Interspecies Interactions of Single- and Mixed-Species Biofilms of Candida albicans and Aggregatibacter actinomycetemcomitans.

Polymicrobial biofilms involving fungal and bacterial species are increasingly recognized as contributors to persistent infections, particularly in the oral cavity. Candida albicans and Aggregatibacter actinomycetemcomitans are two commensals that can turn into opportunistic pathogens and are able to form robust biofilms. Objectives: This study aimed to assess the interaction dynamics between these two microorganisms and to evaluate their susceptibility to fluconazole and azithromycin in single- and mixed-species forms. Methods: Biofilm biomass was quantified using crystal violet assays, while biofilm cell viability was assessed through CFU enumeration (biofilm viability assay). To assess the resistance properties of single versus mixed-species coincubations, we applied the antimicrobial susceptibility test (AST) to each drug, and analysed spatial organization with confocal laser scanning microscopy, using PNA-FISH. Results: The results indicated that both species can coexist without significant mutual inhibition. However, a non-reciprocal synergism was also observed, whereby mixed-species biofilm conditions promoted the growth of A. actinomycetemcomitans, while C. albicans growth remained stable. As expected, antimicrobial tolerance was elevated in mixed cultures, likely due to enhanced extracellular matrix production and potential quorum-sensing interactions, contributing to increased resistance against azithromycin and fluconazole. Conclusions: This study provides novel insights into previously rarely explored interactions between C. albicans and A. actinomycetemcomitans. These findings underscore the importance of investigating interspecies interactions within polymicrobial biofilms, as understanding their mechanisms, such as quorum-sensing molecules and metabolic cooperation, can contribute to improved diagnostics and more effective targeted therapeutic strategies against polymicrobial infections.