A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores

Abstract

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen-1. This amount corresponds to 10(4) to 10(5) CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10(8) CFU ml-1. The detection limit for food was 3 g of rice containing 10(6) to 10(7) CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen-1, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.

FIG. 1

Thin sections of the middle piece of a boar spermatozoon exposed to cell extracts of B. cereus 4810/72 and ATCC 14579^T for 7 days. (A) Mitochondrial damage in the middle piece of a spermatozoon exposed to cell extracts of 2 mg (wet weight) of cells ml⁻¹ of strain 4810/72. The frequency of similarly swollen mitochondria was 75% to over 90% after exposure to extracts from 4 mg (wet weight) of cells ml⁻¹ and <20% after exposure to extracts from 0.5 mg (wet weight) of cells ml⁻¹ (not shown). (B) Middle piece of a spermatozoon exposed to cell extracts of B. cereus ATCC 14579^T (2 mg [wet weight] ml⁻¹). Mitochondria of ordinary size with intact membranes are seen. A spermatozoon exposed to the same extract for 25 days exhibited no change of morphology, and those of unexposed spermatozoa were identical to that shown in panel B. Bars, 200 nm.

FIG. 2

Mass fragmentation of the purified sperm toxin of B. cereus 4810/72. (A) m/z of the molecular ions as determined by ESI-MS. (B) ESI-MS/MS fragmentation of the protonated ion, m/z of 1,153.85, shown in panel A. Peak numbers correspond to the fragment ions assigned in Table 3. Peaks marked with asterisks represent loss of water.