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New lipopeptide homologues (AF3 , AF4 , and AF5 ) with antifungal activities againstCandida andCryptococcus spp. were purified from a cell-free supernatant ofBacillus 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 , andiso -C18 , respectively). Upon comparing the three homologues for MICs against 81Candida (n = 64) andCryptococcus (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 againstCandida albicans ,Candida tropicalis ,Candida auris , andCryptococcus neoformans , respectively, with low hemolysis values (&lt;6%) and 50% inhibitory concentrations (13.31 μg/ml). The additive effects among the homologues AF3 , AF4 , and AF5 were evaluated against threeCandida 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 &gt;70% cell survival with low hemolysis (&lt;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.

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