In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.Food poisoning by emetic Bacillus cereus strains has been shown to cause fatal liver failure (6,15,25). Cereulide, the emetic toxin of B. cereus, is a cyclic dodecadepsipeptide composed of three repetitions of two amino acids and two hydroxyIts chemical structure is closely related to that of valinomycin, which is produced by various Streptomyces spp. (16). Cereulide is a heat-stable toxin (4,21,24,27) and is synthesized enzymatically by a nonribosomal peptide synthesis (NRPS) whose genetic determinants are located on the pCERE01 plasmid of the B. cereus emetic strain Kinrooi 5975c (9), the pBC270 element from B. cereus strain AH187 (23), and the pBCE4810 plasmid from B. cereus reference strain F4810/72 (8). These elements contain a 23-kb gene cluster (ces) involved in the cereulide synthesis. The two largest genes, named cesA (ϳ10 kb) and cesB (ϳ8 kb), lead to the incorporation and modification of D-O-Leu, D-Ala (cesA), L-O-Val, and D-Val (cesB), which compose the basic tetradepsipeptide motif of cereulide (7,14).Cereulide can cause stomach pain and vomiting, respiratory distress, and occasional loss of consciousness, possibly leading to coma and ultimately death of the individual (6). This toxin is also an ionophoric molecule with a high degree of affinity for K ϩ ions (26). It is lipophilic and specifically targets mitochondrial membranes (4), where it leads to cellular dysfunctions, such as swelling mitochondria and blocking oxidative phosphorylation (2, 17).It has been shown that valinomycin and cereulide display similar activities, since they block the motility of boar spermatozoa through the dissipation of mitochondrial internal membrane potential, a property often used for the detection of cereulide (3, 4, 22, 23a). Given these similarities, it was interesting to consider the known activities of valinomycin to hypothesize on the actual "raison d'être" of cereulide. Valinomycin is toxic for some insects, nematodes, and Gram-positive bacteria at very low concentrations (18,19,20). Although the antifungal activity of valinomycin has been reported (18), Altayar and Sutherland (1) showed that cereulide does not have any antifungal activity on rich medium. Since fungi are ubiquitous organisms living in the same environment as B. cereus (11, 12), a possible competition between these organisms could, however, be expected. The purpose of this study was to assess the potential antifungal activity of three emetic B. cereus strains and to highlight the specific contribution of cereulide using isogenic cereulide-producing and cereulide-minus (cured or mutated) strains. Comparison with the antifungal activity spectrum of valinomycin was also ...