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ARS Home Plains Area Clay Center, Nebraska U.S. Meat Animal Research Center Meat Safety and Quality Research





- Bosilevac, Joseph - Mick
- Kalchayanand, Norasak - Nor
- Schmidt, John
- Shackelford, Steven
- Wang, Rong
- Wheeler, Tommy
- Arthur, Terrance
- 2023
- 2022
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Holistic Tactics to Advance the Microbiological Safety and Quality of the Red Meat Continuum **Location:
                            Meat Safety and Quality** 2023 Annual Report **Objectives**
                                

Objective 1: Identify pre- and post-harvest interventions that reduce foodborne pathogen prevalence and levels in the animal, on carcasses, or in meat products.
Sub-objective 1.A: Identify pre-harvest interventions that impact the concentrations of pathogens colonizing animals and present in feed lot pens and barns. 
Sub-objective 1.B: Identify tactics to overcome sanitizer resistance shown by stress resistant (and antimicrobial resistant) bacteria present in niches. 
Sub-objective 1.C: Identify post-harvest interventions that are environmentally friendly and conserve natural resources. 

Objective 2: Advance red meat sampling and detection technologies to more accurately identify microbial contaminates with greater sensitivity. 
Sub-objective 2.A: Validate and implement new sampling technologies for foodborne pathogens associated with red meat.
Sub-objective 2.B: Identify improved detections technologies for foodborne pathogens associated with red meat.
Sub-objective 2.C: Characterization of bacterial biofilms contributing to product contamination at meat processing plants.

Objective 3: Examine pre-harvest and post-harvest environmental factors that cause microbiological populations (foodborne pathogens, antimicrobial resistant bacteria, and spoilage bacteria) to fluctuate and identify candidate mitigation tactics.
Sub-objective 3.A: Determine effects of season and management practices on occurrence of foodborne pathogens associated with meat animal production. 
Sub-objective 3.B: Determine the microbiomes associated with spoilage of case-ready meat products and the impact of trim applied interventions that may improve shelf life through changes to the community profiles. **Approach**
                                

Livestock and their surroundings are sources of microbial contaminants that threaten the safety of meat across the continuum of production. Production and processing practices influence the emergence and persistence of pathogens and spoilage organisms as well as the transmission of stress or antimicrobial resistance traits among various bacterial populations. This project addresses microbiological contaminants that occur across the meat production continuum. Objective 1 identifies pre- and post- harvest interventions directed at pathogens through experiments examining the effects of feed lot surface treatments and the use of slatted floor barns; examines the persistence of stress resistant E. coli in processing; appraises the efficacy as antimicrobial interventions of high pressure processing (HPP) and cold atmospheric plasma (CAP) with organic acids; and establishes methods to control Clostridium blown pack spoilage.  Objective 2 focuses on improving sampling and detection of microbial contaminates by extending the use of a mobile sampling device for beef trim, and the manual (MSD) and continuous (CSD) sampling devices in pork processing; detecting Salmonella with a diagnostic (Dx) bacteriophage, and detecting Shiga toxin producing E. coli (STEC) via virulence factors. Further, Objective 2 determines how the community structure of biofilms protect and promote the transfer of contaminants throughout the processing plant. Objective 3 examines pre- and post-harvest factors that have an effect on microbiological populations with the goal of identifying candidate mitigation tactics. These experiments examine seasonal effects on E. coli O157:H7; feedlot management practices on Salmonella in lymph nodes; and the use of antimicrobials (AMR) on Salmonella in cattle. Post-harvest, Objective 3 aims to identify populations of bacteria in vacuum packaged and modified atmosphere packaged (MAP) ground meat leading to shortened shelf life and identify treatments applied before grinding that can alleviate this problem.  Outcomes from the research will provide improved methods to monitor, detect, and mitigate pathogens on farm and in processing facilities. Further, the developments will be relevant, environmentally friendly, cost effective, and implementable without impeding current processes. **Progress Report**
                                

