The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group

Abstract

The prevalence of the hemolytic enterotoxin complex HBL was determined in all species of the Bacillus cereus group with the exception of Bacillus anthracis. hblA, encoding the binding subunit B, was detected by PCR and Southern analysis and was confirmed by partial sequencing of 18 strains. The sequences formed two clusters, one including B. cereus and Bacillus thuringiensis strains and the other one consisting of Bacillus mycoides, Bacillus pseudomycoides, and Bacillus weihenstephanensis strains. From eight B. thuringiensis strains, the enterotoxin gene hblA could be amplified. Seven of them also expressed the complete HBL complex as determined with specific antibodies against the L(1), L(2), and B components. Eleven of 16 B. mycoides strains, all 3 B. pseudomyoides strains, 9 of 15 B. weihenstephanensis strains, and 10 of 23 B. cereus strains carried hblA. While HBL was not expressed in the B. pseudomycoides strains, the molecular assays were in accordance with the immunological assays for the majority of the remaining strains. In summary, the hemolytic enterotoxin HBL seems to be broadly distributed among strains of the B. cereus group and relates neither to a certain species nor to a specific environment. The consequences of this finding for food safety considerations need to be evaluated.

FIG. 1

Southern blot analysis of selected strains. The genomic DNA was digested with EcoRI. Southern analysis was performed using a DIG-labelled oligonucleotide probe recognizing the B. cereus hblA. Lane 1, WS2621; lane 2, WS2623; lane 3, WS2641; lane 4, WSBC10278; lane 5, WSBC10204; lane 6, WSBC10201; lane 7, WSBC10027; lane 8, WSBC10042. MWS, molecular weight standard. Molecular size is given in kilobases.

FIG. 2

Similarity network based on the nucleotide sequences of the B component of the hemolytic enterotoxin of the B. thuringiensis (B. t.) strains WS2734, WS2629, and WSBC28002; the B. mycoides (B. m.) strains WS2641, WSBC10277, WSBC10360, and WSBC10256; the B. pseudomycoides (B. pm.) strains WS3118, WS3119, and WS3120; the B. weihenstephanensis (B. w.) strains WSBC10202, WSBC10204, WSBC10206, and WSBC10364; and the B. cereus (B. c.) strains WSBC10027, WSBC10028, WSBC10249, and WSBC10312 as compared to the B. cereus L20441 (26). The neighbor-joining method (38) was used, and bootstrap analysis was performed with 100 repeats. For strain designations see Table 1.