Location: Meat Safety and Quality
2024 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. Active packaging film was evaluated to reduce the onset of blown pack spoilage (BPS) of intact beef due to Clostridium estertheticum, and antimicrobial interventions were evaluated to reduce pathogenic and spoilage bacteria on variety meats. From 16 U.S. Food and Drug Administration (FDA) approved commercially available antimicrobials, 2 antimicrobials (nisin and zinc oxide nanoparticles) were identified to reduce the onset of BPS. Each antimicrobial and its combination were incorporated to form active packaging films. The finding indicated that the onset of BPS fresh beef from the film without antimicrobial compound(s) occurred in 16 days at refrigeration temperature. In contrast, the onset BPS from the fresh beef with nisin active packaging film occurred at 36 days. Nisin was the most effective in controlling onset of BPS. Diluted organic acids, ice-chilled (conventional treatment), and cold atmospheric plasma (CAP) were applied to reduce pathogens and spoilage bacteria on variety meats. The findings indicated that ice-chilled did not reduce bacteria on the surface of variety meats. The application of organic acids or CAP immediately reduced bacteria at least 90%.
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 for raw beef trimmings by Agency inspectors in the field. We have been working to extend the product range for the MSD sampling method. Our recent work has focused on using the Mitt form of the MSD to collect samples of beef variety meats, specifically head meat, cheek meat, and hearts. The preliminary data from these trials shows that collecting variety meat samples using the Mitt is as good or better than the current method, which utilizes grab samples. In addition, we have conducted experiments to determine the efficacy of the MSD methods for sampling chicken carcasses at rehang and post-chill as well as chicken parts. Detection targets were Salmonella, Campylobacter and indicator count recovery. From the results it was concluded that the MSD sampling method employing the Mitt had as good or better recovery of target organisms then the rinse method currently used by FSIS and the poultry processing industry. Replacing the rinse method with the MSD method will reduce the cost of sampling and the time it takes to collect samples.
Further work under Objective 2 related to detection and confirmation of pathogens in meats examined the use of digital droplet polymerase chain reaction (ddPCR) to identify regulatory Shiga toxin producing E coli (STEC) in beef and pork samples. The ddPCR identified linked virulence factors (Shiga toxin and the adherence factor intimin) in a much narrower set of samples than those identified by the more common PCR screening methods that cannot identify linked targets. Further, the ddPCR linked positive samples correlated with more culture confirmed samples that the common PCR screening methods. In regards to isolating STEC, a new media that indicates Shiga toxin producing colonies was examined and compared to other more commonly used agar media. The new media is showing promise to help increase the rates of culture confirmation.
Under Objective 3, Sub-objective 3.C: Characterization of bacterial biofilms contributing to product contamination at meat processing plants. We have continued making significant progress toward understanding the contribution of bacterial biofilms to meat contamination. In particular, we investigated the impact of intense sanitization (IS), one action taken by many processors annually or semiannually to address contamination incidence, on environmental biofilms and the subsequent pathogen colonization and stress tolerance. We collected processing plant environmental biofilm samples before and after the IS procedure and tested biofilm formation by these microorganisms as well as the ability of the multispecies biofilms to recruit and/or protect co-inoculated S. enterica strains from sanitization. Overall, post-IS samples formed significantly stronger biofilms than the respective pre-IS samples, even though S. enterica colonization was not different between the pre- and post-IS biofilms. Importantly, a higher S. enterica survival was observed in post-IS mixed biofilms which was associated with the stronger biofilm matrix. Furthermore, 16S sequencing results exhibited a community species diversity decrease 1 week after IS but followed by a significant increase 4 weeks after the treatment. Therefore, IS significantly altered the community composition and the higher presence of certain species in the post-IS community may be associated with the stronger mixed biofilm formation and pathogen tolerance. Our study suggested that the IS procedure might disrupt the existing environmental microbial community and alter the natural population composition, which might lead to unintended consequences as a result of a lack of competition within the multispecies mixture. The survival and recruitment of species with high colonizing capability to the post-IS community may play crucial roles in shaping the ensuing ecological dynamics.
