Cereulide synthetase gene cluster from emetic Bacillus cereus: structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1.

BACKGROUND: Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown. RESULTS: The complete sequence of the cereulide synthetase (ces) gene cluster, which encodes the enzymatic machinery required for the biosynthesis of cereulide, was dissected. The 24 kb ces gene cluster comprises 7 CDSs and includes, besides the typical NRPS genes like a phosphopantetheinyl transferase and two CDSs encoding enzyme modules for the activation and incorporation of monomers in the growing peptide chain, a CDS encoding a putative hydrolase in the upstream region and an ABC transporter in the downstream part. The enzyme modules responsible for incorporation of the hydroxyl acids showed an unusual structure while the modules responsible for the activation of the amino acids Ala and Val showed the typical domain organization of NRPS. The ces gene locus is flanked by genetic regions with high homology to virulence plasmids of B. cereus, Bacillus thuringiensis and Bacillus anthracis. PFGE and Southern hybridization showed that the ces genes are restricted to emetic B. cereus and indeed located on a 208 kb megaplasmid, which has high similarities to pXO1-like plasmids. CONCLUSION: The ces gene cluster that is located on a pXO1-like virulence plasmid represents, beside the insecticidal and the anthrax toxins, a third type of B. cereus group toxins encoded on megaplasmids. The ces genes are restricted to emetic toxin producers, but pXO1-like plasmids are also present in emetic-like strains. These data might indicate the presence of an ancient plasmid in B. cereus which has acquired different virulence genes over time. Due to the unusual structure of the hydroxyl acid incorporating enzyme modules of Ces, substantial biochemical efforts will be required to dissect the complete biochemical pathway of cereulide synthesis.

Hemolytic-uremic syndrome associated with enterohemorrhagic Escherichia coli O26:H infection and consumption of unpasteurized cow's milk.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26 has emerged as a significant cause of hemolytic-uremic syndrome (HUS). The source and the vehicle of contamination with EHEC O26 are not often identified. We report two Austrian cases of HUS due to E. coli O26:H- affecting an 11-month-old boy and a 28-month-old girl in which transmission through unpasteurized cow's milk was positively identified. METHODS AND RESULTS: Using automated ribotyping and pulsed-field gel electrophoresis (PFGE), the isolates (which yielded the virulence genes stx2, eae, and hly) were indistinguishable from each other. An epidemiologic investigation revealed that the children had stayed in the same hotel. Both patients had consumed unpasteurized cow's milk from the breakfast buffet. Fecal samples were taken from the cows of the farm producing the incriminating milk, and one of three cattle EHEC O26:H- isolates had a PFGE pattern indistinguishable from that of the patients' strains. CONCLUSIONS: These two cases of E. coli O26 infection illustrate the hazards associated with the consumption of raw milk, and underline the importance of microbiological diagnostic approaches able to detect sorbitol-fermenting, non-O157 EHEC.