Emetic Toxin Research Articles

Enterotoxins and emetic toxins production by Bacillus cereus and other species of Bacillus isolated from Soumbala and Bikalga, African alkaline fermented food condiments

The ability of various species of Bacillus from fermented seeds of Parkia biglobosa known as African locust bean (Soumbala) and fermented seeds of Hibiscus sabdariffa (Bikalga) was investigated. The study included screening of the isolates by haemolysis on blood agar, detection of toxins in broth and during the fermentation of African locust bean using the Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA) and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (BDEVIA). Detection of genes encoding cytotoxin K (CytK), haemolysin BL (Hbl A, Hbl C, Hbl D), non-hemolytic enterotoxin (NheA, NheB, NheC) and EM1 specific of emetic toxin producers was also investigated using PCR with single pair and multiplex primers. Of 41 isolates, 29 Bacillus belonging to the species of B. cereus, Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus showed haemolysis on blood agar. Using RPLA, enterotoxin production was detected for three isolates of B. cereus in broth and all B. cereus (9) in fermented seeds. Using BDEVIA, enterotoxin production was detected in broth as well as in fermented seeds for all B. cereus isolates. None of the isolates belonging to the other Bacillus species was able to produce enterotoxins either by RPLA or BDEVIA. Nhe genes were detected in all B. cereus while Hbl and CytK genes were detected respectively in five and six B. cereus strains. A weak presence of Hbl (A, D) and CytK genes was detected in two isolates of B. subtilis and one of B. licheniformis but results were inconsistent, especially for Hbl genes. The emetic specific gene fragment EM1 was not detected in any of the isolates studied.

Toxin Gene Profiling of Bacillus cereus Food Isolates by PCR

Seventy-one Bacillus cereus strains (12 references and 59 food isolates) were analyzed for the occurrence of five different enterotoxin genes (nheABC, hblCDA, entFM, cytK, and bceT) and one emetic toxin cereulide synthetase gene (ces) by PCR (polymerase chain reaction). PCR analysis revealed eight toxigenic patterns in all B. cereus strains tested; they all carried both entFM and nheABC. The presence of hblCDA, cytK, and bceT varied according to the enterotoxin-producing strains, among which hblCDA was the least frequently detected in the food-isolated strains. Only five B. cereus strains harbored ces, associated with the emetic type of food poisoning; however, these strains were devoid of hblCDA, cytK, and bceT.

Real-time-PCR-System zum Nachweis von Bacillus cereus (emetischer Typ) in Lebensmitteln

Bacillus cereus is a Gram-positive, facultative anaerobic, spore-performing bacterium. Some B. cereus strains have the ability to produce two different types of toxins: (a) diarrhoeic toxin: the disease is similar to a C. perfringens toxin-infection; it is caused by a heat-labile protein, (b) emetic toxin: the disease is similar to Staphylococcus-aureus intoxikation; it is caused by a heat-stable protein.

Differentiation and Characterization by Molecular Techniques of Bacillus cereus Group Isolates from Poto Poto and Dégué, Two Traditional Cereal-Based Fermented Foods of Burkina Faso and Republic of Congo

Poto poto (a maize sourdough) and dégué (a pearl millet–based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.

Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain

Isolates of the Bacillus cereus group (396 in total) from farms, silo tanks and production lines for pasteurised milk were tested for toxin production potential, and by polymerase chain reaction (PCR) for the presence of toxin genes. Comparison between the tests indicated the presence of gene polymorphisms. Highly toxigenic strains, based on production of subunit A of the nonhemolytic enterotoxin, NHE (NheA) and subunit C of the haemolytic enterotoxin, HBL (HblC), were less common among dairy isolates compared with farm and silo isolates. No producer of high levels of both toxins was found among 156 psychrotrophic dairy isolates ( B. weihenstephanensis) and only 3% of all psychrotrophs were high producers of NheA. Psychrotrophic B. cereus from pasteurised milk appeared to have a low enterotoxin production potential, and they were not producers of emetic toxin or cytotoxin K and therefore may be less likely to cause illness than mesophilic strains.

