Characterization of Toxin Gene Distribution and Toxin Production Provides Insight to the Potential Differentiation of Pathogenic and Non-pathogenic Bacillus cereus Group Strains

Introduction: The Bacillus cereus Group consists of 9 highly genetically related species, some causing foodborne illness, and some being used as insecticidal agents or as probiotics for livestock. Current differentiation methods based on colony morphology and standard biochemical methods do not accurately differentiate between species, therefore methods allowing for better differentiation between potentially pathogenic and non-pathogenic strains within the Bacillus cereus Group is needed. 

Purpose: The purpose of this study was to characterize the distribution of genes encoding toxins among Bacillus cereus strains, and to determine the expression of various virulence factors, including their expression at human body temperature.

Methods: A total of 66 bacterial isolates previously identified as belonging to the B. cereus Group using a 632 nt segment of the rpoB gene were characterized in this study. Briefly, the presence of 8 different toxin encoding genes were detected using PCR. The colony morphology of strains representing 43 unique rpoB allelic types was observed using Brain Heart Infusion and Bacara agar plates. The activity of phosphoinositide phospholipase C(PI-PLC) was characterized using Bacillus cereus/Bacillus thuringiensis chromogenic plating medium. Hemolysis activity was determined using the FDA BAM method. Growth at refrigerated temperatures was determined following 21 days incubation at 6-7°C. Finally, production of enterotoxins Nhe and Hbl was detected using Duopath® Cereus Enterotoxins (Merck) kits.

Results: Overall, the majority of isolates screened encoded all 8 toxin genes.  Out of 43 strains, 40 were hemolytic, 39 showed PI-PLC enzymatic activity, and 13 were able to grow at cold temperatures. Morphology on both BHI and Bacara agar did not allow for differentiation among species within the group. Preliminary results indicate that the isolates tested produce toxins when strains are grown at 37°C.

Significance: These results provide detailed information for strains within the B. cereus Group, and may potentially contribute to improved differentiation of pathogenic and non-pathogenic strains within the B. cereus Group.