Modelling the number of viable vegetative cells of Bacillus cereus passing through the stomach

Aims:  Model the number of viable vegetative cells of B. cereus surviving the gastric passage after experiments in simulated gastric conditions.
Materials and Methods:  The inactivation of stationary and exponential phase vegetative cells of twelve different strains of Bacillus cereus , both mesophilic and psychrotrophic strains isolated from food and faeces from healthy and ill individuals, in simulated gastric conditions was determined using decimal reduction times at low pH ( D _pH). Subsequently inactivation rates were calculated. Inclusion of the inactivation rates into models describing the course of the gastric pH after the consumption of meal of solid food and the transfer of food from the stomach to the small intestine resulted in numbers of viable Bacillus cereus vegetative cells able to pass the stomach.
Conclusions:  According to the model, 3–26% of the ingested vegetative cells from Bacillus cereus may survive the gastric passage, dependent on the growth phase of the vegetative cells, the type of strains, and the age of the consumer.
Significance and Impact of the Study:  Vegetative cells of Bacillus cereus may be involved in the onset of diarrhoeal disease to a greater extent than expected since up to 26% of the ingested cells survive simulated gastric conditions.     

Bacillus cereus can attack the cell membranes of the alga Chara corallina by means of HlyII

We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.     

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.     

Bacillus cereus pore-forming toxins hemolysin II and cytotoxin K: polymorphism and distribution of genes among representatives of the cereus group

Abstract-Phylogenetic interrelation between 40 strains of the Bacillus cereus group has been established using BcREP fingerprinting. The PCR method has shown that the frequency of occurrence of the genes of cytotoxin K (cytK) and hemolysin II (hlyII) is 61% and 56%, respectively, and the gene of the hemolysin II regulator (hlyIIR) occurs together with hlyII. Comparison of the results of fingerprinting, PCR, and RFLP of the toxin genes showed that bacteria with the hlyII+ and cytK+ genotypes did not form separate clusters. However, microorganisms with the similar fingerprints were shown to have toxin genes of the same type. The proposed variant of RFLP analysis made it possible to clearly distinguish between the cytK1 and cytK2 genes. Twenty-three strains having the cytK genes carried no cytK1 dangerous for mammals. Additionally, the entire collection of microorganisms was tested for the ability to grow at 4 degrees C. This property was revealed for five strains, which should most likely be classified as B. weihenstephanensis. Two of the five psychrotolerant microorganisms carried the hemolysin II gene variant of the same type according to RFLP. None of the five strains had the cytK gene. These strains did not form close groups upon clustering by the applied method of Bc-REP fingerprints.     

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.     

Transformation of Bacillus cereus vegetative cells by electroporation

Transformation of untreated vegetative cells of Bacillus cereus 569 with plasmid pC194 (1.8 megadaltons) by high-voltage electroporation resulted in a maximum of 2 x 10(-5) transformants per viable cell. Transformation of a 130-megadalton plasmid occurred at a comparable frequency. The method was simple, rapid, and yielded transformant colonies in 14 to 24 h. Transformation was obtained with unpurified total plasmid DNA.     

Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities

The role played by alpha-helix 4 of the Bacillus thuringiensis toxin Cry1Aa in pore formation was investigated by individually replacing each of its charged residues with either a neutral or an oppositely charged amino acid by using site-directed mutagenesis. The majority of the resulting mutant proteins were considerably less toxic to Manduca sexta larvae than Cry1Aa. Most mutants also had a considerably reduced ability to form pores in midgut brush border membrane vesicles isolated from this insect, with the notable exception of those with alterations at amino acid position 127 (R127N and R127E), located near the N-terminal end of the helix. Introducing a negatively charged amino acid near the C-terminal end of the helix (T142D and T143D), a region normally devoid of charged residues, completely abolished pore formation. For each mutant that retained detectable pore-forming activity, reduced membrane permeability to KCl was accompanied by an approximately equivalent reduction in permeability to N-methyl-D-glucamine hydrochloride, potassium gluconate, sucrose, and raffinose and by a reduced rate of pore formation. These results indicate that the main effect of the mutations was to decrease the toxin's ability to form pores. They provide further evidence that alpha-helix 4 plays a crucial role in the mechanism of pore formation.     

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.     

Fluorescence of Trypan blue in frozen-dried embryos of the rat

Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.     

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.     

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.     

A downstream process for production of a viable and stable Bacillus cereus aquaculture biological agent

Biological products offer advantages over chemotherapeutics in aquaculture. Adoption in commercial application is lacking due to limitations in process and product development that address key end user product requirements such as cost, efficacy, shelf life and convenience. In previous studies, we have reported on the efficacy, physiological robustness and low-cost spore production of a Bacillus cereus isolate (NRRL 100132). This study examines the development of suitable spore recovery, drying, formulation and tablet production from the fermentation product. Key criteria used for such downstream process unit evaluation included spore viability, recovery, spore balance, spore re-germination, product intermediate stability, end product stability and efficacy. A process flow sheet comprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yielded a suitable product. The formulation included corn steep liquor and glucose to enhance subsequent spore re-germination. Viable spore recovery and spore balance closure across each of the process units was high (>70% and >99% respectively), with improvement in recovery possible by adoption of continuous processing at large scale. Spore re-germination was 97%, whilst a product half-life in excess of 5 years was estimated based on thermal resistance curves. The process resulted in a commercially attractive product and suitable variable cost of production.