Unveiling the In Vitro Antibiofilm Efficacy of Antifungal Lipopeptides Purified from <i>Bacillus</i> sp. against Mixed-Species Biofilms of <i>Candida</i>

Polymicrobial fungal infections are often associated with significant invasive mycosis. <i>Candida</i> non-<i>albicans</i> species such as <i>Candida glabrata</i> and <i>Candida tropicalis</i> are most frequent organisms after <i>Candida albicans</i> especially in biofilm-associated fungal infections with significant mortality and morbidity. In the present investigation, the antibiofilm efficacy of <i>Bacillus</i>-derived lipopeptides AF₄ and AF₅ was assessed against <i>Candida</i> dual species mixed biofilms at different stages such as adherence, developmental, and mature phase in vitro experiments. Interestingly, in this study, <i>C. glabrata</i> was found to be predominant over <i>C. tropicalis</i>, whereas AF₄ and AF₅ exposures significantly reduced both biofilm biomass and metabolic activity as was revealed through various experiments such a quantification assays and morphological analyses. The advanced microscopy suggests the strong biofilm-busting activity of both lipopeptides. Additionally, the elucidation of their mode of action against the planktonic phase of <i>C. glabrata</i> and <i>C. tropicalis</i> was performed to investigate the presence of stages of apoptosis with Annexin V-FITC/propidium iodide staining and TUNEL assays. Two strains were exposed to sub-MIC and minimum inhibitory concentration (MIC), also with 2× MIC of AF₄ and AF₅. A significant shift from live cells to early stage and late-stage apoptotic induction percentage was increased along with increasing the concentration of 1× MIC to 2× MIC. TUNEL results confirmed the DNA fragmentation by the enhanced green fluorescence intensity in cells in TUNEL-positive nuclei. Taken together, our results demonstrate that <i>Bacillus</i>-derived lipopeptides AF₄ and AF₅ hold great promise with strong antibiofilm properties. The overall antibiofilm properties and mode of action characterization studies unequivocally confirm the antifungal potential of the two investigational lipopeptides, with the ability to challenge several fungal infections.