Abstract
Candida species, particularly Candida albicans and C. tropicalis, are critical members of the human microbiota and are associated with systemic infections, including candidaemia. The pathogenicity is largely attributed to virulence factors such as biofilm formation, which enhances antifungal resistance and immune evasion. Despite extensive research on single-species biofilms, the dynamics of polymicrobial biofilms, especially those involving fungal‒fungal interactions, remain poorly understood. This study investigated the virulence of monomicrobial and polymicrobial biofilms of C. albicans and C. tropicalis formed in vitro, using Zophobas morio larvae as a model. Biofilms were formed from C. albicans and C. tropicalis obtained from the gastric mucosa of a patient with an intragastric balloon (IGB). The biomass and structure of the monomicrobial and mixed-species biofilms were characterized via crystal violet staining and fluorescence microscopy techniques. The virulence of suspended, adhered, and planktonic Z. morio larvae was evaluated via survival assays, monitored over 10 days. C. albicans single biofilms presented greater biomass and structural organization than C. tropicalis, while mixed-species biofilms produced the highest biomass and density. Fluorescence microscopy revealed enhanced interspecies interactions in mixed biofilms, suggesting synergistic effects. Yeasts from single biofilms impacted less on survival rates, particularly under suspended and adhered cell conditions. These findings suggest that mixed-species biofilms exhibit increased virulence due to synergistic interactions between both species. Moreover, they also suggest distinct functional roles within biofilms, where C. tropicalis contributes to cellular proliferation and C. albicans supports matrix production, collectively enhancing biofilm robustness and pathogenic potential. This study underscores the pathogenic significance of fungal‒fungal interactions in biofilms, particularly under mixed-species conditions. The enhanced virulence observed in mixed biofilms highlights the importance of targeting interspecies dynamics in antifungal strategies. The use of alternative models such as Z. morio larvae provides valuable insights into biofilm-mediated infections and potential therapeutic interventions.