Complete genome sequence of Bacillus cereus NC7401, which produces high levels of the emetic toxin cereulide.

We report the complete and annotated genome sequence of Bacillus cereus NC7401, a representative of the strain group that causes emetic-type food poisoning. The emetic toxin, cereulide, is produced by a nonribosomal protein synthesis (NRPS) system that is encoded by a gene cluster on a large resident plasmid, pNCcld.

Sensitivity of Hep G2 cells to Bacillus cereus emetic toxin.

We herein examined the sensitivity of Hep G2 human hepatoma cells to Bacillus cereus emetic toxin. Hep G2 cells were treated with the emetic toxin, and the cell shape was observed. The same experiments were performed for comparison purposes, using HEp-2 cells, which are currently used by most laboratories for a bioassay of the emetic toxin. Hep G2 cells showed clearer vacuolation in the cytosol within 2 hr and required a shorter incubation period than HEp-2 cells (10 hr). The number of vacuoles in the Hep G2 cells was greater, and the size of the vacuoles was larger than those observed in HEp-2 cells. The minimal concentration of the emetic toxin required to induce the vacuolation of Hep G2 cells was 0.04 ng/ml. The concentration for the HEp-2 cells was 1 ng/ml. These findings indicate that Hep G2 cells show higher sensitivity to the emetic toxin. Hep G2 cells may be superior to the currently used HEp-2 cells for the bioassay of the emetic toxin.

Rapid detoxification of cereulide in Bacillus cereus food poisoning.

Bacillus cereus is recognized as a major pathogenic bacterium that causes food poisoning and produces gastrointestinal diseases of 2 types: emetic and diarrheal. The emetic type, which is often linked to pasta and rice, arises from a preformed toxin, cereulide, in food. Rapid and accurate diagnostic methods for this emetic toxin are important but are limited. Here we describe 3 patients with B cereus food poisoning in which cereulide was detected and measured sequentially. Three family members began to vomit frequently 30 minutes after consuming reheated fried rice. After 6 hours, a 1-year-old brother died of acute encephalopathy. A 2-year-old sister who presented with unconsciousness recovered rapidly after plasma exchange and subsequent hemodialysis. Their mother recovered soon by fluid therapy. From leftover fried rice and the children's stomach contents, B cereus was isolated. Serum cereulide was detected in both children; it decreased to an undetected level in the sister. These cases highlight the importance of measuring the value of cereulide, which would reflect the severity of B cereus emetic food poisoning. The cases also suggest the possible role of blood-purification therapy in severe cases.

A new phylogenetic cluster of cereulide-producing Bacillus cereus strains.

Phenotypic and molecular studies have established that cereulide-producing strains of Bacillus cereus are a distinct and probably recently emerged clone within the Bacillus population. We analyzed a set of B. cereus strains, both cereulide producers and nonproducers, by multilocus sequence typing. Consistent with earlier reports, nonproducers demonstrated high heterogeneity. Most cereulide-producing strains and all flagellar antigen type H1 strains were allocated to the known sequence type of exclusively emetic B. cereus strains. Several cereulide-producing strains, however, were recovered at a new phylogenetic location, all of which were serotype H3 or H12. We hypothesize that the group of cereulide producers is diversifying progressively, probably by lateral transfer of the corresponding gene complex.

Phylogenetic analysis of Bacillus cereus isolates from severe systemic infections using multilocus sequence typing scheme.

Bacillus cereus strains from cases of severe or lethal systemic infections, including respiratory symptoms cases, were analyzed using multilocus sequence typing scheme of B. cereus MLST database. The isolates were evenly distributed between the two main clades, and 60% of them had allele profiles new to the database. Half of the collection's strains clustered in a lineage neighboring Bacillus anthracis phylogenetic origin. Strains from lethal cases with respiratory symptoms were allocated in both main clades. This is the first report of strains causing respiratory symptoms to be identified as genetically distant from B. anthracis. The phylogenetic location of the presented here strains was compared with all previously submitted to the database isolates from systemic infections, and were found to appear in the same clusters where clinical isolates from other studies had been assigned. It seems that the pathogenic strains are forming clusters on the phylogenetic tree.

Quantitative analysis of cereulide, an emetic toxin of Bacillus cereus, by using rat liver mitochondria.

An emetic toxin cereulide, produced by Bacillus cereus, causes emetic food poisonings, but a method for quantitative measurement of cereulide has not been well established. A current detection method is a bioassay method using the HEp-2 cell vacuolation test, but it was unable to measure an accurate concentration. We established a quantitative assay for cereulide based on its mitochondrial respiratory uncoupling activity. The oxygen consumption in a reaction medium containing rat liver mitochondria was rapid in the presence of cereulide. Thus uncoupling effect of cereulide on mitochondrial respiration was similar to those of uncouplers 2,4-dinitrophenol (DNP), carbonylcyanide m-chlorophenylhydrazone (CCCP), and valinomycin. This method gave constant results over a wide range of cereulide concentrations, ranging from 0.05 to 100 microg/ml. The minimum cereulide concentration to detect uncoupled oxygen consumption was 50 ng/ml and increased dose-dependently to the maximum level. Semi-log relationship between the oxygen consumption rate and the cereulide concentration enables this method to quantify cereulide. The results of this method were highly reproducible as compared with the HEp-2 cell vacuolation test and were in good agreement with those of the HEp-2 cell vacuolation test. The enterotoxin of B. cereus or Staphylococcus aureus did not show any effect on the oxygen consumption, indicating this method is specific for the identification of cereulide as a causative agent of emetic food poisonings.

gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group.

Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.

Complexation of cyclic dodecadepsipeptide, cereulide with ammonium salts.

Cereulide is a principal toxin causing emetic syndrome which is produced by Bacillus cereus and has been known as potassium selective ionophore. This paper deals with its complexation with inorganic and organic ammonium ions to assign the higher structures similar to the complex with potassium ion by means of NMR and ESI-MS spectroscopy. Of particular interest, the detectable ions are not only at m/z 1191.8 as K(+) complex but also (or sometimes exclusively) at m/z 1170.8 as NH(4)(+) complex in its LC-MS analyses depending upon the conditions. This difference is due to the sample preparation and measurement condition.