Enterotoxin production in natural isolates of Bacillaceae outside the Bacillus cereus group

Thirty-nine Bacillus strains obtained from a variety of environmental and food sources were screened by PCR for the presence of five gene targets (hblC, hblD, hblA, nheA, and nheB) in two enterotoxin operons (HBL and NHE) traditionally harbored by Bacillus cereus. Seven isolates exhibited a positive signal for at least three of the five possible targets, including Bacillus amyloliquefaciens, B. cereus, Bacillus circulans, Bacillus lentimorbis, Bacillus pasteurii, and Bacillus thuringiensis subsp. kurstaki. PCR amplicons were confirmed by restriction enzyme digest patterns compared to a positive control strain. Enterotoxin gene expression of each strain grown in a model food system (skim milk) was monitored by gene-specific reverse transcription-PCR and confirmed with the Oxoid RPLA and Tecra BDE commercial kits. Lecithinase production was noted on egg yolk-polymyxin B agar for all strains except B. lentimorbis, whereas discontinuous beta hemolysis was exhibited by all seven isolates grown on 5% sheep blood agar plates. The results of this study confirm the presence of enterotoxin genes in natural isolates of Bacillus spp. outside the B. cereus group and the ability of these strains to produce toxins in a model food system under aerated conditions at 32 degrees C.