Streptococcus oralis maintains homeostasis in oral biofilms by antagonizing the cariogenic pathogen Streptococcus mutans

Bacteria residing in oral biofilms live in a state of dynamic equilibrium with one another. The intricate synergistic or antagonistic interactions between them are crucial for determining this balance. Using the six‐species Zürich “supragingival” biofilm model, this study aimed to investigate interactions regarding growth and localization of the constituent species. As control, an inoculum containing all six strains was used, whereas in each of the further five inocula one of the bacterial species was alternately absent, and in the last, both streptococci were absent. Biofilms were grown anaerobically on hydroxyapatite disks, and after 64 h they were harvested and quantified by culture analyses. For visualization, fluorescence in situ hybridization and confocal laser scanning microscopy were used. Compared with the control, no statistically significant difference of total colony‐forming units was observed in the absence of any of the biofilm species, except for Fusobacterium nucleatum, whose absence caused a significant decrease in total bacterial numbers. Absence of Streptococcus oralis resulted in a significant decrease in Actinomyces oris, and increase in Streptococcus mutans (P < .001). Absence of A. oris, Veillonella dispar or S. mutans did not cause any changes. The structure of the biofilm with regards to the localization of the species did not result in observable changes. In summary, the most striking observation of the present study was that absence of S. oralis resulted in limited growth of commensal A. oris and overgrowth of S. mutans. These data establish highlight S. oralis as commensal keeper of homeostasis in the biofilm by antagonizing S. mutans, so preventing a caries‐favoring dysbiotic state.     

Microbial dynamics during conversion from supragingival to subgingival biofilms in an in vitro model

The development of dental caries and periodontal diseases result from distinct shifts in the microbiota of the tooth‐associated biofilm. This in vitro study aimed to investigate changes in biofilm composition and structure, during the shift from a ‘supragingival’ aerobic profile to a ‘subgingival’ anaerobic profile. Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar were aerobically grown in saliva‐containing medium on hydroxyapatite disks. After 64 h, Campylobacter rectus, Prevotella intermedia and Streptococcus anginosus were further added along with human serum, while culture conditions were shifted to microaerophilic. After 96 h, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola were finally added and the biofilm was grown anaerobically for another 64 h. At the end of each phase, biofilms were harvested for species‐specific quantification and localization. Apart from C. albicans, all other species gradually increased during aerobic and microaerophilic conditions, but remained steady during anaerobic conditions. Biofilm thickness was doubled during the microaerophilic phase, but remained steady throughout the anaerobic phase. Extracellular polysaccharide presence was gradually reduced throughout the growth period. Biofilm viability was reduced during the microaerophilic conversion, but was recovered during the anaerobic phase. This in vitro study has characterized the dynamic structural shifts occurring in an oral biofilm model during the switch from aerobic to anaerobic conditions, potentially modeling the conversion of supragingival to subgingival biofilms. Within the limitations of this experimental model, the findings may provide novel insights into the ecology of oral biofilms.     

SMU.940 regulates dextran‐dependent aggregation and biofilm formation in Streptococcus mutans

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence‐stimulating peptide. Eight competence‐stimulating peptide‐dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran‐dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild‐type. GbpC is known to be involved in the dextran‐dependent aggregation of S. mutans. An SMU.940‐gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran‐dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran‐dependent aggregation and biofilm formation.     

Anti‐adherence and bactericidal activity of sphingolipids against Streptococcus mutans

