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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Food Safety and Intervention Technologies Research » Research » Research Project #429829

Research Project: Bacterial Pathogens in Regulated Foods and Processing Technologies for Their Elimination

Location: Food Safety and Intervention Technologies Research

2021 Annual Report

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 and levels of L. monoctyogenes, STEC, and Salmonella spp. in RTE foods at retail, as well as at abattoirs/processing plants. 1.2. Determine the relatedness of L. monoctyogenes, STEC, and Salmonella spp. recovered from foods using molecular typing methods such as PFGE and MLGT. 1.3. Assess perceptions, food safety attitudes, and self-reported behaviors related to observed food safety hazards by consumers who shop at grocery stores. 2: Develop, optimize, and validate processing technologies for eliminating pathogens. 2.1 - Determine the transfer and survival of STEC and Salmonella spp. in ground and tenderized (i.e., non-intact) red meat, pork, pet, and poultry products. 2.2 - Determine cook dwell times for ground poultry products using common consumer preparation methods such as cooking on gas or electric grills at internal instantaneous temperatures ranging from 100° to 160°F for lethality towards Salmonella and STEC and for consumer acceptability. 2.3 - Determine the effectiveness of food grade antimicrobials applied via electrostatic spray and Sprayed Lethality in Container (SLIC®) methods on pork offal and on chicken necks and frames for control of Salmonella and STEC. 2.4 - Validate fermentation and cooking of dry-fermented sausages for control of STEC, Salmonella, and other pathogens. 3: Develop and/or validate strategies to deliver antimicrobials to raw and packaged foods from production through to consumption to control L. monocytogenes, STEC, Salmonella spp., and other pathogens.

We will exploit the tools of microbiology, molecular biology, and food science to recover, characterize, and control food borne pathogens from production through to consumption for a variety of foods, with emphasis on specialty/ethnic and higher volume, higher risk foods. We will identify where pathogens enter the food supply, determine how they persist, and investigate biological, chemical, and physical interventions to eliminate or better manage them to improve public health. The target pathogens of greatest concern for this project are Listeria monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli, Trichinella spiralis, and Toxoplasma gondii. Targeted foods would include, but not be limited to, raw and ready-to-eat (RTE) meat, poultry, pet, and dairy foods, as well as raw and further processed non-intact meats. One focus of the proposed research is to identify sources and niches of the above mentioned pathogens in foods and food processing environments, as well as at retail and food service establishments, to gain insight on factors contributing to their survival and persistence. Multiple isolates recovered from each sample testing positive from such surveys will be retained for further characterization by phenotypic and genotypic (e.g., pulsed-field gel electrophoresis, PCR-based methods, and/or whole genome sequencing) methods to establish relatedness of isolates and their source and succession. As another focus of our research, efforts will be made to validate processes and interventions such as fermentation, high pressure processing, food grade chemicals, and heat, alone or in combination, to inhibit/remove undesirable bacteria from the food supply and to better manage their presence, populations, and/or survival during manufacture and storage of target foods/feed. The proposed research to find, characterize, and kill pathogens along the food chain continuum will expand our knowledge of the most prevalence/potent food borne pathogens and help us to elaborate better methods for controlling them in foods prior to human contact or consumption, thereby enhancing the safety of our global food supply.

Progress Report
This is the final report for Project 8072-41420-019-00D, which ended January 18, 2021. New NP108 approved project is entitled “Incidence of bacterial pathogens in regulated foods and applied processing technologies for their destruction.” We addressed all milestones for Project 8072-41420-019-00D via productive collaborations with Cooperative Research and Development Agreement/Material Transfer Agreement (CRADA/MTRA) partners and food safety professionals from academia, government, industry, and consumer groups. Programmatically, we quantified the prevalence, levels, and types of target pathogens along the food chain continuum from farm to flush and developed and validated biological, chemical, and physical interventions to control Listeria monocytogenes (Lm), Shiga toxin-producing Escherichia coli (STEC), Salmonella spp. (Sal), Trichinella spiralis (Ts) and Toxoplasma gondii (Tg) in a variety of foods. Regarding pathogen presence, we established the comparative recovery rate of STEC in raw, non-intact veal and beef purchased at food retailers in the Mid-Atlantic states in the U.S.: in general, STEC was recovered more frequently from raw veal than beef, and non-O157 STEC were more common than serotype O157:H7 cells. In related experiments, the inability to recover viable cells of STEC displaying serogroup-specific surface antigens for at least one of the seven regulated serogroups of STEC in combination with the stx and eae virulence genes suggests that STEC are not common in the raw ground pork or in marinades (fresh and spent) from specialty grocers or food retailers in the Mid-Atlantic states in the U.S. We also quantified the prevalence of Sal in raw chicken livers from food retailers, research farms, and abattoirs. Whereas the pathogen was recovered quite often from raw chicken livers purchased at food retailers (ca. 60%; 6.4 MPN to 2.4 log CFU/g), Sal was recovered less frequently from livers harvested from birds on a research farm (ca. 5.8%; 0.4 to 2.2 MPN/g) or from livers obtained at a poultry slaughter facility (6.7%). Studies are ongoing to subtype the isolates retained from the abovementioned surveys. As part of a large-scale market basket survey, we used whole-genome sequencing (WGS) to characterize 201 isolates of Lm recovered from 102 of 27,389 (RTE) foods purchased at grocery stores in the U.S. over two years. Although significant differences in genetic diversity were not found following pairwise comparisons of isolates, differences in virulence potential and possible pathogen sources were observed. Collectively, these data provide insight on the true prevalence, levels, and types of pathogens in higher volume and/or higher risk foods, as well as their relatedness, persistence, and source(s) and this, in turn, should lead to better management of pathogens in foods and lower public health risks. Regarding pathogen control and interventions, we monitored the viability or improved the safety and extended the shelf life of raw, further processed, or fermented foods by applying high pressure or heat or treating such products with food-grade chemicals surface agents or ingredients. Antimicrobials were effectively and efficiently delivered into foods as ingredients or onto foods via SLIC® or electrostatic spraying. Examples include the use of organic acids or buffered vinegar, a “clean label” food grade chemical, to control Lm in rotisserie chicken salad, uncured turkey breast, mortadella, or ham, as well as monitoring the fate of STEC, Lm, or Sal on slices of prosciutto and pancetta. We also tested if dry/fermented sausage or dry-cured hams provided a favorable environment for persistence or outgrowth of Ts or Tg during manufacture or during extended (up to 12 months) shelf life. For specialty/ethnic products, we assessed the viability of STEC in “soupie”, a homemade soppressata, and in soppressata that we prepared with certified Angus beef, and both sliced retail and bresaola were prepared to validate safe processes for these specialty/ethnic products. We also validated post-fermentative heating temperatures and times to control pathogens in several dry/fermented sausage types, including pepperoni- and Genoa-type sausage. We also demonstrated that well-established cooking and pressure parameters required to eliminate STEC, Sal, and Lm from ground beef should be as effective for controlling cells of these same pathogens in plant-sourced meat. We also analyzed the effect of experimental parameters such as cooking appliance, volume and type of cooking oil, product formulation, levels and types of antimicrobials, and cooking/fermentation times and temperatures on thermal inactivation of target pathogens in red meat and poultry products. As expected, the higher the temperature and the longer the time for applying heat or fermentation/drying, the greater the reduction in pathogen levels. Lastly, in collaboration with several of our academic partners, we developed a message for the masses media/marketing campaign (aka “160° is Good”) to inform consumers about proper thermometer use and that burgers should be cooked to 160°F. Social media was also used to better educate consumers about real versus perceived food safety risks in retail. These data have real-world application for maintaining the safety of our Nation’s food supply via our frequent input from regulators and the food industry and our use of pathogenic strains and pilot-scale processing equipment.

1. Inactivation of foodborne pathogens within plant burgers in response to heat and pressure. Although plant-based burgers have become increasing popular and more readily available little is known about the safety of such products. Thus, ARS researchers at Wyndmoor, Pennsylvania, conducted research to quantify inactivation of Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes (Lm) within hand-formed plant- and beef-based burgers (ca. 114 g each) subjected to high pressure (HPP; 350 or 600 MPa) or cooked in a saute pan (62.8, 68.3, or 73.9C). Levels of both pathogens were lowered by to 400,000 cells/g via HPP and by ca. 25,000 to 10 million cells/g via cooking. Since both pathogens responded similarly to heat and pressure in plant-based as in beef-based burgers, well-established cooking and HPP parameters required to eliminate STEC or Lm from ground beef should be as effective for controlling cells of these same pathogens in burgers made from a plant-sourced protein.

