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.

An aerobic endospore-forming bacillus ) was isolated during a severe food poisoning outbreak in France in 1998, and four other similar strains have since been isolated, also mostly from food poisoning cases. Based on 16S rRNA gene sequence similarity, these strains were shown to belong to the Bacillus cereus Group (over 97 % similarity with the current Group species) and phylogenetic distance from other validly described species of the genus Bacillus was less than 95 %. Based on 16S rRNA gene sequence similarity and MLST data, these novel strains were shown to form a robust and well-separated cluster in the B. cereus Group, and constituted the most distant cluster from species of this Group. Major fatty acids (iso-C 15 : 0 , C 16 : 0 , iso-C 17 : 0 , anteiso-C 15 : 0 , iso-C 16 : 0 , iso-C 13 : 0 ) supported the affiliation of these strains to the genus Bacillus, and more specifically to the B. cereus Group. NVH 391-98 T taxon was more specifically characterized by an abundance of iso-C 15 : 0 and low amounts of iso-C 13 : 0 compared with other members of the B. cereus Group. Genome similarity together with DNA-DNA hybridization values and physiological and biochemical tests made it possible to genotypically and phenotypically differentiate NVH 391-98 T taxon from the six current B. cereus Group species.NVH 391-98 T therefore represents a novel species, for which the name Bacillus cytotoxicus sp. Abbreviations: DDH, DNA-DNA hybridization; MLST, multilocus sequence typing.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene of strains are described in Tables 1 and S1. The sequences of the MLST genes can be found at http://mlstoslo.uio.no/index.html.A supplementary figure and three supplementary tables are available with the online version of this paper.

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 : His 6 -NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

The antibacterial effect of disinfectants is crucial for the control of Listeria monocytogenes in food processing environments. Tolerance of L. monocytogenes to sublethal levels of disinfectants based on quaternary ammonium compounds (QAC) is conferred by the resistance determinants qacH and bcrABC. The presence and distribution of these genes have been anticipated to have a role in the survival and growth of L. monocytogenes in food processing environments where QAC based disinfectants are in common use. In this study, a panel of 680 L. monocytogenes from nine Norwegian meat- and salmon processing plants were grouped into 36 MLVA profiles. The presence of qacH and bcrABC was determined in 101 isolates from the 26 most common MLVA profiles. Five MLVA profiles contained qacH and two contained bcrABC. Isolates with qacH and bcrABC showed increased tolerance to the QAC Benzalkonium chloride (BC), with minimal inhibitory concentrations (MICs) of 5-12, 10-13 and <5ppm for strains with qacH (two allele variants observed), bcrABC, and neither gene, respectively. Isolates with qacH or bcrABC were not more tolerant to BC in bactericidal tests in suspension or in biofilms compared with isolates lacking the genes. Water residue samples collected from surfaces in meat processing plants after QAC disinfection had bactericidal effect against L. monocytogenes when the sample BC levels were high (>100ppm). A sample with lower BC concentrations (14ppm of chain length C-12 and 2.7ppm of chain length C-14) inhibited growth of L. monocytogenes not containing bcrABC or qacH, compared to strains with these genes. The study has shown that L. monocytogenes harbouring the QAC resistance genes qacH and bcrABC are prevalent in the food industry and that residuals of QAC may be present in concentrations after sanitation in the industry that result in a growth advantage for bacteria with such resistance genes.

The mechanism by which Bacillus cereus causes diarrhoea is unknown. Three putative enterotoxins have been proposed, haemolysin BL (Hbl), cytotoxin K and non-haemolytic enterotoxin (Nhe). Both Hbl and Nhe are three-component cytotoxins and maximal cytotoxicity of Nhe against epithelia is dependent on all three components. However, little is known of the mechanism of cytotoxicity. Markers of plasma membrane disruption, namely propidium iodide uptake, loss of cellular ATP and release of lactate dehydrogenase (LDH), were observed in epithelia exposed to Nhe from culture supernatants of B. cereus, but not in those exposed to supernatants from a mutant strain lacking NheB and NheC. Consistent with an exogenous cause of membrane damage, purified Nhe components combined to form large conductance pores in planar lipid bilayers. The inhibition of LDH release by osmotic protectants and the increase in cell size caused by Nhe indicate that epithelia lyse following osmotic swelling. Nhe and Hbl show sequence homology, and Hbl component B has remarkable structural similarities to cytolysin A (ClyA), with both structures possessing an a-helix bundle and a unique subdomain containing a hydrophobic b-hairpin. Correspondingly, we show that Nhe has haemolytic activity against erythrocytes from a variety of species. We propose that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins.