Toxigenic Strains of Bacillus licheniformis Related to Food Poisoning

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the B. licheniformis isolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol⁻¹. The toxic B. licheniformis isolates inhibited growth of Corynebacterium renale DSM 20688^T, but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55°C but not at 10°C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13^T, by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and among six when cut with EcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with PvuII, most toxin-producing isolates shared bands at 2.8 ± 0.2, 4.9 ± 0.3, and 11.7 ± 0.5 or 13.1 ± 0.8 kb.     

Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.     

Toxic Bacillus pumilus from indoor air, recycled paper pulp, Norway spruce, food poisoning outbreaks and clinical samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.     

Bacillus subtilis and B. mojavensis strains connected to food poisoning produce the heat stable toxin amylosin

Aim:  To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group.
Methods and Results:  Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21–37°C for B. subtilis and 11–21°C for B. mojavensis . Both species produced amylosin in air as well as in 7–8% CO₂ with 8–9% O_2.
Conclusions:  Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin.
Significance and Impact of the Study:  This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus .     

Production of an Emetic Toxin,Cereulide, Is Associated with a Specific Class of Bacillus cereus

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. Received: 4 December 1995 / Accepted: 30 January 1996     

Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis.

Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated. Toxins were purified from methanol extracts of the B. licheniformis biomass using boar sperm cells as the toxicity indicator. The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS. The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues L-Gln, L-Leu, D-Leu, L-Val, L-Asp, D-Leu and L-Ile in that order. Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13-15 carbons in length. We showed that the toxins from food and food poisoning isolates of B. licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa. Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity.     

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.     

Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants

Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar ribotypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.     

Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.     

Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 °C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1–10 μg ml^–1 (EC₅₀) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.     

Cereulide-producing strains of <Emphasis Type="Italic">Bacillus cereus</Emphasis> show diversity

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600–1,800 ng cereulide mg⁻¹ wet weight biomass), seven average producers (180–600 ng cereulide mg⁻¹ wet weight biomass), four low cereulide producers (20–160 ng cereulide mg⁻¹ wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.     

Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay

Bacillus cereus causes two types of gastrointestinal diseases: emesis and diarrhea. The emetic type of the disease is attributed to the heat-stable depsipeptide cereulide and symptoms resemble Staphylococcus aureus intoxication, but there is no rapid method available to detect B. cereus strains causing this type of disease. In this study, a polymerase chain reaction (PCR) fragment of unknown function was identified, which was shown to be specific for emetic toxin producing strains of B. cereus. The sequence of this amplicon was determined and a PCR assay was developed on this basis. One hundred B. cereus isolates obtained from different food poisoning outbreaks and diverse food sources from various geographical locations and 29 strains from other species belonging to the B. cereus group were tested by this assay. In addition, 49 non-B. cereus group strains, with special emphasis on food pathogens, were used to show that the assay is specific for emetic toxin producing B. cereus strains. The presented PCR assay is the first molecular tool for the rapid detection of emetic toxin producing B. cereus strains.     

Identification of aerobic mesophilic bacilli isolated from board and paper products containing recycled fibres

AIMS: To identify aerobic mesophilic bacteria isolated from coreboard, kitchen roll paper and food packaging boards containing recycled fibres and to create a rapid fingerprint-based database for their identification. METHODS AND RESULTS: A total of 197 isolates and 20 relevant type strains were characterized by automated ribotyping and as far as possible identified by the similarities of their riboprints to the relevant type strains. One strain from each unidentified ribotype, a total of 87 strains, was subjected to partial 16S rDNA sequencing and in most cases also to fatty acid analysis and physiological tests. From the isolates 113 and seven different ribotypes were generated belonging to the genera Bacillus and Paenibacillus, respectively. The dominating species, or closest related to them, were B. simplex (22.8% of isolates), B. licheniformis (18.3%) and B. amyloliquefaciens (12.7%); 5.1% of the isolates were identified as B. cereus, a potential food-borne pathogen. In particular, this species was present in one food packaging board (26.3% of isolates). Based on these results, 40.1% of the isolates and 45.0% of ribotypes were so different from the relevant type strains that they may represent novel species. CONCLUSIONS: All isolates were aerobic spore-formers, indicating that all non-spore-formers were eliminated during the drying stage of the processes. Although many isolates could be affiliated to described species of Bacillus or Paenibacillus, a significant proportion of the isolates could not be identified unambiguously as members of a described species. SIGNIFICANCE AND IMPACT OF THE STUDY: A RiboPrint identification database, composed of 120 composite patters, was established for bacteria originating from the pulp and paper industry. Considering the discrimination power of ribotyping, this database will be extremely useful in future for the reliable and rapid identification of bacteria isolated from pulp and paper industrial sources.     

Detection and characterization of naturally occurring plasmids in Bacillus licheniformis

Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.     

