Enterotoxin Production byBacillus cereusUnder Gastrointestinal Conditions and Their Immunological Detection by Commercially Available Kits

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.

Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

“…These results are consistent with those for different B. cereus strains in lasagna verde or tryptone soy broth medium with intestinal components (18). Ceuppens et al (15) also did not detect enterotoxins (Nhe or Hbl) during in vitro ileal growth of B. cereus in the absence of competitive microbiota.…”

“…Bacterial concentrations between 5 ϫ 10 6 and 10 7 CFU ml Ϫ1 were required for the detection of Hbl and Nhe by an enzyme-linked immunosorbent assay (ELISA) (21). However, no enterotoxin was previously detected in the presence or absence of intestinal proteases when the B. cereus concentration was Ͻ7 log CFU ml Ϫ1 (15,18). In this study, 6.6 log CFU ml Ϫ1 of luminal bacteria was present only at the end of the ileal simulation, a value close to the limit mentioned above.…”

“…Nevertheless, the intensity of the Nhe signal was clearly reduced during the incubation of the culture and supernatant with pancreatin and bile (CULϩ and SUPϩ). Rapid degradation of Nhe in intestinal media (Ͻ1 h) has been shown previously (18,19). Our results demonstrated a degradation rate much lower than those in these studies, possibly due to differences in the setup (e.g., different concentrations and strengths of host proteases, differences in pH, and a different starting concentration of Nhe) or the different potency of strains to produce toxin.…”

“…Other studies have shown that spore germination and growth are possible during gastrointestinal simulations; however, enterotoxin production was not assessed (17). Enterotoxins, when produced, are unstable (6) and can be degraded rapidly in the presence of digestive enzymes (18,19). Evidently, B. cereus has to withstand and adapt to the environmental hurdles posed by the host (20) and requires a subset of specific conditions to express its virulence.…”

Potential probiotic attributes of a new strain of Bacillus coagulans CGMCC 9951 isolated from healthy piglet feces

“…Compared with mass-spectrometry and immunological assays, the biological assays, including rabbit ileal loop, vascular permeability reaction and cytotoxicity assays, are generally employed in determining the enterotoxins, resulting in the advantage of detecting all biologically active toxins with high sensitivity (Ceuppens et al 2012). To ensure the safety of B. coagulans CGMCC 9951 strain, assessment of bacterial toxins in rabbit ileum was carried out.…”