Synergism of Streptococcus mutans and Candida albicans Reinforces Biofilm Maturation and Acidogenicity in Saliva: An In Vitro Study

Hye-Eun Kim, Yuan Liu, Atul Dhall, Marwa Bawazir, Hyun Koo, Geelsu Hwang, Hye-Eun Kim, Yuan Liu, Atul Dhall, Marwa Bawazir, Hyun Koo, Geelsu Hwang

Abstract

Early childhood caries, a virulent-form of dental caries, is painful, difficult, and costly to treat that has been associated with high levels of Streptococcus mutans (Sm) and Candida albicans (Ca) in plaque-biofilms on teeth. These microorganisms appear to develop a symbiotic cross-kingdom interaction that amplifies the virulence of plaque-biofilms. Although biofilm studies reveal synergistic bacterial-fungal association, how these organisms modulate cross-kingdom biofilm formation and enhance its virulence in the presence of saliva remain largely unknown. Here, we compared the properties of Sm and Sm-Ca biofilms cultured in saliva by examining the biofilm structural organization and capability to sustain an acidic pH environment conducive to enamel demineralization. Intriguingly, Sm-Ca biofilm is rapidly matured and maintained acidic pH-values (~4.3), while Sm biofilm development was retarded and failed to create an acidic environment when cultured in saliva. In turn, the human enamel slab surface was severely demineralized by Sm-Ca biofilms, while there was minimal damage to the enamel surface by Sm biofilm. Interestingly, Sm-Ca biofilms exhibited an acidic environment regardless of their hyphal formation ability. Our data reveal the critical role of symbiotic interaction between S. mutans and C. albicans in human saliva in the context of pathogenesis of dental caries, which may explain how the cross-kingdom interaction contributes to enhanced virulence of plaque-biofilm in the oral cavity.

Keywords: Candida albicans; Streptococcus mutans; acidogenicity; cross-kingdom biofilm; enamel demineralization; human saliva.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Kim, Liu, Dhall, Bawazir, Koo and Hwang.

Figures

Figure 1
Figure 1
Microbiological and biochemical properties of Sm and Sm-Ca biofilms in saliva contained media (0–100%). (A) Biofilm experimental design and composition of culture media. (B) Biomass (Dry-weight) of biofilms. (C) Amounts of insoluble polysaccharides (EPS) in biofilms. The biomass of Sm biofilm in SAL100 was below the detection limit, thus it was not able to measure the content of EPS-glucans in Sm cultured in SAL100. (D) CFU of S. mutans and C. albicans in Sm and Sm-Ca biofilms at final phases (42 h). Dry weight, EPS, and CFU experiments were performed (n>3) separately for each donor saliva, and then the average values were calculated. Asterisk indicates that the p-values are significantly different from SAL0 (whole UFYTE) (ANOVA with Dunnett; *P < 0.05). Hash indicates that the p-values are significantly different between two groups (Student’s t-test; #P < 0.05). ND indicates not detected.
Figure 2
Figure 2
Effect of human saliva on the pH of biofim supernatants in Sm and Sm-Ca biofilms. pH value at early (18 h), middle (28 h), and late (42 h) phases of Sm (A) and Sm-Ca (B) biofilms in different saliva ratios. Sm and Sm-Ca showed similar pH patterns under most culture conditions (SAL0, SAL25, and SAL50), while Sm biofilm showed a completely different pH pattern when cultured under SAL100. Time-lapsed pH measurements of Sm and Sm-Ca biofilms in SAL0(C) and SAL100(D) during the middle phase (from 18 h to 28 h). Asterisk indicates that the p-values are significantly different from the pH of the starting point (ANOVA with Dunnett; *P < 0.05). Hash indicates that the p-values are significantly different among groups (Student’s t-test; #P < 0.05).
Figure 3
Figure 3
Confocal images of biofilms and quantification of biofilm components. (A) Representative top (X–Y) and orthogonal (Y–Z) views of confocal images of Sm and Sm-Ca biofilms at 2, 4, 6, 8, and 18 h cultured under SAL100. Bacterial cells are labeled with SYTO 9 (green), fungal cells with concanavalin A-tetramethylrhodamine (Cyan), and EPS α-glucan with Alexa Fluor 647 (red). Quantified biovolume of (B)S. mutans, (C) EPS, (D)C. albicans, and (E) total (sum of S. mutans, C. albicans, and EPS) in the biofilm. Asterisk indicates that the p-values are significantly different from the biovolume of 2 h (ANOVA with Dunnett; *P < 0.05). Hash indicates that the p-values are significantly different between two groups (t-test #P < 0.05).
Figure 4
Figure 4
Effect of hyphal transformation ability on the properties of Sm-Ca biofilms in saliva. (A)C. albicans colonies of SC5314, SN152, efg1ΔΔ, UR13, and UR18 strains on Spider agar plate. Morphology of fungal colony was photographed after incubation for 4 days at 37°C. (B) Representative top (X–Y) and orthogonal (Y–Z) views of confocal images of each mixed-species biofilm under SAL100 at 18 h. Bacterial cells are labeled with SYTO 9 (green), fungal cells with concanavalin A-tetramethylrhodamine (Cyan), and EPS α-glucan with Alexa Fluor 647 (red). (C) pH value at each time point (18, 28, and 42 h). Data were analyzed by descriptive analysis and one-way analysis of variance (ANOVA) using GraphPad Prism 8. (D) Total biomass (dry weight) of each Sm-Ca biofilm and (E) CFU of S. mutans and C. albicans at the endpoint (42 h). Asterisk indicates that the p-values are significantly different from wild type biofilm (Sm-Ca_SC5314) (ANOVA with Dunnett; *P < 0.05).
Figure 5
Figure 5
Demineralization of human enamel surface by Sm and Sm-Ca biofilm in SAL100. (A) pH changes in Sm and Sm-Ca biofilm throughout the biofilm experiment. (B) Biomass (Dry-weight) of biofilms and (C) CFU of S. mutans and C. albicans in Sm and Sm-Ca biofilms at final phases (114 h). (D) Representative confocal surface-topography and roughness of enamel surfaces (Scale bar: 50 µm). Asterisk indicates that the p-values are significantly different from Sm biofilm (Student’s t-test; *P < 0.05).

