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. <i>Candida albicans</i> and <i>Aggregatibacter actinomycetemcomitans</i> are two commensals that can turn into opportunistic pathogens and are able to form robust biofilms. <b>Objectives</b>: 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. <b>Methods</b>: 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. <b>Results</b>: 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 <i>A. actinomycetemcomitans</i>, while <i>C. albicans</i> 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. <b>Conclusions</b>: This study provides novel insights into previously rarely explored interactions between <i>C. albicans</i> and <i>A. actinomycetemcomitans</i>. 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.