Location: Characterization and Interventions for Foodborne Pathogens
2024 Annual Report
Objectives
Objective 1: Determine the recovery rate, population levels, relatedness, persistence, and harborage sites of target pathogens in raw, further processed, and/or ready-to-eat foods from production through to consumption to assist in risk assessments and communication. [C1, PS1]
Sub-Objective 1.A: Determine the prevalence and levels of Lm, STEC, and Salmonella in RTE foods at retail, raw organ meats from abattoirs, and frozen bakery products containing meat and vegetables from food retailers.
Sub-Objective 1.B: Determine the relatedness of Lm, STEC, and Salmonella recovered from foods using molecular typing methods such as PFGE and WGS.
Sub-Objective 1.C: Assess perceptions, food safety attitudes, and self-reported behaviors related to observed food safety hazards by consumers who shop at grocery stores.
Objective 2: Validate lethality (heating) and stabilization (cooling) processes for ready-to-eat (RTE) and not ready-to-eat (NRTE) meat and poultry products. [C1, PS5]
Sub-objective 2.A: Validate lethality and stabilization processes to control of Salmonella, Lm, and Cperf in non-intact and specialty/ethnic pork and beef products.
Sub-objective 2.B: Validate consumer-relevant cooking times, temperatures, and appliances to control target pathogens in (multi-species) bakery products containing meat and vegetables.
Sub-objective 2.C: Validation of cooking and cooling profiles for large mass meat products to achieve stabilization performance standards that prevent growth of Cperf.
Objective 3: Develop, optimize, and validate biological, physical, and chemical interventions and processes to control target pathogens in raw, RTE, and specialty/ethnic foods. [C1, PS5]
Sub-objective 3.A: Apply interventions to control target pathogens in RTE meats and multi-component meat-based salads and salsas.
Sub-objective 3.B: Validate food-relevant interventions to control target pathogens in plant-sourced meat alternatives.
Sub-objective 3.C: Evaluation of the impact of processing parameters of dry-cured fermented meat on lethality towards STEC, Salmonella, and Lm.
Sub-objective 3.D: Develop and/or validate strategies to control pathogens in forcemeats.
Approach
The overarching theme of this research plan is to identify where pathogens enter the food supply, where, how, and why they persist in foods, and/or what can be done to reduce their levels or to eliminate them along the farm to fork continuum. Target pathogens will include Listeria monocytogenes (Lm), Salmonella, Clostridium perfringens (Cperf), and Shiga toxin-producing cells of Escherichia coli (STEC). Target foods will include raw and ready-to-eat meats, dairy, baked foods, and vegetables, as well as simulated meats and specialty/ethnic foods, targeted for human and animal consumption. A primary focus will be to identify entry points, sources, and levels of target pathogens in foods or within food processing, food service, and retail environments, and to elucidate factors contributing to their survival and persistence. Phenotypic and molecular methods, including pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS), will be used to identify and differentiate isolates from the farm through distribution and at retail to determine pathogen relatedness, niche, persistence, and succession. Another focus will be to validate processes and interventions such as fermentation, drying, high pressure, biopreservatives, food grade chemicals, and heat (e.g., grilling and sous vide), alone or in combination, to inhibit/remove undesirable bacteria and better manage pathogen presence, populations, and/or survival during manufacture and/or subsequent storage of target foods. We will also develop and optimize methods to deliver antimicrobials to food systems, including electrostatic spraying and various strategies to introduce interventions into/onto foods or food containers/packaging (e.g., SLIC®). Our findings will assist numerous producers and processors with meeting current regulatory guidelines and assist regulators such as the DHHS FDA and the USDA FSIS with making science-based policy decisions, thereby enhancing the safety of the Nation’s food supply.
Progress Report
Further progress was made to deliver on our programmatic goals to recover, characterize, and control target pathogens in raw, further processed, and ready-to-eat (RTE) foods. The ability to address our milestones and validate processes and chronicle pathogen presence and fate from production through to consumption of foods was aided by longstanding partnerships with food safety professionals in government, industry, and academia. Target bacterial pathogens included Salmonella (Sal), Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes (Lm), whereas target foods included raw, further processed, and/or fermented beef, chicken, or pork products. Food grade lactates, acetate, and diacetate delivered significant antilisterial activity in various RTE sausage formulated by an industry partner, including sausage stuffed into a natural casing. In general, inclusion of salts of organic acids as ingredients in RTE cooked sausage lowered levels of Lm (i.e., a reduction of 3.5 to 10 cells per package) during extended refrigerated storage compared to otherwise similar sausage without antimicrobials (i.e., an increase of greater than 250,000 cells per package). A series of experiments was also conducted to validate USDA FSIS recommended cooking parameters, as listed in Appendix A, to inactivate target pathogens in large mass meats. These data showed that cooking parameters prescribed in Appendix A were sufficient to deliver reductions of about 10 to 100 million cells of Sal and Lm in/on ham, pork bellies, and pork necks. In related studies conducted with a regulatory partner, time temperature parameters elaborated in Appendix A were evaluated to eliminate Sal in smoked sausage stuffed into natural or synthetic casings. The validated process delivered reductions of greater than 3.2 million cells of Sal in a smoked Hungarian-type sausage and confirmed that equivalent reductions in pathogen levels were achieved for sausage stuffed within non-permeable (cellulose) and permeable (natural) casings irrespective of whether steam was introduced into the smokehouse during cooking. These data ensured product safety while saving processing times/monies, and without adversely affecting product quality. Also, in collaboration with a regulatory partner and collaborating with ARS scientists, a one-day enrichment protocol was developed for Lm presence/absence in deli ham. The validated 24 h protocol using LPT broth decreased the enrichment time from 48 h to 24 h and will reduce costs without compromising sensitivity, specificity, reproducibility, or ease-of-use. Lastly, based on our prior experience and publications we were tasked to confirm that USDA FSIS recommended cooking parameters were sufficient to inactivate avian influenza virus (AIV) in beef patties: cooking patties on a commercial gas grill to 62.8° or 71.1°C resulted in a reduction to non-detectable levels from initial levels of greater than 400,000 50% egg infectious doses (EID50) per 300 g. Collectively, our data are used by industry to validate their processes and by regulators to make science-based policy decisions. Our unique ability to work with real pathogens using pilot-scale equipment ensures that our data can be used directly by our various partners.
