2012 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. Specifically, efforts were directed to determine the prevalence, levels, and types of Listeria monocytogenes (Lm) in 10 categories of ready-to-eat foods purchased from retail establishments from within FoodNet sites in GA, CT, CA, and MD. This research is being conducted in collaboration with the FDA and FSIS. Of about 14,000 foods tested, 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; pathogen levels ranged from <0.3 cells/g as estimated statistically to 251 cells/g. Efforts are also underway to conduct biochemical and molecular characterization of multiple isolates (up to 10 colonies) from each positive food sample. These efforts to sample retail RTE foods will continue in 2013 and will be complimented by studies just 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 Lm, 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 Lm included delicatessen-style ham, roast beef, and turkey, as well as specialty-ethnic products such as goetta. For some studies products were specifically formulated by a commercial producer to contain unique blends of food grade organic acids as ingredients. Antimicrobials were also delivered to packaged foods using the Sprayed Lethality in Container method. Depending on the type and concentration of the antimicrobial being tested, it was possible to achieve reductions of 100 -10 million cells and/or prevent pathogen outgrowth during extended refrigerated shelf life. Lastly, in collaboration with food safety professionals in government, academia, and industry, we quantified the translocation and/or thermal inactivation of Shiga toxin-producing E. coli in raw ground and non-intact beef. Our data established that mechanical or chemical tenderization of beef transfers pathogens from the surface into the internal portions of the meat and confirmed that cooking resulted in reductions of 30-100,000 cells of the pathogen. We also measured the inactivation of Shiga –toxin producing E. coli following cooking in flattened (ca. 1 mm) 3-gram wafers of beef heated in a water bath and monitored their thermal stability following cooking of inoculated ground beef patties on commercial gas or clam-shell electric grills. The data 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 has enhanced the safety and quality of our food supply.
Comparative inactivation of Escherichia coli O157:H7 (ECOH) and non-O157:H7 Shiga toxin-producing E. coli (STEC) in ground beef patties and wafers. Escherichia coli O157:H7 (ECOH) and non-O157:H7 Shiga toxin producing E. coli (STEC) can be recovered from whole-muscle and non-intact raw beef and have also been implicated in costly product recalls and severe human illness, especially from undercooked ground beef. For these reasons, we evaluated the comparative fate of serotype O111:H-, O45:H2, O103:H2, O121:H19, O145:NM, O26:H11, and/or O157:H7 strains in both flattened (ca. 1 mm thick and 3-grams) wafers of ground beef in a heated water bath (54.4 deg, 60 deg, or 65.6 deg C) and in formed ground beef patties (ca. 1 inch thick and 300 grams) cooked on commercial grills (60.0 deg, 65.5 deg, 71.1 deg , or 76.6 deg C). Regardless of the level of fat or type of grill, ARS researchers at Wyndmoor, Pennsylvania validated that cooking ground beef patties to an internal temperature of =71.1 degrees C is effective for destroying ECOH and STEC and, in turn, lessening the threat of illness associated with this food borne pathogen. Our data also established that cooking times/temperatures (54.4 deg, 60 deg, or 65.6 deg C) effective for inactivating ECOH in ground beef are equally effective against the additional STEC serotypes investigated.
Control of Listeria monocytogenes (Lm) in ready-to-eat (RTE) meats. L. monocytogenes can be reintroduced onto the surface of RTE meats via post-process contamination. Thus, ARS researchers at Wyndmoor, Pennsylvania evaluated the efficacy of food grade antimicrobials, alone or in combination, to inhibit L. monocytogenes on the surface of uncured turkey breast at 4 deg C. Inclusion of levulinate, alone or in combination with diacetate and propionate, as ingredients, suppressed outgrowth of L. monocytogenes during extended refrigerated storage. These data were the first to validate the synergistic effect of using levulinate in combination with diacetate and propionate to control L. monocytogenes on an RTE uncured turkey breast product. Thus, in the event of post process contamination, inclusion of this levulinate-diacetate-propionate blend as an ingredient in uncured turkey breast would preclude outgrowth of L. monocytogenes.