Accomplishments
1. New methods that validate the effectiveness of poultry wing rinses for Salmonella. It has become consensus that Salmonella presence/absence testing in poultry is not providing adequate protection against the risk of Salmonella in final products and regulatory requirements may soon include testing whether samples exceed a certain level of Salmonella. To prepare for this eventuality and to improve process control the poultry processing industry requires feasible, rapid methods to identify wing rinses exceeding a certain level of Salmonella. ARS scientists at Clay Center, Nebraska, found that the currently available methods neither provide the required accuracy nor the feasibility of implementation. Therefore, they developed a series of accurate simple threshold tests that can be calibrated to different threshold levels required by the user. This threshold testing approach provides a better combination of cost, speed, ease of use, and accuracy than the currently available methods and can be adapted to many food production sample types to ensure pathogen levels are low and improve food safety.
2. Cattle from different production systems carry different serogroups of Shiga Toxin-producing Escherichia coli in their gut at harvest. There are many different types of Shiga toxin producing E. coli (STEC) that can cause disease of varying severity if they are consumed by humans. Cattle carry numerous STEC which may contaminate beef during harvest and processing. In addition, cattle harvested for beef are raised in diverse production systems, including large and small feedlots, dairies, or on-farm beef herds. ARS scientists at Clay Center, Nebraska, examined how these production systems influenced the amount and type of STEC carried by cattle when they are harvested. Nearly all cattle from each production system carried STEC, but feedlot cattle carried more severe disease related STEC than cull dairy cattle, which carried more than cull beef cattle. Understanding how different STEC populate different production systems will help the beef industry design better risk management strategies for food safety.
3. Manual sampling mitt validated to collect samples from beef manufacturing trimmings for pathogen testing. All raw beef trimmings are sampled and tested for pathogens in federally inspected meat processing facilities. The samples are collected manually using excision methods or using the non-destructive MicroTally (TM) Swab (MT-Swab). A new method using a MicroTally (TM) Mitt (MT-Mitt) has been developed to improve ease and reliability of sample collection. ARS scientists at Clay Center, Nebraska, performed trials and compared using the MT-Swab, excision, or the new method MT-Mitt. Samples were collected on numerous days from five processing plants. The results of these trials demonstrated that manual sampling of raw beef trimmings using the MT-Mitt is equivalent to the MT-Swab, excision, and methods in recovering bacteria. Thus, the MT-Mitt method provides an alternative sampling method with equivalent bacterial recovery and significant implementation advantages for some applications regarding labor and ease of use relative to other approved methods for sampling beef trimmings.
4. Intense sanitization at meat processing plants changes environmental biofilm communities and alters the survival of Salmonella. Salmonella enterica is a leading cause of foodborne illness and is related to the consumption of contaminated foods including meat products. Salmonella may persist in a meat processing plant by living within a community of other bacteria as a biofilm. To combat biofilms meat processors perform intense sanitization of their facilities one or two times per year. ARS scientists at Clay Center, Nebraska, examined the long- and short-term effects of intense sanitization at multiple meat plants on disrupting the types of bacteria present and the biofilms they formed with Salmonella. Results showed that intense sanitization disrupted the existing environmental bacterial community but had unexpected effects by selecting more resilient communities that better supported Salmonella. The study provided insight into the impact of environmental biofilms on Salmonella survival and how they can persistence under sanitizer stress in meat processing facilities. These results will help to design better sanitization protocols to end the persistence of Salmonella in meat plants.
