Inhibition of Biofilm Formation and Virulence Factors of Cariogenic Oral Pathogen Streptococcus mutans by Shikimic Acid.

Streptococcus mutans is known as an important oral pathogen causing dental caries, a widespread oral infectious disease. S. mutans synthesize exopolysaccharide (EPS) using glucosyltransferases (Gtfs), resulting in biofilm formation on the tooth surface. Bacterial cells in the biofilms become strongly resistant to a harsh environment, such as antibiotics and host defense mechanisms, making biofilm-based infections difficult to eliminate. Discovering novel antibiofilm agents, especially from natural products, helps to develop effective strategies against this kind of diseases. The present study investigated the inhibitory effect of shikimic acid (SA), one abundant compound derived from Illicium verum extract, on the biofilm formation of S. mutans. We found SA can reduce the EPS synthesized by this oral pathogen and modulate the transcription of biofilm formation related genes, leading to fewer bacterial cells in its biofilm. SA also interacted with cell membrane and membrane proteins, causing damage to bacterial cells. Ex vivo testing of biofilm formation on bovine teeth showed SA strongly decreased the number of S. mutans cells and the number of EPS accumulated on dental enamel surfaces. Moreover, SA exhibits almost no toxicity to human oral cells evaluated by in vitro biocompatibility assay. In conclusion, shikimic acid exhibits remarkable antibiofilm activity against S. mutans and has the potential to be further developed as a novel anticaries agent. IMPORTANCE Natural products are an important and cost-effective source for screening antimicrobial agents. Here, we identified one compound, shikimic acid, from Illicium verum extract, exhibiting antimicrobial activity against S. mutans proliferation. It also inhibits biofilm formation of this bacteria through decreasing Gtf expression and EPS synthesis. Furthermore, this compound exhibits no significant cytotoxicity at its MIC against S. mutans, providing evidence for its clinical application.

The application of selenium nanoparticles for enhancing the efficacy of photodynamic inactivation of planktonic communities and the biofilm of Streptococcus mutans.

OBJECTIVE: Streptococcus mutans is one of the principal causative agents of dental caries (tooth decay) found in the oral cavity. Therefore, this study investigates whether selenium nanoparticles (SeNPs) enhance the efficacy of photodynamic therapy (PDT) against both planktonic communities and the one-day-old biofilm of S. mutans. In this study, the planktonic and 24-h biofilm of S. mutans have been prepared in 96-cell microplates. These forms were treated by methylene blue (MB) and SeNPs and then were exposed to light-emitting diode (LED) lighting. Finally, the results have been reported as CFU/ml. RESULTS: The outcomes demonstrated that MB-induced PDT and SeNPs significantly reduced the number of planktonic bacteria (P-value < 0.001). The comparison between the treated and untreated groups showed that combining therapy with SeNPs and PDT remarkably decreased colony-forming units of one-day-old S. mutans biofilm (P-value < 0.05). The findings revealed that PDT modified by SeNPs had a high potential to destroy S. mutans biofilm. This combination therapy showed promising results to overcome oral infection in dental science.

The Stability of Dentin Surface Biobarrier Consisting of Mesoporous Delivery System on Dentinal Tubule Occlusion and Streptococcus Mutans Biofilm Inhibition.

BACKGROUND: The dentin exposure always leads to dentin hypersensitivity and/or caries. Given the dentin's tubular structure and low mineralization degree, reestablishing an effective biobarrier to stably protect dentin remains significantly challenging. This study reports a versatile dentin surface biobarrier consisting of a mesoporous silica-based epigallocatechin-3-gallate (EGCG)/nanohydroxyapatite delivery system and evaluates its stability on the dentinal tubule occlusion and the Streptococcus mutans (S. mutans) biofilm inhibition. MATERIALS AND METHODS: The mesoporous delivery system was fabricated and characterized. Sensitive dentin discs were prepared and randomly allocated to three groups: 1, control group; 2, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) group; and 3, the mesoporous delivery system group. The dentin permeability, dentinal tubule occlusion, acid and abrasion resistance, and S. mutans biofilm inhibition were determined for 1 week and 1 month. The in vitro release profiles of EGCG, Ca, and P were also monitored. RESULTS: The mesoporous delivery system held the ability to sustainably release EGCG, Ca, and P and could persistently occlude dentinal tubules with acid and abrasion resistance, reduce the dentin permeability, and inhibit the S. mutans biofilm formation for up to 1 month compared with the two other groups. The system provided prolonged stability to combat oral adverse challenges and served as an effective surface biobarrier to protect the exposed dentin. CONCLUSION: The establishment of the dentin surface biobarrier consisting of a mesoporous delivery system indicates a promising strategy for the prevention and the management of dentin hypersensitivity and caries after enamel loss.

