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<UL> <LI> Determine the growth potential of Bacillus cereus in fish. <LI> Identify strategies for preventing and limiting growth of toxigenic B. cereus in/on fresh seafood. <LI> Identify the signal(s) which initiate sporulation and subsequent enterotoxin formation by Clostridium perfringens. <LI> Determine the ability of spores of each species to grow in inoculated pack studies. <LI> Graduate students will be trained in microbial food safety and results presented at national meetings and submitted for publication in peer-reviewed food microbiology publications for promulgation to food safety professionals.
NON-TECHNICAL SUMMARY: Seafood in increasingly consumed in the U.S. The ability of a particular foodborne pathogen to grow on raw seafood will be determined as will the process leading to the production of a toxin responsible for foodborne illness.
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APPROACH: Cored samples of salmon will be inoculated with active cultures or spores of Bacillus cereus at inoculum levels of 100 to 100 colony forming units per gram. Strains to be used will be those previously isolated from seafood. Samples will be held at temperatures representing mild temperature abuse for 24 and 48 hours at 7 C, 12 C and 20 C. Levels of growth will be determined using a new chromogenic agar following appropriate dilutions. In cases of significant growth, the presence of the hemolysin BL enterotoxin and non-hemolytic enterotoxins will be determined by commercial assay kits. Both enterotoxigenic and emetic toxin types will be used. The role of competitive exclusion will be determined by the co-addition of the predominate bacterial species isolated from salmon. This will be identified by 16S rRNA typing. FDA-approved antimicrobials will be used to determine their effectiveness in preventing the growth of B. cereus. These include lactates and nisin at various concentrations. Other marine species will be assessed for their ability to competitively exclude the growth of this foodborne pathogens as well as the effect of additional temperatures and holding times representing more abusive food service conditions. A separate study will identify the molecule which signals the bacterial foodborne pathogen C. perfringens to sporulate and produce enterotoxin. This molecule will be isolated from spent medium using high-pressure liquid chromatography using various reverse phase columns preceded by low pressure chromatography using Sephadex LH-20, Sephadex G-10 and related columns using eluents to be determined but initially methanol and subsequently aqueous buffers. The identity of the putative peptide will be identified in conjunction with this university's NMR facility. Subsequent research - if time permits - will involve a determination of the mode of action of the isolated molecule. Key milestones will be the ability of B. cereus to grow in large numbers on salmon and, subsequently, separate marine species, in particular those consumed raw. The presence of toxin in such foods will be indicative of the potential hazards of their consumption. In the case of C. perfringens successive milestone will occur at the multiple purification stages with increasing degrees of purity. Knowledge of the events triggering sporulation and toxin of this organism will provide insight on how the process can be inhibited.