Evaluation of Antifungal Efficacy of Three New Cyclic Lipopeptides of the Class Bacillomycin from Bacillus subtilis RLID 12.1.

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.