The complement system as a key modulator of the oral microbiome in health and disease.

In this review, we address the interplay between the complement system and host microbiomes in health and disease, focussing on oral bacteria known to contribute to homeostasis or to promote dysbiosis associated with dental caries and periodontal diseases. Host proteins modulating complement activities in the oral environment and expression profiles of complement proteins in oral tissues were described. In addition, we highlight a sub-set of bacterial proteins involved in complement evasion and/or dysregulation previously characterized in pathogenic species (or strains), but further conserved among prototypical commensal species of the oral microbiome. Potential roles of these proteins in host-microbiome homeostasis and in the emergence of commensal strain lineages with increased virulence were also addressed. Finally, we provide examples of how commensal bacteria might exploit the complement system in competitive or cooperative interactions within the complex microbial communities of oral biofilms. These issues highlight the need for studies investigating the effects of the complement system on bacterial behaviour and competitiveness during their complex interactions within oral and extra-oral host sites.

Effect of Extracts, Fractions, and Isolated Molecules of Casearia sylvestris to Control Streptococcus mutans Cariogenic Biofilm.

The effects of extracts, fractions, and molecules of Casearia sylvestris to control the cariogenic biofilm of Streptococcus mutans were evaluated. First, the antimicrobial and antibiofilm (initial and pre-formed biofilms) in prolonged exposure (24 h) models were investigated. Second, formulations (with and without fluoride) were assessed for topical effects (brief exposure) on biofilms. Third, selected treatments were evaluated via bacterium growth inhibition curves associated with gene expression and scanning electron microscopy. In initial biofilms, the ethyl acetate (AcOEt) and ethanolic (EtOH) fractions from Brasília (BRA/DF; 250 µg/mL) and Presidente Venceslau/SP (Water/EtOH 60:40 and Water/EtOH 40:60; 500 µg/mL) reduced ≥6-logs vs. vehicle. Only the molecule Caseargrewiin F (CsF; 125 µg/mL) reduced the viable cell count of pre-formed biofilms (5 logs vs. vehicle). For topical effects, no formulation affected biofilm components. For the growth inhibition assay, CsF yielded a constant recovery of surviving cells (≅3.5 logs) until 24 h (i.e., bacteriostatic), and AcOEt_BRA/DF caused progressive cell death, without cells at 24 h (i.e., bactericidal). CsF and AcOEt_BRA/DF damaged S. mutans cells and influenced the expression of virulence genes. Thus, an effect against biofilms occurred after prolonged exposure due to the bacteriostatic and/or bactericidal capacity of a fraction and a molecule from C. sylvestris.

Polymicrobial biofilms related to dental implant diseases: unravelling the critical role of extracellular biofilm matrix.

Biofilms are complex tri-dimensional structures that encase microbial cells in an extracellular matrix comprising self-produced polymeric substances. The matrix rich in extracellular polymeric substance (EPS) contributes to the unique features of biofilm lifestyle and structure, enhancing microbial accretion, biofilm virulence, and antimicrobial resistance. The role of the EPS matrix of biofilms growing on biotic surfaces, especially dental surfaces, is largely unravelled. To date, there is a lack of a broad overview of existing literature concerning the relationship between the EPS matrix and the dental implant environment and its role in implant-related infections. Here, we discuss recent advances in the critical role of the EPS matrix on biofilm growth and virulence on the dental implant surface and its effect on the etiopathogenesis and progression of implant-related infections. Similar to other biofilms associated with human diseases/conditions, EPS-enriched biofilms on implant surfaces promote microbial accumulation, microbiological shift, cross-kingdom interaction, antimicrobial resistance, biofilm virulence, and, consequently, peri-implant tissue damage. But intriguingly, the protagonism of EPS role on implant-related infections and the development of matrix-target therapeutic strategies has been neglected. Finally, we highlight the need for more in-depth analyses of polymicrobial interactions within EPS matrix and EPS-targeting technologies' rationale for disrupting the complex biofilm microenvironment with more outstanding translation to implant applications in the near future.

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 ß-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.

Farnesol delivery via polymeric nanoparticle carriers inhibits cariogenic cross-kingdom biofilms and prevents enamel demineralization.