Anticariogenic activities of Libidibia ferrea, gallic acid and ethyl gallate against Streptococcus mutans in biofilm model.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, ethnopharmacological studies show that Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz is commonly used in folk medicine as an antifungal, antimicrobial and anti-inflammatory. In the Amazon region, the dried fruit powder of L. ferrea are widely used empirically by the population in an alcoholic tincture as an antimicrobial mouthwash in oral infections and the infusion is also recommended for healing oral wounds. However, there are few articles that have evaluated the antimicrobial activity against oral pathogens in a biofilm model, identifying active compounds and mechanisms of action. AIM OF THE STUDY: The aim of this study was to evaluate the antimicrobial and anti-adherence activities of the ethanolic extract, fractions and isolated compounds (gallic acid and ethyl gallate) of the fruit and seed of L. ferrea against Streptococcus mutans. The inhibition of acidicity/acidogenicity and the expression of the S. mutans GTF genes in biofilms were also evaluated. MATERIALS AND METHODS: Minimal Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Minimum Inhibitory Concentration of Cell Adhesion (MICA) were evaluated with ethanolic extract (EELF), fractions, gallic acid (GA) and ethyl gallate (EG) against S. mutans. Inhibition of biofilm formation, pH drop and proton permeability tests were conducted with EELF, GA and EG, and also evaluated the expression of the GTF genes in biofilms. The compounds of dichloromethane fraction were identified by GC-MS. RESULTS: This is the first report of shikimic, pyroglutamic, malic and protocatechuic acids identified in L. ferrea. EELF, GA and EG showed MIC at 250 µg/mL, and MBC at 1000 µg/mL by EELF. EELF biofilms showed reduced dry weight and acidogenicity of S. mutans in biofilms. GA and EG reduced viable cells, glucans soluble in alkali, acidogenicity, aciduricity and downregulated expression of gtfB, gtfC and gtfD genes in biofilms. SEM images of GA and EG biofilms showed a reduction of biomass, exopolysaccharide and microcolonies of S. mutans. CONCLUSIONS: The ethanolic extract of fruit and seed of L. ferrea, gallic acid and ethyl gallate showed great antimicrobial activity and inhibition of adhesion, reduction of acidogenicity and aciduricity in S. mutans biofilms. The results obtained in vitro validate the use of this plant in ethnopharmacology, and open opportunities for the development of new oral anticariogenic agents, originated by plants that can inhibit pathogenic biofilm that leads to the development of caries.

Effect of LongZhang Gargle on Dual-Species Biofilm of Candida albicans and Streptococcus mutans.

Bioactive natural products have become a hot spot for oral disease treatments. At the present study, LongZhang Gargle was investigated for its effects on single-species biofilms of Candida albicans and dual-species biofilms of Candida albicans and Streptococcus mutans. Two different models of single and dual-species biofilms were grown in YNBB medium under appropriate conditions. Biofilm biomass, biofilm architecture, and cell activity in biofilms were assessed using Crystal Violet Staining, MTT, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Significant reductions of biofilm biomass and fungus activity were obtained when treated with LongZhang Gargle at 2% (P < 0.05), 4% (P < 0.05), and 8% (P < 0.05) in single-species biofilms of C. albicans, and at 4% (P < 0.05) and 8% (P < 0.05) in double-species biofilms. Suppression of density, thickness, and the proportion of hyphae and fungal spores were obtained under SEM and CLSM. In conclusion, LongZhang Gargle affects single and dual-species biofilms by inhibiting biofilm biomass, cell activity, and formation of hyphae, but it does not affect the production of Extracellular polysaccharides (EPS). We speculate that LongZhang Gargle would be a promising natural drug, which can be used in treatment against C. albicans and S. mutans in oral diseases.

Potential for direct application of blue light for photo-disinfection of dentine.

