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Background: An appropriate photosensitizer (PS) for photodynamic inactivation should have a pronounced antimicrobial efficacy but low dark toxicity. The aim of this study is to investigate the concentration-dependent antimicrobial efficacies of methylene blue (MB) and chlorin e6 (Ce6), against Streptococcus mutans biofilms and to compare the efficacies of these two PSs., Methods: The 48-h S. mutans UA159 biofilms, grown on glass coverslips, were subjected to MB or Ce6 at 25, 50, 100 and 200 μM with or without irradiation by 660 nM LED light (L). Control groups (-PS-L and -PS + L) were also included. Viability of the biofilm was analyzed by CFU/biofilm and biofilm lactic acid production was quantified by an enzymatic assay., Results: With irradiation, MB under 25 μM resulted in 2-log reduction in biofilm viability and 30-fold reduction in biofilm lactic acid production. However, this biofilm killing efficacy did not change with increasing MB concentration. The biofilm killing efficacy of Ce6 increased with increasing Ce6 concentrations and resulted in 5-log reduction in biofilm viability. The lactic acid inhibitory effect of Ce6 was significantly lower than MB at 25 μM (p<0.01) but higher than MB at 200 μM (p=0.05), although the difference at 200 μM did not reach statistical significance. No dark toxicity could be observed for MB whereas low dark toxicity could be seen for Ce6 when the concentration is above 50 μM., Conclusion: Ce6 under 200 μM showed to be a more powerful PS for photodynamic inactivation than MB. Both Ce6- and MB-based photodynamic inactivation are useful methods for biofilm control in caries prevention., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Streptococcus mutans SpaP mediates the binding of this cariogenic bacteria to tooth surfaces. It was reported that the SpaP of S. mutans clinical isolates could be classified to 2 genotypes, type A and B. Our aims are to examine spaP genotypes in often-used S. mutans laboratory strains as well as clinical isolates and to explore the relationship between the genotypes of S. mutans strains and their adherence to salivary-agglutinin (SAG). The sequences of SpaP of 11 S. mutans strains were analyzed with alignment tools. Out of these strains, 9 strains were examined for their adherence to SAG-coated surfaces. The SpaP expression on the cell surfaces and in the spent media of 9 strains were examined by a dot-blot assay. Based on the alignment of the variable V region of SpaP, 9 strains were classified as previously-defined type-A and 3 strains type-B. Among type-B strains, the SpaPs of GS5 and HG723 contain a premature stop codon which resulted in loss of adherence and absence of SpaP expression on the cell surfaces. However, clear SpaP expression was observed in the spent media of both strains. The type-B strain UA159 demonstrated low SpaP expression on the cell surface, but it showed similar adherence ability as the type-A strains. In conclusion, the presence of SpaP on the cell surface determines the adherence of S. mutans to SAG. No difference in SAG-mediated adherence could be seen between type A and B strains, probably due to the limited number of type B strain tested.

Previously, we identified a single nucleotide mutation in the promoter (mutp) of the fluoride antiporter-coding genes in a naturally fluoride-resistant Streptococcus mutans strain. Here, we studied the role of this mutation in a defined genetic background. The results confirmed that this mutation alone confers fluoride resistance on S. mutans, as shown by growth and lactic acid production assays. This resistance was explained by constitutively higher mutp promoter activity and upregulation of the fluoride antiporter-coding genes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

