to 10% sucrose. The medium was changed 2 times per day (after the 8 cariogenic challenges and after the overnight period of famine), and aliquots were analyzed to determinate the pH (indicator of biofilm acidogenicity). After 96 h, the biofilms were collected to determine the wet weight, colony-forming units, and the amounts of extracellular polysaccharides (soluble and insoluble). Dentine demineralization was assessed by surface hardness loss (% SHL). The architecture of the biofilms was analyzed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Data were analyzed by ANOVA followed by Tukey’s test (α = 0.05). The dual-species C. albicans + S. mutans biofilm provoked higher % SHL on dentine (p < 0.05) than the S. mutans and C. albicans biofilm. This was supported by the results of biofilm acidogenicity and the amounts of soluble (6.4 ± 2.14 vs. 4.0 ± 0.94 and 1.9 ± 0.97, respectively) and insoluble extracellular polysaccharides (24.9 ± 9.22 vs. 18.9 ± 5.92 and 0.7 ± 0.48, respectively) (p < 0.05). The C. albicans biofilm alone presented low cariogenicity. The images by CLSM and TEM, respectively, suggest that the C. albicans + S. mutans biofilm is more voluminous than the S. mutans biofilm, and S. mutans cells interact with C. albicans throughout polysaccharides from the biofilm matrix. These findings show that C. albicans enhances the cariogenic potential of the S. mutans biofilm, increasing dentine demineralization.

In situ effect of fluoride toothpaste supplemented with nano-sized sodium trimetaphosphate on enamel demineralization prevention and biofilm composition

ObjectiveTo evaluate the effect of a fluoride toothpaste containing nano-sized sodium trimetaphosphate (TMPnano) on enamel demineralization in situ and composition of the biofilm. DesignThis crossover double-blind study consisted of four phases (seven days each) and 12 volunteers who wore oral appliances containing four enamel bovine blocks. The cariogenic challenge was performed by 30% sucrose solution (6x/day). The toothpaste treatments (3x/day) were as follows: no F/TMP/TMPnano (Placebo), 1100 ppm F (1100F), 1100F plus 3% micrometric or nano-sized TMP (1100F/TMP; 1100F/TMPnano). Percentage of surface hardness loss (%SH), and integrated loss of subsurface hardness (ΔKHN), as well as enamel calcium (Ca), phosphorus (P), and fluoride (F) were determined. Moreover, biofilm formed on the blocks were analyzed for F, Ca, P, and insoluble extracellular polysaccharide (EPS) concentrations. Data were analyzed using one-way ANOVA, repeated measures followed by Fisher LSD test (p < 0.001). Results1100F/TMPnano promoted the lowest %SH and ΔKHN among all groups (p < 0.001). Regarding the F concentrations in the enamel and in the biofilm, there were no significant differences between 1100 F and 1100 F/TMPnano, but significantly increased enamel Ca concentrations (p < 0.001). 1100F/TMPnano showed lower values of EPS concentration when compared with 1100F (∼80%) (p < 0.001). Conclusion1100F/TMPnano promoted a greater protective effect against enamel demineralization and significantly affected the composition of biofilm formed in situ when compared to 1100F toothpaste.

Effect of sucrose on biofilm formed in situ on titanium material.

Because sucrose may change the composition of biofilms formed on dental surfaces, the aim of this study was to evaluate in situ the effect of this dietary sugar on biofilm formation on titanium surface. In this blind, crossover, in situ study, 10 volunteers wore, in 3 phases of 7days each, a palatal appliance containing titanium specimens. In each phase, the specimens were treated extraorally with 20% sucrose solution at a frequency of 4 or 8times per day. As control, no treatment was rendered (0×). At the end of each phase, the biofilms were collected for biochemical analysis of biofilm wet weight (biomass), protein concentration, soluble (S-EPS), and insoluble (I-EPS) extracellular polysaccharides and intracellular polysaccharides (IPS), and for microbiologic analysis by checkerboard DNA-DNA hybridization (for levels and proportions of 40 bacterial species). Biochemical data were analyzed by linear regression and microbiological findings by Friedman and Dunn tests (α= .05). A positive significant linear relationship was found among sucrose exposure (0×, 4×, and 8×) and biomass, S-EPS, I-EPS and IPS (p< 0.05). The biofilms treated with sucrose (4× and/or 8×) presented higher mean total levels of the 40 bacterial species evaluated, higher proportions of red complex species and lower proportions of the host-compatible green complex species, in comparison with the control group (p< 0.05). The findings of the present study suggest that daily sucrose exposure has a harmful effect on the composition of biofilms formed on titanium surfaces.

