Insoluble Exopolysaccharide Research Articles

Antimicrobial photodynamic therapy against a dual-species cariogenic biofilm using a ruthenium-loaded resin-based dental material

BackgroundStreptococcus mutans and Candida albicans are associated with caries recurrence. Therefore, this study evaluated the combination of a Ru(II)-loaded resin-based dental material (RDM) and antimicrobial photodynamic therapy (aPDT) against a dual-species biofilm of S. mutans and C. albicans. MethodsAn aPDT protocol was established evaluating Ru(II)'s photocatalytic activity and antimicrobial potential under blue LED irradiation (440–460 nm, 22.55 mW/cm2) at different energy densities (0.00, 6.25, 20.25, 40.50 J/cm2). This evaluation involved singlet oxygen quantification and determination of minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). The biofilm was grown (72 h) on resin disks prepared with Ru(II)-doped RDM (0.00, 0.56, or 1.12 %) and samples were exposed to aPDT or dark conditions. The biofilm was then harvested to analyze cell viability (CFU counts) and formation of soluble and insoluble exopolysaccharides. ResultsThe photocatalytic activity of Ru(II) was concentration and energy density dependent (p < 0.05), and MIC/MBC values were reduced for the microorganisms after LED irradiation (40.5 J/cm2); therefor, this energy density was chosen for aPDT. Although incorporation of Ru(II) into RDM reduced the biofilm growth compared to Ru(II)-free RDM for both species in dark conditions (p < 0.05), aPDT combined with an Ru(II)-loaded RDM (0.56 or 1.12 %) potentialized CFU reductions (p < 0.05). Conversely, only 1.12 % Ru(II) with LED irradiation showed lower levels of both soluble and insoluble exopolysaccharides compared to Ru(II)-free samples in dark conditions (p < 0.05). ConclusionsWhen the Ru(II)-loaded RDM was associated with blue LED, aPDT reduced cell viability and lower soluble and insoluble exopolysaccharides were found in the cariogenic dual-species biofilm.

Distinct Agents Induce Streptococcus mutans Cells with Altered Biofilm Formation Capacity.

ABSTRACTDental caries is a multifactorial biofilm- and sugar-dependent disease. This study investigated the influence of different agents on the induction of surviving Streptococcus mutans cells after successive treatment cycles and characterized the biofilms formed by these cells recovered posttreatment. The agents (with their main targets listed in parentheses) were compound 1771 (lipoteichoic acids), 4′ hydroxychalcone (exopolysaccharides), myricetin (exopolysaccharides), tt-farnesol (cytoplasmatic membrane), sodium fluoride (enolase—glycolysis), chlorhexidine (antimicrobial), and vehicle. Recovered cells from biofilms were generated from exposure to each agent during 10 cycles of consecutive treatments (modeled on a polystyrene plate bottom). The recovered cell counting was different for each agent. The recovered cells from each group were grown as biofilms on saliva-coated hydroxyapatite discs (culture medium with sucrose/starch). In S. mutans biofilms formed by cells recovered from biofilms previously exposed to compound 1771, 4′ hydroxychalcone, or myricetin, cells presented higher expression of the 16S rRNA, gyrA (DNA replication and transcription), gtfB (insoluble exopolysaccharides), and eno (enolase—glycolysis) genes and lower quantities of insoluble dry weight and insoluble exopolysaccharides than those derived from other agents. These findings were confirmed by the smaller biovolume of bacteria and/or exopolysaccharides and the biofilm distribution (coverage area). Moreover, preexposure to chlorhexidine increased exopolysaccharide production. Therefore, agents with different targets induce cells with distinct biofilm formation capacities, which is critical for developing formulations for biofilm control.IMPORTANCE This article addresses the effect of distinct agents with distinct targets in the bacterial cell (cytoplasmatic membrane and glycolysis), the cell’s extracellular synthesis of exopolysaccharides that are important for cariogenic extracellular matrix construction and biofilm buildup in the generation of cells that persisted after treatment, and how these cells form biofilms in vitro. For example, if preexposure to an agent augments the production of virulence determinants, such as exopolysaccharides, its clinical value may be inadequate. Modification of biofilm formation capacity after exposure to agents is critical for the development of formulations for biofilm control to prevent caries, a ubiquitous disease associated with biofilm and diet.

