Location: Meat Safety and Quality
2021 Annual Report
Objectives
Objective 1: Develop and validate novel pre- and post-harvest intervention strategies to reduce or eliminate foodborne pathogen colonization and persistence in the animal and on carcasses and meat products.
Sub-objective 1.A: Identify effective control measures to reduce pathogens and in the pre-harvest environment.
Sub-objective 1.B: Identify and/or improve efficacious non-thermal post-harvest interventions to reduce contamination of processing plant surfaces, hides, carcasses, and meat products.
Sub-objective 1.C: Determine if current processing interventions are equally effective on AMR bacteria and foodborne pathogens.
Objective 2: Develop improved sampling, detection, and tracking technologies to identify points, including biofilms, where pathogens persist and contaminate in the production of red meat.
Sub-objective 2.A: Characterization of bacterial and environmental components contributing to high event periods (HEP) of E. coli O157:H7 contamination at beef processing plants.
Sub-objective 2.B: Identify improved sampling and detections technologies for foodborne pathogens associated with red meat.
Sub-objective 2.C: Develop and evaluate indicator organisms as surrogates for tracking pathogens through beef processing.
Objective 3: Identify environmental and management practices that influence antimicrobial resistance, colonization of lymph nodes, and colonization rates of cattle, veal, and swine.
Sub-objective 3.A: Determine effects of season and production system on occurrence of antimicrobial resistance and foodborne pathogens associated with food animal production.
Sub-objective 3.B: Identify environmental and management practices that influence Salmonella in lymph nodes.
Sub-objective 3.C: Determine the prevalence of STEC and AMR in veal production systems and identify factors contributing to colonization.
Approach
Cattle and swine can serve as reservoirs of foodborne pathogens that can spread through the environment or to meat during harvest. Further, pharmacologic antimicrobial use in meat animal production is a concern due to the perceived possibility of emergence and transmission of antimicrobial resistant (AMR) bacteria to the environment and food supply. Research to develop ways to reduce the levels of foodborne pathogens such as Shiga-toxin producing Escherichia coli (STEC) and Salmonella on farms and in foods is important, as is understanding and reducing the risk posed to food safety by AMR bacteria present in the meat production system. To this end, the effects of animal vaccines and direct fed microbial feed additives will be investigated to reduce or eliminate foodborne pathogens in the pre-harvest environment. During the harvest process, chlorine dioxide gas, cold atmospheric plasma, and a unique nano-technology sprayer will be assessed to reduce contamination. Novel methods to detect and track pathogens will be designed and tested including examining processing plants for biofilms and determining their roles during times of widespread pathogen contamination. Environmental and animal management practices that influence antimicrobial resistance and colonization of meat animals by pathogens will be studied, with the goal of identifying management practices that influence Salmonella in beef carcass lymph nodes and the prevalence of STEC in veal production. Successful completion of the project objectives will increase the ability of producers and processors to monitor production and use improved interventions to control contamination and product loss, and clarify the risk of antimicrobial resistance in meat production, while providing meat consumers a decreased risk of foodborne illness.
Progress Report
Under Objective 1, blown pack spoilage (BPS) of refrigerated vacuum-packaged intact fresh meat due to bacterial spores was investigated. Spores can grow without oxygen and can resist antimicrobial interventions. BPS is a significant problem in the meat industry and has caused considerable economic loss to meat processors. The onset of spoilage is a challenge because the specific level of spore contamination that leads to BPS is not known and there are no specific interventions available to prevent BPS. Our finding indicated that BPS depends on the contaminated spore levels and the onset of BPS is as fast as 12 days at refrigeration temperature. Therefore, hygienic carcass dressing is critical to keep contamination to a minimum and maximize storage life of the vacuum-packaged chilled product.
Since the meat processing plants harbor a wide variety of environmental microorganisms along with the pathogens, and the environmental mixed biofilms may enhance pathogen stress tolerance, we collected floor drain samples to investigate the impact of mixed biofilm formation by environmental microorganisms on Escherichia coli (E. coli) O157:H7 survival and prevalence. Results showed that E. coli O157:H7 strains obtained significantly greater sanitizer tolerance when formed mixed biofilms with drain microorganisms from the facility with historic recurrence of this pathogen than those mixed with drain samples from the other facility. Analysis of 16S rRNA sequencing results indicated that the E. coli O157:H7 protecting biofilms had higher species diversity with certain unique families and the percentages of the species in the mixture were altered significantly after sanitization, suggesting the community composition affects the role and tolerance level of each individual species. To further our mixed biofilm-pathogen interaction study to address Salmonella contamination, we collected additional environmental samples from various meat plants with different Salmonella prevalence histories. Multiple Salmonella strains of serovars frequently identified in meat product contamination were isolated from the facility with the history of Salmonella recurrence. Our results further revealed various mechanisms underlying the survival advantages of these Salmonella strains, including strong biofilm formation, outcompeting capability against background microorganisms, strain – specific high stress tolerance, and the interspecies interactions with environmental microbials resulting in enhanced sanitizer tolerance via mixed biofilm formation. Our study therefore indicates the unique and highly diverse environmental microflora is critical for pathogen recruitment, tolerance, and survival, that can result in high prevalence and recurrent contamination events by certain pathogens.
Under Objective 2, training on the Continuous and Manual Sampling Devices (CSD/MSD) was performed in three commercial beef processing plants. In-plant validations of these methods were completed for four commercial beef processing plants, with two of the plant validations being part of the U. S. Department of Agriculture Agricultural Marketing Service (AMS) approval process pilot program. A new application of the MSD technology for sampling boxed beef trimmings was evaluated and the proof-of-concept trial was completed successfully. In addition, new parameters regarding the minimum beef trim combo bin surface area required for adequate MSD sampling and efficacy of repeated MSD sampling on the same beef trim combo bin were determined.
Under Objective 3, to determine if concentrations of high-risk Salmonella in beef cattle production pen surface materials correlate with contaminated lymph nodes during processing, the participating feedyards were contacted to confirm that sampling will begin in fiscal year (FY) 2022. Limited pilot experiments were performed to ensure detection, enumeration, and molecular confirmation methods to detect high-risk Salmonella are suitable. In addition, under Objective 3, fecal and blood samples were collected from the same 240 head of feedlot cattle during winter and summer to identify microbiological and immunological changes that associate with seasonal shedding of E. coli O157:H7. DNA isolation and metagenomic DNA sequencing have begun.
Accomplishments
1. Antimicrobial resistance in U.S. retail ground beef products with and without label claims regarding antibiotic use is not different. Ground beef products with “raised without antibiotics” label claims are generally perceived to have lower antibiotic resistance levels than conventional products with no label claims regarding antibiotic use. USDA-ARS scientists in Clay Center, Nebraska, in collaboration with scientists from Colorado State University compared antibiotic resistances between products with these different label claims. Four antibiotic resistance levels were higher in raised without antibiotics ground beef while three were higher in conventional ground beef. This also was the first use of metagenomic sequencing to measure the antibiotic resistance in retail ground beef products. These results show that retail ground beef products that had label claims of meat from animals raised without antibiotics were not different from meat without those label claims in terms of antimicrobial resistance. In all, this research adds to the growing body of knowledge showing antibiotic use in livestock production does not have a significant impact on antimicrobial resistance of bacteria found on food products and highlighted the utility of using metagenomic sequencing for future research.
2. Heat resistant Escherichia coli in meat are not a food safety threat. Some Escherichia coli (E. coli) are extremely heat resistant (XHR), and this resistance is due to a genetic element called the Locus of Heat Resistance (LHR). The LHR helps the bacteria deal with stresses such as those that might occur during meat processing or cooking preparation. It is not known if XHR E. coli are more common in certain types of meat such as beef, veal, pork, or lamb so ARS scientists in Clay Center, Nebraska, examined XHR E. coli from these different meats. All XHR E. coli turned out to be non-pathogens with no specific meat type source. Thus, heat resistance is not currently a threat to meat safety because all heat resistance E. coli detected thus far are non-pathogenic.
3. Approaches and applications for using continuous (CSD) and manual (MSD) sampling devices for raw beef trim. New beef trim sampling methods for pathogen detection were recently commercialized and marketed as MicroTally swabs. Users of the MicroTally swabs were in need of validated protocols that addressed commonly encountered implementation variation in commercial beef processing. USDA-ARS scientists in Clay Center, Nebraska, conducted validation experiments for various alternative MicroTally sampling implementation procedures comparing MicroTally-based sampling to established methods. The results showed that the various alternative applications of MicroTally-based trim sampling for pathogen detection were equivalent or better than previous methods and provide additional benefits in reduced labor and other costs, and improved worker safety.
4. Exposure to antimicrobial-resistant bacteria among U.S. ground beef consumers. Some ground beef is made from cattle with the label claim "raised without antibiotics" and is marketed accordingly. Public perception is that this ground beef reduces exposure to antibiotic resistant bacteria compared to ground beef made from conventionally raised cattle that may have been treated with antibiotics for illness. ARS scientists in Clay Center, Nebraska, in collaboration with scientists from the University of Nebraska, Lincoln, performed a retail-to-fork quantitative exposure assessment for eight different antibiotic resistant bacteria and determined that typical consumer ground beef handling and cooking practices resulted in probabilities of ingestion of antibiotic resistant bacteria of less than 1.7%. Substitution of raised without antibiotics ground beef had a negligible effect on reducing antibiotic resistant bacteria exposure compared to proper use of recommended safe cooking and food preparation practices. This quantitative risk assessment determined that consuming ground beef from conventionally raised cattle does not increase the probability of exposure to antibiotic resistant bacteria.
5. Development and validation of assays for detection of Shiga toxin-producing Escherichia coli (STEC) O26 and O111. STEC are dangerous foodborne pathogens that may contaminate meat and fresh produce and are responsible for approximately 150,000 infections each year. For that reason, sensitive and accurate tests are needed to detect them in food. After the widely known and highly pathogenic STEC O157:H7, STEC-O26 and STEC-O111 cause the most cases of severe human illness. Unfortunately, there are also, non-pathogen E. coli O26 and O111 that do not cause human illness but can cause false positive test results for these pathogens. These false positive test results force processors to condemn otherwise wholesome and safe food at great cost. ARS scientists in Clay Center, Nebraska, with colleagues at Florida State University designed two new tests that detect STEC-O26 and STEC-O111 and distinguishes them from the non-pathogen O26 and O111. The two new tests were demonstrated to be more accurate on samples of beef, pork, and spinach at identifying non-pathogens than the current official regulatory method. The use of these two tests will result in fewer false positive pathogen tests on meat and produce. More accurate tests will enable food safety regulators to more accurately identify pathogen-positive samples and reduce food product condemnation.
Review Publications
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.
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.
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.
Guragain, M., Brichta-Harhay, D.M., Bono, J.L., Bosilevac, J.M. 2021. Locus of Heat Resistance (LHR) in meat-borne Escherichia coli: Screening and genetic characterization. Applied and Environmental Microbiology. 87:e02343-20. https://doi.org/10.1128/AEM.02343-20.
Velez, F.J., Bosilevac, J.M., Singh, P. 2021. Validation of high-resolution melting assays for the detection of virulent strains of Escherichia coli O26 and O111 in beef and pork enrichment broths. Food Control. 128. Article 108123. https://doi.org/10.1016/j.foodcont.2021.108123.