2. Whole genome comparisons of Listeria monocytogenes isolates recovered from ready-to-eat retail foods. Since Listeria monocytogenes (Lm) is responsible for numerous illnesses and recalls of ready-to-eat (RTE) foods, research is needed to determine the origin, persistence, and relatedness of Lm associated with high-volume, higher-risk RTE foods at retailers. Between 2010 and 2013, ARS researchers at Wyndmoor, Pennsylvania, in collaboration with Food and Drug Administration (FDA) researchers at College Park, Maryland, characterized 201 isolates of Lm recovered from 102 of 27,389 RTE foods purchased at grocery stores in the U.S. as part of the Interagency Market Basket Survey (Lm MBS). To better understand their potential genetic diversity, ARS and FDA scientists performed whole genome sequencing (WGS) analyses of these isolates. Results demonstrated the presence of 29 clones among the 201 isolates, and the full-length virulence gene inlA was present in 89.4% of the isolates. For 91 of 96 food samples tested, both isolates from the same sample were shown to be indistinguishable by WGS. These unique insights into source, clonal groups, and virulence gene profiles in a well-defined set of 201 isolates recovered from selected categories of refrigerated, RTE foods purchased at retail will foster the development of strategies to lower the occurrence of Lm in RTE foods.

3. Inactivation of Toxoplasma gondii in dry-cured ham. Consumption of raw and uncooked pork meat has been associated with transmission of toxoplasmosis; an illness caused by Toxoplasma gondii bradyzoites. Due to the lack of information regarding inactivation of T. gondii bradyzoites during processing of RTE dry-cured meat products, ARS researchers at Wyndmoor, Pennsylvania, in collaboration with ARS researchers in Beltsville, Maryland, investigated the viability of T. gondii in experimentally-infected, dry-cured whole hams processed using methods approved in the U.S. Code of Federal Regulations (9 CFR 318.10) for inactivation of Trichinella spiralis. Results showed that T. gondii bradyzoites were inactivated during the salting and curing step (33 days) for ham manufacture; viable T. gondii were not detected via a mouse bioassay during periodic sampling over the 12-month duration of this experiment. These results demonstrated that the approved protocols for production of dry-cured hams can inactivate T. gondii, lower the risk of toxoplasmosis to consumers, and help industry/processors meet existing regulatory requirements.

4. Post-fermentation heating to control Salmonella in salami. Pathogens such as Salmonella can be recovered from Genoa salami and other fermented sausage, and consumption of fermented meats harboring pathogens has sporadically caused human illness. Thus, ARS researchers at Wyndmoor, Pennsylvania, evaluated the effect of post-fermentation heating times and temperatures in combination with drying on the fate of Salmonella in Genoa salami. After fermentation, chubs were heated to an internal temperature of 46.3 or 48.9C and held for up to 5 h and then dried at 17C for 25 days. Regardless of the post-fermentation heating temperatures and holding times or casing diameter, the endpoint pH after fermentation was ca. pH 4.7. Fermentation alone lowered levels of Salmonella by about 100 cells per gram. Reductions of at least 100,000 cells of Salmonella were achieved in 2-5 h of post-fermentation heating (46.3 or 48.9C) without adversely affecting product quality. These data provide the industry with time/temperature options to achieve reductions of Salmonella in Genoa salami and meet regulatory requirements without appreciably compromising product quality.

Review Publications
Levine, K., Luchansky, J.B., Porto Fett, A.C., Bryant, V., Herring, C., Chapman, B. 2020. How food safety savvy are shoppers? Investigating and impacting consumers’ risk identification skills at retail. Food Protection Trends. 41:21-35.
Cope, S.J., Porto Fett, A.C., Luchansky, J.B., Hochstein, J., Chapman, B. 2020. Utiization of quantitative and qualitative methods to investigate the impacts of a pilot media campaign targeting safe cooking techniques and proper thermometer use. Food Protection Trends. 40:332-348.
Jung, Y., Porto Fett, A.C., Parveen, S., Meredith, J., Shoyer, B.A., Henry, E., Trauger, Z., Shane, L.E., Osoria, M., Schwarz, J., Rupert, C., Chapman, B., Moxley, R., Luchansky, J.B. 2021. Recovery rate of cells of the seven regulated serogroups of shiga toxin-producing Escherichia coli from raw veal cutlets, ground veal, and ground beef from retail stores in the mid-atlantic region of the United States. Journal of Food Protection. 84(2):220-232.