The direct application of light for photo-disinfection potentially provides a safe and novel modality to inhibit or eliminate cariogenic bacteria residing upon and within dentine. This study aimed to both; characterize the pattern of transmission of 405 nm light through molar dentine at different tooth locations, as well as, determine the irradiation parameters that are antibacterial for Streptococcus mutans under various growth conditions, including lawns, planktonic cultures, and biofilms. To determine the amount of light (405 nm) transmitted at different anatomical tooth locations; irradiance values were recorded after blue light (470-4054 mW/cm2) had traversed through occlusal, oblique, and buccal dentine sections; and three thicknesses - 1, 2 and 3 mm were investigated. To determine tubular density; scanning electron micrographs from 2 mm outer (dentine-enamel junction) and inner (pulp) dentine sections were analysed. For photo-disinfection studies; S. mutans was irradiated using the same 405 nm wavelength light at a range of doses (110-1254 J/cm2) in both biofilm and planktonic cultures. The inhibitory effect of the irradiation on bacterial lawns was compared by measuring zones of inhibition; and for planktonic cultures both spectrophotometric and colony forming unit (CFU) assays were performed. A live/dead staining assay was utilised to determine the effect of irradiation on bacterial viability in mature biofilms. Data indicated that increasing dentine thickness decreased light transmission significantly irrespective of its orientation. Occlusal and oblique samples exhibited higher transmission compared with buccal dentine. Oblique dentine 405 nm light transmission was comparable with that of occlusal dentine independent of section thickness. An increased tubule density directly positively correlated with light transmission. Irradiation at 405 nm inhibited S. mutans growth in both biofilm and planktonic cultures and a dose response relationship was observed. Irradiation at doses of 340 and 831 J/cm2 led to significant reductions in bacterial growth and viability; as determined by CFU counting and live/dead staining. Data suggests that phototherapy approaches utilising a 405 nm wavelength have therapeutic potential to limit cariogenic bacterial infections both at the surface and within dentine.

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.

Characterization of the Streptococcus mutans SMU.1703c-SMU.1702c Operon Reveals Its Role in Riboflavin Import and Response to Acid Stress.

Streptococcus mutans utilizes numerous metabolite transporters to obtain essential nutrients in the "feast or famine" environment of the human mouth. S. mutans and most other streptococci are considered auxotrophic for several essential vitamins including riboflavin (vitamin B2), which is used to generate key cofactors and to perform numerous cellular redox reactions. Despite the well-known contributions of this vitamin to central metabolism, little is known about how S. mutans obtains and metabolizes B2 The uncharacterized protein SMU.1703c displays high sequence homology to the riboflavin transporter RibU. Deletion of SMU.1703c hindered S. mutans growth in complex and defined medium in the absence of saturating levels of exogenous riboflavin, whereas deletion of cotranscribed SMU.1702c alone had no apparent effect on growth. Expression of SMU.1703c in a Bacillus subtilis riboflavin auxotroph functionally complemented growth in nonsaturating riboflavin conditions. S. mutans was also able to grow on flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) in an SMU.1703c-dependent manner. Deletion of SMU.1703c and/or SMU.1702c impacted S. mutans acid stress tolerance, as all mutants showed improved growth at pH 5.5 compared to that of the wild type when medium was supplemented with saturating riboflavin. Cooccurrence of SMU.1703c and SMU.1702c, a hypothetical PAP2 family acid phosphatase gene, appears unique to the streptococci and may suggest a connection of SMU.1702c to the acquisition or metabolism of flavins within this genus. Identification of SMU.1703c as a RibU-like riboflavin transporter furthers our understanding of how S. mutans acquires essential micronutrients within the oral cavity and how this pathogen successfully competes within nutrient-starved oral biofilms.IMPORTANCE Dental caries form when acid produced by oral bacteria erodes tooth enamel. This process is driven by the fermentative metabolism of cariogenic bacteria, most notably Streptococcus mutans Nutrient acquisition is key in the competitive oral cavity, and many organisms have evolved various strategies to procure carbon sources or necessary biomolecules. B vitamins, such as riboflavin, which many oral streptococci must scavenge from the oral environment, are necessary for survival within the competitive oral cavity. However, the primary mechanism and proteins involved in this process remain uncharacterized. This study is important because it identifies a key step in S. mutans riboflavin acquisition and cofactor generation, which may enable the development of novel anticaries treatment strategies via selective targeting of metabolite transporters.

Green tea extract solutions can control bacterial biofilms formed on space maintainers.

