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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** 2022 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.A: Cattle pen surface and lymph node samples were obtained. Based on samples obtained we expect the fiscal year (FY) 2023 milestone will be fully met and the goal of determining the relationship between Salmonella present in cattle feedyards during finishing and Salmonella present in lymph nodes that may contaminate final products will be achieved.
Under Sub-objective 1.C.1: To test the effect of high-pressure processing (HPP) on reduction of Salmonella in ground beef, we have completed screening of ground beef samples to make sure that they do not contain any Salmonella before use and are scheduled to perform high pressure treatments with our collaborators at the University of Nebraska Lincoln (UNL). 
Under Sub-objective 1.C.2: To evaluate cold atmospheric plasma (CAP), lactic acid, and peracetic acid on reducing pathogenic and spoilage bacteria on variety meats, a preliminary study was completed that indicated that CAP reduced approximately 90% of spoilage and pathogenic bacteria on surfaces of cheek meat without effect on color or lipid oxidation.
Under Sub-objective 1.C.3: Thirteen antimicrobial intervention compounds to reduce “Blown Pack” spoilage (BPS) of intact beef due to psychrophilic Clostridium spp. were studied and results indicate that zinc oxide nanoparticles (ZnO-NP) and nisin (an antimicrobial peptide) inhibited the growth of clostridial spores. Next, these compounds will be combined and incorporated into packaging materials to determine any synergistic effects that improve the efficacy of the antimicrobials.
Under Sub-objective 2A: Work revolved around improving methods of continuous and manual sample collection (CSD and MSD) using the MicroTally Swab (MT-Swab). Some users found the MT-Swab to be a bit cumbersome when performing the MSD technique.To provide users with more control during MSD sample collection, a MicroTally Mitt (MT-Mitt) was developed. The MT-Mitt allows the user to collect the sample with more scrubbing force without concern for grip strength. A series of trials were conducted comparing manual sampling of raw beef trim using the MT- Swab vs. the MT-Mitt. These data came from samples collected on numerous days across multiple processing plants, and multiple lean types. The results of the trials collectively demonstrated that sampling beef trim using the MT-Mitt provided organism recovery that is equivalent to that of the MT-Swab.
MSD and CSD validation data for these novel methods using pork as the matrix for sampling were collected. Samples were collected during raw pork processing at multiple hot-boned sow operations using the CSD and MSD and analyzed for indicator counts and pathogen index targets. The CSD and MSD methods were compared to traditional pork grab sampling for ease-of-use, cost, and bacterial recovery. The comparison showed that the MSD and CSD methods were equivalent to the grab method for bacterial recovery, however they could be performed for less cost due to their non-destructive sampling nature. Additional studies will be done using the CSD and MSD methods at fed pork processing facilities.
Under Sub-objective 2.B: To identify improved detection technologies for foodborne pathogens associated with red meat, droplet polymerase chain reaction (PCR) technology was investigated as a means of discriminating Escherichia coli (E. coli) that possess both Shiga toxin (stx) and intimin (eae) genes from mixtures of E. coli that each possess only stx or eae. Inoculation studies with beef trim provided evidence supporting droplet PCR, then regulatory beef enrichment broths were examined as a test of real-world utility. The droplet PCR has so far been able to identify all culture positive samples and identify others that were initially miscategorized as negative using alternate methods. The studies are ongoing to build up a larger set of data. 
Under Sub-objective 2.C: For characterization of bacterial biofilms contributing to product contamination at meat processing plants, significant progress has been made. Floor drain samples were collected from multiple beef and pork plants as representatives of the microorganisms present in the local environment. Biofilm-forming ability of the drain samples was examined under processing conditions and their bacterial species composition was investigated with metagenomic analysis. Importantly, multiple pathogenic strains (Salmonella enterica) were isolated from a processing plant environment with a history of Salmonella recurrence. The isolated Salmonella strains were either strong biofilm formers able to outcompete the companion bacteria and became the major component of the mixed biofilm community or exhibited strain-specific high tolerance against sanitization. Salmonella survival after sanitizer treatment was also positively correlated with the total mixed biofilm matrix. 