During caries formation, dental biofilms function not only as acid producers but also as reservoirs and diffusion barriers for active caries-preventive components. The aim of this study was to investigate the influence of biofilms as a stagnant layer on the efficacy of NaF and nano-hydroxyapatite (nHA). Biofilms of Streptococcus mutans C180-2 were formed on the surfaces of artificially demineralized enamel in an active attachment biofilm model. After 2 days of biofilm formation, the model was subjected to a pH-cycling schedule, together with a control group without biofilms. Specimens were treated for 5 min twice daily with water, a 10% nHA slurry, or 18.4 mM NaF. At the end of the pH-cycling period, the biofilms were removed for the determination of the viable counts, the lactic acid production, and the calcium content. The mineral changes in the demineralized enamel blocks were analyzed by transversal microradiography. No differences in the biofilm viable counts and lactic acid production were found in the different treatment groups. The mean calcium content of the biofilms in the nHA group was 60.7 ± 15.3 mmol/g wet weight, which was approximately 8-fold higher than in the other 2 groups. The application of NaF resulted in net remineralization, but in the presence of a biofilm, net demineralization was observed. In contrast, nHA treatment reduced further demineralization compared with the water treatment, but the presence of a biofilm enhanced this effect. In conclusion, the presence of biofilms clearly influenced the treatment outcomes of anticaries products. Biofilms could either enhance or impede their efficacy. This result implies that biofilms should be included in the in vitro tests for the preclinical screening of caries-protective agents., (© International & American Associations for Dental Research 2015.)

Biofilms are matrix-enclosed microbial population adhere to each other and to surfaces. Compared to planktonic bacterial cells, biofilm cells show much higher levels of antimicrobial resistance. We aimed to investigate Streptococcus mutans strain diversity in biofilm formation and chlorhexidine (CHX) resistance of single S. mutans and dual S. mutans-Enterococcus faecalis biofilms. Four clinical S. mutans strains (C180-2, C67-1, HG723 and UA159) formed 24-h biofilms with or without an E. faecalis strain. These biofilms were treated for 10 min with 0.025% CHX. Biofilm formation, CHX resistance and S.mutans-E. faecalis interactions were evaluated by biomass staining, resazurin metabolism, viable count and competition agar assays. The main finding is that the presence of E. faecalis generally reduced all dual-species biofilm formation, but the proportions of S. mutans in the dual-species biofilms as well as CHX resistance displayed a clear S. mutans strain dependence. In particular, decreased resistance against CHX was observed in dual S. mutans C67-1 biofilms, while increased resistance was found in dual S. mutans UA159 biofilms. In conclusion, the interaction of S. mutans with E. faecalis in biofilms varies between strains, which underlines the importance of studying strain diversity in inter-species virulence modulation and biofilm antimicrobial resistance., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: The aim of this study was to investigate the influence of oral implant surface roughness on bacterial biofilm formation and antimicrobial treatment efficacy., Materials and Methods: Titanium disks with low-roughness pickled surfaces and with moderately rough sandblasted, acid-etched surfaces were used as substrata. Streptococcus mutans biofilms (1 and 3 days old) and Porphyromonas gingivalis biofilms (3 days old) were grown on the two types of substrata and then treated with 0.2% chlorhexidine. Biofilm viability was evaluated by a resazurin metabolism assay and by sonication-colony-forming unit counts., Results: Surface roughness had no influence on the amount of biofilm formation by S mutans or P gingivalis in this in vitro biofilm model. However, it strongly affected the treatment efficacy of chlorhexidine on the biofilms formed by both species. Higher roughness resulted in lower efficacy. Furthermore, treatment efficacy was significantly reduced in older biofilms., Conclusion: A moderately roughened surface did not enhance biofilm formation but reduced treatment efficacy of the biofilms. This finding indicates that efforts should be directed toward optimizing implant surface properties for effective antimicrobial treatment without compromising osseointegration.

