Extensive Description and Comparison of Human Supra-Gingival Microbiome in Root Caries and Health

Knowledge of the polymicrobial etiology of root caries is limited. To conduct a comprehensive research study on root caries, we utilized 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to compare supra-gingival bacterial communities from healthy sites and carious sites of 21 patients with root caries (Patient-controls and Patient-cases) and the sites of 21 healthy individuals (Healthy-controls) from two nursing homes. Healthy-controls and Patient-cases showed no significant differences in terms of biomass, species richness, and species diversity. However, as for beta diversity based on either community membership metric (unweighted UniFrac) or community structure metric (weighted UniFrac), Healthy-controls and Patient-cases were clearly distinguished from each other, appearing more variable in the community membership and structure in root caries microbiome but relatively conserved in the health microbiome. The Patient-controls group was at an intermediate stage between Healthy-controls and Patient-cases, but was more inclined to the former. Demonstrated in both relative abundance and prevalence of species in health and root caries, Propionibacterium acidifaciens, Streptococcus mutans, Olsenella profusa, Prevotella multisaccharivorax, and Lactobacillus crispatus were found to be most associated with root caries, whereas Delftia acidovorans, Bacteroidetes[G-2] sp., Lachnospiraceae[G-3] sp., and Prevotella intermedia are most associated with health. Our study provides a basis for further elucidating the microbial etiology of root caries in the elderly.     

Oral microbiota of children in a school-based dental clinic

ObjectivesDental caries disproportionately affects disadvantaged subjects. This study hypothesized that there were greater caries extent and higher levels of caries-associated and anaerobic subgingival bacterial species in oral samples of Hispanic and immigrant children compared with non-Hispanic and US born children.MethodsChildren from a school-based dental clinic serving a community with a large Hispanic component were examined, and the extent of caries was recorded. Microbial samples were taken from teeth and the tongues of children. Samples were analyzed using DNA probes to 18 oral bacterial species.ResultsSeventy five children were examined. Extent of caries increased with child age in immigrant, but not in US born or Hispanic children. There were no differences in the microbiota based on ethnicity or whether the child was born in US or not. There was a higher species detection frequency from teeth than tongue samples. Levels of Streptococcus mutans and other Streptococcus spp increased with caries extent. Prevotella intermedia, Tannerella forsythia and Selenomonas spp were detected at low levels in these children.ConclusionsWe conclude that, while there was a high rate of dental caries in disadvantaged school children, there were no differences in the caries-associated microbiota, including S. mutans, based on ethnicity or immigration status. Furthermore, while anaerobic subgingival, periodontal pathogens were also detected in children, there was no difference in species detection based on ethnicity or immigration status. Increased levels of streptococci, including S. mutans, however, were detected with high caries levels. This suggested that while it is beneficial to target preventive and treatment programs to disadvantaged populations, there is likely no additional benefit to focus on subgroups within a population already at high risk for dental disease.     

Antimicrobial effects of herbal extracts on Streptococcus mutans and normal oral streptococci

Streptococcus mutans is associated with dental caries. A cariogenic biofilm, in particular, has been studied extensively for its role in the formation of dental caries. Herbal extracts such as Cudrania tricuspidata, Sophora flavescens, Ginkgo biloba, and Betula Schmidtii have been used as a folk remedy for treating diseases. The purpose of this study was to evaluate and compare the antibacterial activity of herbal extracts against normal oral streptococci, planktonic and biofilm of S. mutans. Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguinis, and S. mutans were cultivated with brain heart infusion broth and susceptibility assay for the herbal extracts was performed according to the protocol of Clinical and Laboratory Standard Institute. Also, S. mutans biofilm was formed on a polystyrene 12-well plate and 8-well chamber glass slip using BHI broth containing 2% sucrose and 1% mannose after conditioning the plate and the glass slip with unstimulated saliva. The biofilm was treated with the herbal extracts in various concentrations and inoculated on Mitis-Salivarius bacitracin agar plate for enumeration of viable S. mutans by counting colony forming units. Planktonic S. mutans showed susceptibility to all of the extracts and S. mutans biofilm exhibited the highest level of sensitivity for the extracts of S. flavescens. The normal oral streptococci exhibited a weak susceptibility in comparison to S. mutans. S. oralis, however, was resistant to all of the extracts. In conclusion, the extract of S. flavescens may be a potential candidate for prevention and management of dental caries.     