Topical Application of 4'-Hydroxychalcone in Combination with tt-Farnesol Is Effective against Candida albicans and Streptococcus mutans Biofilms.

Candida albicans and Streptococcus mutans interaction in the presence of dietary sucrose yields a complex biofilm with an organized and structured extracellular matrix that increases the tolerance to environmental stress, including antimicrobials. Both species are found in severe early childhood caries lesions. Thus, compounds 4′-hydroxychalcone (C135) (flavonoid intermediate metabolites), tt-farnesol (Far) (terpenoid), and sodium fluoride (F) were tested either isolated or combined as topical treatments (5 min twice daily) against C. albicans and S. mutans dual-species biofilms grown on saliva-coated hydroxyapatite discs. The biofilms were evaluated for gene expression, microbial population, biochemical components, and three-dimensional (3D) structural organization via confocal microscopy and scanning electron microscopy (SEM). The cytotoxicity of formulations was tested on the keratinocyte monolayer. C135 + Far + F promoted lower gene expression of fungal genes associated with β-glucan synthesis (BGL2, FKS1) and remodeling (XOG1, PHR1, PHR2), oxidative stress (SOD1), and drug tolerance (CDR1, ERG11) and higher expression of bacterial nox1 (oxidative and acidic stress tolerance). C135 + Far yielded less insoluble exopolysaccharides, biomass, and proteins (insoluble portion) and lower expression of BGL2, ERG11, SOD1, and PHR2. C135 + F, C135 + Far + F, and C135 rendered lower biomass, thickness, and coverage percentage (confocal microscopy). C135 + Far and C135 + Far + F maintained C. albicans as yeast morphology (SEM). Therefore, the formulations with C135 affected fungal and bacterial targets but exerted a more pronounced effect against fungal cells.

Evaluation of the application of water kefir grains in the decolouration of solutions containg textile dye

One of the main environmental problems presented by the textile industry is the generation of effluents containing synthetic dyes. Because of that, studies have been carried out in order to remove or degrade the dyes present in wastewater and thus, the adsorption process in polymeric matrices has been highlighted due to its low cost and high efficiency in bioremediation. In this context, it was verified the potential of biodegradation of the anthraquinone C. I. Reactive Blue 4 HFG textile dye (Dianativo®) by water kefir grains, as well as the application of the insoluble exopolysaccharide produced by the grains as an adsorbent of the dye, evaluating the effect of pH and initial concentration of dyes in the reduction of staining in a model system. The grains and their insoluble polysaccharide matrix were presented as capable of decolourizing dye solutions under the evaluated conditions, reducing the dye concentration in the solution by up to 92%.

A Novel Gene vp0610 Negatively Regulates Biofilm Formation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is an important foodborne pathogen and its biofilm formation ability facilitates its colonization and persistence in foods by protecting it from stresses including environmental variation and antibiotic exposure. Several important proteins are involved in biofilm formation; however, the identity and function of many remain unknown. In this study, we discovered a hypothetical protein, VP0610 that negatively regulates biofilm formation in Vibrio parahaemolyticus, and we found that the loss of vp0610 typically results in pleiotropic phenotypes that contribute toward promoting biofilm formation, including significantly increased insoluble exopolysaccharide production and swimming motility, decreased soluble exopolysaccharide production, and decreased bis-(3′-5′)-cyclic dimeric guanosine monophosphate production. Pull-down assays revealed that VP0610 can interact with 180 proteins, some of which (Hfq, VP0710, VP0793, and CyaA) participate in biofilm formation. Moreover, deleting vp0610 enhanced the expression of genes responsible for biofilm component (flaE), the sugar phosphotransferase system (PTS) EIIA component (vp0710 and vp0793), and a high-density regulator of quorum sensing (opaR), while reducing the expression of the bis-(3′-5′)-cyclic dimeric guanosine monophosphate degradation protein (CdgC), resulting in faster biofilm formation. Taken together, our results indicate that vp0610 is an integral member of the key biofilm regulatory network of V. parahaemolyticus that functions as a repressor of biofilm formation.

Differential miRNA Expression in Human Macrophage-Like Cells Infected with Histoplasma capsulatum Yeasts Cultured in Planktonic and Biofilm Forms

Histoplasma capsulatum affects healthy and immunocompromised individuals, sometimes causing a severe disease. This fungus has two morphotypes, the mycelial (infective) and the yeast (parasitic) phases. MicroRNAs (miRNAs) are small RNAs involved in the regulation of several cellular processes, and their differential expression has been associated with many disease states. To investigate miRNA expression in host cells during H. capsulatum infection, we studied the changes in the miRNA profiles of differentiated human macrophages infected with yeasts from two fungal strains with different virulence, EH-315 (high virulence) and 60I (low virulence) grown in planktonic cultures, and EH-315 grown in biofilm form. MiRNA profiles were evaluated by means of reverse transcription-quantitative polymerase chain reaction using a commercial human miRNome panel. The target genes of the differentially expressed miRNAs and their corresponding signaling pathways were predicted using bioinformatics analyses. Here, we confirmed biofilm structures were present in the EH-315 culture whose conditions facilitated producing insoluble exopolysaccharide and intracellular polysaccharides. In infected macrophages, bioinformatics analyses revealed especially increased (hsa-miR-99b-3p) or decreased (hsa-miR-342-3p) miRNAs expression levels in response to infection with biofilms or both growth forms of H. capsulatum yeasts, respectively. The results of miRNAs suggested that infection by H. capsulatum can affect important biological pathways of the host cell, targeting two genes: one encoding a protein that is important in the cortical cytoskeleton; the other, a protein involved in the formation of stress granules. Expressed miRNAs in the host’s response could be proposed as new therapeutic and/or diagnostic tools for histoplasmosis.

Modulation of Lipoteichoic Acids and Exopolysaccharides Prevents Streptococcus mutans Biofilm Accumulation

Dental caries is a diet–biofilm-dependent disease. Streptococcus mutans contributes to cariogenic biofilms by producing an extracellular matrix rich in exopolysaccharides and acids. The study aimed to determine the effect of topical treatments with compound 1771 (modulates lipoteichoic acid (LTA) metabolism) and myricetin (affects the synthesis of exopolysaccharides) on S. mutans biofilms. In vitro S. mutans UA159 biofilms were grown on saliva-coated hydroxyapatite discs, alternating 0.1% sucrose and 0.5% sucrose plus 1% starch. Twice-daily topical treatments were performed with both agents alone and combined with and without fluoride: compound 1771 (2.6 µg/mL), myricetin (500 µg/mL), 1771 + myricetin, fluoride (250 ppm), 1771 + fluoride, myricetin + fluoride, 1771 + myricetin + fluoride, and vehicle. Biofilms were evaluated via microbiological, biochemical, imaging, and gene expression methods. Compound 1771 alone yielded less viable counts, biomass, exopolysaccharides, and extracellular LTA. Moreover, the combination 1771 + myricetin + fluoride decreased three logs of bacterium counts, 60% biomass, >74% exopolysaccharides, and 20% LTA. The effect of treatments on extracellular DNA was not pronounced. The combination strategy affected the size of microcolonies and exopolysaccharides distribution and inhibited the expression of genes linked to insoluble exopolysaccharides synthesis. Therefore, compound 1771 prevented the accumulation of S. mutans biofilm; however, the effect was more pronounced when it was associated with fluoride and myricetin.

