Laurence Delbrassinne

Sudden death of a young adult associated with Bacillus cereus food poisoning.

ABSTRACT 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 μg/g) were found in the spaghetti meal.

Follow-up of the Bacillus cereus emetic toxin production in penne pasta under household conditions using liquid chromatography coupled with mass spectrometry

Two outbreak-related Bacillus cereus emetic strains were investigated for their growth and cereulide production potential in penne pasta at 4, 8 and 25 °C during 7-day storage. Cereulide production was detected and quantified by LC-MS method (LOD of 1 ng/ml, LOQ of 5 ng/ml) and growth was determined by culture-based enumeration. Inoculated B. cereus strains (10(5) CFU/g) were able to reach counts of more than 10(8) CFU/g and cereulide production of about 500 ng/g already after 3 days of storage at 25 °C. Interestingly, a constant increase of the toxin was noticed during incubation at ambient temperature storage: the cereulide was continuously produced during the bacterial stationary growth phase reaching maximal amounts at the end of the experiment (7 days, concentration of about 1000 ng/g). Strictly respected cold chain temperature as 4 °C did not allow any detectable cereulide production for any of the two tested strains. At the limited temperature abuse of 8 °C, a detectable amount of cereulide was observed after two days for one of the strain (TIAC303) (<LOQ). These results confirm that cereulide production is controlled by multiple factors (from type of strain to temperature) and that prolonged storage time plays a crucial role for consumer safety.

Prevalence and levels of Bacillus cereus emetic toxin in rice dishes randomly collected from restaurants and comparison with the levels measured in a recent foodborne outbreak.

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.

Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test

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.

Antifungal activity displayed by cereulide, the emetic toxin produced by Bacillus cereus.

ABSTRACT In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.

Diversity of pulsed‐field gel electrophoresis patterns of cereulide‐producing isolates of Bacillus cereus and Bacillus weihenstephanensis

Bacillus cereus is an important foodborne pathogen causing diarrhoea, emesis and in, rare cases, lethal poisonings. The emetic syndrome is caused by cereulide, a heat-stable toxin. Originally considered as a rather homogenous group, the emetic strains have since been shown to display some diversity, including the existence of two clusters of mesophilic B. cereus and psychrotolerant B. weihenstephanensis. Using pulsed-field gel electrophoresis (PFGE) analysis, this research aimed to better understand the diversity and spatio-temporal occurrence of emetic strains originating from environmental or food niches vs. those isolated from foodborne cases. The diversity was evaluated using a set of 52 B. cereus and B. weihenstephanensis strains isolated between 2000 and 2011 in ten countries. PFGE analysis could discriminate 17 distinct profiles (pulsotypes). The most striking observations were as follows: (1) more than one emetic pulsotype can be observed in a single outbreak; (2) the number of distinct isolates involved in emetic intoxications is limited, and these potentially clonal strains frequently occurred in successive and independent food poisoning cases; (3) isolates from different countries displayed identical profiles; and (4) the cereulide-producing psychrotolerant B. weihenstephanensis were, so far, only isolated from environmental niches.

Is Cytotoxin K from Bacillus cereus a bona fide enterotoxin

Cytotoxin K (CytK) produced by Bacillus cereus s.l. has generally been considered to be associated with the foodborne diarrhoeal syndrome. Two distinct variants of CytK have been reported: CytK-1 from Bacillus cytotoxicus and CytK-2 from B. cereus. In order to determine whether CytK plays a significant role in the diarrhoeal disease, the occurrence of cytK genes was assessed among 390 B. cereus isolates with different origins including clinical and food poisoning samples and was found to be 46%. Interestingly, the cytK occurrence was slightly lower in food poisoning and clinical isolates than in environmental samples. Seventy cytK-2 positive strains (including 28 isolates from foodborne outbreaks) were then selected in order to assess their genetic diversity. A genetic dendrogram based on the cytK-2 sequences of these 70 strains and on two cytK-1 sequences from strains NVH 391-98 and 883-00 showed an important diversity. However, no strain clustering according to the origin or source of isolation was observed. These observations were confirmed by Multi-Locus Sequences Typing (MLST) based on five different loci of housekeeping genes (ccpA, recF, sucC, purF and gdpD) for which no grouping of foodborne outbreak strains could be identified. Therefore, the choice of cytK as virulence factor for the diarrhoeal pathotype does not seem to be relevant per se, even though the involvement of CytK in the diarrhoeal syndrome cannot be fully excluded. Potential synergistic effects between CytK and other virulence factors, together with their potential variable expression levels should be further investigated.

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

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.

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

ABSTRACT Group II nonproteolytic Clostridium botulinum (gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies with C. botulinum are cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain, bont/E was exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use of pyrE as a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia. IMPORTANCE The nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophic Clostridium botulinum. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.