Zerumbone combined with antimicrobial photodynamic inactivation reduces growth of Candida albicans biofilms and modulates virulence gene expression

Abstract Candida albicans biofilms represent a clinical challenge due to their protective extracellular matrix and the resistance to conventional antifungals. This study investigates the strategy of combining the phytochemical zerumbone (ZER) with antimicrobial photodynamic inactivation (aPDI) mediated by Photodithazine ® (PDZ) against growth and gene expression of mature C. albicans biofilms (ATCC 90028). The biofilms were evaluated across seven experimental conditions, including ZER, aPDI alone, and the combination of ZER + PDI. The efficacy of the treatment was assessed through colony-forming units (CFU), biomass reduction (crystal violet staining), and membrane permeability assays (Propidium Iodide). To evaluate mechanisms related with the results, the Reactive Oxygen Species (ROS) production was quantified, and RT-qPCR was performed to evaluate the expression of genes associated with virulence and biofilm formation ( ALS1 , HWP1 , EFG1 , and SOD1 ), along with the ergosterol biosynthesis gene ERG11 . Additionally, photodegradation was assessed for PDZ alone and in combination with ZER under red-light irradiation (50 J/cm²), with absorbance measured over time. The ZER+aPDI treatment exhibited better results compared with the other groups, achieving a reduction in cell viability and biomass. The combination promoted the induction of ROS production and increased membrane permeabilization. The gene expression was downregulated after the treatment with ZER+aPDI, signaling a comprehensive suppression of adhesion, morphogenesis, antioxidant defense and ergosterol biosynthesis. These findings demonstrate that the association of ZER and aPDI exerts a potent multimodal effect by disrupting the biofilm’s physical architecture, attenuating fungal pathogenicity and antioxidant defenses, and suppressing ergosterol biosynthesis, thereby offering a significant antifungal effect.