Antibacterial and Antibiofilm Activity of Temporin-GHc and Temporin-GHd Against Cariogenic Bacteria, Streptococcus mutans.

Temporin-GHc (GHc) and temporin-GHd (GHd) produced by the frog Hylarana guentheri had shown broad-spectrum antibacterial activities against bacteria and fungi. In this study, we investigated whether they exert antibacterial and antibiofilm activities against cariogenic bacteria, Streptococcus mutans. GHc and GHd adopt the random coil conformation in aqueous solution and convert to α-helix in membrane mimetic environments by using circular dichroism spectroscope. They are positively charged by histidine, with the polar and nonpolar amino acids on opposing faces along the helix. The amphipathicity enabled the peptides to target at bacterial membrane, leading to an increase in membrane permeation and disruption of S. mutans, which allowed the peptides to bind with genomic DNA. GHc and GHd completely impeded the initial attachment of biofilm formation and disrupted preformed S. mutans biofilms. The expression of exopolysaccharide (EPS) biosynthesis genes which synthesize glucosyltransferases in S. mutans was downregulated by exposing to GHc or GHd, contributing to the decrease of soluble and insoluble EPS. GHc and GHd exhibited selectivity toward S. mutans in the presence of human erythrocytes, and no cytotoxicity toward human oral epithelial cells was observed at a concentration of 200 μM. These results laid the foundation for the development of GHc and GHd as potential anti-caries agents.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth.

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J·cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Inactivation of Streptococcus mutans genes lytST and dltAD impairs its pathogenicity in vivo

ABSTRACTBackground: Streptococcus mutans orchestrates the development of a biofilm that causes dental caries in the presence of dietary sucrose, and, in the bloodstream, S. mutans can cause systemic infections. The development of a cariogenic biofilm is dependent on the formation of an extracellular matrix rich in exopolysaccharides, which contains extracellular DNA (eDNA) and lipoteichoic acids (LTAs). While the exopolysaccharides are virulence markers, the involvement of genes linked to eDNA and LTAs metabolism in the pathogenicity of S. mutans remains unclear. Objective and Design: In this study, a parental strain S. mutans UA159 and derivative strains carrying single gene deletions were used to investigate the role of eDNA (ΔlytS and ΔlytT), LTA (ΔdltA and ΔdltD), and insoluble exopolysaccharides (ΔgtfB) in virulence in a rodent model of dental caries (rats) and a systemic infection model (Galleria mellonella larvae). Results: Fewer carious lesions were observed on smooth and sulcal surfaces of enamel and dentin of the rats infected with ∆lytS, ∆dltD, and ΔgtfB (vs. the parental strain). Moreover, strains carrying gene deletions prevented the killing of larvae (vs. the parental strain). Conclusions: Altogether, these findings indicate that inactivation of lytST and dltAD impaired S. mutans cariogenicity and virulence in vivo.

Effect of tt-farnesol and myricetin on in vitro biofilm formed by Streptococcus mutans and Candida albicans