Comment: Low dental caries rate in Neandertals: The result of diet or the oral flora composition?

Dental caries is an infectious disease caused by oral acidophilic bacteria feeding on fermentable sugars, e.g. Streptococcus mutans. The frequency of dental caries in Neandertals was very low. This was usually explained as the result of a low-sugar diet. Recent research, however, revealed some regional differences between European and Near Eastern Neandertals, with the latter consuming considerable amounts of plants including highly cariogenic dates. This discovery, compared with the results of research on genetic diversity of S. mutans, may suggest that this species, and perhaps other most virulent species, were absent in the oral flora of Neandertals.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.     

The oral metagenome in health and disease

The oral cavity of humans is inhabited by hundreds of bacterial species and some of them have a key role in the development of oral diseases, mainly dental caries and periodontitis. We describe for the first time the metagenome of the human oral cavity under health and diseased conditions, with a focus on supragingival dental plaque and cavities. Direct pyrosequencing of eight samples with different oral-health status produced 1 Gbp of sequence without the biases imposed by PCR or cloning. These data show that cavities are not dominated by Streptococcus mutans (the species originally identified as the ethiological agent of dental caries) but are in fact a complex community formed by tens of bacterial species, in agreement with the view that caries is a polymicrobial disease. The analysis of the reads indicated that the oral cavity is functionally a different environment from the gut, with many functional categories enriched in one of the two environments and depleted in the other. Individuals who had never suffered from dental caries showed an over-representation of several functional categories, like genes for antimicrobial peptides and quorum sensing. In addition, they did not have mutans streptococci but displayed high recruitment of other species. Several isolates belonging to these dominant bacteria in healthy individuals were cultured and shown to inhibit the growth of cariogenic bacteria, suggesting the use of these commensal bacterial strains as probiotics to promote oral health and prevent dental caries.     

Antibacterial Peptides: Opportunities for the Prevention and Treatment of Dental Caries

Dental caries is a multifactorial disease that is a growing and costly global health concern. The onset of disease is a consequence of an ecological imbalance within the dental plaque biofilm that favors specific acidogenic and aciduric caries pathogens, namely Streptococcus mutans and Streptococcus sobrinus. It is now recognized by the scientific and medical community that it is neither possible nor desirable to totally eliminate dental plaque. Conversely, the chemical biocides most commonly used for caries prevention and treatment indiscriminately attack all plaque microorganisms. These treatments also suffer from other drawbacks such as bad taste, irritability, and staining. Furthermore, the public demand for safe and natural personal hygiene products continues to rise. Therefore, there are opportunities that exist to develop new strategies for the treatment of this disease. As an alternative to conventional antibiotics, antibacterial peptides have been explored greatly over the last three decades for many different therapeutic uses. There are currently tens of hundreds of antibacterial peptides characterized across the evolutionary spectrum, and among these, many demonstrate physical and/or biological properties that may be suitable for a more targeted approach to the selective control or elimination of putative caries pathogens. Additionally, many peptides, such as nisin, are odorless, colorless, and tasteless and do not cause irritation or staining. This review focuses on antibacterial peptides for their potential role in the treatment and prevention of dental caries and suggests candidates that need to be explored further. Practical considerations for the development of antibacterial peptides as oral treatments are also discussed.     

Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.     

