The pattern of toxin genes and expression of diarrheal enterotoxins in Bacillus thuringiensis strains isolated from commercial bioinsecticides

The pathogenic profile of 13 Bt strains of isolated from commercial bioinsecticides was defined. On the basis of the information on the labels, 10 strains belonged to the subsp. kurstaki and 3 to the subsp. tenebrionis, israeliensis and aizawai. The production of the diarrheal enterotoxins HBL (haemolytic fraction L2) and NHE (proteic fraction A, 45-kDa) was evaluated on the broth culture incubated overnight, using RPLA (BCET-RPLA, Oxoid) and ELISA (Tecra, Australia) commercial kits, following the manufacturers’ instructions. After the DNA extraction, the genes encoding for virulence factors were detected by means of PCR: cerA (phospholipase C); cerB (sphingomyelinase) (Schraft and Griffiths 1995); bceT (enterotoxin T) (Agata et al. 1995); cytK (cytotoxin k) (Lund et al. 2000); operon hbl genes hblA, hblC, and hblD, respectively encoding for the linking component B and the lythic components L 1 and L 2 ; operon nhe genes nheA, nheB, and nheC, encoding for proteins A, B and C respectively (Hansen and Hendriksen 2001); as well as the ces fragment (cereulide), which is common to all the emetic strains (Ehling-Schulz et al. 2005).All the isolates showed the typical features of the Bc group microrganisms. Four of the strains (30.8 %) were identified as Bt by the VITEK system (ID > 70 %), while nine strains were identified as Bc (69.2 %). Genes cerA and cerB were found in all the strains. The three hbl operon genes were identified in 12 isolates while in one (subsp. tenebrionis) the hblD and hblC genes were not found. Of the tested isolates, 12 produced the L2 fraction while this was not the case in the strain where hblC was absent. The concentrations of subunit L2 in the overnight broth culture detected by ELISA were between ≥8 and 512 mg mL−1. All nhe operon genes were found in the 13 isolates, while production of the proteic fraction A was detected in 11 (84.6 %). The two isolates that did not produce the proteic fraction A were the subspecies kurstaki and israeliensis. The bceT gene was detected in 13 isolates (100 %) and the cytK gene in 11 (84.6 %). The ces fragment was not found in any of the isolates. There was concordance between the PCR and RPLA results (100 %), while 84.6 % of agreement was found between PCR and ELISA. In two strains the presence of nheA was not associated with the detection of the A protein.