Computer aided boar semen motility analysis for cereulide detection in different food matrices

Computer Aided Semen Analysis (CASA) study of the boar semen motility has been demonstrated to be an appropriate assay for detection of cereulide ( Bacillus cereus emetic toxin). Application of the boar semen bio-assay to detect cereulide directly in foods requires investigation of potential interference of food components, preservatives and other microbial and chemical food contaminants with the bio-assay. Current study provides evidence that none of included Staphylococcus aureus enterotoxins A, B, C and D nor B. cereus Hemolysin BL (HBL) and non-hemolytic enterotoxin (NHE) and three mycotoxins (Sterigmatocystin, Fumonisin B1 and Patulin) exhibited a toxic impact on semen progressive motility. Aflatoxin M1, M3 and zearalenone impaired semen motility only at concentrations (0.004 mg ml − 1 , 0.1 mg ml − 1 and 10 mg ml − 1 , respectively) much higher than those found in foods and those permitted by legislation, in comparison to cereulide which induces motility cease at concentrations lower than 20 ng ml − 1 . Ten commonly used preservatives, namely potassium sorbate, sodium benzoate, ( dl) malic acid, citric acid, ( l+) tartaric acid, acetic acid, ( dl) lactic acid, ( l+) ascorbic acid, sodium chloride and sucrose induced no cease in spermatozoa motility even at preservative concentrations higher than permitted by legislation. Dioxins, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), and acrylamide had no acute effect on spermatozoa motility at concentrations of 500 and 10,000 mg ml − 1 , respectively. Robustness of computer aided boar semen motility analysis, tested with 14 different foods inoculated with cereulide producing B. cereus, showed distinct cereulide production in seven samples (although B. cereus growth to counts higher than 8 log CFU g − 1 was noted in 11 samples), in amounts close to those reported in foodborne outbreaks. Test evaluation in 33 samples suspected to hold cereulide showed actual cereulide presence in ten samples and no interference of food matrix with the assay.

Toxicological profile of cereulide, the Bacillus cereus emetic toxin, in functional assays with human, animal and bacterial cells

Some strains of the endospore-forming bacterium Bacillus cereus produce a heat-stable ionophoric peptide, cereulide, of high human toxicity. We assessed cell toxicity of cereulide by measuring the toxicities of crude extracts of cereulide producing and non-producing strains of B. cereus, and of pure cereulide, using cells of human, animal and bacterial origins. Hepatic cell lines and boar sperm, with cytotoxicity and sperm motility, respectively, as the end points, were inhibited by ⩽1 nM of cereulide present as B. cereus extract. RNA synthesis and cell proliferation in HepG2 cells was inhibited by 2 nM of cereulide. These toxic effects were explainable by the action of cereulide as a high-affinity mobile K + carrier. Exposure to cereulide containing extracts of B. cereus caused neither activation of CYP1A1 nor genotoxicity (comet assay, micronucleus test) at concentrations below those that were cytotoxic (0.6 nM cereulide). Salmonella typhimurium reverse mutation (Ames) test was negative. Exposure of Vibrio fischeri to extracts of B. cereus caused stimulated luminescence up to 600%, independent on the presence of cereulide, but purified cereulide inhibited the luminescence with an IC 50% (30 min) of 170 nM. Thus the luminescence-stimulating B. cereus substance(s) masked the toxicity of cereulide in B. cereus extracts to V. fischeri.

Complete Sequence Analysis of Novel Plasmids from Emetic and Periodontal Bacillus cereus Isolates Reveals a Common Evolutionary History among the B. cereus -Group Plasmids, Including Bacillus anthracis pXO1

The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical approximately 272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one approximately 270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

Characterization of the Cereulide NRPS α-Hydroxy Acid Specifying Modules: Activation of α-Keto Acids and Chiral Reduction on the Assembly Line

Several nonribosomal peptide natural products are composites of alpha-hydroxy acid and alpha-amino acid monomers. Cereulide, the emetic toxin from the human pathogen Bacillus cereus, and valinomycin, from Streptomyces spp., are closely related macrocyclic K+ ionophores. The macrocyclic core of each natural product contains alternating peptide (six) and ester (six) bonds, and their cyclododecadepsipeptide structures consist of a tetradepsipeptide unit repeated three times. Here we overexpress the cereulide NRPS alpha-hydroxy acid specifying modules from CesA and CesB and demonstrate that each contains an alpha-keto acid activating adenylation domain and a chiral alpha-ketoacyl-S-carrier protein reductase (alpha-KR). The logic used by the cereulide NRPS is likely at work in the valinomycin NRPS and may be the general strategy used in bacterial NRPSs to form alpha-hydroxy acid containing natural products.

