2013 Annual Report
1a.Objectives (from AD-416):
The overall goal of this research is to reduce the occurrence, risk, and severity of illness associated with consumption of foods contaminated with pathogenic microorganisms. This project will focus on the following three main objectives aimed at increasing our understanding of pathogen persistence in foods and, in turn, developing and evaluating effective interventions to enhance the safety and security of our food supply:
1. Determine the prevalence, levels, types, and locations of pathogens at various points from production through to consumption of raw, further processed, and/or RTE foods.
1.1. Determine the prevalence of L. monocytogenes in retail environments to include harborage sites, mechanisms of cross-contamination, and external sources of contamination.
1.2. Determine the relatedness of L. monocytogenes from FSIS- and FDA-regulated foods using molecular typing methods such as PFGE and MLGT.
2. Develop, optimize, and validate processing technologies for eliminating pathogens.
2.1. Determine the transfer and survival of Shiga-toxin producing Escherichia coli in tenderized (non-intact) beef.
2.2. Determine cook dwell times for ground meat products, with and without marinade or other enhancing solutions, using common consumer preparation methods such as cooking on gas or electric grills at internal instantaneous temperatures ranging from 120' to 160°F.
3. Develop and/or validate strategies to deliver antimicrobials to raw and packaged foods from production through to consumption.
3.1. Derive data to aid verification of growth inhibitor effectiveness for L. monocytogenes in RTE products from time of production through consumption.
1b.Approach (from AD-416):
Identify where pathogens enter the food supply, how they persist, and/or what can be done to eliminate or control them. The target pathogens of greatest concern for this proposal are Listeria monocytogenes and Shiga toxin-producing Escherichia coli, but other pathogens may also be evaluated. The targeted foods are raw and ready-to-eat (RTE) meat, poultry, and dairy products, as well as raw and further processed non-intact meats. Identify sources of L. monocytogenes in foods or food processing and retail environments and to elucidate factors contributing to its survival and persistence. Molecular methods such as pulsed-field gel electrophoresis (PFGE) and multilocus genotyping (MLGT) will be used to differentiate isolates from various sources from the farm through distribution and at retail to determine pathogen niche and succession. Validate processes and interventions such as fermentation, high pressure processing, food grade chemicals, and heat (cooking), alone or in combination, to inhibit/remove undesirable bacteria and better manage pathogen presence, populations, and/or survival during manufacture and storage of target foods. The proposed research to find, characterize, and kill pathogens along the food chain continuum will expand our knowledge of important food borne pathogens and lead to better methods for controlling them in foods prior to human contact and consumption, thereby enhancing the safety of our Nation’s food supply.
Research was expanded to recover, enumerate, and control target pathogens in higher-risk, higher-volume foods. We determined the prevalence, levels, and types of Listeria monocytogenes on 15 categories of some 23,000 ready-to-eat (RTE) foods purchased from retail establishments from within FoodNet sites in Georgia, Connecticut, California, and Maryland. This research is being conducted in collaboration with the DHHS-FDA and USDA-FSIS. The pathogen was recovered, albeit at low frequency and low levels, from smoked seafood, seafood salads, low acid cut fruits, raw milk, sandwiches, deli salads, deli meat salads, deli meats, dry/fermented sausages, but not from soft cheese. When present, pathogen levels ranged from <0.03 MPN/gram to greater than 110 MPN/g. We performed biochemical and molecular characterization of multiple isolates (up to 10 colonies) from each food sample testing positive for the pathogen. Studies were also initiated to determine the proximate composition of select foods and to evaluate the fate of the pathogen in inoculated-package challenge studies. In related studies, we evaluated biological, chemical, and physical interventions to control L. monocytogenes, as well as Shiga toxin-producing Escherichia coli or Salmonella spp., in a variety of raw, further processed, and/or RTE red meat, poultry, and dairy products. Products inoculated with L. monocytogenes included delicatessen-style ham, roast beef, and turkey and natural hams preserved with celery salt. For some studies, products were specifically formulated by a commercial producer to contain unique blends of food grade organic acids as ingredients and/or delivered to packaged foods using the Sprayed Lethality in Container (SLIC) method. Reductions of 100 to 10 million cells and/or prevention of pathogen outgrowth during extended refrigerated shelf life were observed. Lastly, we quantified the translocation and/or thermal inactivation of Shiga toxin-producing E. coli in raw ground and non-intact beef, including prime rib, veal cutlets, ground beef patties, wafers of beef, jerky, goetta, and fermented dry sausage. These experiments employed pathogenic strain of E. coli, pilot scale commercial food processing equipment, and entire steaks, subprimals, and patties etc. In general, these data validated that cooking on gas or electric grills/skillets resulted in reductions of 30 to 100,000 cells of the pathogen. The data also confirmed that non-O157 strains of Shiga toxin-producing E. coli behave similar to serotype O157:H7 strains of E. coli with respect to their translocation and thermal inactivation in beef. Our findings assisted manufacturers in meeting existing regulatory requirements and assisted regulators in making science-based policy decisions which, in turn, has enhanced the safety and quality of our food supply.