Fate of Escherichia coli O157:H7 (ECOH) in mechanically tenderized prime rib. The process of mechanical and/or chemical tenderization transfers surface-residing cells of pathogens into the deeper tissues of the meat wherein they may be more resistant to subsequent thermal challenge. Similar to previous work with thermal inactivation of ECOH compared with non-O157:H7 Shiga toxin-producing E. coli (STEC) in steaks prepared from blade/chemical tenderized subprimals following cooking on commercial grills, ARS researchers at Wyndmoor, Pennsylvania evaluated the effect of the commercial practice of low-temperature, long-time heating when preparing prime rib roast for elimination of ECOH from mechanically tenderized beef prime rib. These results demonstrated that to meet the required 5.0-log reduction of ECOH to produce a product that was both safe and of high quality, it would be necessary to sear, cook to internal temperatures of 48.9 deg C, 60.0 deg C, or 71.1 deg C, and then hold previously inoculated and tenderized prime rib roasts in a warming oven at 60.0 deg C for 8 h. These data support regulators in the development of science-based cooking guidelines and assist restaurants and/or food service establishments to enhance the safety of non-intact prime rib served at the point of consumption.
Determination of the true/current prevalence of Listeria monocytogenes in the ready-to-eat (RTE) foods at retail. Although significant efforts have been taken by federal government and food industry to control Lm in RTE foods over the last decade, foodborne illness caused by Lm continues. Thus, a study was undertaken by ARS researchers at Wyndmoor, Pennsylvania to determine the current prevalence and levels of Lm in deli-packaged versus prepackaged RTE foods purchased at retail establishments in four FoodNet sites. About 14,000 RTE comprising the following food categories were purchased: smoked seafood, seafood salad, low acid cut fruits, soft cheese, deli salads (non-meat), raw milk, sandwiches, deli meats, deli salads containing meat, and dried/fermented sausage. Samples were collected in both supermarket chain and independent grocery stores in California, Maryland, Connecticut and Georgia from December 2010 to July 2012. Of the ca. 8,000 FDA regulated samples tested to date, the observed prevalence ranged from ca. 0% to 1.0% for seven product categories. The prevalence data for the ca. 6,000 FSIS-regulated products are currently being analyzed. For the 39 samples testing positive during screening, Lm levels ranged from ca. <0.3 cells/g as estimated statistically to 251 cells/g. This is the most comprehensive survey of Lm in retail RTE foods in the past decade. These findings provide data to assess changes in Lm prevalence and levels in RTE foods, information that is critical for policy decisions on further controls of this pathogen and an understanding of its contribution to the public health burden. The data from this study will also be used to update the 2003 Interagency Risk Assessment on Ready-to-Eat Foods.
Luchansky, J.B., Shoyer, B.A., Call, J., Schlosser, W., Shaw, W., Bauer, N., Latimer, H., Porto-Fett, A. 2012. Fate of shiga-toxin producing 0157:H7 and non-0157:H7 Escherichia coli cells within blade-tenderized beef steaks after cooking on a commerical open-flame gas grill. Journal of Food Protection. 75:62-70.
Pagadala, S., Parveen, S., Rippen, T., Luchansky, J.B., Porto Fett, A.C., Bowers, J., Tamplin, M.L., Wiedmann, M., Call, J.E. 2012. Prevalence and sources of Listeria monocytogenes in blue crab (Callinectus sapidus) meat and blue crab processing plants. Food Microbiology. 31:263-270.
Leggett, L.N., Tomasula, P.M., Van Hekken, D.L., Porto Fett, A.C., Shoyer, B.A., Luchansky, J.B., Renye Jr, J.A., Farkye, N. 2012. Effect of storage at 4 and 10 C on growth of Listeria monocytogenes in Queso Fresco. Journal of Food Safety. 32:236-245.
Kich, J.D., Coldebella, A., Mores, N., Nogueira, M.G., Cardoso, M., Fratamico, P.M., Call, J.E., Cray, P.J., Luchansky, J.B. 2011. Prevalence, distribution, and molecular characterization of Salmonella recovered from swine finishing herds and a slaughter facility in Santa Catarina, Brazil. International Journal of Food Microbiology. 151(3):307-313.
Chaluvadi, S., Hotchkiss, A.T., Call, J.E., Luchansky, J.B., Phillips, J.G., Liu, L.S., Yam, K.L. 2012. Protection of probiotic bacteria in a synbiotic matrix following aerobic storage at 4 deg C. Beneficial Microbes. 3:175-187.