Emetic Toxin Production Research Articles

Emetic toxin production of Bacillus cereus in a biofilm

Bacillus cereus sensu stricto (B. cereus) belongs to the B. cereus group, and is a well-known foodborne pathogen causing human disease including emesis which is caused by an emetic toxin, cereulide, with 105-108 cells per gram required to cause disease. The presence of this highly heat, pH and protease-resistant toxin presents a serious challenge to the food industry, as the bacteria itself may be eliminated during processing but the cereulide toxin will survive most food processing techniques. This study shows that cereulide toxin is associated with cells and biofilm structures rather than suspended in the surrounding liquid phase or environment. This is the first report investigating the cereulide toxin production in the presence of biofilms of B. cereus, showing that the cereulide toxin produced is associated with biofilm complex and also attaches to the substrate such as glass and stainless-steel on which the biofilm grows. The RT-qPCR showed that the expression of cesA and cesB were comparable between planktonic cells and biofilms. This study contributes a better understanding of food safety issues in the industry caused by cereulide toxin produced by B. cereus, and provides valuable information for developing control methods for cereulide toxin in the food industry.

Goat Colostrum—Source of Toxigenic Bacillus Cereus

Abstract The aim of this study was to evaluate the toxigenic potential of Bacillus cereus strains isolated from frozen goat colostrum. Of the 50 phenotypically suspected B. cereus isolates, 39 (78.0 %) were confirmed as B. cereus by the polymerase chain reaction (PCR) method based on the gyrB gene detection. In these isolates, genes encoding the production of haemolysin BL (Hbl), a complex of non-haemolytic enterotoxins (Nhe) and emetic toxin were detected by the PCR method. In 36 (92.3 %) confirmed B. cereus isolates, genes encoding at least one type of toxins of interest were detected. In all toxigenic isolates, we found the presence of genes for Nhe production, and in 16 (41.0 %) of the isolates, genes encoding both Nhe and haemolysin BL were shown. Eight (20.5 %) of the emetic strains of B. cereus were identified. The emetic toxin production gene was always detected simultaneously with genes encoding non-haemolytic enterotoxin production. The ability to produce BL haemolysin and non-haemolytic enterotoxins were confirmed by the immunochromatographic method. In summary, goat colostrum can be a significant source of toxigenic strains of B. cereus.

Analysis of emetic toxin production by Bacillus species using cellular cytotoxicity, molecular, and chromatographic assays

In this study, we analyzed production of the emetic toxin, cereulide, in 30 Bacillus strains by using a cellular cytotoxicity (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MTT) assay, polymerase chain reaction (PCR), and micellar electrokinetic chromatographycapillary electrophoresis (MEKC-CE) analysis. The Bacillus cereus emetic strains produced 60 ∼ 227 µg/mL of cereulide when analyzed using MEKC-CE. Some other Bacillus species, including B. subtilis, B. pumilus, and B. megaterium, produced putative cereulide at levels similar to those produced by emetic B. cereus, whereas B. mycoides and B. thuringiensis did not produce the putative toxin or produced it at a concentration less than 2 µg/mL. Only B. cereus emetic strains were found to be toxic to bovine fibroblast cells, with the exception of one Bacillus diarrheal strain. The PCR results correlated with the MTT assay results, except in the case of one B. cereus diarrheal strain. This strain may produce either unusually heat-stable enterotoxins or is a co-producer of emetic and diarrheal toxins. Collectively, our results indicate that several Bacillus species produce toxin(s) with structures and properties similar to those of cereulide.

Improved multiplex PCR assay for simultaneous detection of Bacillus cereus emetic and enterotoxic strains

Toxin producing Bacillus cereus can cause enterotoxic and/or emetic food poisoning. In the present study, a multiplex PCR assay was developed to detect all toxin genes known to be involved in food poisoning of B. cereus in a single reaction. Specific primers for the detection of enterotoxic (entFM, hblC, nheA, and cytK) genes and emetic toxin production (2 primer pairs: ces, CER) were designed based on the GeneBank sequences. The developed multiplex PCR assay was evaluated in pure culture and artificially inoculated milk, using 43 B. cereus strains and non-target strains. In brief, sensitivity in pure culture was 10-fold or more higher than artificially inoculated milk in multiplex PCR detection limit assay. The presented PCR assay is a developed molecular tool for the rapid simultaneous detection of emetic and enterotoxin producing B. cereus strains.

Follow-up of the Bacillus cereus emetic toxin production in penne pasta under household conditions using liquid chromatography coupled with mass spectrometry

Two outbreak-related Bacillus cereus emetic strains were investigated for their growth and cereulide production potential in penne pasta at 4, 8 and 25 °C during 7-day storage. Cereulide production was detected and quantified by LC-MS method (LOD of 1 ng/ml, LOQ of 5 ng/ml) and growth was determined by culture-based enumeration. Inoculated B. cereus strains (10 5 CFU/g) were able to reach counts of more than 10 8 CFU/g and cereulide production of about 500 ng/g already after 3 days of storage at 25 °C. Interestingly, a constant increase of the toxin was noticed during incubation at ambient temperature storage: the cereulide was continuously produced during the bacterial stationary growth phase reaching maximal amounts at the end of the experiment (7 days, concentration of about 1000 ng/g). Strictly respected cold chain temperature as 4 °C did not allow any detectable cereulide production for any of the two tested strains. At the limited temperature abuse of 8 °C, a detectable amount of cereulide was observed after two days for one of the strain (TIAC303) (<LOQ). These results confirm that cereulide production is controlled by multiple factors (from type of strain to temperature) and that prolonged storage time plays a crucial role for consumer safety.

Transcriptional kinetic analyses of cereulide synthetase genes with respect to growth, sporulation and emetic toxin production in Bacillus cereus

In light of the increasing number of serious food borne outbreaks caused by emetic Bacillus cereus, a better understanding of the cereulide synthetase ( ces) gene expression and toxin synthesis is required. Here, the relative expression levels of three ces genes ( cesP, cesA and cesB) were investigated using quantitative real-time reverse transcription PCR in relation to growth, degree of sporulation and toxin production of the emetic reference strain B. cereus F4810/72 and the weakly emetic strain IH41385. The strict co-transcription of all three genes confirmed the operon structure of the ces gene cluster responsible for cereulide formation. ces transcription turned out to be highly temporal and tightly regulated; ces mRNA was only detectable during mid to late exponential growth in both strains. The low toxigenic potential of the weakly emetic strain IH41385 correlated well with its respective ces transcripts, showing reduced activity at a transcriptional level. Two non-sporulating mutants (F4810/72Δspo0A and F4810/72INsigH) demonstrated that cereulide synthesis is part of the Spo0A regulon but independent of later sporulation processes. Besides strain specific intrinsic factors, ces transcription was found to be significantly influenced by the cellular growth state as well as by extrinsic abiotic factors, like salt. An increase of sodium chloride in the media resulted in lower ces transcription and coincided with lower cereulide toxin levels. Interestingly, at 25 g l −1 NaCl, toxin levels were already reduced without strongly affecting the growth of B. cereus, indicating an inhibitory effect of NaCl on cereulide biosynthesis independent of growth. This illustrates that ces gene expression and toxicity cannot be predicted solely from growth rates or cell numbers, but is influenced by complex interactions of various intrinsic as well as extrinsic factors, which remain to be clarified in detail.

Bacillus cereus emetic toxin production in relation to dissolved oxygen tension and sporulation

Seven emetic toxin-producing strains of Bacillus cereus were examined for growth, sporulation and toxin production in skim milk medium in shaken and unshaken batch cultures under aerobic, microaerobic and anaerobic atmospheres. High levels of toxin production were detected in aerobic and microaerobic cultures. Static cultures yielded 90% less toxin than equivalent shaken cultures. Emetic toxin production was detected in aerobic and microaerobic cultures when bacterial counts reached > 6·0 log 10 cfu ml −1, which also coincided with spore production. However, no correlation was found between spore count and toxin production (R 2 = 0·032). In anaerobic culture, production of emetic toxin was undetectable and the dissolved oxygen tension in the growth medium fell below 125±46 ppm within 6 h of inoculation. In aerobic culture, most toxin was produced between 16 and 22 h of incubation, when the dissolved oxygen tension had decreased to 60−70 ppm. Therefore, while O 2-dependent respiration is fundamentally required for production of the emetic toxin, most toxin production occurs when little free oxygen is available.

Bacillus cereus emetic toxin production in cooked rice

Three mesophilic strains of Bacillus cereus known to produce emetic toxin were used to model germination, growth and emetic toxin production in boiled rice cultures at incubation temperatures ranging from 8°C to 30°C. Minimum temperatures for germination and growth in boiled rice were found to be 15°C for all strains. Toxin production at 15°C was found to be significantly greater ( P<0·01; reciprocal toxin titre of 373±124) than at 20°C and 30°C (reciprocal toxin titres 112±37 and 123±41, respectively). Toxin production became detectable after 48 h incubation at 15°C, with a maximum titre reached by 96 h. At 20°C and 30°C, toxin production was detected at 24 h incubation, with a maximum titre reached by 72 h. Toxin production at 15°C was detectable at lower bacterial counts (6·2 log 10 cfu g −1), than with incubation at 20°C and 30°C (>7·0 log 10 cfu g −1). In this study, the lower temperature limit for germination and growth on solid laboratory medium was found to be 12°C for all strains, i.e. 3°C lower than that observed in boiled rice.

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 μg/g. A vomiting-type strain, B. cereus NC7401, was inoculated into various foods and incubated for 24 h at 20, 30, and 35 °C. In boiled rice, B. cereus rapidly increased to 10 7–10 8 cfu/g and produced emetic toxin at both 30 and 35 °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.

Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables.

In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and 4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.

Semiautomated metabolic staining assay for Bacillus cereus emetic toxin.

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.

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.

各種食品中でのＢａｃｉｌｌｕｓ ｃｅｒｅｕｓの増殖性とおう吐毒素産生性に関する検討

各種食品中でのＢａｃｉｌｌｕｓ ｃｅｒｅｕｓの増殖性とおう吐毒素産生性に関する検討