Control of Listeria monocytogenes (Lm) in ready-to-eat (RTE) meats. ARS researchers at Wyndmoor, Pennsylvania, evaluated the efficacy of buffered vinegar (BV), a food grade antimicrobial, to inhibit Lm on the surface of naturally-cured ham and uncured turkey breast during storage at abuse and refrigeration conditions. Ham formulated with BV at levels likely to be found in retail products (1.0, 1.5, or 2.0%) and cured naturally via the addition of celery powder/salt was stored at 4 or 10C for up to 120 days. Regardless of the ham formulation and storage temperature, pathogen numbers increased by 3.4 to 6.5 log CFU/slice after 120 days of storage. Such significant increases would likely cause listeriosis. For turkey breast formulated with or without BV (2.0, 2.5, or 3.0%) at levels likely to be found at retail, the higher the concentration of the BV in the formulation, the lower the levels of the pathogen during storage. At 10C, Lm numbers increased to extremely high levels, that being by 3.7 to 10.5 log CFU/slice. However at 4C, when turkey breast was formulated without BV, pathogen numbers increased by 6.2 log CFU/slice after 90 days. But, when the product was formulated with 2.0 or 2.5% of BV, pathogen numbers remained unchanged by ca. 45 days, and then increased by 1.6 and 0.8 log CFU/slice, respectively, whereas inclusion of 3.0% of BV reduced Lm numbers by 0.7 log CFU/slice after 90 days of storage, such results would appreciably enhance product safety and consumer confidence. Inclusion of BV in uncured turkey breast suppressed outgrowth of Lm during shelf life. Further work is required to evaluate other antimicrobials applied either as an ingredient of applied to the product surface to better control outgrowth of Lm on naturally-cured ham and/or uncured turkey breast.
Comparative inactivation of Shiga toxin-producing Escherichia coli (STEC) in raw ground and non-intact beef. Illnesses due to Shiga toxin producing E. coli (STEC) has been linked to undercooked ground beef and on occasion to non-intact beef as well. As such, the USDA FSIS now considers strains of serotype O157:H7 and strains form a subset of six non-O157 serotypes of STEC as adulterants in raw ground and non-intact beef. Although much has been published on the former, considerably less information has been published on the fate of other strains/serotypes of STEC in raw, cooked, fermented, and/or further processed beef. For these reasons, ARS researchers at Wyndmoor, Pennsylvania evaluated the comparative fate of serotype O111:H-, O45:H2, O103:H2, O121:H19, O145:NM, O26:H11, and/or O157:H7 strains in prime rib, veal cutlets, ground patties, goetta, jerky, and dry/fermented sausage. In general, the longer the cooking and fermentation times, the higher the internal temperature and the lower the pH of the meat, respectively, along with the greater the reduction in levels of STEC. Regardless of the type of beef and depending on the processing parameters being tested, reductions of about 1.5 to >5.0 log were observed. Moreover, our data confirmed that non-O157 STEC behave similar to serotype O157:H7 and establish that interventions effective against the later should be equally effective against the former. These findings will assist manufacturers by providing validated processes/technologies and these data fill key data gaps for policy making decisions.