Introduction: Endodontic infections are caused by polymicrobial biofilms. Therefore, novel root canal disinfectants should be evaluated not only on single-species biofilms but also on dual- or mixed-species biofilms. A simple, high-throughput assay is urgently needed for this. In this study, the application of the resazurin metabolism assay was investigated for the evaluation of a root canal disinfectant on dual-species biofilms., Methods: Enterococcus faecalis with or without Streptococcus mutans in biofilms were formed in an active attachment biofilm model for 24 hours. Subsequently, the biofilms were treated with various concentrations of NaOCl for 1 minute. After resazurin metabolism by both organisms was confirmed, treatment efficacies using 0.0016% resazurin were evaluated., Results: During NaOCl treatments, resazurin metabolism displays a clear dose response, not only in single-species E. faecalis (or S. mutans) biofilms but also in dual-species biofilms. Notably, the assay revealed that the resistance of dual-species biofilms to NaOCl was 30-fold higher than in single-species E. faecalis biofilms. Viability counts on a selected NaOCl treatment (0.004%) confirmed this result and showed the increased resistance of E. faecalis in dual-species biofilms., Conclusions: Clearly, the high-throughput and low cost resazurin metabolism assay has a great potential for testing novel root canal antimicrobial agents in mixed-species biofilms., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background/aims: The antimicrobial resistance of microorganisms in biofilms and the polymicrobial interactions in these biofilms that modulate resistance require novel strategies to evaluate the efficacy of caries-preventive compounds. The current study aimed to evaluate the effects of a caries-preventive agent in Streptococcus mutans and polymicrobial biofilms., Methods: We developed a novel high-throughput active attachment model. The model consisted of a custom-designed lid containing glass discs that fit on top of standard 24-well plates. Biofilms were formed using either S. mutans C180-2 or saliva. At the end of biofilm formation (up to 96 h) the biofilms were treated with amine fluoride (AmF) solutions. The viability of the biofilms was determined by CFU counts, and metabolic activity was measured via lactate production., Results: The effect of AmF on the viability of the polymicrobial biofilms was significantly less than that on the S. mutans biofilms, indicating a higher resistance in the complex biofilms. Both types of biofilms became more resistant to AmF with age. The higher resistance of the polymicrobial biofilms was not reflected in metabolic activity; in dose-response experiments AmF reduced lactate production in both types of biofilms to the same extent. Moreover, the age-induced increased resistance in the polymicrobial biofilms was less pronounced in terms of the inhibition of metabolic activity., Conclusions: This study clearly shows that when evaluating the efficacy of caries-preventive compounds it is essential to use appropriate polymicrobial biofilm models, and more importantly that efficacy needs to be judged based on the reduction of acid formation (i.e. cariogenic potential) as well as on bacterial viability., (Copyright 2010 S. Karger AG, Basel.)

The oral cavity is colonized by microorganisms growing in biofilms in which interspecies interactions take place. Streptococcus mutans grows in biofilms on enamel surfaces and is considered one of the main etiological agents of human dental caries. Candida albicans is also commonly found in the human oral cavity, where it interacts with S. mutans. C. albicans is a polymorphic fungus, and the yeast-to-hypha transition is involved in virulence and biofilm formation. The aim of this study was to investigate interkingdom communication between C. albicans and S. mutans based on the production of secreted molecules. S. mutans UA159 inhibited C. albicans germ tube (GT) formation in cocultures even when physically separated from C. albicans. Only S. mutans spent medium collected in the early exponential phase (4-h-old cultures) inhibited the GT formation of C. albicans. During this phase, S. mutans UA159 produces a quorum-sensing molecule, competence-stimulating peptide (CSP). The role of CSP in inhibiting GT formation was confirmed by using synthetic CSP and a comC deletion strain of S. mutans UA159, which lacks the ability to produce CSP. Other S. mutans strains and other Streptococcus spp. also inhibited GT formation but to different extents, possibly reflecting differences in CSP amino acid sequences among Streptococcus spp. or differences in CSP accumulation in the media. In conclusion, CSP, an S. mutans quorum-sensing molecule secreted during the early stages of growth, inhibits the C. albicans morphological switch.

