Sperm bioassay for rapid detection of cereulide-producing Bacillus cereus in food and related environments

A novel in vitro method, sperm micro assay for rapidly distinguishing cereulide, the emetic toxin producing Bacillus cereus from non-producers is described and its use for quantitating cereulide and screening large numbers of B. cereus strains/colonies evaluated. The assay is non-laborious and can be executed with equipment present in most laboratories. Boar spermatozoa, purchased as standard semen from artificial insemination suppliers, are used to detect toxicity. Boar sperms respond within 5 min by cessation of motility when exposed at 37 °C to heat-treated (100 °C) extract prepared from a cereulide containing B. cereus. The assay can be done on individual colonies on the primary plate, with no need for pure culture and the qualitative result is obtained within 30 min. The assay is robust, not sensitive to age or storage of the culture plates. The use of the sperm micro assay for semiquantitative estimation of cereulide in B. cereus was validated with 14 different B. cereus strains using as reference the specific chemical assay for cereulide, based on liquid chromatography-ion trap mass spectrometry (LC-ion trap MS). The cereulide contents calculated from endpoint dilutions of the sperm micro assay matched the result of the chemical analysis closely. The detection threshold of the sperm micro assay was measured as 0.3±0.1 ng of cereulide per 5.4x10(6) sperm cells in 0.2 ml or 0.9 ng of cereulide per mg of B. cereus biomass (wet wt.). Food-related B. cereus strains contained 4-400 ng of cereulide per mg (wet wt.). When a large number of B. cereus of food, non-food, clinical and environmental origins were screened and 107 independent strains/isolates were identified as cereulide producers, it was observed that all of these had low or no haemolytic activity when cultivated on bovine blood agar. None of the strains/isolates with wide, clear zones of haemolysis, considered typical of B. cereus, produced cereulide.