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Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease.

It is crucial for the quality and safety of ready-to-eat (RTE) foods to maintain the cold chain from production to consumption. The effect of temperature abuse related to daily meals and elevated refrigerator temperatures on the growth and toxin production of Bacillus cereus, Bacillus weihenstephanensis and Staphylococcus aureus and the growth of Listeria monocytogenes and Yersinia enterocolitica was studied. A case study with temperature loggings in the domestic environment during Easter and Christmas holidays was performed to select relevant time and temperature courses. A model for bacterial surface growth on food using nutrient agar plates exposed to variations in temperatures was used to simulate food stored at different temperatures and exposed to room temperature for short periods of time. The results were compared with predicted growth using the modeling tool ComBase Predictor. The consumers exposed their cold cuts to room temperatures as high as 26.5°C with an average duration of meals was 47 min daily for breakfast/brunch during the vacations. Short (≤ 2 h) daily intervals at 25°C nearly halved the time the different pathogens needed to reach levels corresponding to the levels associated with human infection or intoxication, compared with the controls continuously stored at refrigerator temperature. Although the temperature fluctuations affected growth of both B. weihenstephanensis and S. aureus, toxin production was only detected at much higher cell concentrations than what has been associated with human intoxications. Therefore, growth of L. monocytogenes and Y. enterocolitica was found to be the limiting factor for safety. In combination with data on temperature abuse in the domestic environment, modeling programs such as ComBase Predictor can be efficient tools to predict growth of some pathogens but will not predict toxin production.

Besides Bacillus cereus, some strains of the psychrotolerant, potentially foodborne pathogen Bacillus weihenstephanensis can produce the emetic toxine (cereulide). This toxin is a heat- and acid-stable cyclic dodecadepsipeptide that causes food intoxication with vomiting. However, some severe clinical cases with lethal outcomes have been described. If cereulide can be produced during refrigerated storage, it will not be inactivated by reheating food, representing an important risk of food intoxication for consumers. In this paper, we determined the capacity of the B. weihenstephanensis strains BtB2-4 and MC67 to grow and produce cereulide on agar media at temperatures from 8 °C to 25 °C and at a pH from 5.4 to 7.0. At 8 °C, strain BtB2-4 produced quantifiable amounts of cereulide, whereas the limit of detection was reached for strain MC67. For BtB2-4, cereulide production increased 5-fold between 8 °C and 10-15 °C and by more than 100-fold between 15 °C and 25 °C. At temperatures of 10 °C and higher, cereulide concentrations were within the range of those reported by previous works in foods implicated in emetic poisoning. At 25 °C, decreasing the pH to 5.4 reduced cereulide production by strain BtB2-4 by at least 20-fold.

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted β-tongue were replaced with glycine (NheC cys− ) or where the entire hydrophobic stretch was deleted (NheC hr− ), the predicted hydrophobic β-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

Clostridium perfringens enterotoxin binds to receptors on Vero cells. This process does not depend on the presence of divalent cations (Ca++, Mg++). Binding of enterotoxin causes inhibition of 14C-leucine incorporation into proteins, probably because of depression of amino acid transport. The presence of Mg++ speeds up this effect of the enterotoxin. The enterotoxin produces membrane leakage only in the presence of Ca++, but additional Mg++ increases the rate of this process. These results indicate that the dissociation constant of the enterotoxin receptor interaction is reduced in the presence of Mg++. A model for the mode of action of the enterotoxin is proposed.

During the course of screening Bacillus species from food and water in Norway, we isolated a strain of Bacillus sphaericus of DNA homology group V, not previously recognized to contain entomopathogenic strains, that was cytotoxic to Vero cell epithelia. Peptide mass fingerprinting of a protein purified from the culture supernatant of B. sphaericus B354 identified a cholesterol-dependent cytolysin (CDC) with high amino acid sequence identity with sphaericolysin, a CDC identified recently in B. sphaericus DNA homology group IIA. The toxin was haemolytic against erythrocytes from a range of species. Haemolysis was potentiated by dithiothreitol and inhibited by preincubation with cholesterol. The toxin induced lactate dehydrogenase release from Vero cells and formed pores in planar lipid bilayers. The distribution of CDC genes in B. sphaericus was examined, with CDC gene products obtained in 13 out of 17 strains representing four of the six DNA homology groups. Thus, we demonstrate the presence of a CDC in a nonentomopathogenic DNA homology group of B. sphaericus (group V) with typical CDC characteristics. CDCs appear to be present in a high proportion of B. sphaericus strains and are not restricted to group IIA insecticidal strains.

Characteristics concerning diarrhoeal potential were investigated in B. cereus dairy strains. The thirty-nine strains, isolated from whipping cream, were tested for cytotoxicity after culturing at human body temperature as well as 25 degrees C and 32 degrees C. At 37 degrees C, none of the strains were highly cytotoxic. This observation suggests that those strains should be considered to pose a minor risk with regard to diarrhoeal food poisoning. However, some strains were moderately or highly cytotoxic when grown at 25 degrees C and 32 degrees C. While the majority of the strains were able to grow at refrigeration temperatures, only four B. weihenstephanensis strains were identified among them when subjected to discriminative PCR assays and growth temperatures which delimit this species.

