Interactions between non- albicans Candida and Streptococcus mutans enhance cariogenic potential of dual-species interkingdom biofilms

Abstract

Background: Non-albicans Candida species (NACs) are commonly found in carious lesions, yet their specific role in caries progression remains unclear. Hence, we conducted an in vitro study to explore how NACs interactions with Streptococcus mutans affect cariogenicity.

Materials and methods: Dual-species interkingdom biofilms were developed with S.mutans and six Candida species, C.albicans and the NACs: C.dubliniensis, C.glabrata, C.krusei, C.parapsilosis and C.tropicalis. Biofilm mass, viable cell counts, and pH were evaluated in mono- and dual-species biofilms. Quantitative RT-PCR was used to assess the expression of S.mutans genes associated with cariogenicity.

Results: Co-culturing S.mutans with either C.albicans, C.glabrata, or C.tropicalis significantly increased biofilm mass. While S.mutans numbers either increased or remained stable in dual-species biofilms, C.krusei, C.parapsilosis, and in particular C.tropicalis numbers significantly increased. All dual-species biofilms exhibited a pH below the critical demineralization level of enamel, akin to S.mutans mono-species biofilms. The expression of a battery of cariogenic genes in S.mutans was upregulated, particularly in dual-species biofilms with C.krusei and with C.tropicalis.

Conclusion: NACs influence the biofilm production and the cariogenic gene expression of S.mutans. The dual-species biofilm of S.mutans and NACs, particularly C.tropicalis, likely possess heightened cariogenic potential. Further research is warranted to unravel these intriguing interactions within interkingdom biofilms.

Keywords: Biofilms; Candida albicans; Candida tropicalis; Streptococcus mutans; dental caries; non-albicans Candida species.

Figure 1.

The mass of S. mutans and NAC species, along with the gram-staining of Candida spp., in mono- and dual-species interkingdom biofilms. (a) Biofilm mass of mono- and dual-species relative to that of the mono-species counterparts. The data are means and standard deviations of five separate experiments (n = 5). Asterisks denote statistical significance (*p < 0.05 and **p < 0.01). (b) Gram staining of Candida species in mono- (upper row) and dual-species biofilms with S. mutans (lower row). Filamentation and yeast forms were observed in C. albicans mono- and dual-species biofilms, while NACs predominantly remained in yeast form, except for C. tropicalis, which formed a small number of hyphae. Sm: S. mutans, Ca: C. albicans, Cd: C. dubliniensis, Cg: C. glabrata, Ck: C. krusei, Cp: C. parapsilosis, Ct: C. tropicalis.

Figure 2.

A comparison of the numbers of viable S. mutans and NAC species cells in mono- and dual-species biofilms. The data are mean and standard deviations of five separate experiments (n = 5). The viable (a) S. mutans and (b) Candida species cell numbers in mono- (light color) and dual-species biofilms (dark color). Asterisks denote statistical significance; *p < 0.05, **p < 0.01, ***p ≤ 0.001 and ****p < 0.0001. Sm: S. mutans, Ca: C. albicans, Cd: C. dubliniensis, Cg: C. glabrata, Ck: C. krusei, Cp: C. parapsilosis, Ct: C. tropicalis.

Figure 3.

The pH values of mono- and dual-species biofilms at 36 hours. The data are mean and standard deviations of five separate experiments (n = 5). The white bar represents S. mutans, the light-colored bars represent Candida spp. in mono-species biofilms, and the dark-colored bars represent S. mutans and Candida spp. in dual-species biofilms. The dotted line indicates the critical enamel demineralization pH of 5.5. Sm: S. mutans, Ca: C. albicans, Cd: C. dubliniensis, Cg: C. glabrata, Ck: C. krusei, Cp: C. parapsilosis, Ct: C. tropicalis.

Figure 4.

The pH kinetics of mono- and dual-species, inter-kingdom biofilms during 36 hours experimental period. The data are means and standard deviations of three separate experiments (n = 3). The dark-colored lines indicate the pH of mono-species biofilms, whereas the light-colored lines denote the pH of dual-species biofilms. Sm: S. mutans, Ca: C. albicans, Cd: C. dubliniensis, Cg: C. glabrata, Ck: C. krusei, Cp: C. parapsilosis, Ct: C. tropicalis.

Figure 5.

The expression of genes associated with S. mutans cariogenicity in dual-species biofilms (presented as a log2-fold change relative to S. mutans mono-species biofilms). A log2-fold change over 1 was defined as upregulation, while a value less than − 1 was considered as downregulation (label value). The experiments were performed on three separate occasions.