BackgroundDental caries is considered a multifactorial disease, in which microorganisms play an important role. The diet is decisive in the biofilm formation because it provides the necessary resources for cellular growth and exopolysaccharides synthesis. Exopolysaccharides are the main components of the extracellular matrix (ECM). The ECM provides a 3D structure, support for the microorganisms and form diffusion-limited environments (acidic niches) that cause demineralization of the dental enamel. Streptococcus mutans is the main producer of exopolysaccharides. Candida albicans is detected together with S. mutans in biofilms associated with severe caries lesions. Thus, this study aimed to determine the effect of tt-farnesol and myricetin topical treatments on cariogenic biofilms formed by Streptococcus mutans and Candida albicans.MethodsIn vitro dual-species biofilms were grown on saliva-coated hydroxyapatite discs, using tryptone-yeast extract broth with 1% sucrose (37 °C, 5% CO2). Twice-daily topical treatments were performed with: vehicle (ethanol 15%, negative control), 2 mM myricetin, 4 mM tt-farnesol, myricetin + tt-farnesol, myricetin + tt-farnesol + fluoride (250 ppm), fluoride, and chlorhexidine digluconate (0.12%; positive control). After 67 h, biofilms were evaluated to determine biofilm biomass, microbial population, and water-soluble and -insoluble exopolysaccharides in the ECM.ResultsOnly the positive control yielded a reduced quantity of biomass and microbial population, while tt-farnesol treatment was the least efficient in reducing C. albicans population. The combination therapy myricetin + farnesol + fluoride significantly reduced water-soluble exopolysaccharides in the ECM (vs. negative control; p < 0.05; ANOVA one-way, followed by Tukey’s test), similarly to the positive control.ConclusionsTherefore, the combination therapy negatively influenced an important virulence trait of cariogenic biofilms. However, the concentrations of both myricetin and tt-farnesol should be increased to produce a more pronounced effect to control these biofilms.

Effect of methylene blue-induced photodynamic therapy on a Streptococcus mutans biofilm model

BackgroundSeveral studies have reported the use of antimicrobial photodynamic therapy (aPDT) to control biofilm but its efficacy depends on several factors, such as biofilm model used. This study aims to examine whether exposure to diode laser combined with methylene blue affects the bacterial viability and polysaccharide content in a Streptococcus mutans cariogenic biofilm model, which simulated ‘feast-famine’ episodes of exposure to sucrose that occur in the oral cavity. Materials and methodsS. mutans biofilms were formed on acrylic resin discs and exposed to a 10% sucrose solution for 1min, eight times/day. After growing for 48h, the biofilms were submitted to the following treatments, twice daily (n=4): (i) 0.9% NaCl (NaCl) as the negative control; (ii) 0.12% chlorhexidine digluconate (CHX) as the positive antibacterial control; (iii) diode laser combined with methylene blue, using an energy density of 320J/cm2 (aPDT). After 120h of growth, the biofilm formed on each disc was collected to determine the viable bacterial counts and concentration of insoluble exopolysaccharides (IEPS) and intracellular polysaccharides (IPS). ResultsBacterial counts in the biofilms formed differed among the treatments. Compared with NaCl, aPDT significantly destabilized biofilm (p<0.0001). aPDT and CHX equally lowered the concentration of IEPS and IPS in biofilms. ConclusionUnder the experimental conditions assessed, our findings indicate that a twice-daily treatment with diode laser combined with methylene blue effectively decreased bacterial viability and the intra- and extracellular polysaccharide concentration in biofilms of S. mutans, a cariogenic bacterium.

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ∆lytS and ∆lytT; LTA – ∆dltA and ∆dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

L-Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms.

l-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenous l-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However, l-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenous l-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol) l-arginine (also a clinically effective concentration) exposure suppressed the outgrowth of S. mutans, favored S. gordonii dominance, and maintained Actinomyces naeslundii growth within biofilms (versus vehicle control). In parallel, topical l-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly, l-arginine repressed S. mutans genes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, while spxB expression (H2O2 production) by S. gordonii increased sharply during biofilm development, which resulted in higher H2O2 levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while the in situ pH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties of l-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor. Dental caries is one of the most prevalent and costly infectious diseases worldwide, caused by a biofilm formed on tooth surfaces. Novel strategies that compromise the ability of virulent species to assemble and maintain pathogenic biofilms could be an effective alternative to conventional antimicrobials that indiscriminately kill other oral species, including commensal bacteria. l-Arginine at 1.5% has been shown to be clinically effective in modulating cariogenic biofilms via alkali production by arginolytic bacteria. Using a mixed-species ecological model, we show new mechanisms by which l-arginine disrupts the process of biofilm matrix assembly and the dynamic microbial interactions that are associated with cariogenic biofilm development, without impacting the bacterial viability. These results may aid in the development of enhanced methods to control biofilms using l-arginine.