BACKGROUND: Microorganisms in the mouth are protected from negative environmental conditions by forming biofilms; however, the use of anti-plaque agents in children is not preferred due to toxic side effects. Green tea has been reported to have anti-microbial and anti-dental caries properties. AIMS: The aim of this study was to assess the ability of green tea extract to prevent the formation of biofilm on the teeth of children using space maintainers. METHODS: Bacteria were isolated from samples obtained from children aged between 8 and 10 years. The micro-titer plate method and Congo red agar were used to assay biofilm formation. Green tea leaves were obtained from Rize, Turkey. Methanol, hexane and distilled water were used for preparing the extracts. The effects of green tea extract and chlorhexidine on biofilm formation were examined using scanning electron microscopy. RESULTS: Presence of S. mutans 3,3, S. anginosus 2.1.b, S. dysgalactie 6.1.4.1, and E. faecium 10.2. was measured in the biofilm samples. The extracts showed a bacteriostatic effect on the test bacteria, and among the green tea extracts, the methanol extract was found to exhibit the highest efficacy against biofilm formation by S. mutans 3.3. CONCLUSION: Green tea extract showed good efficacy in controlling bacterial growth, and is recommended as a better-tasting alternative for daily oral hygiene due to a lack of known side effects.

Casein phosphopeptide combined with fluoride enhances the inhibitory effect on initial adhesion of Streptococcus mutans to the saliva-coated hydroxyapatite disc.

BACKGROUND: Recent preventive strategies for dental caries focus on targeting the mechanisms underlying biofilm formation, including the inhibition of bacterial adhesion. A promising approach to prevent bacterial adhesion is to modify the composition of acquired salivary pellicle. This in vitro study investigated the effect and possible underlying mechanism of pellicle modification by casein phosphopeptide (CPP) on Streptococcus mutans (S. mutans) initial adhesion, and the impact of fluoride on the efficacy of CPP. METHODS: The salivary pellicle-coated hydroxyapatite (s-HA) discs were treated with phosphate buffered saline (negative control), heat-inactivated 2.5% CPP (heat-inactivated CPP), 2.5% CPP (CPP) or 2.5% CPP supplemented with 900 ppm fluoride (CPP + F). After cultivation of S. mutans for 30 min and 2 h, the adherent bacteria were visualized by scanning electron microscopy (SEM) and quantitatively evaluated using the plate count method. Confocal laser scanning microscopy (CLSM) was used to evaluate the proportions of total and dead S. mutans. The concentrations of total, free, and bound calcium and fluoride in the CPP and fluoride-doped CPP solutions were determined. The water contact angle and zeta potential of s-HA with and without modification were measured. The data were statistically analyzed using one-way ANOVA followed by a Turkey post hoc multiple comparison test. RESULTS: Compared to the negative control group, the amount of adherent S. mutans significantly reduced in the CPP and CPP + F groups, and was lowest in the CPP + F group. CLSM analysis showed that there was no statistically significant difference in the proportion of dead S. mutans between the four groups. Water contact angle and zeta potential of s-HA surface significantly decreased in the CPP and CPP + F groups as compared to the negative control group, and both were lowest in the CPP + F group. CONCLUSIONS: Pellicle modification by CPP inhibited S. mutans initial adhesion to s-HA, possibly by reducing hydrophobicity and negative charge of the s-HA surface, and incorporating fluoride into CPP further enhanced the anti-adhesion effect.

Antibacterial activity of phellodendron bark against Streptococcus mutans.

Streptococcus mutans is a major cause of tooth decay due to its promotion of biofilm formation and acid production. Several plant extracts have been reported to have multiple biological activities such as anti-inflammation and antibacterial effects. This study investigated the antibacterial activity of three plant extracts, phellodendron bark (PB), yucca, and black ginger, and found that PB had a stronger effect than the other extracts. Then, the minimum inhibitory concentration (MIC) of PB against 100 S. mutans strains was investigated. The MIC range of PB was 9.8-312.5 µg/mL. PB suppressed the growth kinetics of S. mutans in a dose-dependent manner, even at sub-MICs of PB. Then, we investigated the effect of PB on S. mutans virulence. The PB suppressed biofilm formation at high concentrations, although PB did not affect the expression of glucosyltransferase genes. Additionally, PB suppressed the decrease in pH from adding an excess of glucose. The expression of genes responsible for acid production was increased by the addition of excess glucose without PB, whereas their expression levels were not increased in the presence of 1× and 2× MIC of PB. Although PB showed a bacteriostatic effect on planktonic S. mutans cells, it was found that more than 2× MIC of PB showed a partial bactericidal effect on biofilm cells. In conclusion, PB not only showed antibacterial activity against S. mutans but also decreased the cariogenic activity in S. mutans.