Antimicrobial and anti-biofilm effect of Bac8c on major bacteria associated with dental caries and Streptococcus mutans biofilms

Dental caries is a common oral bacterial infectious disease. Its prevention and treatment requires control of the causative pathogens within dental plaque, especially Streptococcus mutans (S. mutans). Antimicrobial peptides (AMPs), one of the promising substitutes for conventional antibiotics, have been widely tested and used for controlling bacterial infections. The present study focuses on evaluating the potential of the novel AMPs cyclic bactenecin and its derivatives against bacteria associated with dental caries. The results indicate that Bac8c displayed highest activity against the bacteria tested, whereas both cyclic and linear bactenecin had weak antimicrobial activity. The cytotoxicity assay showed that Bac8c did not cause detectable toxicity at concentrations of 32–128 μg/ml for 5 min or 32–64 μg/ml for 60 min. S. mutans and Lactobacillus fermenti treated with Bac8c showed variable effects on bacterial structure via scanning electron microscopy and transmission electron microscopy. There appeared to be a large amount of extracellular debris and obvious holes on the cell surface, as well as loss of cell wall and nucleoid condensation. The BioFlux system was employed to generate S. mutans biofilms under a controlled flow, which more closely resemble the formation process of natural biofilms. Bac8c remarkably reduced the viability of cells in biofilms formed in the BioFlux system. This phenomenon was further analyzed and verified by real-time PCR results of a significant suppression of the genes involved in S. mutans biofilm formation. Taken together, this study suggests that Bac8c has a potential clinical application in preventing and treating dental caries.     

Direct interactions with commensal streptococci modify intercellular communication behaviors of Streptococcus mutans

The formation of dental caries is a complex process that ultimately leads to damage of the tooth enamel from acids produced by microbes in attached biofilms. The bacterial interactions occurring within these biofilms between cariogenic bacteria, such as the mutans streptococci, and health-associated commensal streptococci, are thought to be critical determinants of health and disease. To better understand these interactions, a Streptococcus mutans reporter strain that actively monitors cell–cell communication via peptide signaling was cocultured with different commensal streptococci. Signaling by S. mutans, normally highly active in monoculture, was completely inhibited by several species of commensals, but only when the bacteria were in direct contact with S. mutans. We identified a novel gene expression pattern that occurred in S. mutans when cultured directly with these commensals. Finally, mutant derivatives of commensals lacking previously shown antagonistic gene products displayed wild-type levels of signal inhibition in cocultures. Collectively, these results reveal a novel pathway(s) in multiple health-associated commensal streptococci that blocks peptide signaling and induces a common contact-dependent pattern of differential gene expression in S. mutans. Understanding the molecular basis for this inhibition will assist in the rational design of new risk assessments, diagnostics, and treatments for the most pervasive oral infectious diseases.Subject terms: Biofilms, Microbial ecology, Bacteriology, Bacterial genetics     

Improving the Efficiency of the Modified Hodge Test in KPC-Producing Klebsiella pneumoniae Isolates by Incorporating an EDTA Disk

Streptococcus mutans (S. mutans) is the main etiological agent of dental caries, and adheres to the tooth surface through the sortase A (SrtA)-mediated cell wall-anchored protein Pac. Inhibition of SrtA activity results in a marked reduction in the adhesion potential of S. mutans, and the frequency of dental caries. Morin is a natural plant extract that was previously reported to inhibit Staphylococcus aureus SrtA activity. Here, we demonstrate that morin has an inhibitory effect against S. mutans UA159 SrtA, with an IC₅₀ of 27.2 ± 2.6 μM. Western blotting demonstrated that 30 μM morin induced the partial release of the Pac protein into the supernatant. The biofilm mass of S. mutans was reduced in the presence of 30 μM morin, which was not caused by a decrease in S. mutans viability. These results indicate that morin might be important as a new agent to prevent caries.     

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

The ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novel Streptococcus strain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogen Streptococcus mutans. A12 produced copious amounts of H₂O₂ via the pyruvate oxidase enzyme that were sufficient to arrest the growth of S. mutans. A12 also produced a protease similar to challisin (Sgc) of Streptococcus gordonii that was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production by S. mutans. Wild-type A12, but not an sgc mutant derivative, could protect the sensitive indicator strain Streptococcus sanguinis SK150 from killing by the bacteriocins of S. mutans. A12, but not S. gordonii, could also block the XIP (comX-inducing peptide) signaling pathway, which is the proximal regulator of genetic competence in S. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar to Streptococcus australis and Streptococcus parasanguinis but sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.     

Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

Background

Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.

Results

As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.

Conclusions

These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.     