Introduction: An important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of E. faecalis in the presence of other microorganisms., Methods: Eight clinical strains of E. faecalis were tested for biofilm formation on hydroxyapatite disks in the presence and absence of a Streptococcus mutans biofilm., Results: Significantly more E. faecalis viable cells were found in biofilms in the presence of S. mutans. This phenomenon was, however, strain-dependent. Of the 8 strains tested, biofilm formation of strains AA-OR34, ER5/1, and V583 was not influenced by S. mutans biofilms., Conclusions: The results from this study, especially the strain difference, underline the importance of studying biofilm formation in a more realistic multispecies setting.

Antimicrobial resistance of micro-organisms in biofilms requires novel strategies to evaluate the efficacy of caries preventive agents in actual biofilms. Hence we investigated fluorescence intensity (FI) in Streptococcus mutans biofilms constitutively expressing green fluorescent protein (GFP). Upon addition of glucose FI in these biofilms increased significantly to steady state levels. FI-increase could be inhibited by oral care products in a dose-responsive manner. Lactic acid produced in these biofilms was measured at the end of the FI-recording. A linear correlation with a coefficient of 0.96 (p<0.01) was observed between FI-increase and lactate production, irrespective of the inhibitor used. The viability of biofilm cells after chlorhexidine (CHX) titration was also examined. Reduction of FI-increase was observed at low concentrations of CHX whereas a loss in viability was only seen at high concentrations. In conclusion, GFP synthesis can be used as a metabolic activity indicator in S. mutans biofilms.

The aim of this study was to investigate the antimicrobial activity of vanadium chloroperoxidase (VCPO) reaction products on planktonic and biofilm cells of Streptococcus mutans C180-2. Planktonic and biofilm cells were incubated in a buffered reaction mixture containing VCPO, halide (either chloride or bromide) and hydrogen peroxide, and the killing efficacy was assessed by CFU counts. The enzymatic products formed by VCPO significantly reduced the viability of planktonic and biofilm cells compared to their negative controls and the effect on the biofilm cells was more effective than a 0.2% chlorhexidine digluconate treatment. We conclude that VCPO and its reaction products form a potent antimicrobial system against S. mutans.

The genome sequence of the oral pathogen Streptococcus mutans predicts the presence of two putative polysaccharide deacetylases. The first, designated PgdA in this paper, shows homology to the catalytic domains of peptidoglycan deacetylases from Streptococcus pneumoniae and Listeria monocytogenes, which are both thought to be involved in the bacterial defense mechanism against human mucosal lysozyme and are part of the CAZY family 4 carbohydrate esterases. S. mutans cells in which the pgdA gene was deleted displayed a different colony texture and a slightly increased cell surface hydrophobicity and yet did not become hypersensitive to lysozyme as shown previously for S. pneumoniae. To understand this apparent lack of activity, the high-resolution X-ray structure of S. mutans PgdA was determined; it showed the typical carbohydrate esterase 4 fold, with metal bound in a His-His-Asp triad. Analysis of the protein surface showed that an extended groove lined with aromatic residues is orientated toward the active-site residues. The protein exhibited metal-dependent de-N-acetylase activity toward a hexamer of N-acetylglucosamine. No activity was observed toward shorter chitooligosaccharides or a synthetic peptidoglycan tetrasaccharide. In agreement with the lysozyme data this would suggest that S. mutans PgdA does not act on peptidoglycan but on an as-yet-unidentified polysaccharide within the bacterial cell surface. Strikingly, the pgdA-knockout strain showed a significant increase in aggregation/agglutination by salivary agglutinin, in agreement with this gene acting as a deacetylase of a cell surface glycan.

