Insights into the ecology and genetic diversity of cereulide-producing bacillus cereus strains

The so-called 'Bacillus cereus sensu lato group' of Gram-positive spore-forming bacteria comprises six highly related species. Among them stand the opportunist B. cereus sensu stricto, Bacillus anthracis which is the causative agent of anthrax, and the entomopathogen Bacillus thuringiensis, largely used on crops as bioinsecticide. Classically, these species can be distinguished based on phenotypical traits, mostly associated with large plasmids responsible for the distinct B. cereus s.l. lifestyles. B. cereus s.s. is responsible for two distinct types of food poisoning, namely the diarrhoeal and the emetic types. The emetic syndrome is caused by a cyclic dodecadepsipeptide, called cereulide, produced by certain strains of B. cereus and often associated with particularly severe intoxications, sometimes leading to fatalities as it was the case in Kinrooi (Belgium) in 2003. Cereulide is synthesized by a multienzymatic Non-Ribosomal Peptide Synthetase (NRPS) complex whose genetic determinants are organized in an operon. Cereulide genetic determinants were shown to be located on a large plasmid, named pCERE01 (270 kb in size), that turned out later to be related to the B. anthracis virulence plasmid, pXO1. Transfer and mobilization experiments showed that the cereulide-producing strain B. cereus 5975c was able to act as a conjugation partner in biparental matings with B. thuringiensis strains containing the conjugative plasmids pXO16 or pAW63 and achieved 102-fold higher transfer frequencies in milk than in standard laboratory LB medium. A large distribution study on more than 2,000 B. cereus s.l. strains confirmed that cereulide-producing strains are rare in environmental microcosms, representing only 1.5 % of the strains tested. It also emphasized the absence of putative emetic strains among more than 450 well-identified B. thuringiensis, B. mycoides and B. pseudomycoides. Moreover, two distinct clusters were identified among cereulide-producing strains, with isolates belonging to the second cluster identified as psychrotolerant able to grow at 8°C. Strikingly, in this cluster, the location of the cereulide genetic determinants varied depending on the strain. Indeed, they were located either on plasmid(s) unrelated to the pXO1-replicon, or on the chromosome, indicating a probable genomic mobility.Doctorat en sciences agronomiques et ingénierie biologique (AGRO 3)--UCL, 200