The Contribution of Photodynamic Inactivation vs. Corsodyl Mouthwash to the Control of Streptococcus mutans Biofilms.

This work compared the inhibition effect of the commercially available mouthwash Corsodyl, containing 0.1% chlorhexidine digluconate, and photodynamic inactivation (PDI) employing methylene blue (MB) with irradiation from a red laser on 24-h biofilms formed by Streptococcus mutans strains on hydroxyapatite surfaces. The cytotoxicity of Corsodyl and MB was evaluated by Galleria mellonella surviving assay. The viability of biofilm cells after exposure to mouthwash and PDI was determined by counting colony-forming units. The inhibitory effect of antimicrobial agents was confirmed by confocal scanning laser microscopy. MB did not exhibit a cytotoxic effect on larval survival. Non-diluted Corsodyl slightly decreased the survival of larvae. Using our PDI parameters achieved better inhibition than with non-PDI, proving a significant effect on the eradication of S. mutans biofilms and therefore could be an appropriate supplement for the eradication of dental caries.

The impact of photobiomodulation of major salivary glands on caries risk.

Dental caries is a complex multifactorial chronic infectious disease guided by several risk or protective factors. Saliva has an important role in caries and the remineralization process. Caries risk assessment is defined as the probability of new caries lesion development or the existing lesion progression in a given time period. Caries diagnostics and risk factor assessment are followed by targeted elimination of risk factors and less conservative but abundant preventive therapeutic measures. The aim of our prospective randomized study was to elucidate on how photobiomodulation of major salivary glands with polychromatic light or LED light affects caries risk factors in high caries-risk patients. Thirty-six patients were assigned to one of the following three experimental groups: the first, irradiated with polarized polychromatic light (40 mW/cm2, wavelengths 480-3400 nm); the second, a continuous LED light (16 mW/cm2, wavelengths 625, 660, 850 nm); the third, same LED light in a pulsed mode. The fourth group was the control, for which a non-therapeutic visible light was used. Light was administered extra-orally bilaterally above the parotid and submandibular glands for 10 min and intra-orally above the sublingual glands for 5 min, 3 times a week, for 4 consecutive weeks. Each patient's caries risk was assessed according to Cariogram before and after therapy. Caries risk factors were determined from samples of saliva before therapy, two weeks after it commenced, at the end of therapy, and four weeks after the end of therapy. At the end of treatment, the following findings were obtained: In the group irradiated with polarized polychromatic light and in the group irradiated with continuous LED light, the Streptococcus mutans and Lactobacillus counts decreased and salivary buffering capacity increased (p < 0.05). In the group irradiated with pulsed LED light, Streptococcus mutans counts decreased and unstimulated salivary flow and salivary buffering capacity increased (p < 0.05). In all three experimental groups, caries risk was lower (p < 0.05). In the placebo control group, there were no statistically significant differences between parameters before and after therapy. We concluded that photobiomodulation of major salivary glands in high caries-risk patients can reduce the cariogenic bacteria in saliva and improve some salivary parameters, thus reducing caries risk.

Tart cherry (Prunus cerasus L.) fractions inhibit biofilm formation and adherence properties of oral pathogens and enhance oral epithelial barrier function.

Dental caries, candidiasis, and periodontal disease are the most common oral infections affecting a wide range of the population worldwide. The present study investigated the effects of two tart cherry (Prunus cerasus L.) fractions on important oral pathogens, including Candida albicans, Streptococcus mutans, and Fusobacterium nucleatum, as well as on the barrier function of oral epithelial cells. Procyanidins and quercetin and its derivatives were the most important constituents found in the tart cherry fractions. Although the fractions showed poor antimicrobial activity, they inhibited biofilm formation by the three oral pathogens in a dose-dependent manner. The tart cherry fractions also attenuated the adherence of C. albicans and S. mutans to a hydroxylapatite surface as well as the adherence of F. nucleatum to oral epithelial cells. Treating oral epithelial cells with the tart cherry fractions significantly enhanced the barrier function as determined by monitoring the transepithelial electrical resistance. In conclusion, this study showed that the tart cherry fractions and their bioactive constituents could be promising antiplaque compounds by targeting biofilm formation and adherence properties of oral pathogens. Furthermore, its property of increasing the epithelial barrier function may protect against microbial invasion of the underlying connective tissue.

Antimicrobial and antibiofilm activities of Casearia sylvestris extracts from distinct Brazilian biomes against Streptococcus mutans and Candida albicans.

BACKGROUND: Dental caries is a biofilm-diet-dependent worldwide public health problem, and approaches against microorganisms in cariogenic biofilms are necessary. METHODS: The antimicrobial and antibiofilm activities of 12 Casearia sylvestris extracts (0.50 mg/mL) from different Brazilian biomes (Atlantic Forest, Cerrado, Caatinga, Pampa, and Pantanal) and varieties (sylvestris, lingua, and intermediate) were tested against two species found in cariogenic biofilms (Streptococcus mutans and Candida albicans). The extracts effective against S. mutans were used to evaluate the "adhesion strength" of this bacterium to the salivary pellicle and initial glucan matrix and the S. mutans-GtfB activity. Also, the antimicrobial activity against S. mutans of three fractions (methanol, ethyl acetate, and hexane; 0.25 mg/mL) from the extracts was evaluated. RESULTS: Three extracts from the Atlantic Forest variety sylvestris (FLO/SC, GUA/CE, PRE/SP) reduced ≥50% (> 3 logs) S. mutans viable population (p < 0.0001 vs. vehicle), while two extracts from the same biome and variety (PAC/CE, PRE/SP) decreased ≥50% of the viable counts of C. albicans (p < 0.0001 vs. vehicle). For S. mutans biofilms, three extracts (GUA/CE, PAC/CE, PRE/SP) reduced the biomass by ≥91% (p > 0.0001 vs. vehicle) and 100% of the microbial population (p < 0.0001 vs. vehicle). However, for the fungal biofilm, two extracts (PAC/CE, PRE/SP) reduced the viable counts by ≥52% (p < 0.0001 vs. vehicle), but none reduced biomass. The extracts with higher antimicrobial and antibiofilm activities presented higher content of clerodane-type diterpenes and lower content of glycosylated flavonoids than the less active extracts. The extracts had no effect on the removal of cells adhered to the pellicle (p > 0.05 vs. vehicle) while promoted the detachment of a larger number of S. mutans cells from GtfB-glucan matrix (p < 0.0031 vs. vehicle), and FLO/SC, GUA/CE and PRE/SP reduced the quantity of glucans (p < 0.0136 vs. vehicle). Only the ethyl acetate fractions reduced the microbial population of S. mutans (p < 0.0001 vs. vehicle), except for one (PAC/CE). Among the ethyl acetate fractions, three from var. lingua (two from Cerrado, and one from Cerrado/Caatinga) reduced ≥83% of the microbial population. CONCLUSIONS: C. sylvestris extracts from Atlantic Forest var. sylvestris and ethyl acetate fractions from Cerrado and Cerrado/Caatinga var. lingua may be used as a strategy against cariogenic microorganisms.

Diacetylcurcumin: a new photosensitizer for antimicrobial photodynamic therapy in Streptococcus mutans biofilms.

This study evaluated the effect of antimicrobial photodynamic therapy (aPDT) on S. mutans using diacetylcurcumin (DAC) and verified DAC toxicity. In vitro, S. mutans biofilms were exposed to curcumin (CUR) and DAC and were light-irradiated. Biofilms were collected, plated and incubated for colony counts. DAC and CUR toxicity assays were conducted with Human Gingival Fibroblast cells (HGF). In vivo, G. mellonella larvae were injected with S. mutans and treated with DAC, CUR and aPDT. The hemolymph was plated and incubated for colony counts. Significant reductions were observed when DAC and CUR alone were used and when aPDT was applied. HGF assays demonstrated no differences in cell viability for most groups. DAC and CUR reduced the S. mutans load in G. mellonella larvae both alone and with aPDT. Systematic toxicity assays on G. mellonella demonstrated no effect of DAC and CUR or aPDT on the survival curve.