References

    1. Ahn S.-J., Ahn S.-J., Wen Z. T., Brady L. J., Burne R. A. (2008). Characteristics of biofilm formation by Streptococcus mutans in the presence of saliva. Infect. Immun. 76, 4259–4268. 10.1128/IAI.00422-08
    1. Baillie G. S., Douglas L. J. (1999). Role of dimorphism in the development of Candida albicans biofilms. J. Med. Microbiol. 48, 671–679. 10.1099/00222615-48-7-671
    1. Carlisle P. L., Kadosh D. (2013). A genome-wide transcriptional analysis of morphology determination in Candida albicans . Mol. Biol. Cell 24, 246–260. 10.1091/mbc.e12-01-0065
    1. Cheaib Z., Rakmathulina E., Lussi A., Eick S. (2015). Impact of acquired pellicle modification on adhesion of early colonizers. Caries Res. 49, 626–632. 10.1159/000442169
    1. Cocco A. R., Cuevas-Suárez C. E., Liu Y., Lund R. G., Piva E., Hwang G. (2020). Anti-biofilm activity of a novel pit and fissure self-adhesive sealant modified with metallic monomers. Biofouling 36, 245–255. 10.1080/08927014.2020.1748603
    1. Colombo N. H., Ribas L. F., Pereira J. A., Kreling P. F., Kressirer C. A., Tanner A. C., et al. . (2016). Antimicrobial peptides in saliva of children with severe early childhood caries. Arch. Oral. Biol. 69, 40–46. 10.1016/j.archoralbio.2016.05.009
    1. Cross B. W., Ruhl S. (2018). Glycan recognition at the saliva–oral microbiome interface. Cell. Immunol. 333, 19–33. 10.1016/j.cellimm.2018.08.008
    1. Dale B. A., Fredericks L. P. (2005). Antimicrobial peptides in the oral environment: expression and function in health and disease. Curr. Issues Mol. Biol. 7, 119–133. 10.1093/jac/dki103
    1. Dawes C., Wong D. (2019). Role of saliva and salivary diagnostics in the advancement of oral health. J. Dental Res. 98, 133–141. 10.1177/0022034518816961
    1. de Carvalho F. G., Silva D. S., Hebling J., Spolidorio L. C., Spolidorio D. M. P. (2006). Presence of mutans streptococci and Candida spp. in dental plaque/dentine of carious teeth and early childhood caries. Arch. Oral. Biol. 51, 1024–1028. 10.1016/j.archoralbio.2006.06.001
    1. Falsetta M. L., Klein M. I., Colonne P. M., Scott-Anne K., Gregoire S., Pai C.-H., et al. . (2014). Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect. Immun. 82, 1968–1981. 10.1128/IAI.00087-14
    1. Gregoire S., Xiao J., Silva B., Gonzalez I., Agidi P., Klein M., et al. . (2011). Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces. Appl. Environ. Microbiol. 77, 6357–6367. 10.1128/AEM.05203-11
    1. Hajishengallis E., Parsaei Y., Klein M. I., Koo H. (2017). Advances in the microbial etiology and pathogenesis of early childhood caries. Mol. Oral. Microbiol. 32, 24–34. 10.1111/omi.12152
    1. Hallett K. B., O’Rourke P. K. (2002). Early childhood caries and infant feeding practice. Community Dental Health 19, 237–242.
    1. Hara A. T., Zero D. T. (2014). “The potential of saliva in protecting against dental erosion,” in Erosive tooth wear (Basel, Switzerland: Karger Publishers; ), 197–205.
    1. Helmerhorst E. J., Hodgson R., Van’t Hof W., Veerman E., Allison C., Nieuw Amerongen A. (1999). The effects of histatin-derived basic antimicrobial peptides on oral biofilms. J. Dental Res. 78, 1245–1250. 10.1177/00220345990780060801
