Abstract
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.