Several powdered food products were screened using repetitive element palindrome PCR (rep-PCR) for the presence of enterotoxin producing species of Bacillus. Samples from these products were screened by being placed into a tryptone-peptoneglucose-yeast enrichment medium (TPGY), heat-treated, and shake-incubated. DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified through rep-PCR using extragenic sequence-targeting primers and optimized for each food product. Amplified PCR products were analyzed electrophoretically and viewed using an ultraviolet photodocumentation system. Bacillus cereus positive control DNA fingerprints were compared to banding patterns from enriched food samples, revealing the presence of the typical diagnostic 1,230 bp band in non-fat dry milk (NFDM). Restriction Fragment Length Polymorphism (RFLP) with Alu I restriction enzyme was performed on the 1230 bp diagnostic band from NFDM and displayed a profile consistent with Bacillus cereus positive control. RPLA (Reverse Passive Latex Agglutination) and BDE ELISA (Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay - Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples. / Department of Biology

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology