Heat resistance of Bacillus cereus emetic toxin, cereulide

Aims: The study describes the effects of heating temperature and exposure time on the thermal stability of cereulide under different conditions (pH, presence/absence of oil phase and cereulide concentration). Methods and Results: Cereulide heat inactivation was investigated at 100, 121 and 150°C under different alkaline pH values (8.6–10.6) and in the presence of oil phase (0.6–1.4%). Three different cereulide concentrations (0.5, 5 and 6 μg ml^?1) were used. Cereulide detection was performed with computer‐aided semen analyzer and with HPLC–MS. Highly alkaline pH was needed to achieve inactivation. At lower cereulide concentrations less drastic conditions were needed. Removal of alkaline buffer after the heat treatment resulted in the recovery of toxic activity. Conclusions: Heat stability of cereulide has been proved to be remarkable, even at highly alkaline pH values, at all temperatures tested. The loss of activity appeared to be reversible. Significance and Impact of the Study: The study demonstrates the inability of any heat treatment used in the food industry to inactivate cereulide. Food safety has to rely on prevention and cold chain maintenance. Cleaning practices also need to be adapted as cereulide may remain in its active form upon sterilization of used material.     

Toxin gene profiling of enterotoxic and emetic Bacillus cereus

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.     

Phenotypic homogeneity of emetic Bacillus cereus isolates in China

Emetic Bacillus cereus strains produce a potent cereulide cytotoxin, which can cause acute and fatal cases of food poisoning. We isolated 18 emetic B. cereus strains from a food poisoning event, and from clinical and non-random food surveillance in China and phenotypic characteristics of haemolysis, starch hydrolysis, salicin fermentation, gelatin liquefaction, cytotoxicity, and susceptibility to antibiotics were assessed. All isolates were positive for haemolysis and gelatin liquefaction, and negative for starch hydrolysis and salicin fermentation. Their haemolytic potentials were intermediate to Bacillus anthracis and B. cereus ATCC 14579 (a non-emetic strain). All isolates were cytotoxic to CHO, Hep-2, and Vero cells, and were sensitive to ampicillin. The homogeneous phenotypes of emetic isolates from China are similar to the corresponding traits of European and Japanese isolates that have been characterized, suggesting highly similar phenotypes of emetic B. cereus worldwide.     

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.     

A method for the isolation of asporogenic mutants of Bacillus thuringiensis.

Asporogenic and oligosporogenic mutants of Bacillus thuringiensis can be isolated by streaking the bacteria onto a solid medium, incubating at 42 degrees C, and picking small, raised areas of growth which appear on the streaks after 48 h.     

Genome organization of temperate phage 11143 from emetic Bacillus cereus NCTC11143

A temperate phage was isolated from emetic Bacillus cereus NCTC 11143 by mitomycin C and characterized by transmission electron microscopy and DNA and protein analyses. Whole genome sequencing of Bacillus phage 11143 was performed by GS-FLX. The phage has a dsDNA genome of 39,077 bp and a 35% G+C content. Bioinformatic analysis of the phage genome revealed 49 putative ORFs involved in replication, morphogenesis, DNA packaging, lysogeny, and host lysis. Bacillus phage 11143 could be classified as a member of the Siphoviridae family by morphology and genome structure. Genomic comparisons at the DNA and protein levels revealed homologous genetic modules with patterns and morphogenesis proteins similar to those of other Bacillus phages. Thus, Bacillus phages might have a mosaic genetic relationship.     

Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk

The Bacillus cereus sensu lato group includes potentially pathogenic bacteria that are ubiquitous in the environment and, importantly, could also be present in food products. This study focuses on emetic isolates which presumably could cause acute food poisoning and emetic syndrome. Here, we evaluate the ability of psychrotolerant Bacillus weihenstephanensis MC118 (isolated from soil) and mesophilic B. cereus BOD3/9 isolated from milk to germinate and multiply at 7 and 30 °C. Whereas the rates of germination at 30 °C in milk and nutrient broth of MC118 and BOD3/9 were similar, MC118, but not BOD3/9, proliferated to achieve relatively high numbers (～10⁶?colony-forming units/g) within 7 days of incubation at 7 °C. Mesophilic BOD3/9 showed a slight decrease of cell concentration in similar studies at 7 °C. Genotyping with repetitive extragenic palindromic sequence-based PCR and pulsed field gel electrophoresis revealed significant similarities between BOD3/9 and emetic reference B. cereus F4810/72 strain, while the B. weihenstephanensis MC118 isolate was more similar to the B. weihenstephanensis non-emetic reference DSMZ11821 strain. Our data suggest that emetic isolates that are also psychrotolerant, such as MC118, could constitute a hazard in the dairy industry, where milk could be a suitable medium for germination and growth.     

A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus

Abstract A vacuole-formation substance, cereulide of Bacillus cereus , is an emetic toxin in animals. Both oral administration and intraperitoneal injection of cereulide caused dose-dependent emesis in Suncus murinus , a new animal model of emesis. Vagotomy or a 5-HT₃ receptor antagonist completely abolished this emetic effect. Therefore, cereulide causes emesis through the 5-HT₃ receptor and stimulation of the vagus afferent. We also found that our purified cereulide caused swelling of mitochondria of HEp-2 cells.     

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.     

Antifungal activity displayed by cereulide, the emetic toxin produced by Bacillus cereus

In this study, the fungistatic activity of Bacillus cereus cereulide-producing strains was demonstrated against nine fungal species. The role of cereulide was confirmed using plasmid-cured derivatives and ces knockout mutants. The fungistatic spectra of cereulide and valinomycin, a chemically related cyclododecadepsipeptide, were also compared and found to be similar but distinct.     

High incorporation of L-amino acids to cereulide, an emetic toxin from Bacillus cereus

Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus. This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13C labeled L-amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on cereulide and its hydrolysate dipeptides. The incorporation of the 13C atom was 95% in each O-Val, O-Leu and L-Val, while 40% in D-Ala of cereulide.     

Appearance of spore coat protein in the cell extracts of Bacillus subtilis asporogenic mutants.

By use of the antigen-antibody techniques we have studied whether asporogenic mutants of Bacillus subtilis can synthesize the spore coat protein. Antibody specific to spore coat protein was prepared and used to demonstrate that the spore coat protein was synthesized at the early stage of sporulation. We report here that asporogenic mutants synthesize the spore coat protein.     

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.     

Comparative analysis of antimicrobial activities of valinomycin and cereulide, the Bacillus cereus emetic toxin

Cereulide and valinomycin are highly similar cyclic dodecadepsipeptides with potassium ionophoric properties. Cereulide, produced by members of the Bacillus cereus group, is known mostly as emetic toxin, and no ecological function has been assigned. A comparative analysis of the antimicrobial activity of valinomycin produced by Streptomyces spp. and cereulide was performed at a pH range of pH 5.5 to pH 9.5, under anaerobic and aerobic conditions. Both compounds display pH-dependent activity against selected Gram-positive bacteria, including Staphylococcus aureus, Listeria innocua, Listeria monocytogenes, Bacillus subtilis, and Bacillus cereus ATCC 10987. Notably, B. cereus strain ATCC 14579 and the emetic B. cereus strains F4810/72 and A529 showed reduced sensitivity to both compounds, with the latter two strains displaying full resistance to cereulide. Both compounds showed no activity against the selected Gram-negative bacteria. Antimicrobial activity against Gram-positive bacteria was highest at alkaline pH values, where the membrane potential (ΔΨ) is the main component of the proton motive force (PMF). Furthermore, inhibition of growth was observed in both aerobic and anaerobic conditions. Determination of the ΔΨ, using the membrane potential probe DiOC(2)(3) (in the presence of 50 mM KCl) in combination with flow cytometry, demonstrated for the first time the ability of cereulide to dissipate the ΔΨ in sensitive Gram-positive bacteria. The putative role of cereulide production in the ecology of emetic B. cereus is discussed.