Altered protein expression of Streptococcus oralis cultured at low pH revealed by two-dimensional gel electrophoresis

Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database for Streptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.     

Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.     

Genotypic Heterogeneity of Streptococcus oralis and Distinct Aciduric Subpopulations in Human Dental Plaque

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (χ² = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.     

Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.     

Analysis of Streptococcus mutans Proteins Modulated by Culture under Acidic Conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

Genotypic heterogeneity of Streptococcus oralis and distinct aciduric subpopulations in human dental plaque

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (chi(2) = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.     

Low pH-induced membrane fatty acid alterations in oral bacteria

Four oral bacterial strains, of which two are considered aciduric and two are considered acid-sensitive, were grown under glucose-limiting conditions in chemostats to determine whether their membrane fatty acid profiles were altered in response to environmental acidification. Streptococcus gordonii DL1, as well as the aciduric strains S. salivarius 57.I, and Lactobacillus casei 4646 increased the levels of mono-unsaturated membrane fatty acids. The non-aciduric strain S. sanguis 10904 did not alter its membrane composition in response to pH values examined here. Thus, in response to low pH, aciduric oral bacteria alter their membrane composition to contain increased levels of long-chained, mono-unsaturated fatty acids. This suggests that membrane fatty acid adaptation is a common mechanism utilized by bacteria to withstand environmental stress.     

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.     

Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

Antimicrobial Effects of Peptides from Human Beta-Defensin-3 on Planktonic and Biofilm States of Streptococci

Human beta-defensin-3 (hBD3) acts as a first line of defense against both Gram-positive and Gram-negative bacteria infection. Streptococci are the significant cause for oral biofilm associated diseases. We synthesized three fragments (hBD3-1, hBD3-2, hBD3-3) from the hBD3 and evaluated the antibacterial efficacy on oral streptococci. All of the three fragments from hBD3 had good estimated solubility and hBD3-3 had a higher net positive charge than others. Structure analysis showed that the three fragments shared stable β-sheet structure, but tyrosine were not found in hBD3-2 and hBD3-3 by using Raman and circular dichroism spectroscopy. The inhibition ability of the peptides was examined on the bioactivity of Streptococcus oralis (S.oralis), Streptococcus sanguinis (S. sanguinis) and Streptococcus gordonii (S. gordonii) by minimal inhibitory concentration, minimum bactericidal concentration and anti-biofilm formation test. Three fragments had antimicrobial activity on planktonic state of streptococci, and S. oralis had much more sensitive to the three peptides. Results of antibiofilm experiment showed that streptococci biofilm formation was more sensitive to hBD3-3. Confocal laser scanning microscopy and scanning electron microscopy showed the decrease of biomass and bacterial morphology destruction, which indicated that the antimicrobial mechanism of hBD3-3 might involve an electrostatic charge-based impact on membrane permeability. In conclusion, hBD3-3 possessed the potential capacity for depressing the growth of bacteria, especially first colonizers during the development of oral biofilm. Powerful, endogenous antimicrobial peptide provides the potential to interfere with biofilm by disorganizing early biofilm formation and thereby inhibiting biofilm-associated diseases.     

pH landscapes in a novel five-species model of early dental biofilm

Background

Despite continued preventive efforts, dental caries remains the most common disease of man. Organic acids produced by microorganisms in dental plaque play a crucial role for the development of carious lesions. During early stages of the pathogenetic process, repeated pH drops induce changes in microbial composition and favour the establishment of an increasingly acidogenic and aciduric microflora. The complex structure of dental biofilms, allowing for a multitude of different ecological environments in close proximity, remains largely unexplored. In this study, we designed a laboratory biofilm model that mimics the bacterial community present during early acidogenic stages of the caries process. We then performed a time-resolved microscopic analysis of the extracellular pH landscape at the interface between bacterial biofilm and underlying substrate.

Methodology/Principal Findings

Strains of Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Streptococcus downei and Actinomyces naeslundii were employed in the model. Biofilms were grown in flow channels that allowed for direct microscopic analysis of the biofilms in situ. The architecture and composition of the biofilms were analysed using fluorescence in situ hybridization and confocal laser scanning microscopy. Both biofilm structure and composition were highly reproducible and showed similarity to in-vivo-grown dental plaque. We employed the pH-sensitive ratiometric probe C-SNARF-4 to perform real-time microscopic analyses of the biofilm pH in response to salivary solutions containing glucose. Anaerobic glycolysis in the model biofilms created a mildly acidic environment. Decrease in pH in different areas of the biofilms varied, and distinct extracellular pH-microenvironments were conserved over several hours.

Conclusions/Significance

The designed biofilm model represents a promising tool to determine the effect of potential therapeutic agents on biofilm growth, composition and extracellular pH. Ratiometric pH analysis using C-SNARF-4 gives detailed insight into the pH landscape of living biofilms and contributes to our general understanding of metabolic processes in in-vivo-grown bacterial biofilms.     

Phenotypic and Genotypic Selection of Microbiota Surviving under Dental Restorations

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, β-N-acetylglucosaminidase, and β-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P = 0.002 and P = 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.     

Pathogenic and antimicrobial resistance genes in Streptococcus oralis strains revealed by comparative genome analysis

Streptococcus oralis is an early colonizer bacterium in dental plaques and is considered a potential pathogen of infective endocarditis (IE) disease. In this study, we built a complete genome map of Streptococcus oralis strain SOT, Streptococcus oralis strain SOD and Streptococcus infantis strain SO and performed comparative genomic analysis among these three strains. The results showed that there are five genomic islands (GIs) in strain SOT and one CRISPR in strain SOD. Each genome harbors various pathogenic genes related to diseases and drug resistance, while the antibiotic resistance genes in strains SOT and SOD were quite similar but different from those in strain SO. In addition, we identified 17 main virulence factors and capsule-related genes in three strains. These results suggest the pathogenic potential of Streptococcus strains, which lay a foundation for the prevention and treatment of a Streptococcus oralis infection.     

Elevated incidence of dental caries in a mouse model of cystic fibrosis

Background

Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model.

Methodology/Principal Findings

We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value<0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16±2 mM vs. 31±2 mM, CF and wildtype mice, respectively; p value<0.01; t test).

Conclusions/Significance

Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H₂O₂) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H₂O₂ may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H₂O₂-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H₂O₂, inhibited S. oralis cytotoxicity, and H₂O₂ alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H₂O₂ production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H₂O₂ induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H₂O₂ is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.