This study evaluated the anti‐biofilm activity of sphingosine, phytosphingosine (PHS), and sphinganine for: (i) anti‐adherence activity on hydroxyapatite (HA) surfaces; and (ii) bactericidal activity on different Streptococcus mutans phenotypes (i.e. planktonic cells and cells from a disrupted biofilm). For this, HA discs treated with sphingolipids were incubated with S. mutans and the number of adherent cells was evaluated by both culture and confocal microscopy. Sphinganine strongly inhibited bacterial adherence by 1000‐fold compared with an untreated surface. Phytosphingosine and sphingosine inhibited bacterial adherence by eight‐ and five‐fold, respectively, compared with an untreated surface. On saliva‐coated HA, sphinganine and PHS inhibited bacterial adherence by 10‐fold. Bactericidal activity of sphingolipids was evaluated by culture. For biofilms, the strongest bactericidal activity was exhibited by sphingosine compared with PHS and sphinganine. At a concentration of 12.5 μg ml^?1, PHS and sphingosine were profoundly effective against planktonic and disrupted biofilms; and sphinganine reduced the number of cells in planktonic form by 100‐fold and those derived from a disrupted biofilm by 1000‐fold. Atomic force microscopy studies suggested that mechanical stability does not appear to be a factor relevant for anti‐fouling activity. The results suggest that sphingolipids may be used to control oral biofilms, especially those loaded with S. mutans.     

Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans

Objective

The aim of this study was to evaluate the acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans.

Materials and methods

The following strains were tested: Bifidobacterium dentium DSM20436, Parascardovia denticolens DSM10105, and Scardovia inopinata DSM10107. Streptococcus mutans UA159 and Lactobacillus acidophilus ATCC4356 were used as control. Bifidobacteria were studied planktonically as they were not able to form monospecies biofilm, they were grown in biofilms associated with S. mutans. Endogenous polysaccharide reserves of cultures at log phase were depleted. Standardized suspensions of the microorganisms were incubated in growth media supplemented with 10 mM glucose, lactose, raffinose, glucose, or xylitol. S. mutans biofilms were grown on glass cover slips for 24 h to which bifidobacteria were added. After 24 h, the dual-species biofilms were exposed to the same carbon sources, and after 3 h, the pH of spent culture media and concentrations of organic acids were measured. Statistical analyses were carried out using ANOVA and Tukey’s test (α = 0.05).

Results

A higher pH drop was observed when S. mutans was associated with P. denticolens or S. inopinata, in either planktonic or biofilm cultures, than with S. mutans alone. Bifidobacteria showed a higher pH drop in the presence of raffinose than S. mutans or L. acidophilus.

Conclusions

Dual-species biofilms of bifidobacteria and S. mutans produced more acid and greater pH drops than biofilms of S. mutans alone.

Clinical relevance

New insights on the complex process of caries pathogenicity contribute to the establishment of preventive and therapeutic measures, in particular in specific cases, such as in early childhood caries.

     

Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum

Adhesin‐mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species‐specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non‐mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram‐positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD‐SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species‐ and subspecies‐dependent adhesin interactions.     

Influence of surface properties of resin‐based composites on in vitro Streptococcus mutans biofilm development

The aim of this in vitro study was to evaluate the influence of physicochemical surface properties of resin‐based composites on Streptococcus mutans biofilm formation. Specimens were prepared from each of four resin‐based composites by polymerization against Mylar strips. Half of the number of specimens received no further surface treatment, whereas the other half were subjected to a polishing treatment. Surface roughness (SR) and topography were assessed using profilometry and atomic force microscopy. Surface free‐energy (SFE) was determined, and the chemical surface composition was analysed by X‐ray photoelectron spectroscopy (XPS). S. mutans biofilms were formed on the surface of the resin‐based composite specimens for either 48 or 96 h using an artificial mouth system (AMS). Polishing caused a significant decrease in SFE, and XPS analysis indicated an increase of surface silicon and a decrease of surface carbon. Only for Grandio was a significant increase in SR identified after polishing, which was probably related to the higher concentration of filler particles on its surface. Significantly less S. mutans biofilm formation was observed on polished resin‐based composites than on unpolished resin‐based composites. These results indicate that the proportions of resin matrix and filler particles on the surface of resin‐based composites strongly influence S. mutans biofilm formation in vitro, suggesting that minimization of resin matrix exposure might be useful to reduce biofilm formation on the surface of resin‐based composites.     