Influence of Type of Food on the Kinetics and Overall Production of Bacillus cereus Emetic Toxin

Potato puree and penne pasta were inoculated with cereulide producing B. cereus 5964a and B. cereus NS117. Static incubation at 28°C proved these two foods to be a better substrate for higher cereulide production (4,080 ng/g in puree and 3,200 ng/g in penne were produced by B. cereus 5964a during 48 h of incubation) compared with boiled rice (2,000 ng/g). This difference occurred despite B. cereus counts of more than 108 CFU/g in all three products. Aeration of cultures had a negative effect on cereulide production, causing concentrations more than 10-fold lower than in some statically incubated samples. Cereulide production remained undetectable in shaken milk, whereas it reached 1,140 ng/ml in statically incubated milk. At 12 and 22°C, presence of background flora was also a determinative factor. A total B. cereus count of more than 106 CFU/ml did not necessarily lead to uniform cereulide production and was also dependent on the B. cereus strain involved. In this study, we confirm that a number of factors play a crucial role in the determination of the extent to which, if at all, cereulide will be produced. Among those, type of the food, temperature, pH, and whether additional aeration (via incubation on an orbital shaker) is induced had an important role. An important effect was also induced by the cereulide-producing strain involved.

Probiotics for animal nutrition in the European Union. Regulation and safety assessment

Probiotics are alive micro-organisms, generally bacteria but also yeasts than, when ingested alive in sufficient amount, they have a positive effect on the health going beyond the nutritional ones commonly known. Probiotics may operate through a nutritional and/or health or sanitary effect. Micro-organisms used in animal feed in the EU are mainly bacterial strains of Gram-positive bacteria belonging to the types Bacillus, Enterococcus, Lactobacillus, Pediococcus, Streptococcus and strains of yeast belonging to the Saccharomyces cerevisiae species and kluyveromyces. While most of the species and genera are apparently safe, certain micro-organisms may be problematic, particularly the enterococci, which may harbour transmissible antibiotic resistance determinants and bacilli, specially those belonging to the Bacillus cereus group that are known to produce enterotoxins and an emetic toxin. The history and the current legislation in the European Union on probiotics feed additives including the requirements for the safety assessment for the target animal species, consumers, workers, and environment are presented.

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

BackgroundCereulide, 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.ResultsThe 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.ConclusionThe 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.

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains ( n = 17), strains connected to diarrheal foodborne outbreaks ( n = 40) and food–environment strains ( n = 43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 °C. In contrast, 11% (9 food–environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 °C and 49% at 7 °C (28 diarrheal and 13 food–environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 °C, but only 39% (16 diarrheal and 16 food–environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 °C and a lower germination, particularly at 7 °C, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 °C, 37 °C, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.

Potential of selected infant food formulas for production of Bacillus cereus emetic toxin, cereulide

Cereulide producing Bacillus cereus was isolated from randomly chosen commercial infant foods. The cereulide production in infant food formulas was investigated. When the reconstituted foods were inoculated with > 10 5 cfu ml − 1 of cereulide producing B. cereus, 2 to 200 μg of cereulide per 100 ml of food accumulated during 24 h of non-refrigerated storage. The amount of cereulide measured in the foods by the accurate chemical assay (LC-MS) matched with that found by sperm micro assay, proving the cereulide was the sole heat stable toxin in the foods and present in its toxic form. The infant formulas containing both cereal and dairy ingredients were the most supportive for cereulide production. Cereulide accumulation was affected by the infant food composition as well as by the handling of the food. Diluting the reconstituted food with water resulted in increased toxin production expressed as μg per volume. More cereulide was accumulated when the food was incubated stationary compared with moderate shaking. The amount of cereulide accumulated within 24 h at room temperature per 100 ml of cereal and dairy or in rice–nondairy reconstituted infant formulas, inoculated with ≥ 10 5 cfu ml − 1 of B. cereus strain F4810/72, was higher or similar to the amounts reported for foods implicated in emetic type of food poisonings. Thus mishandling and temperature abuse of infant foods may cause food poisoning when emetic B. cereus is present.

ＬＣ‐ＭＳを用いたセレウス菌嘔吐毒産生試験に関する検討

Two types of food poisoning from Bacillus cereus, the emetic type caused by emetic toxins and the diarrhea type caused by enterotoxins, have been reported . Most cases in Japan are the emetic type, therefore, the cereulide needs to be proven when B. cereusfood poisoning is suggested. The original detection method for cereulide is performed by microscopic assessment of vacuole formation in HEp-2 cells, but it involves some judgement problems that require adept skills as well as reproducibility of the result. Furthermore, it also requires considerable labor to obtain and maintain the cells. Recently, an analysis method using liquid chromatography-mass spectrometry (LC-MS), which shows excellent accuracy, quantification, specificity and reproducibility is often used to detect cereulide. In this paper, we compared Nutrient broth containing manganese and glucose (NB+Mn) medium as previously recommended and 10% skim milk medium for the production of cereulide, and further investingated the influence of heat-treatment process of culture fluid on the recovery of cereulide measured by LC-MS.