Baccharis dracunculifolia-based mouthrinse alters the exopolysaccharide structure in cariogenic biofilms

Baccharis dracunculifolia is a native plant from Brazil with antimicrobial activity. The purpose of this study was to investigate whether a B. dracunculifolia-based mouthrinse (Bd) changes the structure of insoluble exopolysaccharides (IEPS) in Streptococcus mutans UA159 cariogenic biofilm. Biofilms were grown on glass slides and treated with Bd, its vehicle (VC), chlorhexidine digluconate (CHX), or saline solution (NaCl). Among the treatments, only CHX significantly reduced the biofilm biomass and bacterial viability (p<0.05). Gas chromatography–mass spectrometry and nuclear magnetic resonance analyses revealed that IEPS from the four biofilm samples were α- glucans containing different proportions of (1→6) and (1→3) glycosidic linkages. The structural differences among the four IEPS were compared by principal component analysis (PCA). PCA analysis indicated that IEPS from VC- and NaCl-treated biofilms were structurally similar to each other. Compared with the control, IEPS from Bd- and CHX-treated biofilms were structurally different and had distinct chemical profiles. In summary, the fact that Bd changed the IEPS chemical composition indicates that this mouthrinse may affect the cariogenic properties of the S. mutans biofilm formed.

Surface-induced changes in the conformation and glucan production of glucosyltransferase adsorbed on saliva-coated hydroxyapatite.

Glucosyltransferases (Gtfs) from S. mutans play critical roles in the development of virulent oral biofilms associated with dental caries disease. Gtfs adsorbed to the tooth surface produce glucans that promote local microbial colonization and provide an insoluble exopolysaccharides (EPS) matrix that facilitates biofilm initiation. Moreover, agents that inhibit the enzymatic activity of Gtfs in solution often have reduced or no effects on surface-adsorbed Gtfs. This study elucidated the mechanisms responsible for the differences in functionality that GtfB exhibits in solution vs surface-adsorbed. Upon adsorption to planar fused-quartz substrates, GtfB displayed a 37% loss of helices and 36% increase of β-sheets, as determined by circular dichroism (CD) spectroscopy, and surface-induced conformational changes were more severe on substrates modified with CH3- and NH2-terminated self-assembled monolayers. GtfB also underwent substantial conformation changes when adsorbing to hydroxyapatite (HA) microspheres, likely due to electrostatic interactions between negatively charged GtfB and positively charged HA crystal faces. Conformational changes were lessened when HA surfaces were coated with saliva (sHA) prior to GtfB adsorption. Furthermore, GtfB remained highly active on sHA, as determined by in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, producing glucans that were structurally different than GtfB in solution and known to increase the accumulation and virulence of biofilms. Our data provide the first insight into the structural underpinnings governing Gtf conformation and enzymatic function that occur on tooth surfaces in vivo, which may lead to designing potent new inhibitors and improved strategies to combat the formation of pathogenic oral biofilms.

Inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on exopolysaccharide production by Streptococcus mutans under microfluidic conditions

Plaque-forming microorganisms produce insoluble exopolysaccharide (EPS), which allows the microorganisms to attach strongly to the tooth surface. Then, they start forming biofilm, which is responsible for plaque formation. The green tea polyphenol epigallocatechin gallate (EGCG) is known to inhibit biofilm formation of oral bacteria. However, its inhibitory effects on biofilm formation between the teeth have not been well studied due to the lack of devices that mimic the space between the teeth. In this study, we used a microfluidic device packed with glass beads (250–300 µm in diameter) to mimic the small cavities between the teeth in order to test the inhibitory effects of EGCG on the biofilm formation of Streptococcus mutans, one of the plaque-forming microorganisms. EPS production by S. mutans was more effectively inhibited by EGCG in the microfluidic device, compared to on agar plates, suggesting that it is more effective against biofilm formation by S. mutans cells under flow conditions than under non-flow conditions, such as conditions achieved by agar plates. These results suggest that our microfluidic device may be highly useful for studying the actual effects of antimicrobial compounds against plaqueforming microorganisms.

