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Physiology and Control of Foodborne Disease Agents

Labbe, Ronald
University of Massachusetts
Start date
End date
  1. Isolate, purify and characterize the C. perfringens sporulation factor.
  2. Evaluate the effectiveness of extracts of cloned herbs on inhibition of gram positive foodborne pathogens.
  3. Determine the synergistic effectiveness of phenolic phytochemical extracts on inhibition of gram positve foodborne organisms.
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NON-TECHNICAL SUMMARY: Foodborne illness continues to be a significant and preventable public health issue in the U.S. This project will determine the cellular events leading to the production of an enterotoxin responsible for foodborne disease as well as possible use of plant (natural) products to inhibit growth of selected bacteria which are causes of such illnesses.

APPROACH: C. perfringens produces an enterotoxin during sporulation. The toxin is responsible for the symptoms associated with foodborne illness due to this organism. A previously-identified sporulation factor produced by C. perfrigens will be purified by a variety of biochemical methods. These include FPLC, HPLC, and ambient pressure column chromatography. The working assumption is that the factor is a peptide. Several peptide-specific columns are available for chromatographic procedures. Absorbance of suspect fractions will be measured at 214 nm. Activity of fractions will be monitored by the ability of suspect fractions to stimulate the sporulation of C. perfringens in a complex sporulation medium. Purity will be monitored by analytical HPLC using C18 columns. Stucture of active isolated material will be determined by amino acid composition, N-terminal amino acid analysis, and NMR. Once the presumptive structure has been determined a commercially prepared product will be used to confirm stucture. In another aspect of the proposal the antimicrobial activity of extracts of cloned rosemary and oregano will be determined. These clones are high in phenolic compounds. Ethanol and water extracts of crushed leaves will be concentrated by evaporation, filter-sterilized and used as a stock solution after standardization on a phenolics/ml basis. Antimicrobial activity will be determined using Staphylococcus aureus, Clostridium perfringens and Bacillus cereus. The effect of pH will be determined using lactic and acetic acids. Antimicrobial activity will be determined by the broth inhibition test using tryptose soy broth with yeast extract. Similarly the synergistic effects of multiple phenolic compounds will be determined. Preliminary results have shown cranberry powder extracts, also high in phenolic compounds, possess antimicrobial activity and these will be used as aother source of plant phenoics. The use of multiple-hurdle plant-based products may avoid unwanted adverse sensory issues associated with dependence on single plant extracts.

PROGRESS: 2007/10 TO 2008/09
OUTPUTS: Bacillus cereus is a gram-positive, endospore forming pathogenic bacterium that is ubiquitous in the environment and is frequently associated with emetic and diarrheal types of foodborne illness due to the production of emetic or diarrheal-type toxins. In this study, 178 samples of raw rice from retail food stores were analyzed for the presence of B. cereus spores. Spores of Bacillus species were found in 52.8% of the rice samples with an average concentration of 32.6 g. Eighty three of the 94 isolates were identified as B. cereus and 11 were identified as B. thuringiensis. Using PCR the isolates were checked for the presence of (1) the cereulide synthetase gene (ces), which produces the emetic toxin, (2) the hblA and hblD genes of the hemolysin BL (HBL) enterotoxin complex and (3) the nheA and nheB genes of the nonhemolytic (NHE) enterotoxin complex. The ces gene was not identified in any of the isolates. By contrast 56.6% B. cereus isolates possessed the hblA and hblD genes and 89.1% isolates possessed the nheA and nheB genes. As determined by commercial assay kits, 53.0% of the 83 B. cereus isolates produced both NHE and HBL enterotoxins whereas 93.9% were positive for either one or the other. Protein toxin crystals were detected visually in the 11 B. thuringiensis isolates. All the B. thuringiensis isolates were positive for NHE and HBL enterotoxins. Results of this work were presented at the annual meeting of the Institute of Food Technologists and submitted for publication.

