Development of an in vitro gastrointestinal simulation experiment to assess Bacillus cereus NVH 1230-88 survival during gastrointestinal passage (2012)

Introduction : Viable and enterotoxin producing B. cereus are required in the small intestine to cause diarrhoeal food poisoning, since these toxins are not resistant to most food processing activities and gastrointestinal passage [1]. The aim of this study was to developing an in vitro gastrointestinal simulation experiment to investigate the survival and toxin production of B. cereus in the gastrointestinal tract. Materials and methods : The food used in the simulation consisted of mashed potato flakes (Mousline classic, Maggi) reconstituted with whole milk and twofold diluted (50:50, w/w) with feed solution that was derived from the dynamic gut simulator SHIME (Simulator of the Human Intestinal Microbial Ecosystem) [2]. B. cereus NVH 1230-88 spores (7 log spores/mL) were produced in this mashed potato medium after 2 weeks at 30 °C. The composition and volume ratios of simulation media for saliva, gastric and intestinal fluid were based on the RIVM in vitro digestion model for the fed state [3]. Dialysis was performed with a Diacap® Polysulfone high-flux dialyzer (Diacap HiFlo18, B. Braun). Enumeration of B. cereus was done by plating on Tryptone Soya Agar (TSA) and with quantitative PCR (qPCR) for mixed bacterial cultures [4]. Enterotoxin production was assessed with the Duopath® Cereus Enterotoxins (Merck). Results and discussion : The 9 h gastrointestinal simulation experiment comprised 5 phases: 1) the mouth phase, 2) the stomach phase, 3) the duodenum phase, 4) dialysis and 5) the ileum phase. The gastric phase simultaneously comprised continuous acidification and fractional emptying in 5 fractions into the next intestinal vessel, in correspondence with the human gastrointestinal physiology. To mimic bile reabsorption in the small intestine, dialysis was performed to obtain ≥ 90 % bile (Oxgall, Difco) removal. During the ileum phase, competitive pressure was exerted by addition of the mixed intestinal bacterial community from SHIME. The B. cereus spore inoculum was unaffected by the mouth, stomach and duodenum phase. At the end of the duodenum phase and during the dialysis, the spore germination started. During the final 4 h ileum phase, only survival without outgrowth was observed. The lack of outgrowth can be fully attributed to the competitive human intestinal bacteria. No enterotoxin production was detected, which was to be expected for ≤ 5 log CFU/mL metabolically active B. cereus [1]. Conclusion : A dynamic gastrointestinal simulation experiment was developed in correspondence with the gastrointestinal physiology of healthy people. No B. cereus proliferation occurred during the simulation of gastrointestinal passage, despite the high spore concentration in the ingested food (7 log spores/g). The preliminary results of this study show that diarrheal food poisoning is not caused by proliferation in the intestinal lumen, but probably by interaction of B. cereus and its enterotoxins with the host (epithelium). References [1] Ceuppens S. et al. (2011). Crit Rev Microbiol, 37: 188-213. [2] Possemiers S. et al. (2004). FEMS Microbiol Ecol, 49: 495-507. [3] Hagens W.I. et al. (2008). RIVM Letter report, 711701080/2008. [4] Ceuppens S. et al. (2010). J Microbiol Meth, 83: 202-210.