Understanding Heterogeneity and Tolerance of Dual <scp> <i>Candida albicans–Staphylococcus aureus</i> </scp> Biofilms to Cold Atmospheric Plasma and Antimicrobial Combinations

Interkingdom polymicrobial biofilms of Staphylococcus aureus and Candida albicans increase pathogenicity and complicate treatment strategies, contributing to greater morbidity and mortality. These infections are typically investigated using the C. albicans strain SC5314, despite its uncertain clinical relevance. Here, we evaluate how different C. albicans (high, intermediate, low biofilm formers) and S. aureus strains influence biofilm tolerance to both conventional antimicrobials and cold atmospheric plasma (CAP). CAP is an emerging technology that has been shown, including in our prior work, to disrupt biofilm biomass via reactive oxygen and nitrogen species, positioning it as a promising adjunct to antimicrobial therapy. We determined how strain variation influenced biofilm structure and tolerance to vancomycin, amphotericin B and CAP. Interkingdom biofilm architecture was primarily influenced by the C. albicans strain, particularly its ability to form hyphae. Despite this, all strains conferred protection to S. aureus against vancomycin. CAP eradicated S. aureus biofilms within 120 s, but this effect was lost in dual-species biofilms. However, CAP pretreatment enhanced the efficacy of both antimicrobials in interkingdom biofilms. These findings highlight the role of hyphal morphology in vancomycin tolerance and support further clinical evaluation of CAP as a biofilm-targeting adjunct.