Detection of EnterotoxicBacillus cereusandBacillus thuringiensisStrains by PCR Analysis

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Abstract
Many strains ofBacillus cereuscause gastrointestinal diseases, and the closely related insect pathogenB. thuringiensishas also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (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 22B. cereusand 41B. thuringiensisstrains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of theB. thuringiensisstrains, while sixB. cereusstrains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate thatbceTis widely distributed amongB. cereusandB. thuringiensisstrains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected thebceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceTgene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereusandB. thuringiensisbased on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection ofbceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.