Under Sub-objective 3.B: To 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, a comparison of metagenomic methods was performed. A set of samples were submitted for 16S  sequencing and for shallow shotgun metagenomic sequencing. The shallow shotgun approach was examined because it is not biased by the PCR amplification 16S  sequencing relies on and because it can provide greater discrimination of  organisms present. The shallow shotgun results were superior and further studies of metagenomics during spoilage will be examined using this approach, rather than 16S metagenomics. **Accomplishments**
                                

1. Salmonella biofilms transfer from food contact surfaces to beef products. Beef contamination by Salmonella is a serious public health concern that can cause foodborne illness outbreaks and significant meat industry financial losses due to product recalls. Bacterial biofilms are surface-attached microbial communities that can contribute to meat contamination by directly transferring from a food contact surface to a meat product. USDA-ARS scientists at Clay Center, Nebraska, determined that the biofilm transfer rates of Salmonella strains were greater from stainless steel than from polyvinylchloride to surfaces of lean meat and fat tissues of beef trim. Salmonella biofilms could repeatedly transfer from contact surfaces to both lean and fat surfaces of beef products. These data highlight the need for effective sanitation processes that address the potential for biofilms in meat processing plants containing Salmonella to cause meat contamination. 2. Genome comparisons of Escherichia coli O113:H21 reveal why some strains cause severe human illness. Shiga toxin-producing Escherichia coli (E. coli) of serotype O113:H21 are found around the globe and can be divided into two groups. One group called ST-820 is found in Australia and can cause very severe disease while the other group called ST-223 is found outside Australia and does not cause severe illness. To investigate why these two groups of E. coli O113:H21 differ in their ability to cause disease, USDA-ARS scientists at Clay Center, Nebraska, partnered with scientists at the University of Texas at San Antonio to sequence strains from these two groups and compare them to other E. coli O113:H21 in public databases. Results revealed how the strains were evolutionarily related to one another and identified many virulence markers that set the ST-820’s apart from the ST-223’s. This whole genome sequencing project provided new information about pathogenic E. coli that will now be applied to improve risk assessment, surveillance, and public health. 3. Escherichia coli found in beef may be the real culprit in colorectal cancer. Some Escherichia coli (E. coli) produce a toxin called colibactin that can lead to colorectal cancer in people. Since E. coli can be found on red meat and red meat diets have been associated with colorectal cancer, USDA-ARS scientists at Clay Center, Nebraska, examined E. coli isolated from cattle and beef products to see if any were colibactin producers. Although colibactin E. coli were found in cattle and on beef, current beef processing interventions effectively control them. Consequently, the presence of these E. coli on beef may be confounding the association of colorectal cancer and red meat. More specifically based on these results, the colibactin E. coli on contaminated beef may actually be the cause of colorectal cancer in beef consumers rather than the beef per se. **Review Publications**
                            *Allué-Guardia, A., Koenig, S.S., Martinez, R.A., Rodriguez, A.L., Bosilevac, J.M., Feng, P., Eppinger, M. 2022. Pathogenomes and variations in Shiga toxin production among geographically distinct clones of Escherichia coli O113:H21. Microbial Genomics. 8. Article 000796. https://doi.org/10.1099/mgen.0.000796.*
                                *Wang, R., King, D.A., Kalchayanand, N. 2022. Evaluation of Salmonella biofilm cell transfer from common food contact surfaces to beef products. Journal of Food Protection. 85(4):632-638. https://doi.org/10.4315/JFP-21-334.*