Streptococcus mutans and Candida albicans are frequently detected together in the plaque from patients with early childhood caries (ECC) and synergistically interact to form a cariogenic cross-kingdom biofilm. However, this biofilm is difficult to control. Thus, to achieve maximal efficacy within the complex biofilm microenvironment, nanoparticle carriers have shown increased interest in treating oral biofilms in recent years. Here, we assessed the anti-biofilm efficacy of farnesol (Far), a hydrophobic antibacterial drug and repressor of Candida filamentous forms, against cross-kingdom biofilms employing drug delivery via polymeric nanoparticle carriers (NPCs). We also evaluated the effect of the strategy on teeth enamel demineralization. The farnesol-loaded NPCs (NPC+Far) resulted in a 2-log CFU/mL reduction of S. mutans and C. albicans (hydroxyapatite disc biofilm model). High-resolution confocal images further confirmed a significant reduction in exopolysaccharides, smaller microcolonies of S. mutans, and no hyphal form of C. albicans after treatment with NPC+Far on human tooth enamel (HT) slabs, altering the biofilm 3D structure. Furthermore, NPC+Far treatment was highly effective in preventing enamel demineralization on HT, reducing lesion depth (79% reduction) and mineral loss (85% reduction) versus vehicle PBS-treated HT, while NPC or Far alone had no differences with the PBS. The drug delivery via polymeric NPCs has the potential for targeting bacterial-fungal biofilms associated with a prevalent and costly pediatric oral disease, such as ECC.

Nanoparticle carrier co-delivery of complementary antibiofilm drugs abrogates dual species cariogenic biofilm formation in vitro.

BACKGROUND: Dental caries is a multifactorial disease caused by pathogenic biofilm. In particular, Streptococcus mutans synthesizes biofilm exopolysaccharides, while Candida albicans is associated with the development of severe carious lesions. AIM: This study aimed to prevent the formation of S. mutans and C. albicans biofilms by exploiting pH-sensitive nanoparticle carriers (NPCs) with high affinity to exopolysaccharides to increase the substantivity of multi-targeted antibiofilm drugs introduced topically in vitro. METHODS: Dual-species biofilms were grown on saliva-coated hydroxyapatite discs with sucrose. Twice-daily, 1.5 min topical treatment regimens of unloaded and drug-loaded NPC were used. Drugs included combinations of two or three compounds with distinct, complementary antibiofilm targets: tt-farnesol (terpenoid; bacterial acid tolerance, fungal quorum sensing), myricetin (flavonoid; exopolysaccharides inhibitor), and 1771 (lipoteichoic acid inhibitor; bacterial adhesion and co-aggregation). Biofilms were evaluated for biomass, microbial population, and architecture. RESULTS: NPC delivering tt-farnesol and 1771 with or without myricetin completely prevented biofilm formation by impeding biomass accumulation, bacterial and fungal population growth, and exopolysaccharide matrix deposition (vs. control unloaded NPC). Both formulations hindered acid production, maintaining the pH of spent media above the threshold for enamel demineralization. However, treatments had no effect on pre-established dual-species biofilms. CONCLUSION: Complementary antibiofilm drug-NPC treatments prevented biofilm formation by targeting critical virulence factors of acidogenicity and exopolysaccharides synthesis.

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial "adhesion strength" to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix' glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

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.

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.

DNase increases the efficacy of antimicrobial photodynamic therapy on Candida albicans biofilms.

Antimicrobial Photodynamic Therapy (aPDT) has been proposed as a means to treat Candida infections. However, microorganisms in biofilms are less susceptible to aPDT than planktonic cultures, possibly because the matrix limits the penetration of the photosensitizer. Therefore, the goals here were: (1) to target biofilm matrix components of a fluconazole-susceptible (S) and a fluconazole-resistant (R) C. albicans (Ca) strains using the hydrolytic enzymes ß-glucanase and DNase individually or in combination; (2) to apply the best enzyme protocol in association with aPDT mediated by Photodithazine® (PDZ); (3) to verify under confocal microscope the penetration of PDZ in biofilms pre-treated or not with DNase at different periods of incubation. CaS and CaR 48h-old biofilms were incubated with the hydrolytic enzymes (5 min) and evaluated by cell viability, biomass, and matrix components. DNase showed the best outcomes by significantly reducing extracellular DNA (eDNA) and soluble proteins from the matrix of both strains; and water-soluble polysaccharides from CaR matrix. Subsequently, 48h-old biofilms were incubated with DNase for 5 min, followed by incubation with PDZ for 20 min and exposure to LED light (660 nm, 50 J/cm²). Controls were biofilms treated only with aPDT without DNase, PDZ only, PDZ + DNase, light only, light + DNase, and biofilm without treatment. Pre-treatment with DNase allowed PDZ penetration into deeper biofilm layers, and the aPDT effect was enhanced, showing a significant reduction of the cell viability (p = 0.000) and eDNA amounts (p ≤ 0.047). DNase affected the matrix composition improving the penetration of the photosensitizer, thereby, improving the effectiveness of subsequent aPDT.