Accomplishments
1. Inactivation of avian influenza virus (AIV) in beef patties. Since 2022, approximately 100 million poultry have been lost in the U.S. alone due to AIV. In 2024 AIV was detected in dairy cows and in raw milk from dairy cattle infected with AIV. Three cases of bird flu infection were also confirmed in dairy farm workers. Because cull dairy cows comprise about 10% of U.S. beef production, ARS scientists in Wyndmoor, Pennsylvania, in collaboration with ARS scientists in Athens, Georgia, purposefully inoculated ground beef patties (20% fat; 2.54 cm thick; 300 g each) with high levels of AIV. Cooking inoculated ground beef patties to a rare degree of doneness (about 120°F) reduced AIV by about 300 to 500 fold per gram of patty, whereas cooking patties to a medium degree of doneness (about 145°F) or to well done [about 160°F; the USDA Food Safety and Inspection Service (FSIS) recommended minimum internal temperature for ground beef] reduced infectious virus levels at least 500,000 fold per gram of patty (no infectious virus could be detected). Although limited in both scope and in numbers of patties analyzed, our findings suggest that the current risk for humans becoming infected with AIV from a beef source is negligible when patties are heated to USDA FSIS temperature recommendations.
2. Validation of a 24-hour enrichment protocol to detect Listeria monocytogenes (Lm) in regulated foods. The USDA Food Safety and Inspection Service (FSIS) routinely tests various regulated ready-to-eat (RTE) food samples for Lm. The current protocol requires about two days to obtain results. ARS scientists in Wyndmoor, Pennsylvania, evaluated several broth media to derive a one-day protocol to test for the presence/absence of Lm in deli ham (a regulated food). Prefatory broth studies confirmed that the natural flora of ham had a significant effect on recovery of Lm: of the 5 commercially-available media tested, in the presence of high levels of background microbiota (1 million cells per g), only two broths, namely Fraser and LPT, consistently detected Lm at levels of 1 and 10 CFU per 2.5 g sample of ham. Next, ARS scientists directly compared the 24-h enrichment of inoculated ham in LPT and Fraser broths to the USDA FSIS standard 48-h enrichment protocol. The results validated that enrichment of ham samples inoculated with low levels of Lm and enriched in LPT for 26 h or 28 h performed like enrichment in the USDA FSIS enrichment media over 48, 50, or 52 h. The validated 24 h protocol using LPT broth will reduce the cost and decrease the enrichment time from 48 h to 24 h without compromising sensitivity, specificity, reproducibility, or ease-of-use.
3. Validation of lethality process parameters for pathogen inactivation in large mass ready-to-eat (RTE) meat products. Heating processes commonly used for large mass meat products may not meet USDA Food Safety and Inspection Service (FSIS) recommended lethality (Appendix A) performance guidelines, allowing for pathogen survival during cooking and/or outgrowth during cooling and storage. ARS scientists in Wyndmoor, Pennsylvania, working with an industry partner developed and validated processing conditions for cured, restructured, smoked boneless ham and cured pork necks following Appendix A guidelines. Hams and pork necks were inoculated with multi-strain cocktails of Salmonella or Listeria monocytogenes (Lm) at about 10 to 100 million cells/product, and then cooked within a commercial smokehouse under different temperature, time, smoking, and relative humidity (RH ) cycles. Results populated data gaps related to time, temperature, and RH parameters that are now elaborated in Appendix A. These data established reductions of about 60,000 to 80 million cells of Lm and Sal in hams and pork necks. These findings on thermal inactivation of Lm and Sal in hams and pork necks will help processors meet current regulatory guidelines for lethality of pathogens in large meat products.
4. Validation of heating and cooling processes for partially heat-treated, smoked hams. Although cooked/smoked hams have been enjoyed for centuries without incident, data are lacking on the effect of smoke in combination with heating/cooling parameters to control pathogens in partially heat-treated meats. ARS scientists in Wyndmoor, Pennsylvania, evaluated a commercially-available liquid smoke product for lethality towards Clostridium perfringens (Cperf) or Staphylococcus aureus (Sa) inoculated on boneless/tumbled hams (9-12 lbs.) during partial cooking/smoking and cooling. The multi-step cooking process, with or without added smoke, delivered reductions of about 20,000 and 400 cells per gram of Sa, respectively. This same process, with or without smoke, delivered reductions of about 85 and 2.5 cells per gram of Cperf, respectively. The conditions for cooling also precluded outgrowth (less than 2 cells per gram increase) of Cperf. These results populate data voids in Appendix A and B for lethality and stabilization, respectively, of Sa or Cperf during cooking and cooling of large mass hams. These data are of immediate use to support Hazard Analysis and Critical Control Points (HACCP) plans and may allow some processors to remain in business.