Cereulide Production Research Articles

Cereulide formation by Bacillus weihenstephanensis and mesophilic emetic Bacillus cereus at temperature abuse depends on pre-incubation conditions

Emetic toxin (cereulide) formation was recently identified in a psychrotolerant species, Bacillus weihenstephanensis [Thorsen, L., Hansen, B.M., Nielsen, K.F., Hendriksen, N.B., Phipps, R.K., Budde, B.B., 2006. Characterization of emetic Bacillus weihenstephanensisis, a new cereulide-producing bacterium. Applied and Environmental Microbiology, 72, 5118–5121.]. Although recent findings indicated B. weihenstephanensis as a cereulide producer only limited information is available regarding environmental conditions affecting cereulide production. In the present study a model agar system was used to compare cereulide production during surface growth of B. weihenstephanensis MC67, and two well known mesophilic cereulide producing Bacillus cereus strains, NC7401 and NS117. Cereulide production was quantified by use of Liquid-Chromatography Mass Spectrometry/Mass Spectrometry. Cereulide production of B. weihenstephanensis MC67 occurred in stationary growth phase, as previously observed for B. cereus, and biomass formation and cereulide formation showed a linear correlation. During incubation at 5 °C for 1, 2 and 3 weeks growth was inhibited and as a consequence no detectable cereulide production occurred for any of the three strains. Similar results were obtained for the mesophilic B. cereus strains when incubated at 8 °C, whereas B. weihenstephanensis MC67 grew to stationary phase and produced 0.002 μg cereulide/cm 2 agar surface in 1 week. Raising the temperature from 5 °C to 25 °C for 24 h after 1 week of incubation resulted in growth to stationary phase and production of variable levels of cereulide. B. weihenstephanensis MC67 produced 6.18 μg cereulide/cm 2, B. cereus NS117 0.91 μg cereulide/cm 2 and B. cereus NC7401 0.09 μg cereulide/cm 2. Similar levels of cereulide was produced by the mesophilic strains when raising the temperature from 8 °C (instead of from 5 °C) to 25 °C for 24 h, while a considerably lower level was produced by B. weihenstephanensis MC67 (0.10 μg cereulide /cm 2). If the temperature was raised from 5 °C and 8 °C to 25 °C for 24 h after an increased incubation time for 2 and 3 weeks, all three strains produced considerably less cereulide. B. weihenstephanensis MC67 produced 100–6000 times less and the mesophilic B. cereus strains produced 9–40 times less cereulide. These results can partly be explained by differences in the growth at the temperature abuse. Effect of chill storage on cereulide production at temperature abuse has not been investigated previously . Results of the present study indicate that storage at 5 and 8 °C will not lead to emetic intoxications, however the time at, and choice of chill temperature will determine the amount of cereulide produced in a temperature abuse situation. These results are of relevance for the safety of chilled foods of extended durability.

Cereulide synthesis in emetic Bacillus cereus is controlled by the transition state regulator AbrB, but not by the virulence regulator PlcR

Cereulide, a depsipeptide structurally related to the antibiotic valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that cereulide is produced non-ribosomally by the plasmid-encoded peptide synthetase Ces. Using deletion mutants of the emetic reference strain B. cereus F4810/72, the influence of the well-known transcription factors PlcR, Spo0A and AbrB on cereulide production and on the transcription of the cereulide synthetase gene cluster was investigated. Our data demonstrate that cereulide synthesis is independent of the B. cereus specific virulence regulator PlcR but belongs to the Spo0A-AbrB regulon. Although cereulide production turned out to be independent of sporulation, it required the activity of the sporulation factor Spo0A. The sigma(A)-promoted transcription of spo0A was found to be crucial for cereulide production, while the sigma(H)-driven transcription of spo0A did not affect cereulide synthesis. Overexpression of the transition state factor AbrB in B. cereus F4810/72 resulted in a non-toxic phenotype. Moreover, AbrB was shown to bind efficiently to the main promoter region of the ces operon, indicating that AbrB acts as a repressor of cereulide production by negatively affecting ces transcription.