A clinical,randomized study on the influence of dental whitening on Streptococcus mutans population

Background

Dental whitening with peroxides has been popularized through the at‐home technique, which employs low concentrations of peroxide applied in individual trays. However, there are few clinical trials reporting the effects of its continuous use on oral microbiota. Thus, the purpose of the present clinical, randomized study was to evaluate the influence of at‐home whitening treatment on Streptococcus mutans in saliva, buccal mucosa, and subgingival and supragingival plaque.

Methods

Thirty volunteers were randomly divided into two study groups (N = 15) according to the whitening therapy: G CP, whitening using 10% carbamide peroxide 4 h daily for 21 days; and G HP, whitening using 6% hydrogen peroxide 1.5 h daily for 21 days. Samples from the predetermined locations were collected at three evaluation periods: T1, before; T2, immediately after; and T3, 30 days after the beginning of the treatment. The microbiological evaluation was made using conventional and molecular methods.

Results

Student's t‐test demonstrated a statistically significant decrease in S. mutans population in the subgingival and supragingival plaque for HP samples between T1 and T2 no difference was found between T1 and T3 regardless of the location and the whitening product used (α = 0.05).

Conclusions

Although HP reduced S. mutans during treatment, the levels returned to baseline when assessed 30 days after the treatment.     

Antigen I/II mediates interactions between Streptococcus mutans and Candida albicans

Streptococcus mutans and Candida albicans are frequently co‐isolated from dental plaque of children with early childhood caries (ECC) and are only rarely found in children without ECC, suggesting that these species interact in a manner that contributes to the pathogenesis of ECC. Previous studies have demonstrated that glucans produced by S. mutans are crucial for promoting the formation of biofilm and cariogenicity with C. albicans; however, it is unclear how non‐glucan S. mutans biofilm factors contribute to increased biofilm formation in the presence of C. albicans. In this study we examined the role of S. mutans antigen I/II in two‐species biofilms with C. albicans, and determined that antigen I/II is important for the incorporation of C. albicans into the two‐species biofilm and is also required for increased acid production. The interaction is independent of the proteins Als1 and Als3, which are known streptococcal receptors of C. albicans. Moreover, antigen I/II is required for the colonization of both S. mutans and C. albicans during co‐infection of Drosophila melanogaster in vivo. Taken together, these results demonstrate that antigen I/II mediates the increase of C. albicans numbers and acid production in the two‐species biofilm, representing new activities associated with this known S. mutans adhesin.     

Candida–streptococcal mucosal biofilms display distinct structural and virulence characteristics depending on growth conditions and hyphal morphotypes

Candida albicans and streptococci of the mitis group form communities in multiple oral sites, where moisture and nutrient availability can change spatially or temporally. This study evaluated structural and virulence characteristics of Candida–streptococcal biofilms formed on moist or semidry mucosal surfaces, and tested the effects of nutrient availability and hyphal morphotype on dual‐species biofilms. Three‐dimensional models of the oral mucosa formed by immortalized keratinocytes on a fibroblast‐embedded collagenous matrix were used. Infections were carried out using Streptococcus oralis strain 34, in combination with a C. albicans wild‐type strain, or pseudohyphal‐forming mutant strains. Increased moisture promoted a homogeneous surface biofilm by C. albicans. Dual biofilms had a stratified structure, with streptococci growing in close contact with the mucosa and fungi growing on the bacterial surface. Under semidry conditions, Candida formed localized foci of dense growth, which promoted focal growth of streptococci in mixed biofilms. Candida biofilm biovolume was greater under moist conditions, albeit with minimal tissue invasion, compared with semidry conditions. Supplementing the infection medium with nutrients under semidry conditions intensified growth, biofilm biovolume and tissue invasion/damage, without changing biofilm structure. Under these conditions, the pseudohyphal mutants and S. oralis formed defective superficial biofilms, with most bacteria in contact with the epithelial surface, below a pseudohyphal mass, resembling biofilms growing in a moist environment. The presence of S. oralis promoted fungal invasion and tissue damage under all conditions. We conclude that moisture, nutrient availability, hyphal morphotype and the presence of commensal bacteria influence the architecture and virulence characteristics of mucosal fungal biofilms.     

