Detection of enterotoxins produced by Bacillus cereus strains involved in food poisoning using MALDI-TOF mass spectrometry

Introduction: B. cereus is a foodborne pathogen that can cause diarrhea through secretion of enterotoxins, such as NHE and CytK, in the small intestine of the host. Purpose: Detection of these enterotoxins is critical for successful microbial food safety risk assessment. Among the existing detection methods, serological assays appear to be the most promising but their application is limited by the availability and the appropriate selection of antibodies. Available polyclonal kits for verification of NHE production lack specificity and frequently result in false negatives. At this moment, there are no commercial serological assays for detection of CytK. Therefore, alternative approaches need to be developed. Methods: We have used MALDI-TOF MS and MS/MS to detect enterotoxins in four pure B. cereus cultures using tryptic digests of proteins separated by SDS-PAGE. The selected strains have been involved in foodborne outbreaks. Results: CytK1, a rare but lethal toxin, was detected in B. cereus NVH 0391/98 and the fully cleaved tryptic peptides ANPTLSDAPVDGYPIPGASVTLR and TYPHETDAR are selected as MS biomarkers due to their specificity and abundance in the mass spectra. Forty-five different peptides from all tested strains were matched to the NheA component of the NHE complex, a toxin genetically present in all B. cereus strains. Only three of them were common and abundant in the four strains, i.e. QKELLPLIQK, EWIDEYNPK and LIDLNQEMMR, but they were either incompletely digested or susceptible to modification. These characteristics obscured their application as universal markers, thus detection of NheA in unknown samples should be based on the simultaneous identification of other enterotoxin specific peptides. Our results demonstrated that NheA from B. cereus NVH 0391/98 was very heterogeneous compared to that of the other strains explaining the failed previous attempts to detect this toxin through antibodies or PCR primers. Our method was comparable with the commercial immunological assay in respect to detection of NheA produced by B. cereus NVH 0075/95, but the latter technique is based on polyclonal antibodies which can also react with other proteins, hence limiting its specificity. Significance: We showed that MALDI-TOF mass spectrometry is a powerful tool for detection of enterotoxins because its application is not restricted by toxin sequence diversity among different strains. The recommended MS biomarkers will be used for the screening of environmental and food associated strains that can be involved in food poisoning