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

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.

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

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

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

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

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).