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.     

Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans

Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S. oligofermentans and the ability of S. oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4‐aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S. oligofermentans. S. oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S. mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S. oligofermentans, the effects of the different culture pH values were as follows: pH 7.0 > 6.5 > 6.0 > 7.5 > 5.5 = 8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S. oligofermentans to inhibit the growth of S. mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced.     

Oral implantation of Streptococcus mutans in man with and without prior chlorhexidine mouthrinses

Abstract — Oral implantation of streptomycin resistant S. mutans was enhanced when chlorhexidine mouthrinses were applied before implantation. The difference in recovery of implanted bacteria between implantations with and without prior chlorhexidine mouthrinses was significant 9 days ( P<0.05 ), 15 days ( P <0.05) and 28 days ( P <0.01) after implantation. After 28 days implanted bacteria were no longer detected in any of the 20 test subjects when chlorhexidine was not used prior to implantation, but persisted in 11 subjects when chlorhexidine was used.     

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.     

Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh,YidC1, and YidC2

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild‐type strain and four mutants devoid of protein translocation machinery components, specifically ?ffh, ?yidC1, ?yidC2, or ?ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane‐localized chaperone insertases. Our results suggest that the co‐translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane‐localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ?yidC2 background may be non‐functional, that ?yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ?ffh and ?yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ?yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1.     

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.     

Phenotypic characterization of the foldase homologue PrsA in Streptococcus mutans

Streptococcus mutans is generally considered to be the principal etiological agent for dental caries. Many of the proteins necessary for its colonization of the oral cavity and pathogenesis are exported to the cell surface or the extracellular matrix, a process that requires the assistance of the export machineries. Bioinformatic analysis revealed that the S. mutans genome contains a prsA gene, whose counterparts in other gram‐positive bacteria, including Bacillus and Lactococcus, encode functions involved in protein post‐export. In this study, we constructed a PrsA‐deficient derivative of S. mutans and demonstrated that the prsA mutant displayed an altered cell wall/membrane protein profile as well as cell‐surface‐related phenotypes, including auto‐aggregation, increased surface hydrophobicity and abnormal biofilm formation. Further analysis revealed that the disruption of the prsA gene resulted in reduced insoluble glucan production by cell surface localized glucosyltransferases, and mutacin as well as cell surface‐display of a heterologous expressed GFP fusion to the cell surface protein SpaP. Our study suggested that PrsA in S. mutans encodes functions similar to those identified in Bacillus, and so is likely to be involved in protein post‐export.     

EzrA,a cell shape regulator contributing to biofilm formation and competitiveness in Streptococcus mutans

Bacterial cell division is initiated by tubulin homologue FtsZ that assembles into a ring structure at mid‐cell to facilitate cytokinesis. EzrA has been identified to be implicated in FtsZ‐ring dynamics and cell wall biosynthesis during cell division of Bacillus subtilis and Staphylococcus aureus, the model rod and cocci. However, its role in pathogenic streptococci remains largely unknown. Here, the role of EzrA was investigated in Streptococcus mutans, the primary etiological agent of human dental caries, by constructing an ezrA in‐frame deletion mutant. Our data showed that the ezrA mutant was slow‐growing with a shortened length and extended width round cell shape compared to the wild type, indicating a delay in cell division with abnormalities of peptidoglycan biosynthesis. Additionally, FtsZ irregularly localized in dividing ezrA mutant cells forming angled division planes, potentially contributing to an aberrant cell shape. Furthermore, investigation using single‐species cariogenic biofilm model revealed that deletion of ezrA resulted in defective biofilm formation with less extracellular polysaccharides and altered three‐dimensional biofilm architecture. Unexpectedly, in a dual‐species ecological model, the ezrA mutant exhibited substantially lower tolerance for H₂O₂ and reduced competitiveness against one commensal species, Streptococcus sanguinis. Taken together, these results demonstrate that EzrA plays a key role in regulating cell division and maintaining a normal morphology in S. mutans and is required for its robust biofilm formation/interspecies competition. Therefore, EzrA protein represents a potential therapeutic target in the development of drugs controlling dental caries and other biofilm‐related diseases.     

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.     

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.     

Deletion of csn2 gene affects acid tolerance and exopolysaccharide synthesis in Streptococcus mutans

Csn2 is an important protein of the CRISPR‐Cas system. The physiological function of this protein and its regulatory role in Streptococcus mutans, as the primary causative agent of human dental caries, is still unclear. In this study, we investigated whether csn2 deletion would affect S. mutans physiology and virulence gene expression. We used microscopic imaging, acid killing assays, pH drop, biofilm formation, and exopolysaccharide (EPS) production tests to determine whether csn2 deletion influenced S. mutans colony morphology, acid tolerance/production, and glucan formation abilities. Comparisons were made between quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) data from the UA159 and csn2 deletion strain to determine the impact of csn2 knockout on S. mutans gene expression. The results showed that deletion of S. mutans csn2 changed its colony morphotype and made it more sensitive to acid. The expression levels of aciduricity genes, including leuA, leuB, leuC, and leuD, were significantly down‐regulated. Acid adaptation restored the aciduricity of csn2 mutant and enhanced the ability to synthesize EPS. The expression levels of EPS synthesis‐related genes, including gtfC and gtfD, were significantly up‐regulated after acid adaptation. In summary, deletion of S. mutans csn2 exerted multiple effects on the virulence traits of this pathogen, including acid tolerance and EPS formation, and that these alterations could partially be attributed to changes in gene expression upon loss of csn2. Understanding the function of csn2 in S. mutans might lead to novel strategies to prevent or treat imbalances in oral microbiota that may favor diseases.     

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.