    1. Heydorn A., Nielsen A. T., Hentzer M., Sternberg C., Givskov M., Ersbøll BK., et al. . (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146 (10), 2395–2407.
    1. Hwang G., Marsh G., Gao L., Waugh R., Koo H. (2015). Binding Force Dynamics of Streptococcus mutans-glucosyltransferase B to Candida albicans. J. Dental Res. 94, 1310–1317. 10.1177/0022034515592859
    1. Hwang G., Liu Y., Kim D., Li Y., Krysan D. J., Koo H. (2017). Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo. PloS Pathog. 13, e1006407. 10.1371/journal.ppat.1006407
    1. ISO (2012). 25178-2: 2012—Geometrical Product Specifications (GPS)—Surface Texture: Areal—Part 2: Terms, Definitions and Surface Texture Parameters (Geneva, Switzerland: International Standards Organization; ).
    1. Jurczak A., Kościelniak D., Papież M., Vyhouskaya P., Krzyściak W. (2015). A study on β-defensin-2 and histatin-5 as a diagnostic marker of early childhood caries progression. Biol. Res. 48, 1–9. 10.1186/s40659-015-0050-7
    1. Kadosh D. (2013). Shaping up for battle: morphological control mechanisms in human fungal pathogens. PloS Pathog. 9, e1003795. 10.1371/journal.ppat.1003795
    1. Kim D., Liu Y., Benhamou R. I., Sanchez -H., Simón-Soro A., Li Y., et al. . (2018). Bacterial-derived exopolysaccharides enhance antifungal drug tolerance in a cross-kingdom oral biofilm. ISME J. 12(6), 1427–1442. 10.1038/srep41332
    1. Kim D., Sengupta A., Niepa T. H., Lee B.-H., Weljie A., Freitas-Blanco V. S., et al. . (2017). Candida albicans stimulates Streptococcus mutans microcolony development via cross-kingdom biofilm-derived metabolites. Sci. Rep. 7, 1–14. 10.1038/srep41332
    1. Klein M. I., Scott-Anne K. M., Gregoire S., Rosalen P. L., Koo H. (2012). Molecular approaches for viable bacterial population and transcriptional analyses in a rodent model of dental caries. Mol. Oral. Microbiol. 27, 350–361. 10.1111/j.2041-1014.2012.00647.x
    1. Koo H., Xiao J., Klein M., Jeon J. (2010). Exopolysaccharides produced by Streptococcus mutans glucosyltransferases modulate the establishment of microcolonies within multispecies biofilms. J. Bacteriol. 192, 3024–3032. 10.1128/JB.01649-09
    1. Larsen M., Pearce I. (1997). A computer program for correlating dental plaque pH values, cH+, plaque titration, critical pH, resting pH and the solubility of enamel apatite. Arch. Oral. Biol. 42, 475–480. 10.1016/S0003-9969(97)00044-7
    1. Liu H., Kohler J., Fink G. R. (1994). Suppression of hyphal formation in Candida albicans by mutation of a STE12 homolog. Science 266, 1723–1726. 10.1126/science.7992058
    1. Liu Y., Naha P. C., Hwang G., Kim D., Huang Y., Simon-Soro A., et al. . (2018). Topical ferumoxytol nanoparticles disrupt biofilms and prevent tooth decay in vivo via intrinsic catalytic activity. Nat. Commun. 9, 1–12. 10.1038/s41467-018-05342-x
    1. Marsh P. D. (2010). Microbiology of dental plaque biofilms and their role in oral health and caries. Dental Clinics 54, 441–454. 10.1016/j.cden.2010.03.002
    1. Murakami S., Mealey B. L., Mariotti A., Chapple I. L. (2018). Dental plaque–induced gingival conditions. J. Clin. Periodontol. 45, S17–S27. 10.1002/JPER.17-0095
