New lipopeptide homologues (AF₃, AF₄, and AF₅) with antifungal activities against Candida and Cryptococcus spp. were purified from a cell-free supernatant of Bacillus subtilis RLID 12.1. The lipopeptides AF₃, AF₄, and AF₅ were identified with the same peptide sequence Asn-Pro-Tyr-Asn-Gln-Thr-Ser with variations in the fatty acid branching type and chain length (anteiso-C₁₇, iso-C₁₇, and iso-C₁₈, respectively). Upon comparing the three homologues for MICs against 81 Candida (n = 64) and Cryptococcus (n = 17) clinical isolates and their cytotoxicities, we found that AF₄ was the most promising antifungal lipopeptide, since it demonstrated 100% inhibition at geometric mean MICs of 3.31, 3.41, 3.48, and 2.83 μg/ml against Candida albicans, Candida tropicalis, Candida auris, and Cryptococcus neoformans, respectively, with low hemolysis values (<6%) and 50% inhibitory concentrations (13.31 μg/ml). The additive effects among the homologues AF₃, AF₄, and AF₅ were evaluated against three Candida species, along with the cytotoxicity studies. Five combinations exhibited good additive interaction effects: AF₃/AF₄ (at corresponding concentrations of 4 and 4 μg/ml [4/4 μg/ml]), AF₃/AF₅ (4/4 μg/ml), AF₃/AF₅ (2/4 μg/ml), AF₄/AF₅ (4/4 μg/ml), and AF₄/AF₅ (2/4 μg/ml) in planktonic cell inhibition and AF₃/AF₄ (4/4 μg/ml), AF₃/AF₅ (4/4 μg/ml), and AF₃/AF₅ (2/4 μg/ml) in the inhibition of biofilm formation. However, combinations AF₃/AF₄ and AF₃/AF₅, which showed >70% cell survival with low hemolysis (<5%), were found to be comparatively effective. We describe here the additive effects of lipopeptide homologues showing reduced cytotoxicity against mammalian cells; these combinations might serve as a potent antibiofilm-forming substitute.

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