Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers

Eight new pairs of PCR primers were designed and efficiently detect eight toxin genes (hblC, hblD, hblA, nheA, nheB, nheC, cytK, and entFM) in 411 B. cereus strains (121 food- and 290 soil isolates) and 205 B. thuringiensis strains (43 serovars, 10 food- and 152 soil isolates). According to the presence of these eight toxin genes, they were divided into four groups among the total 616 isolates. In Group I, all eight genes occurred simultaneously in 403 (65.42%) isolates, while Group II (134 isolates or 21.75%) and Group III (46 isolates or 7.47%) were devoid of hblCDA and cytK, respectively. In Group IV, there were thirty-three isolates which lacked both hblCDA and cytK. The presence of hblCDA in B. thuringiensis strains (86.80%) was significantly higher (P < 0.05) than in B. cereus strains (66.18%) whereas no significant difference in nheABC, cytK and entFM occurrence was detected between both bacterial groups. Both nheABC and entFM genes were found in all B. cereus and B. thuringiensis strains (616 strains in total), while the cytK gene could be detected in 365 (88.80%) of the B. cereus and 172 (83.90%) of the B. thuringiensis strains. None of the 616 tested strains showed the presence of only a single or two genes in either the hbl or nhe operons. The eight primer pairs designed for this multiplex PCR allowed rapid detection of eight toxin genes from boiled cells with high sensitivity, gave 100% reproducibility, and did not cross-react to 32 other bacterial strains.