The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black‐lipid membranes in KCl solutions in an ionophore‐like manner. It formed adducts with K⁺, Na⁺, and NH₄⁺ but the conductance was highly selective for K⁺ in relation to Na⁺ and H⁺ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K⁺. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K⁺, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO₃‐ but not in NaNO₃‐containing media, less in acetate. In NaNO₃ media addition of the cereulide caused a transient diffusion potential which was reduced by adding K⁺. It is concluded that the toxic effects of cereulide are due to it being a K⁺ ionophore.

A bacterial isolate with an optimum growth temperature of about 55 degrees C was recovered on a medium composed of one part Kligler's iron agar and four parts of Thermus Agar from the host spring at São Pedro do Sul in central Portugal. Phylogenetic analyses using the 16S rRNA gene sequence of strain SPS-1037T indicated that the new organism represented a new genus and species of beta-Proteobacteria. The major fatty acids of strain SPS-1037T are C16:0 and C17:0. Ubiquinone 8 is the major respiratory quinone, and the major polar lipids are phosphatidylethanolamine and phosphatidylglycerol. The new isolate is aerobic and chemolithoheterotrophic. Thiosulfate and tetrathionate were oxidized to sulfate. The growth yield of the organism was improved by the addition of thiosulfate to media containing organic carbon sources, but the organism did not grow autotrophically under the conditions examined. Heterotrophic growth of strain SPS-1037T occurs on amino acids and organic acids, but this organism does not assimilate carbohydrates. On the basis of the phylogenetic analyses, and physiological and biochemical characteristics, it is proposed that strain SPS-1037T represents a new genus and a new species for which the name Tepidimonas ignava is proposed.

SUMMARY: The lipophilic toxin, cereulide, emitted by emetic food poisoning causing strains of Bacillus cereus, is a powerful mitochondria toxin. It is highly lipophilic and rapidly absorbed from the gut into the bloodstream. We tested how this toxin influences natural killer (NK) cells, which are important effectors in defence against infections and malignancy. Cereulide inhibited cytotoxicity and cytokine production of natural killer cells, caused swelling of natural killer cell mitochondria, and eventually induced natural killer cell apoptosis. The suppressive effect on cytotoxicity was fast and toxic concentration low, 20–30 μg/l. As the emesis causing concentration of cereulide is around 10 μg/kg of total body mass, our results suggest that emesis causing or even lower doses of cereulide may also have a systemic natural killer cell suppressive effect.

Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated. Toxins were purified from methanol extracts of the B. licheniformis biomass using boar sperm cells as the toxicity indicator. The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI‐TOF‐MS. The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino‐acid residues l‐Gln, l‐Leu, d‐Leu, l‐Val, l‐Asp, d‐Leu and l‐Ile in that order. Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3‐hydroxy fatty acids are 13–15 carbons in length. We showed that the toxins from food and food poisoning isolates of B. licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa. Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity.

To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group. Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21-37 degrees C for B. subtilis and 11-21 degrees C for B. mojavensis. Both species produced amylosin in air as well as in 7-8% CO(2) with 8-9% O(2). Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin. This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus.