Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42 degrees C, and no reduction in activity was observed even after 24 h of growth at 42 degrees C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22 degrees C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.     

Escherichia coli strains isolated from pigs with edema disease produce a variant of Shiga-like toxin II

Escherichia coli O157:H7 strains 933 produces elevated levels of 2 phage-encoded, antigenically distinct cytotoxins designated Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II). These toxins kill both HeLa and Vero cells. In this report, the relationship between SLTs and a cytotoxin produced by E. coli strains isolated from pigs with edema disease (ED) was examined. Culture filtrates from 72 out of 81 ED strains were cytotoxic for Vero but not HeLa cells. Cytotoxicity was neutralized by antiserum to SLT-II but not by anti-Shiga toxin. No toxin-converting phage were detected in 20 toxigenic ED strains examined. The cytotoxin of the ED-causing strains appears to be a variant of SLT-II and production of this cytotoxin is not phage-mediated.     

From soil to gut: Bacillus cereus and its food poisoning toxins

Bacillus cereus is widespread in nature and frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insects and mammals. From these habitats it is easily spread to foods, where it may cause an emetic or a diarrhoeal type of food-associated illness that is becoming increasingly important in the industrialized world. The emetic disease is a food intoxication caused by cereulide, a small ring-formed dodecadepsipeptide. Similar to the virulence determinants that distinguish Bacillus thuringiensis and Bacillus anthracis from B. cereus, the genetic determinants of cereulide are plasmid-borne. The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores, thought to produce protein enterotoxins in the small intestine. Three pore-forming cytotoxins have been associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K. Hbl and Nhe are homologous three-component toxins, which appear to be related to the monooligomeric toxin cytolysin A found in Escherichia coli. This review will focus on the toxins associated with foodborne diseases frequently caused by B. cereus. The disease characteristics are described, and recent findings regarding the associated toxins are discussed, as well as the present knowledge on virulence regulation.     

Toxigenic Strains of Bacillus licheniformis Related to Food Poisoning

Toxin-producing isolates of Bacillus licheniformis were obtained from foods involved in food poisoning incidents, from raw milk, and from industrially produced baby food. The toxin detection method, based on the inhibition of boar spermatozoan motility, has been shown previously to be a sensitive assay for the emetic toxin of Bacillus cereus, cereulide. Cell extracts of the toxigenic B. licheniformis isolates inhibited sperm motility, damaged cell membrane integrity, depleted cellular ATP, and swelled the acrosome, but no mitochondrial damage was observed. The responsible agent from the B. licheniformis isolates was partially purified. It showed physicochemical properties similar to those of cereulide, despite having very different biological activity. The toxic agent was nonproteinaceous; soluble in 50 and 100% methanol; and insensitive to heat, protease, and acid or alkali and of a molecular mass smaller than 10,000 g mol⁻¹. The toxic B. licheniformis isolates inhibited growth of Corynebacterium renale DSM 20688^T, but not all inhibitory isolates were sperm toxic. The food poisoning-related isolates were beta-hemolytic, grew anaerobically and at 55°C but not at 10°C, and were nondistinguishable from the type strain of B. licheniformis, DSM 13^T, by a broad spectrum of biochemical tests. Ribotyping revealed more diversity; the toxin producers were divided among four ribotypes when cut with PvuII and among six when cut with EcoRI, but many of the ribotypes also contained nontoxigenic isolates. When ribotyped with PvuII, most toxin-producing isolates shared bands at 2.8 ± 0.2, 4.9 ± 0.3, and 11.7 ± 0.5 or 13.1 ± 0.8 kb.     

A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen⁻¹. This amount corresponds to 10⁴ to 10⁵ CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10⁸ CFU ml⁻¹. The detection limit for food was 3 g of rice containing 10⁶ to 10⁷ CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen⁻¹, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.     

Characterization of Emetic Bacillus weihenstephanensis, a New Cereulide-Producing Bacterium

Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8°C.     

