Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.

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.Bacillus cereus is known to cause two different types of food poisoning (for reviews, see references 9, 15, and 26), which are characterized by either emesis or diarrhea. At present, two different protein complexes, each consisting of three exoproteins, as well as a single protein (cytotoxin K) (19), are discussed as causative agents (9, 13). The enterotoxin described by Beecher and Wong (3, 5), consisting of the components B, L 1 , and L 2 , showed hemolytic activity and was therefore named hemolysin BL (HBL). HBL has been characterized intensively in view of the biological activity (5) as well as genetically (14,25). The nonhemolytic enterotoxin (Nhe) described by Lund and Granum (17) contains the protein components NheA (41.0 kDa), NheB (39.8 kDa), and NheC (36.5 kDa). The genes encoding the components of Nhe have been cloned and characterized, and it has been shown that they are transcribed as one operon (10, 16).Specific monoclonal antibodies (MAbs) for immunochemical studies on the protein level are available only for HBL (6). Due to this limitation, the detection of the B. cereus enterotoxins is still not satisfactory, and a range of in vivo and in vitro tests is used to estimate the toxicity of B. cereus isolates, e.g., the mouse lethality test, the rabbit ileal loop test, the vascular permeability reaction, and cell culture assays (1, 3, 5, 27). These assays, however, do not allow differentiation between the specific activities of the individual toxins. On the other hand, several studies published during recent years showed that nearly all strains of B. cereus harbor nhe, whereas hbl genes were detected in only about 50% of the tested isolates (7,11,12,21,22,28). Although there is some evidence that both c...

This study focuses on the interaction of the three components of the Bacillus cereus

No abstract

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx 1 gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx 1 from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx 2 by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods. Shiga toxins (Stx) are highly potent cytotoxins and essential virulence factors of enterohemorrhagic Escherichia coli (EHEC).Based on antigenic, cytotoxic and genotypic differences, two major types of these toxins, Stx1 and Stx2, are known, both of which are phage encoded. Stx2 includes several subtypes, Stx2c (26), Stx2d (22), Stx2e (30), and Stx2f (25) being the most important ones. In contrast, Stx1 is rather homogenous and basically identical with Shiga toxin of Shigella dysenteriae, only one amino acid is replaced and three nucleotide changes were detected (28). However, during the last decade, several minor variants of Stx1 have been described as well. Paton et al. (20,21) identified three variants which differed by two amino acids in the A subunit. Nucleotide sequence homology of these variants with stx 1 of phage 933J was more than 99%. A more substantial deviation was observed only in strain