Under Objective 1: Identify pre- and post-harvest interventions that reduce foodborne pathogen prevalence and levels in the animal, on carcasses, or in meat products. High-pressure processing (HPP) was evaluated to reduce Salmonella in ground beef, and cold atmospheric plasma (CAP) was evaluated to reduce pathogenic and spoilage bacteria on variety meats. The pressure required to reduce Salmonella in ground beef at least 90% was identified. The minimum pressure is 35,000 psi. The findings indicated that the reduction of Salmonella was higher with lean ground beef (93% lean) than with fatty ground beef (80% lean). HPP reduced antimicrobial resistant (AMR) Salmonella equally as non-AMR Salmonella. One contingency with this study is that the pressure of 35,000 psi caused discoloration of the ground beef. We plan to titrate with less pressure to reduce discoloration once the HPP equipment is repaired. CAP treatment for 30 seconds reduced pathogens from less than 90% to more than 99.99%. The finding indicated that the reduction of pathogens using CAP treatment varied with the type of variety meats. The distance between plasma plume and surfaces of variety meats will be determined to optimize the inactivation effect on the type of variety meats.
Under Objective 2: Advance red meat sampling and detection technologies to more accurately identify microbial contaminates with greater sensitivity. Research was performed to address Sub-objective 2A, Validate and implement new sampling technologies for foodborne pathogens associated with red meat. The Continuous and Manual Sampling Devices (CSD and MSD, respectively) are the methods of choice for pathogen detection sampling in the commercial beef processing industry. In addition, the Food Safety and Inspection Service (FSIS) has adopted the MSD method as the standard sampling method by Agency inspectors in the field. We have conducted several in-plant validations in the past 12 months to assist industry members in implementing the methods in their establishments. Additional work on the MSD focused on new swab and mitt versions and was conducted to determine the efficacy of the MSD methods for sampling turkey carcasses at rehang and post-chill. This project was done in collaboration with FSIS to identify improved methods for collection of turkey carcass samples. Four methods were evaluated during sampling trips to multiple commercial turkey processing plants. The methods were the current FSIS turkey carcass sampling methodology (handheld cellulose sponge and a 5x10 cm template), a polyurethane sponge, half-MicroTally Swab (Half MT-Swab) and MicroTally Mitt (MT-Mitt). Detection targets were Salmonella, Campylobacter and indicator count recovery. From the results it was concluded that the MSD sampling method employing the MT-Mitt had the best recovery of target organisms. Increasing the surface area sampled increases the prevalence of pathogen detection and increasing the force/friction/pressure of the sample improves pathogen recovery. 
Regarding Sub-objective 2.B: Identify improved detections technologies for foodborne pathogens associated with red meat, we began experiments to address stakeholder concerns regarding the reliability of assays designed to quantitate Salmonella in meat samples. Preliminary experiments determined the cold and nutrient stress survival abilities of 12 Salmonella strains isolated from FSIS poultry samples. We then performed pilot experiments for four quantification methods and have begun experiments comparing 4 Salmonella quantification methods with post-chill turkey wings. Highly variable results have been found, as communicated by stakeholders. Sample collection and processing protocols are being examined to improve reliability of assay results. Other work under this sub-objective has focused on evaluating the utility of digital droplet PCR (ddPCR) to identify potentially pathogenic Shiga toxin-producing Escherichia coli (E. coli) (STEC) that possess the virulence factor intimin, and the utility of a STEC indicator agar to allow serogroup independent isolation of STEC. In experiments conducted with FSIS, ddPCR was found to accurately resolve 98% as mixed cultures or containing an STEC in inoculation studies, and when examining FSIS regulatory broths ddPCR agreed with culture isolation 86%. Eleven hundred strains of E. coli (including STEC and non-STEC) were screened on the indicator agar. The agar was highly accurate, however when used for isolation from FSIS beef broths, its inability to prevent background bacteria growth did not allow for isolation of STEC. Reformulation with the addition of selective supplements is underway to improve the agars performance.
Lastly, regarding Sub-objective 2.C: Characterization of bacterial biofilms contributing to product contamination at meat processing plants, we have continued making significant progress toward understanding the potential contribution of environmental microorganisms to pathogen survival and prevalence at processing plants. Floor drain samples at multiple beef and pork plants were collected as representatives of the microorganisms present in the processing environment. The effectiveness of three novel multicomponent sanitizers against Salmonella enterica harbored in biofilms formed from these samples were evaluated. We found that Salmonella strains were able to integrate into the mixed biofilms efficiently even under low temperatures (e.g. 7C), and that foam treatment with the multicomponent sanitizers reduced biofilm and Salmonella in most samples to a non-enumerable level and more significantly, inhibited the pathogen post-sanitization recovery growth. In comparison, application using a fog treatment was less effective. Interestingly, Salmonella had increased survival after sanitization in the pork plant mixed biofilms compared to beef plant biofilms. Metagenomic analysis of the pork and beef biofilm communities showed different relative abundance of species but no significant difference in species diversity. Further analysis is continuing.
Under Objective 3: Examine pre-harvest and post-harvest environmental factors that cause microbiological populations (foodborne pathogens, antimicrobial resistant bacteria, and spoilage bacteria) to fluctuate and identify candidate mitigation tactics, DNA was isolated from fecal and pen surface samples collected from feedlot cattle during winter and summer to identify microbiome changes that associate with seasonal shedding of E. coli O157:H7. Diets for the two seasons were the same to ensure that the changes in intestinal microbiome were not simply due to changes in nutrient availability. Preliminary metagenomic results show potential positive and negative associations between gut microflora and E. coli O157 shedding. Other progress under this objective was made in applying novel dry vinegar ingredients to vacuum packaged (VP) beef and pork blends in order to address spoilage. The addition of these ingredients prevented gas production and blowup of VP packed ground meats and improved other organoleptic qualities and shelf-life. Metagenomic analysis of these products will follow to examine the impact of the ingredients on the microbial communities. **Accomplishments**
                                

