Abstract
Bacillus cereus is a spore-forming ubiquitous bacterium recognized as a food-spoilage hazard. Indeed, many isolates have been shown to be responsible for clinical infections and food-poisoning outbreaks (Ghelardi et al., 2002; Chan et al., 2003; Musa et al., 1998). Their incidence is probably much higher than is generally reported owing to the differences that exist between the reporting procedures of different countries and the symptomatic similarities with other pathogenic bacteria such as Staphylococcus aureus or Clostridium perfringens. Two types of food poisoning are caused by B. cereus, namely diarrhoeal and emetic types. The diarrhoeal syndrome seems to be induced by the simultaneous action of several factors, mainly the haemolytic (Hbl) and non-haemolytic (Nhe) toxins, and is characterized by abdominal pain and diarrhoea (Beecher et al., 1995). The emetic type, characterized by nausea and vomiting, involves a small cyclic dodecadepsipeptide [(D-o-leu-D-ala-L-o-val-L-Val)3] named cereulide (Agata et al., 1995). Due to its chemical structure, cereulide was thought to be synthesized non-ribosomally and, recently, partial identification and characterization of the non-ribosomal peptide synthetase gene have been carried out (Toh et al., 2004; Horwood et al., 2004; Ehling-Schulz et al., 2005). Cereulide acts as an ionophore through mitochondrial membranes and interferes with oxidative phosphorylation. Preferentially produced in starchy food, mainly boiled rice, cereulide induces the emetic syndrome between 1 and 5 h after consumption of the contaminated food. This suggests that the toxin is pre-formed in the food before bacterial sporulation. Cereulide cannot be inactivated by standard heat treatment or by digestion on account of its acid- and protease-resistant properties (Melling et al., 1976; Shinagawa et al., 1995).
Generally, food-borne illnesses caused by B. cereus are relatively mild and do not last more than 24 h. However, the emetic syndrome can have a fatal and fulminant outcome (Mahler et al., 1997). This was the case in Kinrooi (Belgium) in 2003, when a 7-year-old girl died after consuming pasta contaminated by B. cereus. Cereulide is thought to have been responsible for the child's death (Dierick et al., 2005).
To date, the location of the cereulide genetic determinants has remained unknown. A potential extrachromosomal location was thus investigated by plasmid profiling and curing experiments in strains collected from emetic isolates from food-poisoning outbreaks, including Kinrooi 5975c isolated from the Belgian lethal case in 2003. This study shows that the genomic location of genetic determinants involved in cereulide production is extrachromosomal and is found on a plasmid named pCERE01.
Plasmid content profiling of several strains, including Kinrooi 5975c, was performed according to the method developed by Jensen et al. (1995) (Fig. 1a). The initial plasmid isolation revealed important genomic similarities between the five emetic-positive strains (lanes 37). This observation confirmed the genomic similarity among cereulide-producing strains that we had already observed by PFGE (Dierick et al., 2005). Moreover, a large number of plasmids, with sizes ranging from around 2 to about 350 kb, was identified in all the strains tested. The five emetic strains (lanes 37) showed particularly high numbers of plasmid bands, reaching 10 for B. cereus strain Kinrooi 5975c. Interestingly, it was noted that one similar large plasmid was present in all five emetic isolates as well as in the non-emetic strain B. cereus 17532 (Fig. 1a).