Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions

Abstract

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.

FIG. 1.

HPLC-MS analysis of cereulide and valinomycin. (a) HPLC chromatogram (500 to 1,300 m/z) showing separation of cereulide and valinomycin. (b) Mass spectrum of valinomycin. (c) Mass spectrum of cereulide. (d) Calibration curve for valinomycin determined from four injections of two separate dilution series.

FIG. 2.

Comparison of chemical and biological assays for determining cereulide concentrations in methanol extracts from independent preparations of different B. cereus strains. The line indicates a 1:1 correlation. The inset shows the same data drawn to a linear scale.

FIG. 3.

Production of cereulide by B. cereus strains NC7401, F5881, and F4810/72 compared to that by F528, ATCC 14579, and OH599 (mean) grown on tryptic soy agar plates for 11 days at different temperatures.

FIG. 4.

Growth of B. cereus strain NC7401 in Trypticase soy broth and production of cereulide.