5. Peracetic acid reduces pathogens on beef. Production and consumption of ground beef has raised awareness regarding microbiological food safety due to repeated contamination from foodborne pathogens. If the pathogens are present when meat trimmings are ground, then more of the meat is exposed to the harmful bacteria through the grinding process. Consequently, it is important to reduce pathogens on beef trimmings before grinding, while simultaneously meeting regulatory requirements to limit liquid weight gain in the ground product. ARS scientists at Clay Center, Nebraska, demonstrated that peracetic acid treatment of beef trimmings and subprimal beef cuts reduced pathogens compared to untreated fresh beef and met the regulatory requirements for weight gain. These results provided the necessary information to convince ground beef producers they can improve the safety of their ground beef while staying within regulatory limits.
6. Analyses of closed Shiga toxin positive and negative Escherichia coli O157:H7 genomes reveal pathogen evolution. Escherichia coli O157:H7 can cause very severe human disease because it expresses Shiga toxins. Two different E. coli O157:H7 were isolated from an infected person (strains TT12A and TT12B). One strain was the expected disease-causing E. coli O157:H7 that produced Shiga toxins, while the other lacked Shiga toxins. ARS scientists at Clay Center, Nebraska, in collaboration with scientists at the University of Texas San Antonio, used high resolution DNA sequencing to compare full closed genomes of the two strains. It was found that the two strains were identical except for the loss of the DNA elements that provide Shiga toxin. The results revealed one example of how E. coli O157:H7 has evolved and will continue to evolve over time and this work will better inform future genome based methods to improve food safety.
Review Publications
Chen, Q., Palanisamy, V., Wang, R., Bosilevac, J.M., Chitapilly Dass, S. 2024. Salmonella-induced microbiome profile in response to sanitation by quaternary ammonium chloride. Microbiology Spectrum. 12(2). Article 02346-23. https://doi.org/10.1128/spectrum.02346-23.
Arthur, T.M., Reno, F.J., Wheeler, T.L. 2024. Validation of a new method of sampling beef manufacturing trimmings for pathogen testing using a manual sampling mitt approach. Journal of Food Protection. 87(3). Article 100233. https://doi.org/10.1016/j.jfp.2024.100233.
Bosilevac, J.M., Katz, T.S., Arthur, T.M., Kalchayanand, N., Wheeler, T.L. 2024. Proportions and serogroups of enterohemorrhagic Shiga Toxin-producing Escherichia coli in feces of fed and cull beef and cull dairy cattle at harvest. Journal of Food Protection. 87(6). Article 100273. https://doi.org/10.1016/j.jfp.2024.100273.
Wang, R., Guragain, M., Chitlapilly Dass, S., Palanisamy, V., Bosilevac, J.M. 2024. Impact of intense sanitization on environmental biofilm communities and the survival of Salmonella enterica at a beef processing plant. Frontiers in Microbiology. 15. Article 1338600. https://doi.org/10.3389/fmicb.2024.1338600.
Kalalah, A.A., Koenig, S.S., Feng, P.C., Bosilevac, J.M., Bono, J.L., Eppinger, M. 2024. Pathogenomes of Shiga toxin positive and negative Escherichia coli O157:H7 strains TT12A and TT12B: Comprehensive phylogenomic analysis using closed genomes. Microorganisms. 12(4). Article 699. https://doi.org/10.3390/microorganisms12040699.
Kalchayanand, N., Arthur, T.M., Wang, R., Brown, T., Wheeler, T.L. 2024. Evaluation of peracetic acid treatment on beef trimmings and subprimals against Salmonella and E. coli O157:H7 within regulatory retained water limitations. Journal of Food Protection. 87(3). Article 100217. https://doi.org/10.1016/j.jfp.2024.100217.
Bosilevac, J.M., Guragain, M., Barkhouse, D.A., Velez, S.E., Katz, T., Lu, G., Wang, R. 2024. Impact of intense sanitization procedures on bacterial communities recovered from floor drains in pork processing plants. Frontiers in Microbiology. 15. Article 1379203. https://doi.org/10.3389/fmicb.2024.1379203.