<OL> <LI> To determine the incidence and numbers of viable cells of the human infectious bacterium Plesiomonas shigelloides in shellfish and retail fillets using conventional selective cultivation methods and quantitative PCR<LI> To definitively identify strains of Clostridium botulinum recently isolated from retail seafood via 16S rRNA methodology<LI> To determine whether recent isolates of C. perfringens from retail seafood are enterotoxigenic using a commercial enterotoxin assay system<LI> To determine the enterotoxigenic profiles of 78 recent rtail seafood isolates of Bacillus cereus and their ability to grow psychrotrophically<LI> To develop elicitor-inducible pea phenolics active against L. monocytogenes and to develop fungal fermented pea phenolics active against L. monocytogenes<LI> To develop a laboratory model drain system to study colonizaiton of Listeria monocytogenes in the presence of other natural contaminants for evaluation of cleaning and sanitation compounds.
NON-TECHNICAL SUMMARY: The bacterium Plesiomonas shigelloides is known to cause meningitis in addition to gastroenteritis. The mortality rate of patients with resulting Plesiomonas septicemia is high with most seafood borne outbreaks historically derived from oysters since they are eaten raw. However, the increased consumption of raw fish (sushi) can be assumed to greatly enhance the public health threat from this organism which this study will assess. Outbreaks of food infections and intoxications derived from spore forming bacteria still occur in the U.S. annually. Documenting the extent of the public health threat from these organisms in the Northeast will assist in delineating the extent of the problem locally. Outbreaks of listeriosis derived from consumption of fish contaminated with L. monoctogenes still occur annually in the U.S. The development of new methods for inhibiting the refrigerated growth of the organism and destroying L. monocytogenes on seafood in addition to optimizing sanitary practices of seafood processing plants will assist in greatly reducing the numbers of this organism in processing plants and on fish tissue and in eliminating or reducing further seafood derived outbreaks The purpose of these studies is to reduce the public health hazard of human pathogenic bacteria associated with seafood by reducing the number of human pathogenic bacteria in processing plants, identifying optimized plant sanitation practices, and by reduction of psychrotrophic human pathogenic bacteria on seafood.
APPROACH: The incidence and CFU/g of the human pathogenic bacterium Plesiomonas shigelloides in oysters and fresh retail fish fillets will be determined using conventional bacteriological techniques and real-time PCR. A laboratory model drain system will be developed to study colonization of Listeria monocytogenes in the presence of other natural contaminants for evaluation of cleaning and sanitation compounds. The identity of presumptive isolates of C. botulinum from retail seafood will be confirmed by 16S rRNA sequencing. In addition biochemical tests will be performed such as casein hydrolysis. C. botulinum Type E is non-proteolytic and this characteristic can be determined using casein agar. The presence of the enterotoxin gene in isolates of C. perfringens from retail seafood will be determined by PCR. Enterotoxin production by these strains will be determined using a commercial enterotoxin assay system since possessing the gene does not necessarily mean it will be expressed. The enterotoxin profiles of isolates of B. cereus from retail sea food will be determined using separate commercial assay kits. In addition the presence of the hemolysin BL (HBL, nonhemolytic (NHE) enterotoxin gene complexes, and the emetic toxin gene will be determined by PCR. The inhibition of growth of L. monocytogenes on fish fillets dipped in solutions of pea extracts containing high levels of phenolic microbial inhibitors will be determined.
PROGRESS: 2004/09 TO 2007/09<BR>
OUTPUTS: The seaweed Porphyra yezoensis was grown, harvested, and fed to rainbow trout at a level of 30%. Results indicated that the growth rate of the trout was similar to the reference diet. Seaweeds were also found capable of taking up and metabolizing various organic aquatic toxicants. Isolates of The human pathogenic bacterium Plesiomonas shigelloides from various environmental sources were found to exhibit via RAPD analysis significant genetic diversity. These studies also indicated that seafood may be a serious source of potential risk of human infection by this bacterium. A quantitative PCR assay developed for P. shigelloides in shellfish was found capable of detecting 60 CFU/g. Phenolics from oregano and cranberry extract were found to inhibit L.monoytogenes synergistically. This inhibition was enhanced by lactic acid. The establishment of Listeria monocytogenes on environmental surfaces was found not to occur in the presence of competing biofilms microflora. Among a total of 62 strains of Bacillus cereus isolated from seafood, 33 were found to produce enterotoxin. <BR> PARTICIPANTS: Project was split funded between University of Massachusetts, Amherst (Professor R. E. Levin) and Northeastern University (Professor Donald Cheney). Project funded 5 graduate students for further training and professional development. <BR> TARGET AUDIENCES: Target audiences consist of the seafood processing industry, fish farms, state and federal food and drug personnel <P>
IMPACT: 2004/09 TO 2007/09<BR>
The seaweed Porphyra yezoensis was found capable of taking up and metabolizing various organic aquatic toxicants. The development of a rapid quantitative PCR assay for P. shigelloides in shellfish capable of detecting 60 CFU/g of tissue greatly facilitates the rapid detection of this human pathogenic organism in shellfish. The ability of oregano and cranberry extracts to synergistically inhibit Listeria monocytogens has potential for significantly reducing the public health risk of this organism associated with seafood. Our studies on the surface development of L. monocytogenes have significantly contributed to our insight into the occurrence and development of this organism on processing surfaces. The observation that strains of Bacillus cereus from seafood are capable of producing enterotoxin indicates the potential hazard involved with this organism when seafood is subjected to storage temperature abuse.