In vitro study of the effect of a lactoperoxidase-peroxidase-thiocyanate system with iodine on the cariogenicinity of streptococcus mutans

This study aimed to investigate the effect of a lactoperoxidase-peroxidase-thiocyanate (LPO-H2O-SCN-) system with different concentrations of iodine (I-) on Streptococcus mutans (S. mutans), particularly on various parameters, including growth, adhesion, glucosyltransferase (GTF) enzyme activity, and insoluble exopolysaccharide synthesis. S. mutans ATCC 25175 was used as experimental species. Clonal formation unit (CFU) were counted to investigate the inhibitory effect on bacterial growth. The inhibition rate of bacterial adherence was calculated to analyze the effect on adhesion. Anthrone method was used to determine the content of insoluble exopolysaccharides and the amount of reducing saccharides. GTF activity and enzyme activity were then determined. The inhibitory ability of the LPO-H2O2-SCN- system with I- on the cariogenicinity of S. mutans was strengthened as I- concentration was increased. At I- concentration > or = 100 micromol x L(-1) the antibacterial effects were significantly increased compared with those of the control group (P < 0.05). At I- concentration > or = 1,000 micromol x L(-1), the antibacterial effects were significantly improved compared with those of the group with SCN-only (P < 0.05). At I- concentration > or = 100 micromol x L(-1), the inhibition rate of bacterial adherence was > 50%; insoluble exopolysaccharide synthesis and GTF enzyme activity were reduced (P < 0.05). The antibacterial effects of the LPO-H2O2-I- system were enhanced by adding I- to overcome the antagonistic effect of physiological SCN- concentration. LPO-H2O2-SCN- system with different concentrations of I- showed statistically significant inhibitory effects on growth, adhesion, insoluble exopolysaccharide synthesis, and GTF enzyme activity.

Exopolysaccharide matrix of developed candida albicans biofilms after exposure to antifungal agents

This study aimed to evaluate the effects of fluconazole or nystatin exposure on developed Candida albicans biofilms regarding their exopolysaccharide matrix. The minimal inhibitory concentration (MIC) against fluconazole or nystatin was determined for C. albicans reference strain (ATCC 90028). Poly(methlymethacrylate) resin (PMMA) specimens were fabricated according to the manufacturer's instructions and had their surface roughness measured. Biofilms were developed on specimens surfaces for 48 h and after that were exposed during 24 h to fluconazole or nystatin prepared in a medium at MIC, 10 x MIC or 100 x MIC. Metabolic activity was evaluated using an XTT assay. Production of soluble and insoluble exopolysaccharide and intracellular polysaccharides was evaluated by the phenol-sulfuric method. Confocal laser scanning microscope was used to evaluate biofilm architecture and percentage of dead/live cells. Data were analyzed statistically by ANOVA and Tukey's test at 5% significance level. The presence of fluconazole or nystatin at concentrations higher than MIC results in a great reduction of metabolic activity (p<0.001). At MIC or 10 x MIC, fluconazole showed high amounts of intracellular polysaccharides (p<0.05), but did not affect the exopolysaccharide matrix (p>0.05). The exposure to nystatin also did not alter the exopolysaccharide matrix at all the tested concentrations (p>0.05). Biofilm architecture was not affected by either of the antifungal agents (p>0.05). Nystatin promoted higher proportion of dead cells (p<0.05). It may be concluded that fluconazole and nystatin above the MIC concentration reduced the metabolic activity of C. albicans biofilms; however, they were not able to alter the exopolysaccharide matrix and biofilm architecture.

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.