Enterotoxin Production by Bacillus cereus Under Gastrointestinal Conditions and Their Immunological Detection by Commercially Available Kits Siele Ceuppens, 1,2 Andreja Rajkovic, 1,3 Stefanie Hamelink, 1,2 Tom Van de Wiele, 2 Nico Boon, 2 and Mieke Uyttendaele 1 Abstract Currently, three commercial kits for Bacillus cereus enterotoxins Nhe and/or Hbl detection are available, namely, the Bacillus diarrheal enterotoxin visual immunoassay (BDE VIAÔ) kit (3M Tecra), B. cereus enterotoxin reversed passive latex agglutination (BCET-RPLA) kit (Oxoid), and the Duopath Ò Cereus Enterotoxins (Merck). The performance of the kits and their applicability to gastrointestinal simulation samples were evaluated. Then, the stability and production of enterotoxins Hbl and Nhe under gastrointestinal conditions were investigated. Enterotoxin production was absent or impaired at acidic pH, i.e., in gastric medium with pH 5.0 and lasagne verde with pH 5.5. B. cereus did produce enterotoxins Nhe and Hbl during anaerobic growth in intestinal medium at pH 7.0, but the toxins were instantly degraded by the enzymes in the host’s digestive secretions. Preformed enterotoxins did not withstand gastrointestinal passage under the simulated conditions, which suggests that preformed enterotoxins in food do not contribute to the diarrheal food poisoning syndrome. In conclusion, diarrhea is probably caused by de novo enterotoxin production by B. cereus cells located closely to the host’s intestinal epithelium. Introduction B acillus cereus can cause emetic and diarrheal food poisoning by production of cereulide and several en- terotoxins such as non-hemolytic enterotoxin (Nhe), hemo- lysin BL (Hbl, cytotoxin K (CytK) and enterotoxin FM and virulence factors such as hemolysins (HlyII and HlyIII), col- lagenases, phospholipases C and cereolysins, respectively (Ceuppens et al., 2011). B. cereus enterotoxins are unstable molecules, susceptible to heating ( > 55°C for ‡ 20 min) and protease activity (pronase, pepsin, trypsin, and chemotrypsin, 1–2 mg/mL, 1–24 h) (Granum et al., 1993; Turnbull et al., 1979). As a result, pre- formed enterotoxins in food are highly unlikely to retain their activity after food preparation and gastrointestinal passage. In contrast, the emetic toxin cereulide is highly resistant to heat (resistant to all normal food processing and food preparation temperatures), acid (resistant to pH values of 2–11), and pro- tease activity (pepsin and trypsin) (Rajkovic et al., 2008; Shi- nagawa et al., 1996). Consequently, cereulide is not inactivated during gastrointestinal passage, and preformed cereulide in food plays an prominent role in emetic food poisoning. Multiple detection methods for enterotoxins exist, includ- ing mass-spectrometry, immunological assays, and biological assays such as the vascular permeability reaction, rabbit ileal loop, and cytotoxicity assays. The biological assays are func- tional assays that determine the overall toxicity, resulting in the advantage of detecting all biologically active toxins with usually high sensitivity. On the other hand, the inherent pit- falls of biological assays are the dubious specificity, resulting in false-positive results for samples which contain other tox- ins. Moreover, enterotoxins Nhe, Hbl, and CytK are all toxic for Vero cells (Wehrle et al., 2009), so a positive cytotoxicity assay requires further analysis to reveal which specific en- terotoxins the B. cereus strain produces. Similar to the liquid chromatography mass spectrometry (LC-MS) assay for B. ce- reus emetic toxin cereulide (Delbrassinne et al., 2011), all B. cereus enterotoxins can be detected by mass spectrometry assays (Gilois et al., 2007). The strong points of these methods include their high specificity and sensitivity, while the main drawbacks are the extensive, labor-intensive sample prepa- ration and high investment, running, and maintenance costs. The relatively fast, easy, and cheap immunological detection of enterotoxins make it suitable for research and routine 1 Faculty of Bioscience Engineering, Laboratory of Food Microbiology and Food Preservation (LFMFP), and 2 Faculty of Bioscience En- gineering, Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium. 3 Department of Food Safety and Food Quality Management, Faculty of Agriculture, Belgrade University, Zemun-Belgrade, Serbia. FOODBORNE PATHOGENS AND DISEASE Volume 9, Number 12, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2012.1230 1130