Food poisoning caused by other Bacillus species than B. cereus has been described, but the toxins involved have rarely been isolated. Endospores will survive heat treatment and will germinate and multiply in cooked foods producing toxins under appropriate conditions. We describe a small food poisoning outbreak where three people became ill after a dinner in a Chinese restaurant. Acute symptoms including dizziness, headache, chills and back pain developed during the meal, and a few hours later they got stomach cramps and diarrhoea which lasted for several days. Cooked, reheated rice was the prime suspect of the food poisoning, and from the rice large numbers of Bacillus pumilus were isolated. The isolated B. pumilus strain was found to produce a complex of lipopeptides known as pumilacidins with the highest amounts produced at 15 °C. This is the first report on isolation of a pumilacidin-producing B. pumilus strain from food implicated in food poisoning and characterization of the organism and the toxin complex involved.

The location of the cpe gene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenic Clostridium perfringens strains by means of pulsed field gel electrophoresis of genomic DNA. The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, the HindIII repeat, and an open reading frame, ORF3, that may be part of an insertion sequence. In contrast, when the strains originated from domesticated livestock cpe was located on a large episome where it was often close to a copy of the transposable element IS1151. In these cases, the HindIII repeat was not linked to the cpe gene although this was generally preceded by ORF3.

Humans may play a role in the transmission of gastrointestinal diseases caused by C. perfringens., We found a prevalence of 18% for enterotoxin gene–carrying (cpe+) Clostridium perfringens in the feces of healthy food handlers by PCR and isolated the organism from 11 of 23 PCR-positive persons by using hydrophobic grid membrane filter-colony hybridization. Several different cpe genotypes were recovered. The prevalence was 3.7% for plasmidial IS1151-cpe, 2.9% for plasmidial IS1470-like-cpe, 0.7% for chromosomal IS1470-cpe, and 1.5% for unknown cpe genotype. Lateral spread of cpe between C. perfringens strains was evident because strains from the same person carried IS1470-like cpe but shared no genetic relatedness according to pulsed-field gel electrophoresis analysis. Our findings suggest that healthy humans serve as a rich reservoir for cpe+ C. perfringens type A and may play a role in the etiology of gastrointestinal diseases caused by this organism. The results also indicate that humans should be considered a risk factor for spread of C. perfringens type A food poisoning and that they are a possible source of contamination for C. perfringens type A food poisoning.

The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus. Nhe is composed of NheA, NheB and NheC. The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site. The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coli. Separate expression showed that all three components were required for biological activity. In addition, NheA and NheB were purified from B. cereus culture supernatants. As NheC seems to be expressed in only small amounts by B. cereus, NheC was expressed and purified as a histidine-tagged fusion protein. The maximum cytotoxic activity was obtained when the molar ratio between NheA : NheB : His6-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

A polymerase chain reaction (PCR) fragment from strain NVH 38 (containing bceT) was cloned, sequenced and expressed in Escherichia coli. This sequence showed 50-60% identity to the original. When this bceT clone was expressed in E. coli no biological activity was found in either supernatants or cell extracts. Cell extracts from the Bacillus cereus strains (NVH 38 and B-4ac) were also negative on Vero cells. Neutralisation of supernatant from B. cereus B-4ac using a monoclonal antibody (reacting with NheB and HblD) abolished the activity. A test for cytotoxic enterotoxins was negative for both cell extracts and supernatants. Our data suggest that BcET either has an unknown type enterotoxic action or non at all.

Clostridium perfringens type A food poisoning is one of the more common in the industrialised world. This bacterium is also responsible for the rare but severe food borne necrotic enteritis. C. perfringens enterotoxin (CPE) has been shown to be the virulence factor responsible for causing the symptoms of C. perfringens type A food poisoning. CPE is a single polypeptide chain with a molecular weight of 3.5 kDa that binds to receptors on the target epithelial cells. Through a unique four-step membrane action it finally causes a breakdown in normal plasma membrane permeability properties. Genetic studies of cpe have shown that cpe can be either chromosomal or plasmid-borne and that only a small minority of the global C. perfringens population is cpe positive. CPE expression appears to be transcriptionally regulated during sporulation, at least in part, by regulatory factors that are common to all C. perfringens isolates.

Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain. Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability. In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins. The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Shiga-toxin-2 (stx(2))-encoding bacteriophages were isolated from Norwegian Escherichia coli O157:H7 isolates of cattle and human origin. The phages were characterized by restriction enzyme analysis, hybridization with probes for toxin genes and selected phage DNA such as the P gene, integrase gene and IS1203, and by PCR studies and partial sequencing of selected DNA regions in the integrase to stx(2) region of the phages. The stx(2)-phage-containing bacteria from which inducible phages were detected produced similar amounts of toxin, as shown by a Vero cell assay. The results indicate that the population of stx(2)-carrying phages is heterogeneous, although the phages from epidemiologically linked E. coli O157:H7 isolates were similar. There appears to have been frequent recombination of stx(2) phages with other lambdoid phages.

A sequence of 91 amino acids residues, probably starting from the N-terminal of the mature protein, was determined for the 105-kDa protein of the non-haemolytic enterotoxin of Bacillus cereus. The last part of this sequence was similar to parts of the N-terminal portions of two collagenases of Clostridium histolyticum and Clostridium perfringens. Zymography, with intact collagen fibril and gelatin as substrates, showed that the 105-kDa protein had collagenolytic and gelatinolytic activity. The 105-kDa protein also showed activity against a typical collagenase substrate, azocoll, and was inhibited by EDTA and 1,10-phenanthroline. We conclude that the 105-kDa protein is a collagenase.

Summary Clostridium perfringens type A produces an enterotoxin that induces diarrhoea experimentally in man and animals. The enterotoxin causes increased membrane permeability in susceptible cells which is thought to be due to pore formation in the host cell membrane. The effect of purified C. perfringens enterotoxin on intact intestinal CaCO-2 monolayers was examined in Ussing chambers and on single cells by whole-cell patch clamp. Mucosal application of C. perfringens enterotoxin resulted in prompt increases in short-circuit current coupled with a reduction in transepithelial resistance consistent with movement of sodium and other cations smaller than diethanolamine from mucosa to serosa. These changes were independent of extracellular calcium. Increases in short-circuit current were also observed in the apical membranes of CaCO-2 monolayers permeabilised across the basolateral membrane with nystatin. Currents were blocked by subsequent exposure to mucosal barium and zinc. Zinc also prevented the development of the current increases in apical membranes. Cationic currents were also observed following exposure of single CaCO-2 cells in whole-cell patch clamp recordings. These data indicate that C. perfringens enterotoxin is able to form cation permeant pores in the apical membrane of human intestinal CaCO-2 epithelia and the increases in short-circuit current can be prevented by pre-exposure to zinc ions.

On 21 January 2012, the Norwegian Food Safety Authority was informed about gastrointestinal illness among 111 swimming club members, who were staying at a hotel in Trondheim. A hotel dinner on 20 January was their only common meal. Kitchen staff were interviewed, and food leftovers and kitchen environment were sampled. A case was defined as a swimming team member staying at the hotel from 20 to 22 January, who fell ill with diarrhoea, abdominal pain or nausea during this period. A total of 43 cases were identified, with median duration of symptoms of 35 hours. cpe-positive Clostridium perfringens (3.8 x 108 CFU), but not Bacillus cereus, was isolated from beef stew eaten by cases. cpe-negative C. perfringens was detected in a sample from the kitchen floor. SDS-PAGE showed indistinguishable protein profiles among C. perfringens cultures isolated from the beef stew, but slightly different profiles from the culture isolated from the kitchen floor. Cohort analysis showed that eating beef stew and rice was significantly associated with illness. No pathogens were detected in the rice. The temperature control of the stew, but not of the rice, was poor. Our results strongly indicate that cases were infected by Clostridium perfringens in beef stew that had inadequate temperature control during preparation.

The structure of NheA, a component of the Bacillus cereus Nhe tripartite toxin, has been solved at 2.05 A resolution using selenomethionine multiple-wavelength anomalous dispersion (MAD). The structure shows it to have a fold that is similar to the Bacillus cereus Hbl-B and E. coli ClyA toxins, and it is therefore a member of the ClyA superfamily of α-helical pore forming toxins (α-PFTs), although its head domain is significantly enlarged compared with those of ClyA or Hbl-B. The hydrophobic β-hairpin structure that is a characteristic of these toxins is replaced by an amphipathic β-hairpin connected to the main structure via a β-latch that is reminiscent of a similar structure in the β-PFT Staphylococcus aureus α-hemolysin. Taken together these results suggest that, although it is a member of an archetypal α-PFT family of toxins, NheA may be capable of forming a β rather than an α pore.

There is at present no accepted nomenclature for bacterial protein toxins, although there have been several attempts at dividing them into groups by their mode of action. In this paper we will not try to describe all known bacterial protein toxins, but concentrate on the toxins involved in food poisoning. Although most of these toxins are enterotoxins (protein exotoxins with the site of action on the mucosal cells of the intestinal tract) there are also other toxins involved in food poisoning, like the neurotoxins. In Table 1 the most important food pathogens in Europe are listed. For most, but not all, of these food pathogens, toxins are virulence factors. Generally, we divide food poisoning into infections and intoxications, where Salmonella spp. and Shigella spp. are typical examples of infections and Clostridium botulinum and Staphylococcus aureus for intoxications. We consider it better to make four different groups of food pathogenic bacteria, according to Table 2. Today the first three groups are all defined as infections, although for both group 2 and 3 the bacterium itself does not harm the host directly. The bacterium in such locations is like an 'enterotoxin factory'. The bacteria belonging to group 3 do not even interact with the epithelial cells in the intestine, while the bacteria of group 2 must colonise the epithelial cells prior to enterotoxin production.

The nonhaemolytic enterotoxin (Nhe) of Bacillus cereus plays a key role in cases of B. cereus food poisoning. The toxin is comprised of three different proteins: NheA, NheB and NheC. Here, the expression in Escherichia coli, purification and crystallization of the NheA protein are reported. The protein was crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant. The crystals of NheA diffracted to 2.05 Å resolution and belonged to space group C2, with unit-cell parameters a = 308.7, b = 58.2, c = 172.9 Å, β = 110.6°. Calculation of V(M) values suggests that there are approximately eight protein molecules per asymmetric unit.

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.

Summary: Enterotoxin production in Clostridium perfringens is both strain dependent and sporulation associated. Underlying these phenotypic observations must lie a genetic and molecular explanation and the principal keys will be held within the DNA sequence both upstream and downstream of the structural gene cpe. In accordance with the above we have sequenced 4·1 kbp of DNA upstream of cpe in the type strain NCTC 8239. A region of DNA extending up to 1·5 kb 5′ to cpe is conserved in all enterotoxin-positive strains. This region contains a putative ORF with substantial homology to an ORF in the Salmonella typhimurium IS200 insertion element and, in addition, contains multiple perfect consensus DNA-binding sequences for the Bacillus subtilis transition state regulator Hpr. The detailed structural elements revealed by the sequence analysis are presented and used to develop a new perspective on the molecular basis of enterotoxin production in this important food-poisoning bacterium.

Bacteriophages (phages) carrying Shiga toxin genes constitute a major virulence attribute in enterohemorrhagic Escherichia coli (EHEC). Several EHEC outbreaks have been linked to food. The survival of such strains in different foods has received much attention, while the fate of the mobile Shiga toxin-converting phages (Stx phages) has been less studied. We have investigated the stability of an Stx phage in several food products and examined how storage, food processing, and disinfection influence the infectivity of phage particles. The study involved a recombinant Stx phage (Δ stx::cat ) of an E. coli O103:H25 strain from a Norwegian outbreak in 2006. Temperature, matrix, and time were factors of major importance for the stability of phage particles. Phages stored at cooling temperatures (4°C) showed a dramatic reduction in stability compared to those stored at room temperature. The importance of the matrix was evident at higher temperatures (60°C). Phages in ground beef were below the detection level when heated to 60°C for more than 10 min, while phages in broth exposed to the same heating conditions showed a 5-log-higher stability. The phages tolerated desiccation poorly but were infective for a substantial period of time in solutions. Under moist conditions, they also had a high ability to tolerate exposure to several disinfectants. In a dry-fermented sausage model, phages were shown to infect E. coli in situ . The results show that Stx phage particles can maintain their infectivity in foods and under food-processing conditions.

Chitosans, polysaccharides obtained from the exoskeleton of crustaceans, have been shown to exert antibacterial activity in vitro and their use as a food preservative is of growing interest. However, beyond a consensus that chitosan appears to disrupt the bacterial cell membrane, published data are inconsistent on the chemical characteristics that confer the antibacterial activity of chitosan. While most authors agree that the net charge density of the polymer (reflected in the fraction of positively charged amino groups at the C-2 position of the glucosamine unit) is an important factor in antibacterial activity, conflicting data have been reported on the effect of molecular weight and on the susceptibility among different bacterial species to chitosan. Therefore, we prepared batches of water-soluble hydrochloride salts of chitosans with weight average molecular weights (M(w)) of 2-224kDa and degree of acetylation of 0.16 and 0.48. Their antibacterial activity was evaluated using tube inhibition assays and membrane integrity assays (N-Phenyl-1-naphthylamine fluorescence and potassium release) against Bacillus cereus, Escherichia coli, Salmonella Typhimurium and three lipopolysaccharide mutants of E. coli and S. Typhimurium. Chitosans with lower degree of acetylation (F(A)=0.16) were more active than the more acetylated chitosans (F(A)=0.48). No trends in antibacterial action related to increasing or decreasing M(w) were observed although one of the chitosans (M(w) 28.4kDa, F(A)=0.16) was more active than the other chitosans, inhibiting growth and permeabilizing the membrane of all the test strains included. The test strains varied in their susceptibility to the different chitosans with wild type S. Typhimurium more resistant than the wild type E. coli. Salmonellae lipopolysaccharide mutants were more susceptible than the matched wild type strain. Our results show that the chitosan preparation details are critically important in identifying the antibacterial features that target different test organisms.

Aims: To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms. Methods and Results: Bacterial counts of overnight cultures showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography–mass spectrometry analysis of an acetone–methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2·5 mg ml−1. MIC values of the Sphagnum-specific compound ‘sphagnum acid’ [p-hydroxy-β-(carboxymethyl)-cinnamic acid] were >5 mg ml−1. No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays. Conclusions: Sphagnum-derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves. Significance and Impact of the Study: We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.

During the spring of 2006, a national disease outbreak caused by Shiga toxin-producing Escherichia coli (STEC) O103:H25 was investigated in Norway. At the time of the outbreak the Norwegian School of Veterinary Science was the national reference laboratory for E. coli O157 in food, and the microbiological investigations to identify the food source were performed there. Food- and environmental samples (n = 931) were collected by the Norwegian Food Safety Authorities following two different hypotheses i) that minced meat was the source of STEC, and ii) that fermented sausage was the source of STEC. Twenty seven food samples, all collected following the latter hypothesis contained eae-positive E. coli O103:H25, but none of these were stx-positive. By PFGE it was shown that isolates from one particular type of fermented sausage “morr sausage 1” were identical to the isolates from patients. Samples of sheep meat that were linked epidemiologically to meat used for sausage production also contained isolates identical or closely related to patient strains. The presented study underpins epidemiological indications that fermented sausage was the source of the outbreak, but points specifically to one particular brand of sausage as the source.

Background Three enterotoxins are implicated in diarrhoeal food poisoning due to Bacillus cereus: Haemolysin BL (Hbl), Non-haemolytic enterotoxin (Nhe), and Cytotoxin K (CytK). Toxin gene profiling and assays for detection of toxin-producing stains have been used in attempts to evaluate the enterotoxic potential of B. cereus group strains. B. cereus strain NVH 391/98, isolated from a case of fatal enteritis, was genetically remote from other B. cereus group strains. This strain lacked the genes encoding Hbl and Nhe, but contains CytK-1. The high virulence of this strain is thought to be due to the greater cytotoxic activity of CytK-1 compared to CytK-2, and to a high level of cytK expression. To date, only three strains containing cytK-1 have been identified; B. cereus strains NVH 391/98, NVH 883/00, and INRA AF2. Results A novel gene variant encoding Nhe was identified in these three strains, which had an average of 80% identity in protein sequence with previously identified Nhe toxins. While culture supernatants containing CytK and Nhe from NVH 391/98 and INRA AF2 were highly cytotoxic, NVH 883/00 expressed little or no CytK and Nhe and was non-cytotoxic. Comparative sequence and expression studies indicated that neither the PlcR/PapR quorum sensing system, nor theYvrGH and YvfTU two-component systems, were responsible for the observed difference in toxin production. Additionally, phylogenetic analysis of 13 genes showed that NVH 391/98, NVH 883/00, and INRA AF2 comprise a novel cluster of strains genetically distant from other B. cereus group strains. Conclusion Due to its divergent sequence, the novel nhe operon had previously not been detected in NVH 391/98 using PCR and several monoclonal antibodies. Thus, toxigenic profiling based on the original nhe sequence will fail to detect the toxin in this group of strains. The observation that strain NVH 883/00 carries cytK-1 but is non-cytotoxic indicates that the detection of this gene variant is not a sufficient criterion for identification of highly cytotoxic strains. The presence of the novel nhe operon and the cytK-1 gene variant in this cluster of strains reflect their phylogenetically remote relationship towards other B. cereus group strains.

The bacteria responsible for causing food poisoning have managed to adapt to the new niches presented to them with modem food processing. Much of the adaptation is through assimilation of new genes, including toxin- and invasion-related genes. The virulence factors that are responsible for the direct action on host tissues, and thereby development of disease, are dependent on the physical and genetic background of both the bacteria and the host. The toxins are, for some of thefood poisoning diseases, the only cause of the symptoms but may for others only increase the severity of the disease. This chapter explores some of these toxins that are on mobile elements and can conceivably be transferred to bacteria thathave not yet been identified as potential food pathogens. It is not sufficient for bacteria to possess genes that can encode for toxins or other virulence factors. The bacteria must also be able to survive one or a number of detrimental and changing environmental factors, including dehydration, heat, cold, low pH values (stomach), competing flora, and other intestinal defense mechanisms. There are many bacteria that carry the same or equivalent virulence genes as bacteria known to cause foodborne illness that have not yet been fully recognized as cause of a human gastroenteritis, i.e., Bacteroides fragilis and Citrobacter spp.

International audience; Bacillus cereus is the causative agent of gastrointestinal diarrhoea. At least three known enterotoxins may be involved in this syndrome: nonhaemolytic (Nhe) enterotoxin, Hbl enterotoxin and cytotoxin K. Two different forms were recently described for cytotoxin K, encoded by cytK-1 and cytK-2 genes. The CytK-1 toxin appeared to carry a high toxicity, but there is currently no method available to rapidly detect and discriminate the B. cereus strains able to produce this CytK-1 form. In this study, a duplex PCR assay was developed and validated on 162 known cytotoxin-containing strains. This PCR method is the first molecular tool to provide rapid detection and discrimination of cytK-1- and cytK-2-carrying B. cereus strains.

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.

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°C, and no reduction in activity was observed even after 24 h of growth at 42°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°C. None of the cytotoxic strains produced B. cereus -like enterotoxins as tested by PCR or by immunological methods.

CytK is a cytotoxin isolated from a strain of Bacillus cereus cultured from cases of necrotic enteritis and the amino acid sequence of the protein suggests that it may belong to the family of beta-barrel pore-forming toxins. We show here in planar lipid bilayers the toxin is able to form pores which are weakly anion selective and exhibit an open channel probability close to one. The predicted minimum pore diameter is approximately 7 A. We also show that cytK is a potent cytotoxin against human intestinal Caco-2 epithelia. CytK, like other beta-barrel pore-forming toxins, spontaneously forms oligomers which are resistant to sodium dodecyl sulphate (SDS), but not to boiling. CytK represents a pore-forming toxin linked with human cases of necrotic enteritis.

A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people. A protein of 34 kDa was highly cytotoxic, and the addition of other secreted proteins gave no synergistic effect. CytK was also necrotic and haemolytic. No known B. cereus enterotoxins were produced by this strain. A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced. The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, gamma-haemolysin and alpha-haemolysin, Clostridium perfringens beta-toxin and B. cereus haemolysin II, all belonging to a family of beta-barrel channel-forming toxins. There was no sequence similarity between CytK and enterotoxins of B. cereus. The upstream sequence contained a partial sequence of a putative histidine kinase gene. A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B. cereus, was found in the promoter region of the toxin. This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the beta-toxin of C. perfringens type C.

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.

Chymotrypsin treatment of the enterotoxin from Clostridium perfringens results in the loss of 36 amino acids from the N-terminus and 3 amino acids from the C-terminus. This processing results in a 3.2 fold increase in activity on Vero cells, which is close to what had been found for trypsin treatment following the loss of 25 amino acids from the N-terminus.

Thirty-one isolates of Aeromonas spp. from food and water in Norway were classified and tested for possible virulence factors including cytotoxins (tissue cultures, PCR), enterotoxins (PCR) and invasion ability (Caco-2 cells). Five different species were recorded, A. caviae (9/31), A. hydrophila (15/31), A. schubertii (3/31), A. trota (3/31) and A. veronii biovar veronii (1/31). One of the A. hydrophila strains was probably responsible for a small outbreak of food poisoning caused by ingestion of raw fermented fish. All the A. hydrophila strains produced and secreted cytotoxins at 37 degrees C, as well as two A. trota strains and the single A. veronii biovar veronii strain. In some cases increased cytotoxin secretion was observed under osmotic stress. The majority of the A. caviae strains which produced cytotoxins at 30 degrees C were unable to produce and/or secrete cytotoxins at 37 degrees C. One A. schubertii strain and one A. caviae strain were invasive.

The cytotoxicity of the two different enterotoxin complexes ofBacillus cereuswas compared after isolation from three different strains. Protein components of non-haemolytic enterotoxin (NHE) of 39 kDa, 45 kDa and 105 kDa were isolated from all of the three strains, whilst proteins B, L1and L2of haemolysin BL (HBL) were isolated from supernatants of two strains (F837-76 and 1230-88). These proteins were not detected in strain 0075-95. Inhibition of protein synthesis in Vero cells was used as a measure of cytotoxicity. The HBL complex from strain F837-76 was highly toxic. This strain also produced the NHE complex. However, when purified, at least two of the components of NHE had to be present in higher amounts than those of the components of HBL to cause the same degree of toxicity. Both complexes purified from strain 1230-88 were cytotoxic. The amount required to cause the same degree of cytotoxicity was approximately equal for the components of the two complexes, except that higher amounts of the 105 kDa protein of NHE had to be present than for the other components. None of the purified complexes from strain 1230-88 was toxic in amounts comparable to those of the HBL complex of strain F837-76 and NHE of strain 0075-95. These results indicate that when measuring cytotoxic enterotoxins fromB. cereusat least two different complexes and six different proteins have to be taken into consideration.

Summary: The Clostridium perfringens enterotoxin gene (cpe) is rarely found in naturally isolated strains. In human food poisoning strains, cpe is found on the chromosome, and is located episomally in animal isolates. Observations that the gene was somewhat unstable and could be gained or lost suggested that the gene was on a mobile element. An IS200-like element, IS1469, is almost always upstream of cpe. A new insertion element was identified, IS1470, a member of the IS30 family, which is found both up- and downstream of cpe in the type A strain NCTC 8239. PCR results confirmed that this configuration was conserved in type A human food poisoning strains. The enterotoxin gene was on a 6.3 kb transposon which, in addition to the two flanking copies of IS1470, included IS1469 and two 1 kb stretches, one on each side of cpe, with no open reading frames. Results indicated that 14 bp was copied from the genome during insertion. Details of the configuration of DNA in this transposon are presented, and the possible connection of this transposon with the movement of the enterotoxin gene is discussed.

Three enterotoxic components have been isolated from a strain of Bacillus cereus which was involved in a large food poisoning outbreak in Norway in 1995. The components were purified by chromatography on three different columns. Three proteins of 39, 45 and 105 kDa, respectively, were found to be necessary for maximum cytotoxicity. The amino acid N-terminal sequences of the 39 and 45 kDa proteins were determined. The 45 kDa component was the same protein as the main antigen detected in the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra). The 39 kDa protein showed some similarity to the l1 protein of haemolysin BL from B. cereus. Furthermore, the three toxic components were all recognised by a polyclonal antiserum reported to detect enterotoxin from B. cereus. The proteins were different from the B- and L2-components of haemolysin BL, previously suggested to be a primary virulence factor, and had no detectable haemolytic activity.

Out of 321 strains of Bacillus cereus from several sources and isolated in four different countries, 239 (74%) produced cytotoxins. Only 127 (53%) of the cytotoxic strains were positive for the B-component gene of the haemolysin BL (enterotoxin) by polymerase chain reaction (PCR). Western blots using antiserum produced against enterotoxin(s) gave positive results for 199 (83%) of the cytotoxic B. cereus strains. On closer examination of seven of the strains, involved in food poisoning, we found that two strains completely lacked the L2- and B-components (of the haemolysin BL), and two strains were negative for the B-component gene by PCR, but were positive for the L2-component. From our experiments we concluded that there is at least one enterotoxin complex in addition to the haemolysin BL enterotoxin and enterotoxin T.

Spore-forming bacteria are special problems for the food industry. It is not always possible to apply enough heat during food processing to kill spores, thus we have to take advantage of knowledge of the spore-formers to control them. For the meat industry Clostridium perfringens might become a special problem, although this bacterium mainly causes food poisoning through food served in restaurants, hospitals or homes for elderly people (Cliver, 1987; Reynolds, 1987; Gondrosen et al., 1990). The reason for the food poisoning is always the same: meat-containing dishes stored after cooking with insufficient cooling and reheating (Granum, 1990). Even though it should be relatively easy to control this kind of food poisoning, C. perfringens is still one of the most common sources of foodborne diseases. Proper disinfection is necessary to control this type of food poisoning, as it is now clear that only kitchen strains of C. perfringens are able to produce the large amounts of enterotoxin necessary to cause food poisoning (Granum, 1990; Cornillot et al., 1995). Bacillus cereus is more difficult to control, specifically in the dairy industry, where it is now causing the main problems. Insufficient heating of rice-containing dishes has been known to cause B. cereus food poisoning of the emetic kind for a long time (Kramer and Gilbert, 1989), but will not be dealt with in this paper. There are several reasons for the problems in the dairy industry. First of all it seems to be impossible to completely avoid the presence of B. cereus in all milk samples. Secondly the spores are very hydrophobic (Husmark, 1993), and will attach to the surfaces of the pipelines of the dairy industry, where they might multiply and resporulate. A third problem is that pasteurisation heating is insufficient to kill the spores, while competition from other vegetative bacteria is eliminated. It seems that several B. cereus strains have become psychrotrophic over the years, making possible growth at temperatures as low as 4-6 degrees C (Granum et al., 1993a). None of the methods used to control hygiene in the dairy industry so far are able to control B. cereus. This is a continuously increasing problem for the industry but, with emerging knowledge, we should be able to control it. In this paper we will discuss the problems the food industry is facing with C. perfringens and B. cereus, and how these problems might be solved. We will also give our view on how research might ease these problems in the future.

Of 85 strains of Bacillus cereus isolated in Norway from dairy products, 59% were found to be enterotoxigenic, and 15% were psychotrophic. Six of the isolates (7%) were identified as potential psychotrophic food-poisoning strains as they were both enterotoxigenic and exhibited good growth at 6°C. Enterotoxin production was detected using the Western immunoblot technique, and a commercially available reversed passive latex agglutination (RPLA) assay (Unipath BCET-RPLA TD950). Both methods gave essentially the same results. In a separate study, the Western immunoblot and RPLA assays were used in a conjunction with the in vivo vascular permeability reaction (VPR) assay to determine enterotoxin production among 25 isolates of Bacillus cereus referred to the PHLS Food Hygiene Laboratory from incidents of diarrhoeal- and emetic-syndrome food poisoning and non-gastrointestinal infections. Eighty-four percent of these isolates were found to be enterotoxigenic by the Western immunoblot and the RPLA assays, and these results were in good agreement with those obtained by the VPR assay. In both studies, the BCET-RPLA kit proved to be a simple and reliable means for determining enterotoxin production by strains of Bacillus cereus .

Clostridium perfringens is a large, gram-positive, spore-forming, anaerobic and nonmotile rod belonging to the family Bacillaceae. It is a common inhabitant of the soil and plays an important part in the putrefication process. C. perfringens is also readily isolated from dust, raw meat, water, and the intestinal tract of man and animals, and is the clostridial species most frequently isolated from clinical specimens. Because this organism produces numerous toxins and enzymes, it can cause a spectrum of different diseases in man and animals (for review, see McDonel, 1986). The species is divided into five types based on the production of four major lethal toxins (Table 16.1).

A simple, rapid and sensitive immunoassay, based on immunomagnetic particles (Dynabeads M-280) was developed for detection and quantitation of Clostridium perfringens type A enterotoxin from faecal and food extracts. The assay had a detection limit of 2.5 ng/ml enterotoxin in homogenates of faeces and inoculated meat extracts. The specificity was confirmed by both crossed immunoelectrophoresis and Western immunoblotting techniques, using a purified enterotoxin as standard.

In Clostridium perfringens the acid-soluble spore proteins (ASSPs) of the alpha/beta type are encoded by two genes, sspC1 and sspC2. These genes, encoding ASSP C1 and C2 proteins, respectively, were cloned as parts of 3.5-kb genomic fragments. The genes were sequenced and polypeptides of 59 (C1) and 60 (C2) amino acids (aa) were deduced from the nucleotide sequence. The N-terminal Met is removed from the primary translation products in both cases, yielding mature polypeptides with Mrs of 6306 (C1) and 6553 (C2). The aa sequences of the two polypeptides were very similar and showed a closer relationship with C. bifermentans ASSPs than with ASSPs from Bacillus species.

Clostridium perfringens is a Gram-posit ive spore-forming, anaerobic and nonmotile rod. It is a normal soil inhabitant, and plays an important part in the putrification process. C. perfringens is also readily isolated from dust, raw meat and the intestinal tract of man and animals. It is divided into five types based on the production of four major lethal toxins, as shown in Table I. Table I also shows the different types of diseases caused by C. perfringens. The enterotoxin has been shown to be produced by types A, C and D (Table I), but is not used for typing C. perfringens. C. perfringens is responsible for two very different types of food poisoning (Table I). Type A causes the mild classic form of food poisoning, while the severe type of food poisoning known as necrotic enteritis is caused by type C. The symptoms of the C. perfringens type A food poisoning are: acute abdominal pain and diarrhoea with nausea and fever, with vomiting being rare. The symptoms usually appear 8-12 h (6-24 h) after ingestion of contaminated food (106-107 cells/g) and can last between 12-24 h (Hobbs, 1969). The poisoning is seldom fatal, although a few deaths have been reported in elderly and infirm (Parry, 1963; Sutton and Hobbs, 1965). The true incidence of C. perfringens food poisoning is not known, because of the relative mildness of the syndrome. Nevertheless, in several countries C. perfringens has been reported as the most important cause of food poisoning, only exceeded in incidence by food infections caused by Salmonella (Cliver, 1987; Reynolds, 1987). Necrotic enteritis is a rare and often fatal illness. The symptoms are: acute sudden onset of severe abdominal pain and diarrhoea (often bloody), sometimes with vomiting, and necrotic inflammation of the small intestine.

Growth, sporulation and enterotoxin production have been followed for three different strains of Clostridium perfringens on a vegetative (FTG) medium and a sporulation (DS) medium. Enterotoxin production was followed by use of enzyme-linked immunosorbent assay (ELISA). All strains produced enterotoxin under both vegetative growth and sporulation in spite of the fact that one strain is known to be enterotoxin negative. The presumed negative strain produced about the same amount of enterotoxin on both media (1 ng/ml). The two other strains produced about 1000–2000 times more enterotoxin during sporulation than during vegetative growth.

An acid-soluble spore protein (ASSP) has been isolated from Clostridium perfringens spores. The protein contains 56 amino acids, and 14 of them are homologous to all the other ASSP known. Fourteen other amino acids are also homologous with amino acids from some of the ASSP from Bacillus or Thermoactinomyces.

Far-UV circular-dichroism spectra of the enterotoxin from Clostridium perfringens type A were recorded under different conditions, and conformational analysis was performed. The native enterotoxin (pH 6.8) was shown to contain about 80% pleated sheet, 20% random coil and no helix structure. By adding small amounts of the detergent sodium dodecyl sulfate (0–261 mol SDS/mol enterotoxin), the β-sheet structure could gradually be changed to a more α-helical structure. Treatment with 8 M urea resulted in limited alterations of the secondary structure of the enterotoxin, while heat treatment (60°C, 30 min), treatment with 6.5 M guanidine hydrochloride or 0.01 M NaOH led to a dramatic increase in the amount of random coil.