    1. Murray P., Prakobphol A., Lee T., Hoover C., Fisher S. (1992). Adherence of oral streptococci to salivary glycoproteins. Infect. Immun. 60, 31–38. 10.1128/IAI.60.1.31-38.1992
    1. O’Sullivan J. M., Jenkinson H. F., Cannon R. D. (2000). Adhesion of Candida albicans to oral streptococci is promoted by selective adsorption of salivary proteins to the streptococcal cell surface. Microbiology 146, 41–48. 10.1099/00221287-146-1-41
    1. Parisotto T. M., Steiner-Oliveira C., Silva C. M. S. E., Rodrigues L. K. A., Nobre-Dos-Santos M. (2010). Early childhood caries and mutans streptococci: a systematic review. Oral. Health Prev. Dent. 8, 59–70. 10.3290/j.ohpd.a18828
    1. Paula A. J., Hwang G., Koo H. (2020). Dynamics of bacterial population growth in biofilms resemble spatial and structural aspects of urbanization. Nat. Commun. 11, 1–14. 10.1038/s41467-020-15165-4
    1. Pedersen A. M. L., Belstrøm D. (2019). The role of natural salivary defences in maintaining a healthy oral microbiota. J. Dent. 80, S3–S12. 10.1016/j.jdent.2018.08.010
    1. Pereira D., Seneviratne C., Koga-Ito C., Samaranayake L. (2018). Is the oral fungal pathogen Candida albicans a cariogen? Oral. Dis. 24, 518–526. 10.1111/odi.12691
    1. Phattarataratip E., Olson B., Broffitt B., Qian F., Brogden K. A., Drake D. R., et al. . (2011). Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host antimicrobial peptides. Mol. Oral. Microbiol. 26, 187–199. 10.1111/j.2041-1014.2011.00607.x
    1. van’t Hof W., Veerman E. C., Amerongen A. V. N., Ligtenberg A. J. (2014). “Antimicrobial defense systems in saliva,” in Saliva: Secretion and functions (Basel, Switzerland: Karger Publishers; ), 40–51.
    1. Vorregaard M. (2018). Comstat2-a modern 3D image analysis environment for biofilms. Master's thesis, Master's thesis. Lyngby, Denmark: Technical University of Denmark, DTU, DK-2800 Kgs.
    1. Xiao J., Klein M. I., Falsetta M. L., Lu B., Delahunty C. M., Yates Iii J. R., et al. . (2012). The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm. PloS Pathog. 8, e1002623. 10.1371/journal.ppat.1002623
    1. Xiao J., Moon Y., Li L., Rustchenko E., Wakabayashi H., Zhao X., et al. . (2016). Candida albicans carriage in children with severe early childhood caries (S-ECC) and maternal relatedness. PloS One 11, e0164242. 10.1371/journal.pone.0164242
    1. Xiao J., Hara A. T., Kim D., Zero D. T., Koo H., Hwang G. (2017). Biofilm three-dimensional architecture influences in situ pH distribution pattern on the human enamel surface. Int. J. Oral. Sci. 9, 74–79. 10.1038/ijos.2017.8
    1. Xiao J., Huang X., Alkhers N., Alzamil H., Alzoubi S., Wu T. T., et al. . (2018). Candida albicans and early childhood caries: a systematic review and meta-analysis. Caries Res. 52, 102–112. 10.1159/000481833
    1. Yang X. Q., Zhang Q., Lu L. Y., Yang R., Liu Y., Zou J. (2012). Genotypic distribution of Candida albicans in dental biofilm of Chinese children associated with severe early childhood caries. Arch. Oral. Biol. 57, 1048–1053. 10.1016/j.archoralbio.2012.05.012

Source: PubMed

Patrocinadores e Colaboradores

Condições médicas

Intervenções de drogas

3
Se inscrever