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.

Cereulide is an emetic toxin produced by some strains of Bacillus cereus. This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC–MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of ¹³C₆-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg⁻¹ for food and faeces, respectively. The linearity of the method was very good, with calculated R² values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%–120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC–MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g⁻¹ for food and between 6.32 and 773.37 ng g⁻¹ for faecal samples.

Background: Botulinum toxins (BoNT) type A, B, E, F may induce a serious neuroparalytic illness in humans. Botulism can be transmitted via three routes: foodborne, wound and intestinal (in situ toxemia generally observed in infants and/or in immunocompromised adults) botulism. Case Report: A case of human botulism type B due to the consumption of an imported dried ham is reported. Laboratory results confirmed the diagnosis using the mouse bioassay and the qPCR method. Botulinum toxin was still detected in the serum up to 29 days after food consumption while it was not detected anymore in the faeces. Conclusion: The described case highlights i) prolonged presence of BoNT B in the serum and ii) the risks related to the preparation of artisanal products as well as the importance of an efficient communication to citizens regarding preservation methods of household products.

Although research on foodborne diseases is well documented for certain microorganisms, less research has been undertaken on cross-contamination with Enterobacteriaceae from food within the kitchen environment. In this study, we analysed the presence of Klebsiella spp. and Raoultella spp. and their antibiotic resistance in 100 used kitchen sponges. In a domestic environment, kitchen sponges are commonly used by consumers for doing the dishes and/or cleaning kitchen surfaces. Given their multipurpose use, their often high humidity and presence of organic residuals they are considered a favourable habitat for various groups of microorganisms and could serve as a vehicle in transmission of foodborne pathogens.^1,2 A total of 100 kitchen sponges alongside a questionnaire regarding hygienic parameters were randomly collected from domestic environments and analysed within 24 h after arrival in the lab. Kitchen sponges were immersed in 100 mL buffered peptone water, homogenized and incubated at 37°C for 24 h ± 2 h. Ten microlitres of this enrichment was plated on MacConkey agar (Bio-Rad) and incubated at 37°C for 24 h ± 2 h. Presumptive Klebsiella spp. and Raoultella spp. were isolated and confirmed by MALDI-TOF MS. Antibiotic resistance testing (ART) was performed on all Klebsiella spp. and Raoultella spp. isolates following the EUCAST guidelines. WGS was performed using the Illumina platform. A total of 65% of the kitchen sponges were positive for Klebsiella spp. or Raoultella spp. The species Klebsiella oxytoca was detected in 78.5% of positive samples, followed by Klebsiella pneumoniae (12.31%), Raoultella ornithinolytica (4.62%), Klebsiella variicola (1.54%), Klebsiella aerogenes (1.54%) and Raoultella planticola (1.54%). To the best of our knowledge this is the first study performed on detection of Klebsiella spp. in kitchen sponges from Belgium and has comparable results with previous studies.^1–3 We hypothesize the high positivity rate is caused by cross-contamination. ART revealed the presence of one carbapenem-resistant isolate, with resistance to ertapenem and meropenem. Reduced susceptibility to carbapenems was caused by the presence of bla_SHV-36 combined with porin deficiency. Klebsiella spp. can be part of the commensal flora of human intestines but are considered to be opportunistic pathogens. Klebsiella spp. might present antimicrobial resistance and carbapenem-resistant Enterobacteriaceae are taken up in the WHO global priority list of antibiotic-resistant bacteria. High prevalence of Klebsiella spp. and a carbapenem-resistant isolate in kitchen sponges highlight the occurrence of cross-contamination from food and the possible risks for foodborne disease associated with this kitchen tool. These findings advocate for good hygienic measures within a household setting.