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

Background: 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.

Antimicrobial photodynamic therapy alone or in combination with antibiotic local administration against biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

Antimicrobial photodynamic therapy (aPDT) kills several planktonic pathogens. However, the susceptibility of biofilm-derived anaerobic bacteria to aPDT is poorly characterized. Here, we evaluated the effect of Photodithazine (PDZ)-mediated aPDT on Fusobacterium nucleatum and Porphyromonas gingivalis biofilms. In addition, aPDT was tested with metronidazole (MTZ) to explore the potential antimicrobial effect of the treatment. The minimum inhibitory concentration (MIC) of MTZ was defined for each bacterial species. Single-species biofilms of each species were grown on polystyrene plates under anaerobic conditions for five days. aPDT was performed by applying PDZ at concentrations of 50, 75 and 100 mg/L, followed by exposure to 50 J/cm2 LED light (660 nm) with or without MTZ. aPDT exhibited a significant reduction in bacterial viability at a PDZ concentration of 100 mg/L, with 1.12 log10 and 2.66 log10 reductions for F. nucleatum and P. gingivalis in biofilms, respectively. However, the antimicrobial effect against F. nucleatum was achieved only when aPDT was combined with MTZ at 100× MIC. Regarding P. gingivalis, the combination of PDZ-mediated aPDT at 100 mg/L with MTZ 100× MIC resulted in a 5 log10 reduction in the bacterial population. The potential antimicrobial effects of aPDT in combination with MTZ for both single pathogenic biofilms were confirmed by live/dead staining. These results suggest that localized antibiotic administration may be an adjuvant to aPDT to control F. nucleatum and P. gingivalis biofilms.

Fluconazole impacts the extracellular matrix of fluconazole-susceptible and -resistant Candida albicans and Candida glabrata biofilms.

Background: Fluconazole (FLZ) is a drug commonly used for the treatment of Candida infections. However, ß-glucans in the extracellular matrices (ECMs) hinder FLZ penetration into Candida biofilms, while extracellular DNA (eDNA) contributes to the biofilm architecture and resistance. Methods: This study characterized biofilms of FLZ-sensitive (S) and -resistant (R) Candida albicans and Candida glabrata in the presence or absence of FLZ focusing on the ECM traits. Biofilms of C. albicans American Type Culture Collection (ATCC) 90028 (CaS), C. albicans ATCC 96901 (CaR), C. glabrata ATCC 2001 (CgS), and C. glabrata ATCC 200918 (CgR) were grown in RPMI medium with or without FLZ at 5× the minimum inhibitory concentration (37°C/48 h). Biofilms were assessed by colony-forming unit (CFU)/mL, biomass, and ECM components (alkali-soluble polysaccharides [ASP], water-soluble polysaccharides [WSP], eDNA, and proteins). Scanning electron microscopy (SEM) was also performed. Data were analyzed by parametric and nonparametric tests (α  =  0.05). Results: In biofilms, FLZ reduced the CFU/mL of all strains (p < 0.001), except for CaS (p = 0.937). However, the ASP quantity in CaS was significantly reduced by FLZ (p = 0.034), while the drug had no effect on the ASP levels in other strains (p > 0.05). Total biomasses and WSP were significantly reduced by FLZ in the ECM of all yeasts (p < 0.001), but levels of eDNA and proteins were unaffected (p > 0.05). FLZ affected the cell morphology and biofilm structure by hindering hyphae formation in CaS and CaR biofilms, by decreasing the number of cells in CgS and CgR biofilms, and by yielding sparsely spaced cell agglomerates on the substrate. Conclusion: FLZ impacts biofilms of C. albicans and C. glabrata as evident by reduced biomass. This reduced biomass coincided with lowered cell numbers and quantity of WSPs. Hyphal production by C. albicans was also reduced.

A quest to find good primers for gene expression analysis of Candida albicans from clinical samples.