Although Candida containing biofilms contribute to the development of oral candidosis, the characteristics of multi-species Candida biofilms and how oral bacteria modulate these biofilms is poorly understood. The aim of this study was to investigate interactions between Candida albicans and either Candida glabrata or Streptococcus mutans in biofilms grown on various surfaces, with or without saliva. Hydroxyapatite (HA), polymethylmetacrylate (PMMA) and soft denture liner (SL) discs were used as substratum. Counts of viable micro-organisms in the accumulating biofilm layer were determined and converted to colony forming units per unit surface area. Confocal laser scanning microscopy was used to characterize biofilms and to quantitate the number of hyphae in each condition tested. Viable counts of C. albicans and C. glabrata per mm(2) decreased in the order HA>PMMA>SL (p<0.05). Biofilms grown on saliva-coated specimens harboured fewer C. glabrata than uncoated specimens (p<0.05). Glucose and the presence of S. mutans suppressed C. albicans hyphal formation. Dual Candida species biofilms did not show competitive interaction between the two species. We conclude that Candida biofilms are significantly affected by saliva, substratum type and by the presence of other micro-organisms.

Introduction: Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics., Methods: Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays., Results: When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed., Conclusion: Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens.

Plants naturally produce secondary metabolites that can be used as antimicrobials. The aim of this study was to assess the effects of Psidium cattleianum leaf extract on Streptococcus mutans. The extract (100%) was obtained by decoction of 100 g of leaves in 600 ml of deionized water. To assess killing, S. mutans biofilms were treated with water (negative control) or various extract dilutions [100, 50, 25% (v/v) in water] for 5 or 60 min. To evaluate the effect on protein expression, biofilms were exposed to water or 1.6% (v/v) extract for 120 min, proteins were extracted and submitted to 2-dimensional difference gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry. The effect of 1.6% (v/v) extract on acid production was determined by pH measurements and compared to a water control. Viability was similar after 5 min of treatment with the 100% extract or 60 min with the 50% extract (about 0.03% survival). There were no differences in viability between the biofilms exposed to the 25 or 50% extract after 60 min of treatment (about 0.02% survival). Treatment with the 1.6% extract significantly changed protein expression. The abundance of 24 spots was decreased compared to water (p < 0.05). The extract significantly inhibited acid production (p < 0.05). It is concluded that P. cattleianum leaf extract kills S. mutans grown in biofilms when applied at high concentrations. At low concentrations it inhibits S. mutans acid production and reduces the expression of proteins involved in general metabolism, glycolysis and lactic acid production., ((c) 2008 S. Karger AG, Basel)

In the oral cavity a balanced physiological response is essential for Streptococcus mutans to survive various types of external challenges. In this study we examined the role of the ClpP serine protease in the response of S. mutans towards sodium fluoride, sodium chloride, hydrogen peroxide, and chlorhexidine. By constructing a clpP promoter-green fluorescent protein reporter strain, we showed increased fluorescence intensities under all types of stress, indicating a need for ClpP under all these challenges. We constructed a clpP knockout mutant, which proved to be more sensitive to all the challenges than the wild-type strain. This knockout strain also displayed a reduced growth rate, hyperaggregation, and increased biofilm formation. Furthermore, an increased resistance to toxic levels of hydrogen peroxide and chlorhexidine after pre-incubation with sublethal levels of the corresponding compounds was found in the wild-type strain but not in the knockout mutant. In conclusion, ClpP is involved in the general stress response of S. mutans and assists the bacteria to resist killing through adaptation.

In Streptococcus mutans, virulence and cariogenicity may be modulated via the two-component regulatory system VicRK. Environmental signals, sensed by VicK, inducing this modulation are still unclear, however, and were investigated in the present study. We found that VicRK displays homology with protein-domains that, in other bacteria, are involved in redox-sensing. After constructing a VicRK-promoter GFP-reporter strain, we showed increased fluorescence intensity under oxidative stress. Potential interference of alternative signals and experimental conditions on GFP expression was excluded by the use of negative and positive control strains. Finally, we constructed a clean vicK knockout mutant, which proved to be more sensitive to H(2)O(2) than the wild-type. In conclusion, this study showed that the VicRK system responds to and protects against oxidative stress. As a result, a link between oxidative/redox stress and the cariogenic nature of S. mutans can be hypothesized.