1. Kosher processing of fresh beef controls Shiga toxin-producing Escherichia coli (STEC) and Salmonella. Three-quarters of Americans believe kosher food is safer due to the strict dietary rules of the Jewish faith. However, kosher beef is not immune to contamination by pathogens during harvest and processing. With kosher restrictions, most antimicrobials used in conventional beef processing cannot be applied, leaving only salt as an antimicrobial. Salt has satisfactory antibacterial effects, but its impact on foodborne pathogens and beef quality has not been well determined. ARS scientists in Clay Center, Nebraska, found that salted and chilled treatment of beef improved the microbial safety but caused undesirable color changes, higher salt concentration in the final products, and increased off flavor during storage at refrigeration temperature. These results show that standard kosher processing adequately addresses beef safety concerns, but negatively impacts quality compared to conventionally processed beef. 2. Recurring contamination by Salmonella at a meat plant depends on the strain of Salmonella and the indigenous microbial community present. Consumption of Salmonella contaminated red meat and poultry is a leading cause of foodborne illness. Contamination by Salmonella is generally attributed to cross contamination during harvest or from grinding of contaminated lymph nodes. However, ARS scientists in Clay Center, Nebraska, found that a processing plant with recurrent Salmonella contamination harbored Salmonella strains that formed strong biofilms and that interacted with complex bacterial communities in the processing environment to enhance the Salmonella persistence. The Salmonella exhibited high tolerance against sanitization and enhanced survival through the interactions with local environmental microorganisms. Different types of environmental bacteria can either inhibit or protect Salmonella when it is present. The identification of this alternant pathway of contamination will help improve tactics used to reduce Salmonella contamination of fresh meat. 3. Verification parameters for using the Manual Sampling Device (MSD) for fresh raw beef trim. Methods of sample collection for pathogen testing require verification to ensure the sampling protocols are performed adequately. ARS scientists in Clay Center, Nebraska, determined the parameters for use in verifying proper beef trim sampling for the MSD method. Results were three-part. First, MSD sample collection is adequate for pathogen testing if it covers at least half of the top surface of beef trim in a combo and is collected for a minimum of 90 seconds. Second, if MSD sample collection occurs for not less than 30 seconds per side of the swab, it is adequate for pathogen testing. Finally, when an in-plant MSD sample and a regulatory MSD sample are required from the same combo, two MSD samples can be collected from the same combo bin and both are adequate for pathogen testing. These results describe the acceptable deviations from validated protocols that still provide adequate pathogen detection. These protocols will now serve to prevent plants from having to discard product with minor sampling deviations. 4. Extremely heat-resistant (XHR) Escherichia coli are inversely distributed across the beef production and processing continuum. Some Escherichia coli (E. coli) have genes that allow them to resist high temperatures and other decontamination processes used in beef processing plants. Previous results suggested that E. coli like this were seldom found in cattle but present in beef products. To determine their source, ARS scientists in Clay Center, Nebraska, collected and characterized E. coli from the same cattle at feedlots, at harvest, and after being processed into strip loins. Results confirmed the previous observations but found that the resistant E. coli were effectively controlled by beef processing treatments. Their presence in strip loins is likely due to their ability to persist in the chilled environments of processing plants. This work indicates that controlling extremely heat-resistant (XHR) E. coli will require interventions that focus on the processing environment rather than the cattle or harvest floor. 