Artemisia princeps Inhibits Growth, Biofilm Formation, and Virulence Factor Expression of Streptococcus mutans.

This study was designed to determine whether the ethanol extract of Artemisia princeps could inhibit the cariogenic activity of Streptococcus mutans. The increase in acid production and biofilm formation by S. mutans were evaluated. The expression levels of virulence factor genes were determined by performing the real-time polymerase chain reaction (PCR). The bactericidal effect was tested by confocal laser scanning microscopy. The A. princeps extract was observed to inhibit the growth of S. mutans at concentrations >0.05 mg/mL (P < .05). After using the safranin staining method, we found that the A. princeps extract had an inhibitory effect against biofilm formation at a concentration of >0.05 mg/mL. These experimental results were similar to that observed with the scanning electron microscopy. The results of the confocal microscopy revealed that the A. princeps extract at high concentrations of 0.4-3.2 mg/mL showed a bactericidal effect in a concentration-dependent manner. According to the results of the real-time PCR analysis, it was observed that the A. princeps extract inhibited the expression of virulence factor genes. These results suggest that A. princeps may inhibit the cariogenic activity of S. mutans, and may be useful as an anticariogenic agent.

Cariogenicity of a Milk-Based Drink Used as a Dietary Supplement for Older Adults Using a Root Caries Experimental Model.

The aim of this study was to evaluate the cariogenicity of a milk-based drink intended for older adults that was used as part of a governmental initiative in Chile to improve their nutritional conditions. This drink contains a high concentration of sugars, which can contribute to root caries development. To test this hypothesis, an experimental biofilm/caries model was used. Dentin slabs were used to grow biofilms of Streptococcus mutans UA159. Slabs/biofilms were exposed 3× per day to bovine milk with different fat content, the milk-based drink, and the milk-based drink supplemented with 10 g of sucrose added per serving. Slabs exposed to 10% sucrose or 0.9% NaCl were used as positive and negative controls, respectively. Biofilms were analyzed for bacterial counts and acidogenicity. Dentin demineralization was estimated by the loss of surface microhardness and integrated mineral loss. Results were compared by analysis of variance and Tukey's test. The milk-based drink showed higher acidogenicity than milk with its entire (whole) or reduced total fat content (skim). The milk-based drink supplemented with -sucrose had similar acidogenicity as the 10% sucrose positive control (p = 0.506). Whole milk exposure elicited lower bacterial counts than the positive control, the milk-based drink, and the milk-based drink supplemented with sucrose (p = 0.002; 0.006 and 0.014 respectively). Although skim milk induced higher demineralization than whole milk, both milk types produced lower demineralization than the milk-based drink. Regarding integrated mineral loss, demineralization induced by the milk-based drink and the milk-based drink supplemented with sucrose was similar to that induced by the positive control and skim milk (p > 0.05). Sugar-containing milk-based drinks used as dietary supplements for older adults may be highly cariogenic and could represent a potential risk for root caries.

Mediterranean herb extracts inhibit microbial growth of representative oral microorganisms and biofilm formation of Streptococcus mutans.

In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against oral infections. The aim of this study was to investigate the antimicrobial and antibiofilm activity of Mediterranean herb extracts against representative oral microorganisms. The extraction procedures and the analysis of the obtained extracts were performed under established experimental conditions. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of the methanol extracts of Cistus creticus ssp. creticus, Cistus monspeliensis, Origanum vulgare, Rosmarinus officinalis, Salvia sclarea and Thymus longicaulis against eight typical oral bacteria and the fungus Candida albicans were determined. The antibiofilm activity against Streptococcus mutans was also quantified using the microtiter plate test. Overall, all tested extracts inhibited effectively the screened obligate anaerobic microorganisms and in concentrations ≥0.3 mg ml-1 had moderate to high antibiofilm activity comparable to that of chlorhexidine (CHX) against S. mutans. In particular, R. officinalis (MIC: 0.08-5.00 mg ml-1) and S. sclarea (MIC: 0.08-2.50 mg ml-1) showed the highest antibacterial activity, while Cistus spp., R. officinalis and S. sclarea significantly inhibited S. mutans biofilm formation at 0.60, 1.25 and 2.50 mg ml-1, respectively. Porphyromonas gingivalis and Parvimonas micra were high susceptible to O. vulgare (MIC = 0.30 mg ml-1), whereas T. longicaulis eradicated all oral bacteria (MBC: 0.15-2.50 mg ml-1). Nevertheless, C. albicans showed no sensitivity to the tested extracts. In conclusion, the tested plant extracts could serve as alternative natural antibacterial and antibiofilm components against oral infections.