Salivary Mucins Protect Surfaces from Colonization by Cariogenic Bacteria

Understanding how the body''s natural defenses function to protect the oral cavity from the myriad of bacteria that colonize its surfaces is an ongoing topic of research that can lead to breakthroughs in treatment and prevention. One key defense mechanism on all moist epithelial linings, such as the mouth, gastrointestinal tract, and lungs, is a layer of thick, well-hydrated mucus. The main gel-forming components of mucus are mucins, large glycoproteins that play a key role in host defense. This study focuses on elucidating the connection between MUC5B salivary mucins and dental caries, one of the most common oral diseases. Dental caries is predominantly caused by Streptococcus mutans attachment and biofilm formation on the tooth surface. Once S. mutans attaches to the tooth, it produces organic acids as metabolic by-products that dissolve tooth enamel, leading to cavity formation. We utilize CFU counts and fluorescence microscopy to quantitatively show that S. mutans attachment and biofilm formation are most robust in the presence of sucrose and that aqueous solutions of purified human MUC5B protect surfaces by acting as an antibiofouling agent in the presence of sucrose. In addition, we find that MUC5B does not alter S. mutans growth and decreases surface attachment and biofilm formation by maintaining S. mutans in the planktonic form. These insights point to the importance of salivary mucins in oral health and lead to a better understanding of how MUC5B could play a role in cavity prevention or diagnosis.     

Effects of antimicrobial peptide L-K6, a temporin-1CEb analog on oral pathogen growth,Streptococcus mutans biofilm formation,and anti-inflammatory activity

Dental caries and periodontitis are common bacterial mouth infections. As a potentially attractive substitute for conventional antibiotics, antimicrobial peptides have been widely tested and used for controlling bacterial infections. In this study, we tested the efficacy of the peptides from the skin secretions of Rana chensinensis for killing several major cariogenic and periodontic pathogens as well as Candida albicans. L-K6, a temporin-1CEb analog, exhibited high antimicrobial activity against the tested oral pathogens and was able to inhibit Streptococcus mutans biofilm formation and reduce 1-day-old S. mutans biofilms with a minimum biofilm inhibitory concentration and reducing concentration of 3.13 and 6.25 μM, respectively. The results of confocal laser scanning microscopy demonstrated that the peptide significantly reduced cell viability within oral biofilms. Furthermore, as little as 5 μM L-K6 significantly inhibited lipopolysaccharide (LPS)- and interleukin-1β-induced productions of interleukin-8 and tumor necrosis factor-α from THP-1 monocytic cells. This anti-inflammatory activity is associated with the binding of L-K6 to LPS and neutralizing LPS-induced proinflammatory responses in THP-1 cells, as well as dissociating LPS aggregates. Our results suggest that L-K6 may have potential clinical applications in treating dental caries by killing S. mutans within dental plaque and acting as anti-inflammatory agents in infected tissues.     

Exopolysaccharides Produced by Streptococcus mutans Glucosyltransferases Modulate the Establishment of Microcolonies within Multispecies Biofilms

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyltransferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces.Oral diseases related to dental biofilms afflict the majority of the world''s population, and dental caries is still the single most prevalent and costly oral infectious disease (12, 32). Dental caries results from the interaction of specific bacteria with constituents of the diet within a biofilm formed on the tooth surface known as plaque (5, 36). Streptococcus mutans is a key contributor to the formation of biofilms associated with dental caries disease, although other microorganisms may also be involved (3); S. mutans (i) effectively utilizes dietary sucrose (and possibly starch) to rapidly synthesize exopolysaccharides (EPS) using glucosyltransferases and a fructosyltransferase that adsorb to surfaces, (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is acidogenic and acid tolerant (5, 30).In general, biofilms develop after initial attachment of microbes to a surface, followed by formation of highly structured cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which are in a complex extracellular matrix (6, 49). The majority of biofilm matrices contain exopolysaccharides, and dental biofilms are no exception; up to 40% of the dry weight of dental plaque is composed of polysaccharides (depending on the type of carbohydrate consumption and the time of plaque collection), which are mostly glucans synthesized by microbial glucosyltransferases (Gtfs) (for a review, see reference 36). S. mutans plays a major role in the development and establishment of the EPS matrix in dental biofilms. This bacterium produces at least three Gtfs, which are products of the gtfB, gtfC, and gtfD genes; GtfB synthesizes mostly insoluble glucans containing elevated amounts of α-1,3-linked glucose, GtfC synthesizes a mixture of insoluble and soluble glucans (rich in α-1,6-linked glucose), and GtfD synthesizes predominantly soluble glucans (for reviews, see references 30 and 36). The Gtfs secreted by S. mutans bind avidly to the pellicle formed on the tooth surface and to bacterial surfaces and are enzymatically active; when they are exposed to sucrose, glucans are formed in situ within minutes (17, 33, 38, 40, 46). It is noteworthy that most nonstreptococcal oral bacteria (e.g., Actinomyces and Veillonella spp.) do not produce glucans unless Gtfs are adsorbed on their surfaces (33, 46). The glucans synthesized in situ provide binding sites for colonization and accumulation of S. mutans on the apatitic surface and for binding to each other through interactions with several membrane-associated glucan-binding proteins and surface glucans (8, 39, 47). The exopolymers also contribute to the bulk and physical integrity and stability of the biofilm matrix (for a review, see reference 36). The glucan-mediated processes promote tight adherence and coherence of bacterial cells bound to each other and to the apatitic surface, which leads to the formation of microcolonies by S. mutans and thereby modulates the initial steps of cariogenic biofilm development.When dietary sucrose is consumed frequently, S. mutans, as a member of the oral biofilm community, continues to synthesize polysaccharides and metabolize this sugar to form organic acids. The elevated amounts of EPS, which may involve upregulation of gtf genes in response to pH and carbohydrate availability (29), increase the virulence of the biofilms (42, 51). In addition, the ability of S. mutans to utilize some extra- and intracellular polysaccharides as short-term storage compounds provides an additional ecological benefit and simultaneously increases the amount of acid produced and the extent of acidification within the biofilm (5, 7). The persistence of this aciduric environment leads to selection and dominance of highly acid-tolerant (and acidogenic) organisms, such as S. mutans (32, 37); the low-pH environment in the biofilm matrix results in dissolution of enamel, thus initiating the pathogenesis of dental caries (32, 36).Recently, we have shown that EPS produced by S. mutans Gtfs modulate the initial formation, sequence of assembly, and structural organization of microcolonies by this bacterium on apatitic surfaces (50). However, it was unclear which of the Gtf enzymes were associated with these processes. Furthermore, the polysaccharides may also modulate the formation of microcolonies by complex ecological interactions in a multispecies system. In this study, we investigated (i) the role of each of the S. mutans gtf genes in EPS matrix and microcolony development on a saliva-coated hydroxyapatite (sHA) surface and (ii) the influence of exopolysaccharides on establishment of microcolonies at distinct developmental phases during formation of biofilms by S. mutans in the presence of Streptococcus oralis and Actinomyces naeslundii.(This study was presented at 5th ASM Conference on Biofilms, Cancun, Mexico, 15 to 19 November 2009.)     

Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37 °C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference (p>0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus.     

Anti-biofilm activity of a novel pit and fissure self-adhesive sealant modified with metallic monomers

Abstract

Dental plaque is a biofilm composed of a complex oral microbial community. The accumulation of plaque in the pit and fissures of dental elements often leads to the development of tooth decay (dental caries). Here, potent anti-biofilm materials were developed by incorporating zinc methacrylates or di-n-butyl-dimethacrylate-tin into the light-curable sealant and their physical, mechanical, and biological properties were evaluated. The data revealed that 5% di-n-butyl-dimethacrylate-tin (SnM 5%) incorporated sealant showed strong anti-biofilm efficacy against various single-species (Streptococcus mutans or Streptococcus oralis or Candida albicans) and S. mutans-C. albicans cross-kingdom dual-species biofilms without either impairing the mechanical properties of the sealant or causing cytotoxicities against mouse fibroblasts. The findings indicate that the incorporation of SnM 5% in the experimental pit and fissure self-adhesive sealant may have the potential to be part of current chemotherapeutic strategies to prevent the formation of cariogenic oral biofilms that cause dental caries.