Production and Characterization of Antibodies against Each of the Three Subunits of the Bacillus cereus Nonhemolytic Enterotoxin Complex

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by Bacillus cereus. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified B. cereus exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of B. cereus was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several B. cereus strains. For the first time, it could be shown that strains carrying the nhe genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.     

Heat-stable toxin production by strains of Bacillus cereus, Bacillus firmus, Bacillus megaterium, Bacillus simplex and Bacillus licheniformis

Strains of Bacillus cereus can produce a heat-stable toxin (cereulide). In this study, 101 Bacillus strains representing 7 Bacillus species were tested for production of heat-stable toxins. Strains of B. megaterium, B. firmus and B. simplex were found to produce novel heat-stable toxins, which showed varying levels of toxicity. B. cereus strains (18 out of 54) were positive for toxin production. Thirteen were of serovar H1, and it was of interest that some were of clinical origin. Two were of serovars 17B and 20, which are not usually implicated in the emetic syndrome. Partial purification of the novel B. megaterium, B. simplex and B. firmus toxins showed they had similar physical characteristics to the B. cereus emetic toxin, cereulide.     

Production of enterotoxins by strains of Escherichia coli isolated from pigs

The production of enterotoxins by 237 hemolytic strains of Escherichia coli isolated from pigs was determined with the use of CTE in CHO. Vero and Hela cells and ILT. More frequent (p less than 0.01) production of enterotoxins, determined by ILT, was found for the serotypes being pathogenic for the animals (63.8% of the strains). No correlation between intensity of ILT and particular serotype was observed. Both the serotypes pathogenic for pigs and other serotypes produced LT enterotoxins and ST toxin. The frequency of LT enterotoxin production was statistically insignificant compared to the frequency of ST enterotoxin production by strains with serotypes pathogenic for the pigs. Strains of E. coli producing only enterotoxin ST belonged both to the pathogenic serotypes as well as to other hemolytic serotypes. The cytotoxic activity of supernatants of E. coli strains with different serotypes isolated from pigs in Vero and Hela cells and simultaneous CTE in CHO cells was observed. This suggests the production by the strains of enterotoxin LT and cytotoxin VT. Seven out of the 96 isolates showing CTE in CHO cells gave no reaction in the ILT in pigs. This suggests the production by these isolates of a toxin (toxins) differing from the E. coli enterotoxins.     

Effects of Escherichia Coli Subtilase Cytotoxin and Shiga Toxin 2 on Primary Cultures of Human Renal Tubular Epithelial Cells

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubA_A272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.     

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.     

Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis

Twenty-six strains of Bacillus cereus from different sources were determined to be either mesophilic or psychrotrophic by growth at 6 and 42 degrees C. The strains were also screened by two polymerase chain reaction (PCR) methods designed to discriminate between mesophilic and psychrotrophic types. Seventeen of the 26 strains were able to grow at 6 degrees C, but only four conformed to the new psychrotolerant species Bacillus weihenstephanensis. Among the 26 strains were two which caused outbreaks of food poisoning in Norway, and three others that were isolated from food suspected of causing illness. The presence of the gene components encoding production of enterotoxins Nhe, Hbl, EntT and a recently described cytotoxin K was determined by PCR. All the strains possessed genes for at least one of these toxins, and 19 of the 26 strains were cytotoxic in a Vero cell assay. We conclude that there are psychrotrophic B. cereus strains which cannot be classified as B. weihenstephanensis, and that intermediate forms between the two species exist. No correlation between cytotoxicity and the growth temperature of the strains was found.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Structural Insights into Clostridium perfringens Delta Toxin Pore Formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present G_M2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.     