Estudio in vitro del Efecto Antibacteriano de la Oleorresina de Copaifera reticulata y el Aceite Esencial de Origanum majoricum Frente a Streptococcus mutans y Enterococcus Faecalis Bacterias de Importancia en Patologías Orales / In vitro Study of the Antibacterial Effect of Oleoresin of Copaifera reticulata and the Essential Oil of Origanum majoricum against Streptococcus mutans and Enterococcus faecalis Bacteria of Importance in Oral Pathologies

RESUMEN: El objetivo del estudio fue determinar el efecto antibacteriano in vitro de la oleorresina de Copaifera reticulata (C. reticulata) "copaiba" y del aceite esencial de Oreganum majoricum (O. majoricum) "orégano" frente a Streptococcus mutans (S. mutans) y Enterococcus faecalis (E. faecalis). Se desarrollaron pruebas de sensibilidad activando primero las cepas bacterias a enfrentar. La oleorresina de copaiba fue diluida con dimetilsulfósido (DMSO), obteniéndose al final concentraciones a probar de 100 %, 50 %, 25 %, y 12,5 %. En relación al aceite esencial de orégano este se probó solamente al 100 %. Para la prueba de difusión en agar con discos, se tomaron inóculos 100 µL de cada cepa bacteriana a una turbidez de 0,5 de Mc Farlam, para ser sembrados por diseminación en placas de tripticasa soya agar, para luego colocar los discos de forma equidistante cargados con las diferentes concentraciones de los productos naturales, se utilizaron como control positivo a la clorhexidina al 0,12 % y al DMSO como control negativo. Se incubaron las placas por el método de la vela en extinción a 37 °C, por un periodo de 24 horas, pasado el tiempo se realizó la lectura de los halos de inhibición. Los resultados obtenidos por la copaiba, determinaron un efecto antibacteriano en sus cuatro concentraciones, siendo los mayores halos de inhibición a la concentración del 100 %, copaiba genero mayores halos promedios para S, mutans de 30,00 ± 0,00 mm y para E. faecalis de 8,3 ± 0,50 mm. Para el caso del orégano se producen halos a la concentración del 100 % con un promedio de 25,3 ± 0,96 mm para S. mutans y para E. faecalis de 9,5 ± 1,29 mm. Se concluye del estudio que tanto copaiba como el orégano presentan un efecto antibacteriano para ambas bacterias, siendo su mayor efecto antibacteriano para ambos productos naturales sobre S. mutans.

ABSTRACT: The objective of the study was to determine the in vitro antibacterial effect of the oleoresin of Copaifera reticulata (C. reticulata) "copaiba" and of the essential oil of Oreganum majoricum (O. majoricum) "oregano" against Streptococcus mutans (S. mutans) and Enterococcus faecalis (E. faecalis). Sensitivity tests were developed by first activating the bacteria strains to be confronted. The oleoresin of copaiba was diluted with dimethylsulphoside (DMSO), obtaining final concentrations to be tested of 100 %, 50 %, 25 %, and 12.5 %. In relation to the essential oil of oregano, it was only 100 % tested. For the disk agar diffusion test, 100 mL of each bacterial strain was taken at a turbidity of 0.5 of Mc Farlam, to be planted by dissecting trypticase soy agar plates, and then placing the disks equidistantly loaded with the different concentrations of natural products; 0.12 % chlorhexidine was used as a positive control and DMSO as negative control. The plates were incubated by the candle method in extinction at 37 °C, for a period of 24 hours, after which time the inhibition halos were read. The results obtained by the copaiba, determined an antibacterial effect in its four concentrations, being the biggest halos of inhibition at the concentration of 100 %, copaiba genus higher average halos for S. mutans of 30.00 ± 0.00 mm and for E. faecalis of 8.3 ± 0.50 mm. In the case of oregano, haloes are produced at a concentration of 100 % with an average of 25.3 ± 0.96 mm for S. mutans and for E. faecalis 9.5 ± 1.29 mm. It is concluded from the study that both copaiba and oregano present an antibacterial effect for both bacteria, being its greater antibacterial effect for both natural products on S. mutans.