Control of Food-Borne Pathogens in Pre- and Post-Harvest Environments

NON-TECHNICAL SUMMARY: Foodborne pathogens have a significant impact on human health and the food industry in the United States, causing numerous [sometimes fatal] illnesses, along with substantial loss of productivity and increased medical expense. This purpose of this research is to gain a better understanding of pathogen survival as an insight into developing safer and more efficient processing methods and food treatments. We also seek to better understand and eliminate the transfer of antimicrobial resistance from the farm to the foods. <P>APPROACH: Develop methods to eliminate or reduce pathogens on raw food commodities under normal commercial food processing conditions. Development of methods to detect stressed and non -culturable pathogens in pre- and post- harvest environments and assay development and transfer of chemical and antibiotics resistances with emphasis on Listeria.<P>PROGRESS: 2007/01 TO 2007/12<BR>
OUTPUTS: The starvation survival response (SSR) of L. monocytogenes wild type strain 10403 and a sig B mutant were characterized under complete exogenous nutrient deprivation. The effects on cell viability and SSR of adding a protein synthesis inhibitor, chloramphenicol (Chlo) and of inhibitors of substrate - level phosphorylation (SLP) and oxidative level phosphorylation (OLP) on the two strains over 28 days was measured. Survival of buth the wt and mutant were affected by addition of Chlo during the first 4 h of starvation but cells were able to mount a SSR if Chlo were added at 6 h but the mutant was affected more. Neither strain was able to elicit a SSR when exposed to the SLP inhibitors 0.1 M sodium fluoride (SF) or 0.01 M sodium arsenite (SAs), suggesting that reaction downstream from the enolase and pyruvate dehydrogenase enzyme reactions are important in retaining cell viability, possibly through the production of energy needed for cell maintenance and protein synthesis during exogenous nutrient starvation. Conversely, both strains were able to mount a SSR when exposed to two OLP inhibitors, 0.1 M sodium arsenate (SA) or 0.02 M dinitriophenol (DNP). Two possibilities suggested by the latter results are that this organism in using oxygen as a terminal proton and electron acceptor: 1) may not use the classic cytochrome metabolic chain of intermediate enzyme steps but instead may us a direct NADH oxidase system as is reported for a related Gram positive, Enterococcus faecalis; or 2) is not sensitive to these two inhibitors at the levels used. In related work 21 cold sensitive mutants were constructed from above strain 10403 using the plasmid pLTV3 which contains the transposon Tn917. These 21 strains were grouped into 2 categories based on the reduction in growth rates compared to the wild-type strain at 4 degree C: slightly reduced (10-15% reduction) and significantly reduced (20-30% reduction). When examined for survivability at -20 degree C and -80 degree C, both groups showed equal survival at sub-freezing temperatures compared to the wild-type. These findings suggest that bacterial genes involved in low temperature growth of L. monocytogenes may not be important in aiding the adaptation and survival of L. monocytogenes at sub-freezing temperatures. <BR>PARTICIPANTS: Major cooperators in this project were Dr. Gerry Huff of the USDA-ARS Fayetteville Poultry Production and Product Safety Unit along with her M.S. student in Poultry Science, Mr. Vik Dutta who graduated in Dec 2007 and in Jan 2008 moved to North Carolina St U to begin his Ph.D. degree. Two graduate students of MGJ completed their M.S. (Mr. Josh Saldivar, who plans to go on to work for the Ph.D. degree with Dr Ricke) and Ph.D. (Dr. Bwalya Lungu who graduated in Dec 2007 and effective Jan 2008 moved to the U of Georgia to work with Dr. Joe Frank in a post doc position) degrees in Food Science Dept at UAF. Collaboration with Drs Steve Ricke and Arun Muthiyan were fruitful in employing some mutant techniques to look for cold sensitive mutants.<BR>TARGET AUDIENCES: Researchers interested in understanding some of the mechanisms that this pathogen can employ to resist loss of nutrients and cold temperatures. <BR>PROJECT MODIFICATIONS: The use of plasmids to modify the cold tolerance of Listeria monocytogenes was employed using standard procedures. <P>
IMPACT: 2007/01 TO 2007/12<BR>
Above studies indicate that surviving portion of a L. monocytogenes cell population can cope with loss of outside nutrients by living on waste products sloughed off by that portion of the cell population that dies. This mechanism would explain how such surviving cells remain quite viable for long periods of time, up to 1 year or more, without receiving any exogenously added carbohydrate or nitrogen sources.