B. cereus can cause emetic and diarrhoeal and food poisoning by production of resp. emetic (cereulide) and diarrhoeal toxins (non-haemolytic enterotoxin (Nhe), haemolysin BL (Hbl), cytotoxin K (CytK), etc.) . In contrast to the extremely stable toxin cereulide, the enterotoxins are easily degraded by acid and digestive enzymes (proteases) and thus preformed enterotoxins in food do not retain their toxicity during gastrointestinal passage . Therefore, the prerequisite for diarrhoeal food poisoning is enterotoxin production by B. cereus in the small intestine, so the gastrointestinal survival of vegetative B. cereus was investigated. It was previously shown that approx. 10% of the vegetative cells survived gastric passage . Next, the surviving bacteria are confronted with bile in the lumen of the duodenum, the proximal part of the small intestine.
Bile mainly consists of bile acids (approximately 72% of the total lipids), besides phospholipids (approx. 24%) and cholesterol (approx. 4%) . In humans, these bile acids consist mainly of cholic acid (between 50% and 80%) and chenodeoxycholic acid (between 20% and 50%) [5, 6]. They are synthesized in the liver from cholesterol and conjugated to glycine (approx. 75%) or taurine (approx. 25%), with conjugation ratios depending on the diet amongst other factors [6, 7]. Secretion of bile is triggered by fat and acid release from the stomach into the duodenum, which results in 7 to 15 mM bile salts in the small intestine after a meal, corresponding with 5 to 10 g Oxgall/L [7–10]. Deconjugation of bile acids by indigenous intestinal bacteria mainly occurs in the distal ileum, where approx. 95% of the bile acids is reabsorbed, of which approx. 15% is unconjugated [7, 10]. Both conjugated and unconjugated bile acids are absorbed by passive diffusion along the entire gut, but this process is more efficient for unconjugated bile acids. Additionally, specific active transport systems are present in the distal ileum, which are more efficient in the uptake of conjugated bile acids. After absorption from the intestine, the bile acids are transported to the liver via the blood, reconjugated and secreted again into the bile bladder. This recycling process of bile acids is called enterohepatic circulation.
The physiological role of bile acids is to increase the solubility of dietary fat and facilitate its degradation and absorption. Due to their detergent properties, bile acids also alter cell membranes and thus have cytotoxic and bactericidal effects, noticeable by an increased membrane fluidity and permeability [11–14]. Depending on the bile concentration, disruption of the cell membrane integrity occurs nearly instantaneously, causing cell leakage and death, or more slowly and subtly by altering the membrane permeability and fluidity, the activity of critical proteins in the cell membrane and the membrane hydrophobicity . In addition to destruction of the bacterial cell membrane integrity, bile also induces DNA damage, denaturation and misfolding of proteins, leading to the death of bacteria .
Gram-positive bacteria tend to be more sensitive to bile than Gram-negative bacteria, but bile tolerance is very strain specific, so generalized statements for species are not possible [17–19]. Despite the bactericidal effects of bile, some micro-organisms have developed bile resistance by induction of bile metabolizing enzymes and transport systems or by altering membrane permeability, fluidity or charge. Some enteric pathogens may even depend on bile as a host signal for virulence regulation. For example, Salmonella enterica serovar Typhimurium possesses the multidrug efflux pump AcrAB for bile removal and transport through the outer membrane . Moreover, invasion of the host’s epithelial cells by this bacterium is induced by lowered bile concentrations, so after transit to the distal ileum or into the mucus layer .
The co-ingestion of food is an important factor in the antibacterial activity of bile in the intestines. Bile inactivation of bacteria is influenced by the presence of food components, which can create protective micro-environments or bind bile. For example, the maximum bile concentration tolerated by B. cereus during growth in intestinal medium was 3 g/L porcine bile when pea soup was supplemented, while only 0.9 g/L and 0.6 g/L bile were tolerated in the presence of milk and the absence of food, respectively . Also for Lactobacillus curvatus, the presence of a food matrix, namely meat, increased its bile tolerance and subsequently its gastrointestinal survival . Similarly, the bile tolerance of Bifidobacterium breve was enhanced by soy proteins .
In this study, the gastrointestinal survival of vegetative B. cereus cells was investigated and linked to the bactericidal role of bile. The B. cereus inoculum was cultivated in the composite food matrix lasagne verde prior to the in vitro simulation of the gastrointestinal passage to include any potential protective effects of the food particles on the gastrointestinal survival of B. cereus.