A lethal intoxication case, which occurred in Brussels, Belgium, is described. A 20-year-old man died following the ingestion of pasta contaminated with Bacillus cereus. Emetic strains of B. cereus were isolated, and high levels of cereulide (14.8 g/g) were found in the spaghetti meal. CASE REPORTOn 1 October 2008, a 20-year-old man became sick after eating a meal of leftovers of spaghetti with tomato sauce, which had been prepared 5 days before and left in the kitchen at room temperature. After school, he warmed the spaghetti in the microwave oven. Immediately after eating, he left home for his sports activities, but he returned 30 min later because of headache, abdominal pain, and nausea. At his arrival, he vomited profusely for several hours and at midnight had two episodes of watery diarrhea. He did not receive any medication and drank only water. After midnight, he fell asleep. The next morning at 11:00 AM, his parents were worried because he did not get up. When they went to his room, they found him dead.Legal examination determined the time of death, presumably at 4:00 AM, approximately 10 h after ingestion of the suspected meal. An autopsy could not be performed until 5 days later. Macroscopically, brownish and moderately softened liver and ascites (550 ml of citrine liquid) were found. The heart was macroscopically normal. A total lysis of the pancreas was also found, but it could not be excluded that this finding was due to the autopsy delay.Microscopic findings were as follows: moderate centrolobular liver necrosis without inflammatory signs and discrete biliary stasis, significant vascular congestion of the lungs, probably due to acute cardiac insufficiency, significant necrosis from all layers of colon mucosa and submucosa alternating with betterpreserved zones, and mixed intestinal flora but no evidence of invasive bacterial lesions. Significant lysis of the adrenal glands was also reported. The exact cause of death could not be determined by the autopsy because the interpretation of findings was very difficult due to the autopsy delay.Five fecal swabs and two feces samples were taken postmortem, and samples were tested for the presence of Bacillus cereus by growth on mannitol egg yolk polymyxin (MYP) agar. B. cereus was found in only two of the five fecal swabs, and the strains isolated were named ISP321 and ISP322. No B. cereus was cultured from the feces samples. Pasta and tomato sauce samples, the leftovers of the dinner, were also sent for analysis to the National Reference Laboratory for Food-borne Outbreaks (NRLFBO). For enumeration of B. cereus in food samples, the ISO 7932 method (16) was used. Significant B. cereus counts (9.5 ϫ 10 7 CFU/g) were found in the pasta, while B. cereus was absent in the tomato sauce. The strain isolated from the pasta meal was named ISP303.PCR assays that detect the presence of toxin genes were applied to DNA from the pasta isolate (ISP303) and the two human isolates (ISP321 and ISP322), and the results are presented in Table 1. The presence of genes encoding ...

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented.

The detection and identification of botulinum neurotoxins (BoNT) is complex due to the existence of seven serotypes, derived mosaic toxins and more than 40 subtypes. Expert laboratories currently use different technical approaches to detect, identify and quantify BoNT, but due to the lack of (certified) reference materials, analytical results can hardly be compared. In this study, the six BoNT/A1–F1 prototypes were successfully produced by recombinant techniques, facilitating handling, as well as improving purity, yield, reproducibility and biosafety. All six BoNTs were quantitatively nicked into active di-chain toxins linked by a disulfide bridge. The materials were thoroughly characterized with respect to purity, identity, protein concentration, catalytic and biological activities. For BoNT/A1, B1 and E1, serotypes pathogenic to humans, the catalytic activity and the precise protein concentration were determined by Endopep-mass spectrometry and validated amino acid analysis, respectively. In addition, BoNT/A1, B1, E1 and F1 were successfully detected by immunological assays, unambiguously identified by mass spectrometric-based methods, and their specific activities were assigned by the mouse LD50 bioassay. The potencies of all six BoNT/A1–F1 were quantified by the ex vivo mouse phrenic nerve hemidiaphragm assay, allowing a direct comparison. In conclusion, highly pure recombinant BoNT reference materials were produced, thoroughly characterized and employed as spiking material in a worldwide BoNT proficiency test organized by the EQuATox consortium.

Whereas the prevalence of Bacillus cereus emetic strains in the environment has been shown to be very low, there is a lack of information on the prevalence of its toxin, cereulide, in food. Yet, the rice leftovers of a family outbreak which occurred after the consumption of dishes taken away from an Asian restaurant revealed significant amounts of cereulide, reaching up to 13,200 ng/g of food. The occurrence of cereulide in rice dishes collected from various restaurants was therefore evaluated using the liquid chromatography coupled with tandem mass spectrometry method, which allows for the direct quantification of the toxin in food. The cereulide prevalence was found to be 7.4% when samples were analyzed at the day of sampling, but reached 12.9% when exposed to temperature abuse conditions (25°C). The cereulide concentrations observed in cooked rice dishes were low (approximately 4 ng/g of food). However, since little is known yet about the potential chronic toxicity of cereulide, one needs to be very careful and vigilant.