Quantification of the Effect of Culturing Temperature on Salt-Induced Heat Resistance of Bacillus Species

Short- and long-term exposure to mild stress conditions can activate stress adaptation mechanisms in pathogens, resulting in a protective effect toward otherwise lethal stresses. The mesophilic strains Bacillus cereus ATCC 14579 and ATCC 10987 and the psychrotolerant strain B. weihenstephanensis KBAB4 were cultured at 12°C and 30°C until the exponential growth phase (i) in the absence of salt, (ii) in the presence of salt, and (iii) with salt shock after they reached the exponential growth phase and subsequently heat inactivated. Both the first-order model and the Weibull model were fitted to the inactivation kinetics, and statistical indices were calculated to select for each condition the most appropriate model to describe the inactivation data. The third-decimal reduction times (which reflected the times needed to reduce the initial number of microorganisms by three decimal powers) were determined for quantitative comparison. The heat resistance of both mesophilic strains increased when cells were salt cultured and salt shocked at 30°C, whereas these salt-induced effects were not significant for the psychrotolerant strain. In contrast, only the psychrotolerant strain showed salt-induced heat resistance when cells were cultured at 12°C. Therefore, culturing temperature and strain diversity are important aspects to address when adaptive stress responses are quantified. The activated adaptive stress response had an even larger impact on the number of surviving microorganisms when the stress factor (i.e., salt) was still present during inactivation. These factors should be considered when stress-integrated predictive models are developed that can be used in the food industry to balance and optimize processing conditions of minimally processed foods.Bacillus cereus is a widespread, spore-forming pathogen that can be isolated from a range of different food products (4, 27), including pastry, vegetables and vegetable products, milk and milk products, and ready-to-eat foods. This toxin-producing pathogen can cause diarrhea and emesis (13, 25). The diarrheal syndrome is caused by several enterotoxins which are produced by vegetative cells in the small intestine. The emetic toxin, cereulide, causes emesis and is produced in foods before ingestion. Adequate chilling of foods is important to control the growth and toxin production of enterotoxin-producing (17) and emetic toxin-producing (7, 18) B. cereus strains.During processing and storage of mildly processed foods, bacteria are exposed to one or more preservation stresses, known as hurdles (16). While individual hurdles might not be effective in controlling microbial growth, the right combination of hurdles can be powerful in controlling microbial growth in minimally processed foods. However, the potential of Bacillus to become more resistant to stresses challenges the effectiveness of minimal processing. Several studies have demonstrated that exposure to mild stressing conditions can result in the increased resistance of both mesophilic and psychrotolerant members of the B. cereus group (2, 3, 5, 21, 22). These studies used optimal culturing temperature during mild stress exposure to investigate the adaptive stress responses. However, during processing, distribution, and storage, the temperature of foods may be lower because chilling is commonly used in the minimal processing food chain. Therefore, investigation of the effect of low incubation temperature on the adaptive stress responses of food-borne bacteria is of great relevance and could provide valuable information for quantitative exposure assessment studies.In the study described here, three representatives of the B. cereus group (12), namely, the mesophilic strains B. cereus ATCC 14579 and ATCC 10987 and the psychrotolerant strain Bacillus weihenstephanensis KBAB4, were cultured at 30°C in the absence and presence of mild salt stress, after which their heat resistance was assessed. Moreover, the culturing of cells was also performed at 12°C to determine the effect of a lowered culturing temperature on the adaptive salt stress responses. The third-decimal reduction time estimates were determined to evaluate the effects of the various culturing variables on the heat resistance of the three strains.     

Detection of a novel campylobacter cytotoxin

The culture filtrates from 10 Campylobacter species were screened for the presence of cytotoxins on a variety of selected tissue culture cell lines. Some Campylobacter jejuni strains showed no effects on tissue culture cell lines compared with other C. jejuni strains, especially C. jejuni 81116, which consistently produced a cytotoxin that was lethal to tissue culture cells. It was observed that CHO cells were the most sensitive cell line in detecting campylobacter cytotoxins. Samples containing the culture filtrate of C. jejuni 81116 prepared at various growth stages were used to determine the subcellular location of the cytotoxin. This C. jejuni 81116 cytotoxin appears to be a heat-stable toxin that is secreted from the cell during stationary phase; cytotoxin activity can be abolished with proteolytic enzymes.     

Snake venom toxins. The amino-acid sequences of three toxins (9B, 11 and 12A) from Hemachatus haemachatus (Ringhals) venom.

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group.     

Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.