5. Economical detection and genotyping of foodborne pathogens through use of a universal DNA probe. One of the most common methods to test food for contaminating pathogens is to test for the specific segments of DNA of a pathogen. This DNA test requires the use of special reagents for each pathogen. While many of the reagents are inexpensive, the tests that require fluorescent labeled DNA probes are costly and make the DNA tests expensive. ARS scientists in Clay Center, Nebraska, collaborated with scientists at Florida State University, to develop and validate a method that uses a universal replacement for the expensive fluorescent labeled probes. An individual fluorescent probe is normally needed for each real-time DNA test, but the universal replacement can be used for multiple tests. This universal reagent approach simplifies pathogen test development when looking for diverse DNA targets and offers an economical approach that can make real-time pathogen DNA tests less expensive. **Review Publications**
                            *Arthur, T.M., Brown, T., Wheeler, T.L. 2023. Determination of verification parameters for using the manual sampling device for fresh raw beef trim. Journal of Food Protection. 86. Article 100041. https://doi.org/10.1016/j.jfp.2023.100041.*
                                *Kalchayanand, N., Koohmaraie, M., Wheeler, T.L. 2023. Fate of Shiga toxin-producing Escherichia coli (STEC) and Salmonella during kosher processing of fresh beef. Journal of Food Protection. 86(6). Article 100088. https://doi.org/10.1016/j.jfp.2023.100088.*
                                *Haque, M., Bosilevac, J.M., Chaves, B.D. 2022. A review of Shiga-toxin producing Escherichia coli (STEC) contamination in the raw pork production chain. International Journal of Food Microbiology. 377. Article 109832. http://doi.org/10.1016/j.ijfoodmicro.2022.109832.*
                                *Guragain, M., Schmidt, J.W., Dickey, A.M., Bosilevac, J.M. 2023. Distribution of extremely heat-resistant Escherichia coli in the beef production and processing continuum. Journal of Food Protection. 86(1). Article 100031. https://doi.org/10.1016/j.jfp.2022.100031.*
                                *Kalchayanand, N., Wang, R., Brown, T., Wheeler, T.L. 2023. Efficacy of short thermal treatment time against Escherichia coli O157:H7 and Salmonella on the surface of fresh beef. Journal of Food Protection. 86(3). Article 100040. https://doi.org/10.1016/j.jfp.2023.100040.*
                                *Valez, F., Bosilevac, J.M., Mishra, A., Singh, P. 2023. Universal hydrolysis probe-based approach for specific detection and genotyping of foodborne pathogens. Journal of Microbiological Methods. 204. Article 106632. https://doi.org/10.1016/j.mimet.2022.106632.*
                                *Velez, F.J., Bosilevac, J.M., Delannoy, S., Fach, P., Nagpal, R., Singh, P. 2022. Development and validation of high-resolution melting assays for the detection of potentially virulent strains of Escherichia coli O103 and O121. Food Control. 139. Article 109095. https://doi.org/10.1016/j.foodcont.2022.109095.*
                                *Wang, R., Dass, S.C., Chen, Q., Guragain, M., Bosilevac, J.M. 2022. Characterization of Salmonella strains and environmental microorganisms isolated from a meat plant with Salmonella recurrence. Meat and Muscle Biology. 6(1). Article 15442. https://doi.org/10.22175/mmb.15442.*
                                *Agga, G.E., Galloway, H.O., Netthisinghe, A.M., Schmidt, J.W., Arthur, T.M. 2022. Tetracycline-resistant, third-generation cephalosporin–resistant, and extended-spectrum b-lactamase–producing Escherichia coli in a beef cow-calf production system. Journal of Food Protection. 85(11):1522-1530. https://doi.org/10.4315/JFP-22-178.*


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