Psychrotolerant Paenibacillus tundrae isolates from barley grains produce new cereulide-like depsipeptides (paenilide and homopaenilide) that are highly toxic to mammalian cells

Paenilide is a novel, heat-stable peptide toxin from Paenibacillus tundrae, which colonizes barley. P. tundrae produced 20 to 50 ng of the toxin mg(-1) of cells (wet weight) throughout a range of growth temperatures from +5°C to +28°C. Paenilide consisted of two substances of 1,152 Da and 1,166 Da, with masses and tandem mass spectra identical to those of cereulide and a cereulide homolog, respectively, produced by Bacillus cereus NS-58. The two components of paenilide were separated from those of cereulide by high-performance liquid chromatography (HPLC), showing a structural difference suggesting the replacement of O-Leu (cereulide) by O-Ile (paenilide). The exposure of porcine spermatozoa and kidney tubular epithelial (PK-15) cells to subnanomolar concentrations of paenilide resulted in inhibited motility, the depolarization of mitochondria, excessive glucose consumption, and metabolic acidosis. Paenilide was similar to cereulide in eight different toxicity endpoints with porcine and murine cells. In isolated rat liver mitochondria, nanomolar concentrations of paenilide collapsed respiratory control, zeroed the mitochondrial membrane potential, and induced swelling. The toxic effect of paenilide depended on its high lipophilicity and activity as a high-affinity potassium ion carrier. Similar to cereulide, paenilide formed lipocations, i.e., lipophilic cationic compounds, with K(+) ions already at 4 mM [K(+)], rendering lipid membranes electroconductive. Paenilide-producing P. tundrae was negative in a PCR assay with primers specific for the cesB gene, indicating that paenilide was not a product of plasmid pCER270, encoding the biosynthesis of cereulide in B. cereus. Paenilide represents the first potassium ionophoric compound described for Paenibacillus. The findings in this paper indicate that paenilide from P. tundrae is a potential food-poisoning agent.     

Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42 degrees C, and no reduction in activity was observed even after 24 h of growth at 42 degrees C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22 degrees C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.     

Description of heterotrophic bacteria occurring in paper mills and paper products

AIMS: To isolate aerobic mesophilic bacilli and thermophilic bacteria from different paper mill samples and to evaluate their potential harmfulness. METHODS AND RESULTS: A total of 109 mesophilic and 68 thermophilic isolates were purified and characterized by automated ribotyping and partial 16S rDNA sequencing. The mesophilic isolates belonged to the genera Bacillus (13 taxa), Brevibacillus (three taxa) and Paenibacillus (five taxa). The thermophilic bacteria represented seven taxa of Bacillus, Geobacillus or Paenibacillus, four of proteobacteria and one of actinobacteria. The most frequently occurring bacteria were Bacillus cereus, B. licheniformis, Pseudoxanthomonas taiwanensis and bacteria closely related to Paenibacillus stellifer, P. turicensis or Leptothrix sp. One mill was contaminated throughout with bacteria of a novel mesophilic genus most closely related to Brevibacillus centrosporus and another with bacteria of a novel thermophilic genus most closely related to Hydrogenophilus thermoluteolus. One B. cereus isolate producing haemolytic diarrhoeal enterotoxin was detected and all the tested B. licheniformis isolates produced a metabolite toxic to boar sperm cells. CONCLUSIONS: The bacilli and thermophilic bacteria isolated represent species which should not present occupational hazards in paper mill environments. The most harmful bacterium detected was B. licheniformis and potentially also B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the microbial diversity in a paper mill provides a rational basis for development of an effective controlling programme. A database constructed from the fingerprints generated using automated ribotyping helps to identify and trace the contamination routes of bacteria occurring in paper mills.     

Production of diarrheal enterotoxins and other potential virulence factors by veterinary isolates of bacillus species associated with nongastrointestinal infections

With the exceptions of Bacillus cereus and Bacillus anthracis, Bacillus species are generally perceived to be inconsequential. However, the relevance of other Bacillus species as food poisoning organisms and etiological agents in nongastrointestinal infections is being increasingly recognized. Eleven Bacillus species isolated from veterinary samples associated with severe nongastrointestinal infections were assessed for the presence and expression of diarrheagenic enterotoxins and other potential virulence factors. PCR studies revealed the presence of DNA sequences encoding hemolysin BL (HBL) enterotoxin complex and B. cereus enterotoxin T (BceT) in five B. cereus strains and in Bacillus coagulans NB11. Enterotoxin HBL was also harbored by Bacillus polymyxa NB6. After 18 h of growth in brain heart infusion broth, all seven Bacillus isolates carrying genes encoding enterotoxin HBL produced this toxin. Cell-free supernatant fluids from all 11 Bacillus isolates demonstrated cytotoxicity toward human HEp-2 cells; only one Bacillus licheniformis strain adhered to this test cell line, and none of the Bacillus isolates were invasive. This study constitutes the first demonstration that Bacillus spp. associated with serious nongastrointestinal infections in animals may harbor and express diarrheagenic enterotoxins traditionally linked to toxigenic B. cereus.     

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.