Determination of the true/current prevalence of Listeria monocytogenes in ready-to-eat (RTE) foods at retail. ARS researchers at Wyndmoor, Pennsylvania, participated in a multi-agency, multi-disciplinary study to establish the relative occurrence, levels, and subtypes of Listeria monocytogenes (Lm) in ready-to-eat (RTE) in higher-volume, higher-risk foods purchased at retail. Despite appreciable efforts by food safety professionals to eliminate Lm in RTE foods in the 1990’s and early 2000’s, foodborne illness caused by Lm continues. Over a 2-year period, some 23,000 foods were purchased at large and small grocery stores in four FoodNet sites across Maryland, Georgia, Connecticut, and California. The FDA-regulated foods included smoked seafood, seafood salad, low acid cut fruits, soft cheese, deli salads (non-meat), raw milk, sandwiches, cut vegetables (raw), sprouts, artisanal cheese, fresh crab and sushi, and eggs, and the FSIS-regulated foods included deli meats, deli salads containing meat, and dried/fermented sausage. Of the almost 8,000 FDA-regulated foods sampled to date, the estimated prevalence ranged from zero to 1.2%. Moreover, the estimated prevalence of Lm was noticeably higher for foods made/sliced in stores compared with similar foods that were pre-packaged by the manufacturer. For samples testing positive for the pathogen, the levels of Lm ranged from <0.03 MPN/gram to greater than 110 MPN/g. The prevalence data for an additional 9,000 FDA-regulated foods and ca. 6,000 FSIS-regulated products are currently being analyzed. This represent the most comprehensive study on the association of Lm with RTE foods over the past decade and, as such, it is significant form a public health perspective that the recovery rate of Lm reported herein is appreciably lower in comparison to studies of similar scope and magnitude published a decade earlier. The data from this study will also be used to update the 2003 Interagency Risk Assessment on ready-to-eat foods.
Control of Listeria monocytogenes and Escherichia coli O157:H7 in/on specialty/ethnic meats. Although it is a very popular breakfast item, with over 1 million pounds consumed annually, there is a general lack of information on the viability and/or fate of common food-borne pathogens that could become associated with goetta, a specialty/ethnic sausage. ARS researchers at Wyndmoor, Pennsylvania evaluated the viability of Listeria monocytogenes (Lm) and Escherichia coli O157:H7 (ECOH) on goetta both during extended storage and following cooking. Commercial goetta was inoculated onto both the top and bottom surface with ca. 1.4 log CFU/g of a five-strain cocktail of Lm or ECOH. The inoculated slices were stored at 4 or 12C for up to 90 days. For cooking experiments, goetta was inoculated with ca. 7.0 log CFU/g of a five-strain cocktail of Lm or ECOH and placed into a mixer for 2 minutes before patties were formed. The patties were cooked for 2 to 6 min per side on an electric skillet maintained at 176.7C. Results showed that Lm numbers increased from ca. 1.4 log CFU/g to ca. 8.4 log CFU/g over 90 days of storage at 4C, whereas ECOH numbers decreased to =0.4 log CFU/g during storage. At 12C, Lm and ECOH numbers increased from 1.4 log CFU/g to ca. 9.0 log CFU/g over 28 days. After cooking, ca. a 0.7- to 6.6-log reduction of Lm and ECOH was observed. Although goetta supports the growth/survival of Lm or ECOH, about a 5-log reduction of both pathogens can be achieved by cooking the product for about 5 min per side just prior to consumption, thus making this product safe for human consumption, especially in the unlikely event that pathogens would be present at any levels in this product at retail.
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Porto Fett, A.C., Pierre, J., Shoyer, B.A., Luchansky, J.B. 2013. Effect of storage temperature and cooking time on viability of Listeria monocytogenes and Escherichia coli 0157:H7 in/on goetta. Journal of Food Safety. Vol. 33,p.128-136.
Castaneda-Ruelas, M., Castro-Del Campo, N., Felix, J., Valdez Torres, J., Guzman-Uriarte, R., Luchansky, J.B., Porto Fett, A.C., Shoyer, B.A., Chaidez-Quiroz, C. 2013. Prevalence, levels, and relatedness of Listeria monocytogenes isolated from raw and ready-to-eat foods at retail markets in Culiacan, Sinaloa, Mexico. Journal of Microbiology Research. 3(2):92-98.
Santos Villamil, A., Hernandez Anguiana, A., Alberto Eslavo Campo, C., Landa Salgado, P., Mora Aguilera, G., Luchansky, J.B. 2012. Biofilm production and resistance to disinfectants in Salmonella strains isolated from prickly pear, water, and soil. Revista Mexicana de Ciencias Agricolas. 3:1063-1074.
Luchansky, J.B., Porto Fett, A.C., Shoyer, B.A., Phillips, J.G., Eblen, D., Evans, P., Bauer, N. 2013. Thermal inactivation of shiga toxin-producing 0157:H7 and non-0157-H7 cells of Escherichia coli within wafers of ground beef. Journal of Food Protection. 76:1434-1437.
Porto Fett, A.C., Shoyer, B.A., Harshavardhan, T., Luchansky, J.B. 2013. Fate of Escherichia coli O157:H7 in mechanically tenderized beef prime rib following searing, cooking and holding under commercial conditions. Journal of Food Protection. 76:405-412.