IMPACT: 2007/10 TO 2008/09
The presence of Bacillus cereus in rice has never been documented in the U.S. despite the fact that worldwide this food is the principle vehicle for transmission of this foodborne pathogen. We have shown that it is the diarrheal-type biotypes that predominate in U.S. retail rice rather than the emetic biotype type commonly associated with rice in Europe

PROGRESS: 2006/10/01 TO 2007/09/30
OUTPUTS: Three hundred and forty seven fresh and processed seafood samples were examined for the presence of the foodborne pathogens Bacillus cereus, Clostridium perfringens, and Clostridium botulinum. The presence of C. perfringens and C. botulinum was confirmed in one and zero samples respectively. On the other hand 62 B. cereus isolates were confirmed at levels from 3.6 to >1100/gm. Thirty of the isolates produced the two enterotoxins known to be associated with this organism. As determined by PCR the presence of at least one of the three genes of the NHE enterotoxin complex was detected in 99% of isolates while 71% possessed at least one of the three genes of the HBL enterotoxin complex. Fifty of the 62 isolates were from imported seafood. A majority of enterotoxin-producing isolates were resistant to two of 10 antibiotics tested. Results of this work were present at the annual meeting of the International Association of Food Protection.

IMPACT: 2006/10/01 TO 2007/09/30
The presence of toxigenic B. cereus in seafood has never been determined. Our results show that not only is this organism present in this commodity but can be present at relatively high levels. Most of the isolates were from imported seafood including those producing the highest concentration of enterotoxin.

PROGRESS: 2005/10/01 TO 2006/09/30
Ulcer-associated dyspepsia is caused by the Helicobacter pylori. a food-related pathogen that infects the stomach lining. The antimicrobial and antioxidant activity of phytochemical-enriched alcoholic beverages was determined following the release of botanicals enclosed in tea bags. Results indicated that total phenolics increased from 80 ug/ml in control to 186 ug/ml in phenolic-enriched wine with a corresponding increase in antioxidant activity as determined by free radical inhibition. Even more dramatic differences were observed in phenolic-enriched vodka. Raspberry, cinnamon, and peppermint-enriched wines had the highest antimicrobial activity, while in vodka, raspberry was the most effective but no concentation-dependent correlation between phenolics and inhibition was noted. Results indicate that the synergistic contribution of phenolics and antioxidants activity may be more important for inhibition than any specific phenolic concentraion. In another aspect of this grant we are investigating the physiology of the foodborne pathogen Clostridium perfringens. This is a spore-forming microorganism which produces a toxin during sporulation in the small intestine. What triggers sporulation is not known but in the case of non-pathogenic spore-formers small molecules have been isolated which function as triggers. We have isolated a similar peptide from C. perfringens with a molecular weight of 1200 and composed of 5 amino acids. It is effective at picomolar levels in stimulating sporulation. The need for certain cell numbers to detect this molecule suggest a quorum sensing function. Work is in progress to determine the molecular level of its function.

IMPACT: 2005/10/01 TO 2006/09/30
The results of the work indicate the potential for diet-based management of ulcers caused by Helicobacter. The antioxidant and health promoting benefits of wine, especially red wine, are established. The addition well-known and accepted plant extracts suggest their addition to alcoholic beverages may increase the health benefits of such beverages used in moderation. The identification of a molecule controlling toxin formation may lead to methods to inhibit its formation or inactivation.

PROGRESS: 2004/10/01 TO 2005/09/30
Oregano and cranberry extracts demonstrated antimicrobial activity against Listeria when mixed at a ratio of 75% oregano and 25% cranberry; the efficiency was further enhanced by lactic acid. In meat and fish slices the phytochemical and lactic acid synergies were most effective at 4 C. Solid state fermentation by the food-grade fungus Lentinus edodes increased soluble phenolics and enhanced anti-Listeria activity as well as inhibitory activity against E. coli O157:H7 and Vibrio parahemolyticus. Antioxidant activity measured by radical inhibition assays suggest inhibition occurs by disruption of the membrane by localized hyper-acidification.

IMPACT: 2004/10/01 TO 2005/09/30
Current consumer food preferences suggest a trend against chemical food preservatives. The use of natural products with inhibitory activity has been investigated as an alterative. Our results show that a current agricultural waste, cranberry pomace, possesses antimicrobial activity, especially after treatment with a food-grade fungus which induces the release of additional antimicrobial compounds..

Funding Source
Nat'l. Inst. of Food and Agriculture
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Natural Toxins
Escherichia coli
Bacterial Pathogens