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

B. cereus is an opportunistic foodborne pathogen able to cause diarrhoea. However, the diarrhoeal potential of a B. cereus strain remains difficult to predict, because no simple correlation has yet been identified between the symptoms and a unique or a specific combination of virulence factors. In this study, 70 B. cereus strains with different origins (food poisonings, foods and environment) have been selected to assess their enterotoxicity. The B. cereus cell-free supernatants have been tested for their toxicity in vitro, on differentiated (21 day-old) Caco-2 cells, using their ATP content, LDH release and NR accumulation. The genetic determinants of the main potential enterotoxins and virulence factors (ces, cytK, entFM, entS, hbl, nhe, nprA, piplC and sph) have also been screened by PCR. This analysis showed that none of these genes was able to fully explain the enterotoxicity of B. cereus strains. Additionally, in order to assess a possible effect of the mucus layer in vitro, a cytotoxicity comparison between a monoculture (Caco-2 cells) and a co-culture (Caco-2 and HT29-MTX mucus-secreting cells) model has been performed with selected B. cereus supernatants. It appeared that, in these conditions, the mucus layer had no notable influence on the cytotoxicity of B. cereus supernatants.

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.

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.