セレウス菌嘔吐毒検査法の比較検討

Bacillus cereus sometimes causes food poisoning, known as the diarrhoeal type associated with enterotoxin, and the emetic type associated with emetic toxin “Cereulide”. Standard methods for detection of cereulide are oral challenge in mammals, microscopic assessment of vacuole formation in HEp-2 cells and liquid chromatography-mass spectrometry assay; however, each method has some difficulties requiring specific facilities, skilled techniques or an expensive analyzer, respectively. We previously reported a colorimetric cell metabolic assay in HEp-2 cells using 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) - 2 H -tetrazolium, monosodium salt (WST-8), as an simple way to detect cereulide. In the present study, we compared and evaluated four methods: vacuolation, MTT, WST-8, and LC/MS usingB. cereus strains isolated from retail foods. Colorimetric cell metabolic assay in HEp-2 cells using WST-8 was confirmed as sensitive and useful for a general laboratory.

Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food

Extensive analysis of the Staphylococcus aureus genome has allowed the identification of new genes encoding enterotoxin-like superantigens (SEls). Some of these are thought to be involved in staphylococcal food poisoning, while others do not elicit any emetic effect. The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity. In this paper the distribution of the genes coding for enterotoxin-like superantigens in S. aureus isolated from food was studied. Fifty isolates of S. aureus were examined and 27 were shown to be enterotoxigenic. Only 9 of the 27 strains carried genes encoding enterotoxins SEA–SEE. In 18 SEA–SEE-negative strains the presence of newly described enterotoxin genes was detected. All SEA–SEE-positive strains simultaneously carried genes of new SEls. We show that the gene encoding SElH (staphylococcal enterotoxin-like enterotoxin H) was the most frequently detected ( n = 14), while genes encoding SElI together with SElG accompanied by the other genes of the egc locus were detected in three strains. We also detected the presence of three less investigated genes: sep, sel, and sek. These genes were present in eight, two, and one isolate, respectively. In one strain, sep was accompanied by genes of other SEls, while in the remaining seven it was the only enterotoxin-like gene detected. The high prevalence of newly discovered enterotoxin genes, including the genes encoding emetic toxins, was demonstrated in food-derived strains. This supports the need for additional work on its role in food poisoning and, alternatively, to monitor its presence in S. aureus isolated from food. Our results suggest that yet unknown genetic elements encoding enterotoxin genes may exist.

Dynamics of boar semen motility inhibition as a semi-quantitative measurement of Bacillus cereus emetic toxin (Cereulide)

Qualitative and quantitative application of a computer assisted sperm analyzer (CASA) for detection and quantification of cereulide was described. The plot of the decrease of the percentage of boar semen progressive motility (PMOT%) in function of time and the visual inspection of curves provided a qualitative comparison between different samples (curve slope corresponds to the amount of cereulide in the sample). If the change of PMOT% over a time required for achieving PMOT% drop to 10% (ΔPMOT%/Δ τ) is plotted against the standard curve (obtained with known concentrations of valinomycin), a semi-quantitative estimation of the amount of cereulide in the sample is obtained. An optimized CASA method was applied to determine the production of cereulide under different conditions. No cereulide was found in aerated samples and in samples incubated at 12 °C. The amount of cereulide produced depended on the agar medium used, type of Bacillus cereus strain and the amount of oxygen present in the atmosphere.

Establishment of a Novel Multiplex PCR Assay and Detection of Toxigenic Strains of the Species in the Bacillus cereus Group

Five different enterotoxins and one emetic toxin of Bacillus cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex PCR with 12 primer pairs was established. In developing the assay method, a common terminal sequence at the 3′ ends of all primers was chosen and a hot start Taq polymerase was used to overcome primer dimer formation. The assay was successfully applied to analyze the toxigenic potential of 162 food-poisoning and food-related strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of Bacillus mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains (P < 0.05), although the two species were closely related. The results suggest that many B. mycoides strains might be less prone to cause food poisoning. They also indicate the importance of detecting the toxin genes together with the detection of the species in the B. cereus group.

Cereulide-producing strains of Bacillus cereus 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.