                                *Guragain, M., Schmidt, J.W., Kalchayanand, N., Dickey, A.M., Bosilevac, J.M. 2022. Characterization of Escherichia coli harboring colibactin genes (clb) isolated from beef production and processing systems. Nature Scientific Reports. 12. Article 5305. https://doi.org/10.1038/s41598-022-09274-x.*
                                *Wang, R., Zhou, Y., Kalchayanand, N., Harhay, D.M., Wheeler, T.L. 2021. Consecutive treatments with a multicomponent sanitizer inactivate biofilms formed by Escherichia coli O157:H7 and Salmonella enterica and remove biofilm matrix. Journal of Food Protection. 84(3):408-417. https://doi.org/10.4315/JFP-20-321.*
                                *Zhang, Y., Schmidt, J.W., Arthur, T.M., Wheeler, T.L., Zhang, Q., Wang, B. 2022. A farm-to-fork quantitative microbial exposure assessment of beta-lactam resistant Escherichia coli among U.S. beef consumers. Microorganisms. 10(3). Article 661. https://doi.org/10.3390/microorganisms10030661.*
                                *Schmidt, J.W., Murray, S.A., Dickey, A.M., Wheeler, T.L., Harhay, D.M., Arthur, T.M. 2022. Twenty-four-month longitudinal study suggests little to no horizontal gene transfer in situ between third-generation cephalosporin-resistant Salmonella and third-generation cephalosporin-resistant Escherichia coli in a beef cattle feedyard. Journal of Food Protection. 85(2):323-335. https://doi.org/10.4315/JFP-21-371.*
                                *Zhang, Y., Schmidt, J.W., Arthur, T.M., Wheeler, T.L., Wang, B. 2021. A comparative quantitative assessment of human exposure to various antimicrobial-resistant bacteria among U.S. ground beef consumers. Journal of Food Protection. 84(5):736-759. https://doi.org/10.4315/JFP-20-154.*
                                *Moreau, M.R., Kudva, I.T., Katani, R., Cote, R., Li, L., Arthur, T.M., Kapur, V. 2021. Non-fimbrial adhesin mutants reveal divergent Escherichia coli O157:H7 adherence mechanisms on human and cattle epithelial cells. International Journal of Microbiology. 2021. Article 8868151. https://doi.org/10.1155/2021/8868151.*
                                *Katani, R., Kudva, I.T., Srinivasan, S., Schilling, M., Cote, R., Li, L., DebRoy, C., Arthur, T.M., Kapur, V., Stasko, J.A. 2021. Strain and host-cell type dependent role of type 1 fimbriae genes in the adherence phenotype of super-shedder strains of Escherichia coli O157:H7. International Journal of Medical Microbiology. 311(4). https://doi.org/10.1016/j.ijmm.2021.151511.*
                                *Doster, E., Thomas, K.M., Weinroth, M.D., Parker, J.K., Crone, K.K., Arthur, T.M., Schmidt, J.W., Wheeler, T.L., Belk, K.E., Morley, P.S. 2020. Metagenomic characterization of the microbiome and resistome of retail ground beef products. Frontiers in Microbiology. 11. Article 541972.. https://doi.org/10.3389/fmicb.2020.541972.*
                                *Weinroth, M.D., Thomas, K.M., Doster, E., Vikram, A., Schmidt, J.W., Arthur, T.M., Wheeler, T.L., Parker, J.K., Hanes, A.S., Alekoza, N., Wolfe, C., Metcalf, J.L., Morley, P.S., Belk, K.E. 2022. Resistomes and microbiomes of meat trimmings and colon content from culled cows raised in conventional and organic production systems. Animal Microbiome. 4. Article 21. https://doi.org/10.1186/s42523-022-00166-z.*
                                *Edrington, T.S., Arthur, T.M., Loneragan, G.L., Genovese, K.J., Hanson, D.L., Anderson, R.C., Nisbet, D.J. 2020. Evaluation of two commercially-available Salmonella vaccines on Salmonella in the peripheral lymph nodes of experimentally-infected cattle. Therapeutic Advances in Vaccines and Immunotherapy. 8:1-7. https://doi.org/10.1177/2515135520957760.*
                                *Arthur, T.M., Wheeler, T.L. 2021. Validation of additional approaches and applications for using the continuous and manual sampling devices for raw beef trim. Journal of Food Protection. 84(4):536-544. https://doi.org/10.4315/JFP-20-345.*
                                *Kalchayanand, N., Dass, S.C., Zhang, Y., Oliver, E.L., Wang, B., Wheeler, T.L. 2022. Efficacy of antimicrobial interventions used in meat processing plants against antimicrobial tolerant non-antibiotic-resistant and antibiotic-resistant Salmonella on fresh beef. Journal of Food Protection. 85(8):1114-1121. https://doi.org/10.4315/JFP-21-364.*


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