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO 2. The sensitivity to 20% CO 2 was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO 2 was combined with 2% O 2 and in 3 weeks when combined with “0”% O 2 (the remaining atmosphere was made up from N 2). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m 2/24 h and the atmospheres were 2% O 2/20% CO 2 and “0”% O 2/20% CO 2. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m 2/24 h and “0”% O 2/20 % CO 2) inhibited growth of the three strains during 4 weeks storage at 8 °C. Cereulide production was undetectable during storage at 8 °C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 °C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week in MAP with OTR of 1.3 or 40 ml/m 2/24 h and 2% O 2 resulted in cereulide concentrations of 0.816 – 1.353 µg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m 2/24 h, “0”% O 2/20 % CO 2) resulted in minimal cereulide production (0.004 µg/g meat sausage) at abuse condition. Extension of MAP storage at 8 °C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production. Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition . Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.

Environment driven cereulide production by emetic strains of Bacillus cereus

The impacts of growth media and temperature on production of cereulide, the emetic toxin of Bacillus cereus, were measured for seven well characterised strains selected for diversity of biochemical and genetic properties and sources of origin. All strains carried cereulide synthase gene, ces, on a megaplasmid of ca. 200 kb and all grew up to 48–50 °C, but produced cereulide only up to 39 °C. On tryptic soy agar five strains, originating from foods, food poisonings and environment, produced highest amounts of cereulide at 23 to 28 °C, whereas two strains, from human faeces, produced cereulide similarly from 23 to 39 °C, with no clear temperature trend. These two strains differed from the others also by producing more cereulide on tryptic soy agar if supplemented with 5 vol.% of blood, whereas the other five strains produced similarly, independent on the presence of blood. On oatmeal agar only one strain produced major amounts of cereulide. On skim milk agar, raw milk agar, and MacConkey agar most strains grew well but produced only low amounts of cereulide. Three media components, the ratio [K +]:[Na +], contents of glycine and [Na +], appeared of significance for predicting cereulide production. Increase of [K +]:[Na +] (focal variable) predicted ( P < 0.001) high cereulide provided that the contents of glycine and [Na +] (additional variables) were kept constant. The results show that growth medium and temperature up and downregulate cereulide production by emetic B. cereus in a complex manner. The relevance of the findings to production of cereulide in the gut and to the safety of amino acids as additives in foods containing live toxinogenic organisms is discussed.

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.

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.

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.

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.

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

To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). 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. The results indicate that cereulide is produced by a NRPS complex. 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.

Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.

Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains ( n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.

Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography–ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 μg ml −1 or g −1 wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (>99.5% N 2), less than 0.05 μg of cereulide ml −1 or g −1 wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l-leucine and l-valine (0.3 g l −1) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16–24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food.

Atmospheric oxygen and other conditions affecting the production of cereulide by Bacillus cereus in food

Factors influencing the production of cereulide, the emetic toxin of Bacillus cereus in food and laboratory media were investigated, using liquid chromatography–ion trap mass spectrometry and sperm motility inhibition bioassay for detection and quantitation. Oxygen was essential for production of the emetic toxin by B. cereus. When beans, rice or tryptic soy broth were inoculated with cereulide producing strains B203, B116 (recent food isolates) or the strain F-4810/72, high amounts (2 to 7 μg ml −1 or g −1 wet wt) of cereulide accumulated during 4-day storage at room temperature. In parallel cultures and foods, stored under nitrogen atmosphere (>99.5% N 2 ), less than 0.05 μg of cereulide ml −1 or g −1 wet wt accumulated. The outcome of the bioassay matched that of the chemical assay, with no indication of interference by substances in the rice or beans. Boiling for 20 to 30 min did not inactivate cereulide or cereulide producing strains in rice or the beans. Adding l -leucine and l -valine (0.3 g l −1 ) stimulated cereulide production 10- to 20-fold in R2A and in rice water agar. When the B. cereus strains were grown on agar media under permissive conditions (air, room temperature), cereulide was produced overnight with little or no increase when the incubation was extended to 4 days. In broth culture, the production of cereulide started later than 16–24 h. Anoxic storage prevented cereulide production also when the amino acids had been supplied. Packaging with modified atmosphere low in oxygen may thus be used to reduce the risk of cereulide formation during storage of food.

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.