Dynamics of Mono- and Dual-Species Biofilm Formation and Interactions Between Paracoccidioides brasiliensis and Candida albicans

Abstract

The oral cavity is a highly diverse microbial environment in which microorganisms interact with each other, growing as biofilms on biotic and abiotic surfaces. Understanding the interaction among oral microbiota counterparts is pivotal for clarifying the pathogenesis of oral diseases. Candida spp. is one of the most abundant fungi in the oral mycobiome with the ability to cause severe soft tissue lesions under certain conditions. Paracoccidioides spp., the causative agent of paracoccidioidomycosis, may also colonize the oral cavity leading to soft tissue damage. It was hypothesized that both fungi can interact with each other, increasing the growth of the biofilm and its virulence, which in turn can lead to a more aggressive infectivity. Therefore, this study aimed to evaluate the dynamics of mono- and dual-species biofilm growth of Paracoccidioides brasiliensis and Candida albicans and their infectivity using the Galleria mellonella model. Biomass and fungi metabolic activity were determined by the crystal violet and the tetrazolium salt reduction tests (XTT), respectively, and the colony-forming unit (CFU) was obtained by plating. Biofilm structure was characterized by both scanning electronic- and confocal laser scanning- microscopy techniques. Survival analysis of G. mellonella was evaluated to assess infectivity. Our results showed that dual-species biofilm with P. brasiliensis plus C. albicans presented a higher biomass, higher metabolic activity and CFU than their mono-species biofilms. Furthermore, G. mellonella larvae infected with P. brasiliensis plus C. albicans presented a decrease in the survival rate compared to those infected with P. brasiliensis or C. albicans, mainly in the form of biofilms. Our data indicate that P. brasiliensis and C. albicans co-existence is likely to occur on oral mucosal biofilms, as per in vitro and in vivo analysis. These data further widen the knowledge associated with the dynamics of fungal biofilm growth that can potentially lead to the discovery of new therapeutic strategies for these infections.

Keywords: Candida albicans; Galleria mellonella; Paracoccidioides brasiliensis; dual-species biofilm; oral cavity.

FIGURE 1

Quantification of mono and dual-species biofilm biomass by crystal violet methodology. Co-cultivated dual-species biofilms and dual-species biofilms formed by the addition of P. brasiliensis inoculum to preformed 12 h C. albicans biofilms. Three independent experiments with six replicates each were performed. Error bars indicate the standard error. Statistical significances were calculated by using analysis of variance (ANOVA) followed by Bonferroni test.

FIGURE 2

Metabolic activity of mono- and dual-species biofilms. Three independent experiments with six replicates each were performed. Co-cultivated dual-species biofilms and dual-species biofilms formed by the addition of P. brasiliensis inoculum to preformed 12 h C. albicans biofilms. Error bars indicate the standard error. Statistical significances were calculated by using analysis of variance (ANOVA) followed by Bonferroni test.

FIGURE 3

Quantitative analysis of biofilm formation in vitro by CFU/mL (Log10) count for the following groups: (A) Mono-species biofilms formed by C. albicans, co-cultivated C. albicans plus P. brasiliensis biofilms or dual-species biofilms formed by the addition of P. brasiliensis inoculum to preformed 12 h C. albicans biofilms (*p < 0.001). (B) Mono-species biofilms formed by P. brasiliensis, co-cultivated C. albicans plus P. brasiliensis biofilms or dual-species biofilms formed by the addition of P. brasiliensis inoculum to preformed 12 h C. albicans biofilms. Statistical significances were calculated by using analysis of variance (ANOVA) followed by Bonferroni test.

FIGURE 4

Representative confocal microscopy images. P. brasiliensis was marked with CFSE (green) and C. albicans with Calcofluor white (blue). C. albicans (CA) and P. brasiliensis (PB) mono-species biofilms formed for 48 and 144 h, respectively. For dual-species biofilms, P. brasiliensis previously treated with CFSE was added to 12 h preformed C. albicans biofilm, and incubated for 120 h. (A) Dual-species biofilms: overlay of CA and PB (A1), CA (A2), PB (A3) and zoom in image showing dual-species interaction (A4); (B) Mono-species biofilms: CA biofilm (B1) and PB biofilm (B2).

FIGURE 5

Representative scanning electron micrographs of C. albicans and P. brasiliensis mono-species biofilms formed for 48 and 144 h, respectively. For dual-species biofilms, P. brasiliensis was added to 12 h preformed C. albicans biofilm for 120 h. Biofilms formed by C. albicans (A) 1,000× and (B) 5,000× magnification. The white arrows indicate the yeast size (B). Biofilms formed by P. brasiliensis (C) 1,000× and (D) 5,000×. The black arrows denote the measure of yeast (D). Dual-species biofilms grown from P. brasiliensis added to 12 h preformed C. albicans biofilms (E) 1,000× and (F) 5,000×.

FIGURE 6

Survival curve of Galleria mellonella larvae infected with 1 × 10⁶ cells/larvae of planktonic cells, mono-, and dual-species biofilms grown after P. brasiliensis (Pb) was added to preformed C. albicans (Ca) biofilms. Larvae inoculated with PBS was used as control. The values are plotted as Kaplan–Meier survival curves and compared using log-rank tests.