Analysis of emetic toxin production by Bacillus species using cellular cytotoxicity, molecular, and chromatographic assays

In this study, we analyzed production of the emetic toxin, cereulide, in 30 Bacillus strains by using a cellular cytotoxicity (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MTT) assay, polymerase chain reaction (PCR), and micellar electrokinetic chromatographycapillary electrophoresis (MEKC-CE) analysis. The Bacillus cereus emetic strains produced 60 similar to 227 A mu g/mL of cereulide when analyzed using MEKC-CE. Some other Bacillus species, including B. subtilis, B. pumilus, and B. megaterium, produced putative cereulide at levels similar to those produced by emetic B. cereus, whereas B. mycoides and B. thuringiensis did not produce the putative toxin or produced it at a concentration less than 2 A mu g/mL. Only B. cereus emetic strains were found to be toxic to bovine fibroblast cells, with the exception of one Bacillus diarrheal strain. The PCR results correlated with the MTT assay results, except in the case of one B. cereus diarrheal strain. This strain may produce either unusually heat-stable enterotoxins or is a co-producer of emetic and diarrheal toxins. Collectively, our results indicate that several Bacillus species produce toxin(s) with structures and properties similar to those of cereulide.