Early microbial succession in redeveloping dental biofilms in periodontal health and disease

Teles FR, Teles RP, Uzel NG, Song XQ, Torresyap G, Socransky SS, Haffajee AD. Early microbial succession in redeveloping dental biofilms in periodontal health and disease. J Periodont Res 2012; 47: 95–104. © 2011 John Wiley & Sons A/S Background and Objective: The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease. Material and Methods: Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA–DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time‐point. Ecological succession was determined using a modified moving‐window analysis. Results: Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1–4 d. At 4–7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations. Conclusion: There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning.     

Natural antigenic differences in the functionally equivalent extracellular DNABII proteins of bacterial biofilms provide a means for targeted biofilm therapeutics

Bacteria that persist in the oral cavity exist within complex biofilm communities. A hallmark of biofilms is the presence of an extracellular polymeric substance (EPS), which consists of polysaccharides, extracellular DNA (eDNA), and proteins, including the DNABII family of proteins. The removal of DNABII proteins from a biofilm results in the loss of structural integrity of the eDNA and the collapse of the biofilm structure. We examined the role of DNABII proteins in the biofilm structure of the periodontal pathogen Porphyromonas gingivalis and the oral commensal Streptococcus gordonii. Co‐aggregation with oral streptococci is thought to facilitate the establishment of P. gingivalis within the biofilm community. We demonstrate that DNABII proteins are present in the EPS of both S. gordonii and P. gingivalis biofilms, and that these biofilms can be disrupted through the addition of antisera derived against their respective DNABII proteins. We provide evidence that both eDNA and DNABII proteins are limiting in S. gordonii but not in P. gingivalis biofilms. In addition, these proteins are capable of complementing one another functionally. We also found that whereas antisera derived against most DNABII proteins are capable of binding a wide variety of DNABII proteins, the P. gingivalis DNABII proteins are antigenically distinct. The presence of DNABII proteins in the EPS of these biofilms and the antigenic uniqueness of the P. gingivalis proteins provide an opportunity to develop therapies that are targeted to remove P. gingivalis and biofilms that contain P. gingivalis from the oral cavity.     

In silico analysis of the competition between Streptococcus sanguinis and Streptococcus mutans in the dental biofilm

During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries‐free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H₂O₂ by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H₂O₂ levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H₂O₂. S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra‐species communication.     

Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

As a member of subgingival multispecies biofilms, Tannerella forsythia is commonly associated with periodontitis. The bacterium has a characteristic cell surface (S‐) layer modified with a unique O‐glycan. Both the S‐layer and the O‐glycan were analyzed in this study for their role in biofilm formation by employing an in vitro multispecies biofilm model mimicking the situation in the oral cavity. Different T. forsythia strains and mutants with characterized defects in cell surface composition were incorporated into the model, together with nine species of select oral bacteria. The influence of the T. forsythia S‐layer and attached glycan on the bacterial composition of the biofilms was analyzed quantitatively using colony‐forming unit counts and quantitative real‐time polymerase chain reaction, as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. This revealed that changes in the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers. The localization of T. forsythia within the bacterial agglomeration varied depending on changes in the S‐layer glycan, and this also affected its aggregation with Porphyromonas gingivalis. This suggests a selective role for the glycosylated T. forsythia S‐layer in the positioning of this species within the biofilm, its co‐localization with P. gingivalis, and the prevalence of C. rectus. These findings might translate into a potential role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and the immune system, from within or beyond the biofilm.     

Deficiency of PdxR in Streptococcus mutans affects vitamin B6 metabolism,acid tolerance response and biofilm formation

Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA‐binding N‐terminal domain and a C‐terminal domain highly homologous to the pyridoxal phosphate‐dependent aspartate aminotransferases. A pdxR‐deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR‐deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real‐time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B₆ biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR‐deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B₆. Further studies revealed that although S. mutans is known to require vitamin B₆ to grow in defined medium, B₆ vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B₆ metabolism, acid tolerance response and biofilm formation.