Biofilm production contributes to several human diseases, including oral candidiasis. Among the Candida species, Candida albicans is the most prevalent. The expression of virulence genes is implicated in the pathogenic potential of Candida biofilms. However, the evaluation of microbial gene expression from in vivo biofilm samples is not trivial, specifically, assessment via quantitative PCR (qPCR) can be a challenge because of several species present in clinical samples. Hence, the necessity of primers specificity. The aim of this study was to evaluate through in silico and in vitro analyses the specificity of published primers and newly designed primers for C. albicans virulence genes: ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, SAP4, SOD1, SOD5 and ACT1 (normalizing gene). In silico analysis was performed through a PubMed search of articles with primer sequences that evaluated gene expression of C. albicans. Then, the sequence similarity of twenty-eight primers was checked through BLASTn and ClustalW2. The analysis of secondary structures was performed using mfold. When the primers did not present satisfactory characteristics (absence of secondary structures, not discrepant Tm of forward and reverse sequences and specificity) following in vitro analysis (i.e., end point PCR), new primers were designed using Beacon Designer™ and sequences obtained from the "Candida Genome Database". The selected primers were tested in vitro by end point PCR using a panel of genomic DNA from five different Candida species (C. albicans, Candida glabrata, Candida dubliniensis, Candida krusei, and Candida tropicalis). The resulting PCR products were visualized on agarose gel. qPCR reactions were performed to determine primers' optimal concentration and PCR efficiency. End point PCR demonstrated that published primers for the SAP1 and HWP1 were specific for C. albicans and the one for SOD1 reacted with C. albicans and C. dubliniensis. The sequence of primers designed for ACT1, ALS1 and HWP1 genes were specific for C. albicans, while the ones for CAP1, CAT1, EFG1, LIP3, and PLB1 were detected in C. albicans and C. dubliniensis. After optimization, all primers presented a single peak on melt curves, correlation coefficient of ≅1 and qPCR reaction efficiency of 90-110%, with slope of ≅-3.3. Therefore, these primers should be suitable for future gene expression analyses from clinical samples.

Inactivation of genes TEC1 and EFG1 in Candida albicans influences extracellular matrix composition and biofilm morphology.

Background: Infections caused by Candida spp. have been associated with formation of a biofilm, i.e. a complex microstructure of cells adhering to a surface and embedded within an extracellular matrix (ECM). Methods: The ECMs of a wild-type (WT, SN425) and two Candida albicans mutant strains, Δ/Δ tec1 (CJN2330) and Δ/Δ efg1 (CJN2302), were evaluated. Colony-forming units (cfu), total biomass (mg), water-soluble polysaccharides (WSPs), alkali-soluble polysaccharides (ASPs), proteins (insoluble part of biofilms and matrix proteins), and extracellular DNA (eDNA) were quantified. Variable-pressure scanning electron microscopy and confocal scanning laser microscopy were performed. The biovolume (µm3/µm2) and maximum thickness (µm) of the biofilms were quantified using COMSTAT2. Results: ASP content was highest in WT (mean ± SD: 74.5 ± 22.0 µg), followed by Δ/Δ tec1 (44.0 ± 24.1 µg) and Δ/Δ efg1 (14.7 ± 5.0 µg). The protein correlated with ASPs (r = 0.666) and with matrix proteins (r = 0.670) in the WT strain. The population in Δ/Δ efg1 correlated with the protein (r = 0.734) and its biofilms exhibited the lowest biomass and biovolume, and maximum thickness. In Δ/Δ tec1, ASP correlated with eDNA (r = 0.678). Conclusion: ASP production may be linked to C. albicans cell filamentous morphology.

Quantitative assessment of salivary oral bacteria according to the severity of dental caries in childhood.

This study aimed to assess differences in selected oral bacteria in children according to the severity of dental caries. One hundred and thirty-six children, 36-60 months old were divided into three groups according to caries status: caries-free (CF) (n=47), early childhood caries (ECC) (n=40) and severe-early childhood caries (S-ECC) (n=49). Saliva was collected for detection and quantification of selected oral streptococci, Actinomyces naeslundii, Lactobacillus spp., Bifidobacterium spp., and Scardovia wiggsiae by quantitative-polymerase chain reaction. The results showed that the detection and quantitative levels of S. mutans, S. sobrinus, Bifidobacterium spp. and S. wiggsiae were significantly higher in S-ECC children compared to CF and ECC children, while for S. salivarius were significantly higher in CF compared to ECC and S-ECC children. There was no statistical difference among the clinical groups for S. mitis, S. oralis, A. naeslundii and Lactobacillus spp. levels and detection. S-ECC children had a lower monthly family income, started tooth brushing later and were breastfeed for a longer duration compared to CF children. S. mutans levels were positively correlated with S. wiggsiae and Bifidobacterium spp. levels, lower mother's education and child bottle-feeding before sleeping and negatively correlated with S. salivarius. It was concluded that in addition to S. mutans, other bacterial species, including bifidobacteria, Scardovia wiggsiae and S. sobrinus, are associated with severity of early childhood caries, although their role in the progress of dental caries remains unclear.

