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 ofB. 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 104 to 105 CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 108 CFU ml−1. The detection limit for food was 3 g of rice containing 106 to 107 CFU of emetic B. cereusper 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.

Vital fluorescent stains for detection of stress in Pythium ultimum and Rhizoctonia solani challenged with viscosinamide from Pseudomonas fluorescens DR54

“…None of these stains could be loaded into viscosinamide-treated hyphae, however, which in turn indicated that viscosinamide did not induce extensive membrane disorder or destruction. A lipopolypeptide similar to viscosinamide from Bacillus cereus also had no major e¡ects on the membrane integrity in spermatozoa [23]. When extensive pore formation in the membranes could be excluded, an extensive loss of £uorescent stain product from the viscosinamide-treated hyphae could also be ruled out.…”

Identification of emetic toxin producingBacillus cereusstrains by a novel molecular assay

“…Reference strains for the development of the PCR assay were the emetic reference strain F4810/72 [20], three isolates from recent emetic outbreaks in Germany and three emetic isolates from foods (MHI 280, MHI 297, MHI 1305, and MHI 87, MHI 135, MHI 294 [21]), as well as the non-emetic culture collection strains, B. cereus ATCC 14579 T and ATCC 27877, and two non-emetic isolates from foods (MHI 13, MHI 124 [21]). For evaluation of the PCR assay B. cereus strains were obtained from the project culture collection of an ongoing EU project (QLK1-CT-2001-00854).…”

Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis

“…MALDI-TOF-MS was performed as described previously [18]. ESI-MS/MS data were collected as described previously [19]. Samples were dissolved in 50% aqueous methanol containing 1% formic acid before injection into the mass spectrometer.…”

“…The inhibition of the motility and the acrosome reaction of boar spermatozoa caused by toxins, damage to plasma membrane, swelling of mitochondria and loss of cellular ATP and NADH were analyzed as described elsewhere [3,19,21].…”