Norio Agata

Production of Bacillus cereus emetic toxin (cereulide) in various foods

To determine the role of Bacillus cereus as a potential pathogen in food poisoning, the production of an emetic toxin (cereulide) by B. cereus was quantified in various food sources. The amount of emetic toxin in 13 of 14 food samples implicated in vomiting-type food poisoning cases ranged from 0.01 to 1.28 microg/g. A vomiting-type strain, B. cereus NC7401, was inoculated into various foods and incubated for 24 h at 20, 30, and 35 degrees C. In boiled rice, B. cereus rapidly increased to 10(7)-10(8) cfu/g and produced emetic toxin at both 30 and 35 degrees C. In farinaceous foods, the production of emetic toxin was as high as that in the food samples implicated in food poisoning. Low levels of emetic toxin were detectable in egg and meat and their products and a small quantity of toxin was detectable in liquid foods such as milk and soymilk when not aerated. Bacterial growth and toxin production was inhibited in foods cooked with vinegar, mayonnaise, and catsup, supposedly by the decreased pH of acetic acid. This is the first report that has quantified emetic toxin of B. cereus in various foods.

The bceT gene of Bacillus cereus encodes an enterotoxic protein.

A toxin gene (bceT) on a 2.9 kb DNA fragment of Bacillus cereus B-4ac was cloned and expressed in Escherichia coli, and its nucleotide sequence determined. The DNA fragment contained an open reading frame capable of encoding a polypeptide of 336 amino acids with a molecular mass of 41039 Da. The translated product in E. coli exhibited Vero cell cytotoxicity, and was positive in a vascular permeability assay. It also caused fluid accumulation in a ligated mouse ileal loop and was lethal to mice upon injection. These biological activities are considered characteristic of diarrhoeal enterotoxins. We therefore conclude that this gene, designated bceT, encodes one of the enterotoxic proteins of B. cereus which cause food-borne diarrhoea.

Production of an emetic toxin, cereulide, is associated with a specific class of Bacillus cereus.

Abstract. The emetic toxin (cereulide) of Bacillus cereus was quantified in several isolates of B. cereus and in various food sources. When the emetic toxin was produced, vomiting-type food poisoning was observed in humans. We also found that the H-1 serovar phenotype was strongly associated with the production of cereulide and that none of the isolates that hydrolyzed starch or expressed diarrheal enterotoxin activity produced cereulide.

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 childrens 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.

Synthesis and activity of cereulide, a cyclic dodecadepsipeptide ionophore as emetic toxin from Bacillus cereus

Abstract Cereulide is the emetic toxin caused by Bacillus cereus as food contaminant. This cyclic depsipeptide was synthesized from L-O-Val, L-Val, L-O-Leu and D-Ala by coupling the former 2 and the latter 2, with inversion of the configuration of L-O-Leu into D-O-Leu, to form a tetrapeptide having TBDMS and benzyl protection. After selective deprotection of this tetrapeptide into the corresponding alcohol and acid, they were coupled twice and then transformed into a dodecapeptide (seco-acid) that was finally cyclized under high-dilution condition to afford cereulide. The final preparation showed, in fact, emetic toxicity as well as vacuole formation to HEp2 cells as the same level as the natural cereulide.

Identification of Species and Capsular Types of Klebsiella Clinical Isolates, with Special Reference to Klebsiella planticola

In the 77 reference strains for Klebsiella K types, there are 17 strains (22.1%) of Klebsiella planticola, 6 strains (7.8%) of Klebsiella oxytoca, 1 strain (1.3%) of Klebsiella terrigena, and 53 strains (68.8%) of Klebsiella pneumoniae. The species K. planticola, which was originally isolated from botanical and aquatic environments and hence thus named, was also identified at high incidence (81 strains, 18.5%) among the 439 recent clinical isolates of Klebsiella species. Among these K. planticola strains of hospital origin, 52 (64%) were isolated from sputum, 17 (21%) from urine, and the remaining 12 (15%) from other sources. The capsular types of these isolates were determined by the gel precipitation reaction. Seventy of 81 K. planticola isolates (86.4%) were typable by antisera to Klebsiella reference strains for K types and the K types of the clinical isolates distributed to 35 kinds of K types. The proportion of typable strains among clinical isolates of K. planticola was very similar to those in K. pneumoniae (87.5%) and K. oxytoca (86.0%).

A New Phylogenetic Cluster of Cereulide-Producing Bacillus cereus Strains

ABSTRACT 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.

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 μg/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 concentraiton 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.

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 collections 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.

Growth conditions of and emetic toxin production by Bacillus cereus in a defined medium with amino acids.

The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B. cereus. The addition of high levels (50 mM) of leucine, isoleucine and glutamic acid decreased the toxin production. Other amino acids had no effect at this concentration.