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.

Characterization and optimization of pH-responsive polymer nanoparticles for drug delivery to oral biofilms.

We previously reported on cationic, pH-responsive p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PAA) block copolymer micelles with high affinity for dental and biofilm surfaces and efficient anti-bacterial drug release in response to acidic pH, characteristic of cariogenic (tooth-decay causing) biofilm microenvironments. Here, we show that micelle pH-responsive behaviors can be enhanced through alterations in corona:core molecular weight ratios (CCR). Although similarly stable at physiological pH, upon exposure to acidic pH, micelles with CCR of 4.1 were less stable than other CCR examined. Specifically, a ~1.5-fold increase in critical micelle concentration (CMC) and ~50% decrease in micelle diameters were observed for micelles with CCR of 4.1, compared to no changes in micelles with CCR of 0.8. While high CCR was shown to enhance pH-responsive drug release, it did not alter drug loading and dental surface binding of micelles. Diblocks were shown to encapsulate the antibacterial drug, farnesol, at maximal loading capacities of up to ~27 wt% and at >94% efficiencies, independent of CCR or core size, resulting in micelle diameter increases due to contributions of drug volume. Additionally, micelles with small diameters (~17 nm) show high binding capacity to hydroxyapatite and dental pellicle emulating surfaces based on Langmuir fit analyses of binding data. Finally, micelles with high CCR that have enhanced pH-responsive drug release and binding were shown to exhibit greater antibiofilm efficacy in situ. Overall, these data demonstrate how factors essential for nanoparticle carrier (NPC)-mediated drug deliverycan be enhanced via modification of diblock characteristics, resulting in greater antibiofilm efficacy in situ.

Effect of Twice-Daily Blue Light Treatment on Matrix-Rich Biofilm Development.

BACKGROUND: The use of blue light has been proposed as a direct means of affecting local bacterial infections, however the use of blue light without a photosensitizer to prevent the biofilm development has not yet been explored. The aim of this study was to determine how the twice-daily treatment with blue light affects the development and composition of a matrix-rich biofilm. METHODOLOGY/PRINCIPAL FINDINGS: Biofilms of Streptococcus mutans UA159 were formed on saliva-coated hydroxyapatite discs for 5 days. The biofilms were exposed twice-daily to non-coherent blue light (LumaCare; 420 nm) without a photosensitizer. The distance between the light and the sample was 1.0 cm; energy density of 72 J cm-2; and exposure time of 12 min 56 s. Positive and negative controls were twice-daily 0.12% chlorhexidine (CHX) and 0.89% NaCl, respectively. Biofilms were analyzed for bacterial viability, dry-weight, and extra (EPS-insoluble and soluble) and intracellular (IPS) polysaccharides. Variable pressure scanning electron microscopy and confocal scanning laser microscopy were used to check biofilm morphology and bacterial viability, respectively. When biofilms were exposed to twice-daily blue light, EPS-insoluble was reduced significantly more than in either control group (CHX and 0.89% NaCl). Bacterial viability and dry weight were also reduced relative to the negative control (0.89% NaCl) when the biofilms were treated with twice-daily blue light. Different morphology was also visible when the biofilms were treated with blue light. CONCLUSIONS: Twice-daily treatment with blue light without a photosensitizer is a promising mechanism for the inhibition of matrix-rich biofilm development.

Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms.

Biofilms are highly structured microbial communities that are enmeshed in a self-produced extracellular matrix. Within the complex oral microbiome, Streptococcus mutans is a major producer of extracellular polymeric substances including exopolysaccharides (EPS), eDNA, and lipoteichoic acid (LTA). EPS produced by S. mutans-derived exoenzymes promote local accumulation of microbes on the teeth, while forming a spatially heterogeneous and diffusion-limiting matrix that protects embedded bacteria. The EPS-rich matrix provides mechanical stability/cohesiveness and facilitates the creation of highly acidic microenvironments, which are critical for the pathogenesis of dental caries. In parallel, S. mutans also releases eDNA and LTA, which can contribute with matrix development. eDNA enhances EPS (glucan) synthesis locally, increasing the adhesion of S. mutans to saliva-coated apatitic surfaces and the assembly of highly cohesive biofilms. eDNA and other extracellular substances, acting in concert with EPS, may impact the functional properties of the matrix and the virulence of cariogenic biofilms. Enhanced understanding about the assembly principles of the matrix may lead to efficacious approaches to control biofilm-related diseases.