Efficacy of red propolis hydro-alcoholic extract in controlling Streptococcus mutans biofilm build-up and dental enamel demineralization

ObjectiveThe efficacy of a red propolis hydro-alcoholic extract (RP) in controlling Streptococcus mutans biofilm colonization was evaluated. The effect of RP on dental demineralization was also investigated. MethodsChemical composition was determined by High Performance Liquid Chromatography (HPLC). Minimum Inhibitory and Bactericidal Concentration (MIC and MBC, respectively) were investigated against Streptococcus mutans (ATCC 25175). The cytotoxic potential of 3% RP in oral fibroblasts was observed after 1 and 3 min. Bovine dental enamel blocks (N = 24) were used for S. mutans biofilm formation (48 h), simulating ‘feast or famine’ episodes. Blocks/biofilms were exposed 2×/day, for 3 days, to a cariogenic challenge with sucrose 10% (5 min) and treated (1 min) with: 0.85% saline solution (negative control), 0.12% Chlorhexidine (CHX, positive control for biofilm colonization), 0.05% Sodium Fluoride (NaF, positive control to avoid demineralization) and 3% RP. Biofilms were assessed for viability (CFU/mL), and to observe the concentration of soluble and insoluble extracellular polysaccharides (SEPS and IEPS). Dental demineralization was assessed by the percentage of surface hardness loss (%SHL) and through polarized light microscopy (PLM). ResultsThe RP presented 4.0 pH and ºBrix = 4.8. The p-coumaric acid (17.2 μg/mL) and luteolin (15.23 μg/mL) were the largest contents of phenolic acids and flavonoids, respectively. MIC and MBC of RP were 293 μg/mL and 1172 μg/mL, respectively. The 3% RP showed 43% of viably cells after 1 min. Lower number (p < 0.05) of viable bacteria (CFU/mL) was observed after CHX (1.8 × 105) followed by RP (1.8 × 107) treatments. The lowest concentration (μg/CFU) of SEPS (12.6) and IEPS (25.9) was observed in CHX (p < 0.05) followed by RP (17.1 and 54.3), and both differed from the negative control (34.4 and 63.9) (p < 0.05). Considering the %SHL, all groups differed statistically (p < 0.05) from the negative control (46.6%); but NaF (13.9%), CHX (20.1%) and RP (20.7%) did not differ among them (p > 0.05). After all treatments, suggestive areas of caries lesions were observed by PLM, which were lower for CHX and NaF. ConclusionThe 3% RP reduced S. mutans colonization, decreased concentration of extracellular polysaccharides and reduced dental enamel demineralization.

In situ Effect of Arginine-Containing Dentifrice on Plaque Composition and on Enamel Demineralization under Distinct Cariogenic Conditions

There is limited evidence that arginine-containing fluoridated dentifrices (AFD) have a better anticaries effect than regular fluoridated dentifrices (FD), especially in subjects at a higher risk for caries development. This study aimed to assess the effect of AFD on enamel demineralization and on the microbial and biochemical compositions of biofilm formed under different frequencies of sucrose exposure. It consisted of an in situ split-mouth design, where 12 adult volunteers who used FD for at least 2 months prior to the beginning of this study wore acrylic palatal appliances containing 4 bovine enamel specimens (1 pair at each side of the appliance) during 2 phases of 14 days each. FD slurry (3×/day) and 20% sucrose solution (4× and 8×/day) were dripped on the specimens during the first experimental phase. The same volunteers then used AFD during a 2-month washout period, followed by a second experimental phase where the AFD slurry and sucrose solution were applied onto a new subset of specimens. The percentage of enamel surface hardness loss (%SHL), the lesion depth (LD), the integrated mineral loss (IML), microbial counts on biofilms, the biomass, and inorganic and insoluble extracellular polysaccharide (IEPS) biofilm concentrations were determined. Higher %SHL, biomass, and IEPS and lower fluoride values were found at sucrose 8×/day exposure. Lower IEPS were found in the presence of AFD compared to FD. Similar %SHL, LD, and IML values were found between FD and AFD, irrespectively of the cariogenic challenge. The results suggest that AFD have an anticaries effect similar to that of regular FD.