Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis

Many strains of Bacillus cereus cause gastrointestinal diseases, and the cl osely related insect pathogen B. thuringiensis has also been involved in ou tbreaks of diarrhea. The diarrheal types of diseases are attributed to ente rotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) a nd nonhemolytic entero toxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thu ringiensis strains, while six B. cereus strains were devoid of all three HB L genes, three lacked at least two of the three NHE genes, and one lacked a ll three. Five different sets of primers were used for detection of the gen e (bceT) encoding enterotoxin T. The results obtained with these primer set s indicate that bceT is widely distributed among B. cereus and B. thuringie nsis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously dete cted the bceT gene, as confirmed by Southern analysis. The occurrence of th e genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for det ection of enterotoxin-encoding genes in B. cereus and B. thuringiensis base d an PCR analysis with the six primer sets for the detection of genes in th e HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcrib ed spacer region revealed identical patterns for all strains studied.