The emetic syndrome of Bacillus cereus is a food intoxication caused by cereulide (CER) and manifested by emesis, nausea and in most severe cases with liver failure. While acute effects have been studied in the aftermath of food intoxication, an exposure to low doses of cereulide might cause unnoticed damages to the intestines and liver. The toxicity which relies on the mitochondrial dysfunction was assessed on Caco-2 and HepG2 cells after exposure of one, three and ten days to a range of low doses of cereulide. Oxygen consumption rate analyses were used to study the impact of low doses of CER on the bioenergetics functions of undifferentiated Caco-2 and HepG2 cells using Seahorse XF extracellular flux analyzer. Both Caco-2 and HepG2 cells experienced measurable mitochondrial impairment after prolonged exposure of 10 days to 0.25 nM of cereulide. Observed mitochondrial dysfunction was greatly reflected in reduction of maximal cell respiration. At 0.50 nM CER, mitochondrial respiration was almost completely shut down, especially in HepG2 cells. These results corresponded with a severe reduction in the amount of cells and an altered morphology, observed by microscopic examination of the cells. Accurate and robust quantification of basal respiration, ATP production, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration allowed better understanding of the effects of cereulide in underlying respiratory malfunctions in low-dose exposure.

In the last decade, foodborne outbreaks and individual cases caused by bacterial toxins showed an increasing trend. The major contributors are enterotoxins and cereulide produced by Bacillus cereus, which can cause a diarrheal and emetic form of the disease, respectively. These diseases usually induce relatively mild symptoms; however, fatal cases have been reported. With the aim to detected potential toxin producers that are able to grow at refrigerator temperatures and subsequently produce cereulide, we screened the prevalence of enterotoxin and cereulide toxin gene carriers and the psychrotrophic capacity of presumptive B. cereus obtained from 250 food products (cereal products, including rice and seeds/pulses, dairy-based products, dried vegetables, mixed food, herbs, and spices). Of tested food products, 226/250 (90.4%) contained presumptive B. cereus, which communities were further tested for the presence of nheA, hblA, cytK-1, and ces genes. Food products were mainly contaminated with the nheA B. cereus carriers (77.9%), followed by hblA (64.8%), ces (23.2%), and cytK-1 (4.4%). Toxigenic B. cereus communities were further subjected to refrigerated (4 and 7 °C) and mild abuse temperatures (10 °C). Overall, 77% (94/121), 86% (104/121), and 100% (121/121) were able to grow at 4, 7, and 10 °C, respectively. Enterotoxin and cereulide potential producers were detected in 81% of psychrotrophic presumptive B. cereus. Toxin encoding genes nheA, hblA, and ces gene were found in 77.2, 55, and 11.7% of tested samples, respectively. None of the psychrotrophic presumptive B. cereus were carriers of the cytotoxin K-1 encoding gene (cytK-1). Nearly half of emetic psychrotrophic B. cereus were able to produce cereulide in optimal conditions. At 4 °C none of the examined psychrotrophs produced cereulide. The results of this research highlight the high prevalence of B. cereus and the omnipresence of toxin gene harboring presumptive B. cereus that can grow at refrigerator temperatures, with a focus on cereulide producers.

The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.