Sub-Emetic Toxicity of Bacillus cereus Toxin Cereulide on Cultured Human Enterocyte-Like Caco-2 Cells

Abstract

Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied before. In this research, we investigated the effect of low concentrations of CER on the behavior of intestinal cells using the Caco-2 cell line. The MTT (mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the SRB (sulforhodamine B) reactions were used to measure the mitochondrial activity and cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in a MTT reaction of 1 ng/mL after three days of treatment; in an SRB reaction of 0.125 ng/mL after three days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of CER. Additionally, 0.5 and 2 ng/mL of CER increased the lactate presence in the cell culture medium. Proteomic data showed that CER at a concentration of 1 ng/mL led to a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst the first reports to describe the influence of sub-emetic concentrations of CER on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

Screening of Cytotoxic B. cereus on Differentiated Caco-2 Cells and in Co-Culture with Mucus-Secreting (HT29-MTX) Cells

“…This hypothesis may explain why, in this cytotoxicological study, cereulide-producing strains displayed a very low toxicity against fully differentiated (21 days) Caco-2 cells while previous studies described emetic strains as cytotoxic vs. Caco-2 cells and various other cell lines [51,52]. Another explanation of these striking results may be the exposure duration used to assess the cytotoxicity.…”

“…Another explanation of these striking results may be the exposure duration used to assess the cytotoxicity. Indeed, the only study that also used 21 day-aged cells applied a confrontation time with cereulide of 3 days [52] while to reflect more the physiological reality, we decided to let the cells only 3 h in contact with the toxin. However, this specific cytotoxic response of emetic B. cereus should be further investigated, given that Guinebretière et al (2010) have already described other emetic strains non (or weakly) cytotoxic on Caco-2 cells [53].…”

Development and validation of ultra-high-performance liquid chromatography–tandem mass spectrometry methods for the simultaneous determination of beauvericin, enniatins (A, A1, B, B1) and cereulide in maize, wheat, pasta and rice

“…Considering the risks to human and animal health, the determination of the occurrence of these medium-sized cyclic depsipeptides in food and feed is imperative. Their potential presence at low levels is of special relevance to food safety [19,20].…”

Evaluation of the presence of cereulide in food products collected from the Belgian market

“…It is generally assumed that food containing doses <10 5 -10 6 CFU/g of B. cereus and <8 μg cereulide/kg bodyweight is safe for consumption and will not cause acute intoxication (Rajkovic 2014). Another potential threat, however, lies in repeat sub-emetic exposure to the toxin (Rajkovic 2014;Rajkovic et al 2014;Vangoitsenhoven et al 2014). It has already been demonstrated that restaurants pose a potential source of these kinds of exposure, most likely caused by poor storage conditions or inappropriate handling of the food (Delbrassinne et al 2012;Thorsen et al 2009).…”