Two thousand Bacillus cereus sensu lato isolates from food and environmental matrices were screened by PCR for the presence of cereulide-producing strains. This survey identified 73 potential emetic strains, most of which originated from non-random food and clinical samplings. None of the 460 Bacillus thuringiensis, Bacillus mycoides and Bacillus pseudomycoides strains were PCR-positive for the cereulide genetic determinants. The chromosomal and extrachromosomal gene pool diversity of a subset of 30 cereulide-producing strains was then assessed using multilocus sequence typing, large plasmid gel electrophoresis and Southern blot hybridization. The strain toxicity on boar sperm and cereulide production were also analysed. The most striking observation was the identification of two distinct clusters of cereulide-producing strains, with members of the second group (cluster II) identified as psychrotolerant B. weihenstephanensis able to grow at 8°C. Moreover, the location of the cereulide genetic determinants was shown to vary depending on the strain, indicating a probable genomic mobility.

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.

Cereulide is the heat-stable toxin produced by certain strains of Bacillus cereus. It is the main virulence factor of emetic B. cereus strains, which causes the emetic food poisoning syndrome, including rare fatal cases of food intoxications. Due to presumably low intoxication doses, a sensitive, specific, and robust technique is needed for its detection. In 2002, a LC-MS method was developed which allowed absolute quantification of cereulide using valinomycin as standard. This study describes the validation, according to the Commission Decision 2002/657/EC, of the LC-MS² method, a tandem mass spectrometry technique, which guarantees lower detection limit and higher specificity. The LC-MS² method, calibrated with valinomycin, was validated in rice and tested on various matrices (i.e., red beans, spices, and chili con carne) containing cereulide. The process combines a simple extraction step from the food matrix followed by LC-MS² analysis and detection by ion trap mass spectrometer. The detection limit for cereulide in rice was 0.5 ng eq/g, which is 20 to 2,500 times lower than currently understood intoxicative doses between 10 and 1.280 ng/g previously reported for cereulide. The validated method was specific, sensitive, repeatable, and reproducible with recoveries ranging from 77% to 101%.

No abstract

Lactoperoxidase system (LPOS) was incorporated into chitosan solutions (0.5, 1, and 1.5%) to protect mangoes against two strains of fungi. Coating solutions effectiveness in vitro (in Petri dish) and in vivo (on mango) were studied on fungal (Phomopsis sp. RP257 and Pestalotiopsis sp.) growth isolated from mango cv Amelie. In vitro, chitosan concentration at least 1% containing or not LPOS effectively inhibited Pestalotiopsis sp. growth at 100%. Presence of LPOS or Lactoperoxydase system with iodine (LPOSI) in chitosan at 0.5% increased the percentage of inhibition from 26 to 93%. Edible films with LPOS inhibited Phomopsis sp. RP257 particularly when LPOS was incorporated in chitosan concentrations of 1 and 1.5%. Iodine did not influenced antifungal activity of LPOS against Pestalotiopsis sp. but decreased activity antifungal toward Phomopsis sp. RP257. The properties (water vapor permeability and mechanical properties) of chitosan films were not significantly changed by the incorporation of the enzyme system. in vivo condition, chitosan coating at 1 and 1.5% with or without enzyme system was sufficient to inhibit totally (100%) Pestalotiopsis sp. and was 60% efficient against Phomopsis sp. with chitosan only at 1 and 1.5%. However, when coating solution mainly at 1 and 1.5% was enhanced by LPOS with or without iodine, it inhibited totally (100%) Phomopsis sp. RP257. The presence of iodine slightly reduced antifungal activity against Phomopsis sp. RP257.