Toxin Genes Profiles and Toxin Production Ability of Bacillus cereus Isolated from Clinical and Food Samples

Bacillus cereus causes exotoxin-mediated diarrheal food poisoning. Hemolysin BL (HBL) is a well-characterized B. cereus toxin composed of three components (B, L1, and L2) that together possess hemolytic, cytotoxic, dermonecrotic, and vascular permeability activities. Here, we show that HBL causes fluid accumulation in ligated rabbit ileal loops at a dose of 5 micrograms of each component per loop. Maximal fluid responses occurred for combinations of all three components at > or = 25 micrograms of each component per loop. Individual components and binary combinations did not cause significant fluid accumulation at 25 micrograms of each component. Specific antisera to HBL components inhibited the fluid accumulation response of crude culture supernatant from B. cereus F837/76. These antisera were tested against an antiserum to a partially characterized multicomponent diarrheal toxin described previously by Thompson et al. (N. E. Thompson, M. J. Ketterhagen, M. S. Bergdoll, and E. J. Shantz, Infect. Immun. 43:887-894, 1984). Immunoblot and immunoprecipitation analyses indicate that HBL and that toxin are identical. These results confirm previous speculation that HBL is a tripartite enterotoxin that, as for all of its other known activities, requires all three components for maximal activity.

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 non-haemolytic enterotoxin from Bacillus cereus has been sequenced. It is composed of three components, non-haemolytic enterotoxin A, B and C of 41.0, 39.8 and 36.5 kDa, respectively. Transcription of the operon seems to be positively regulated by plcR, a gene that also regulates phospholipase C expression. There is substantial similarity between the three proteins of non-haemolytic enterotoxin and between the non-haemolytic enterotoxin and haemolytic enterotoxin proteins.