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Bacillus-derived lipopeptides AF4 and AF5 significantly reduce biofilm biomass and induce apoptosis and DNA fragmentation in mixed-species biofilms of Candida glabrata and Candida tropicalis. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

November 21, 2025ACS Omega1 citationsOpen Access

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

MMMadhuri MadduriBirla Institute of Technology and Science, Pilani - Goa Campus SRShivaprakash M. RudramurthyHumane Society of the United States URUtpal RoyBirla Institute of Technology and Science, Pilani - Goa Campus

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Abstract

High Resolution Image Download MS PowerPoint Slide Polymicrobial fungal infections are often associated with significant invasive mycosis. Candida non- albicans species such as Candida glabrata and Candida tropicalis are most frequent organisms after Candida albicans especially in biofilm-associated fungal infections with significant mortality and morbidity. In the present investigation, the antibiofilm efficacy of Bacillus -derived lipopeptides AF 4 and AF 5 was assessed against Candida dual species mixed biofilms at different stages such as adherence, developmental, and mature phase in vitro experiments. Interestingly, in this study, C. glabrata was found to be predominant over C. tropicalis, whereas AF 4 and AF 5 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 C. glabrata and C. tropicalis 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 AF 4 and AF 5 . 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 Bacillus -derived lipopeptides AF 4 and AF 5 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.

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