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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Meat Safety and Quality » Research » Research Project #430703

Research Project: Mitigation Approaches for Foodborne Pathogens in Cattle and Swine for Use During Production and Processing

Location: Meat Safety and Quality

2019 Annual Report

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.

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, studies were conducted to determine if the following nonthermal interventions are effective in reducing pathogenic bacteria on surfaces of stainless steel compared with hot water and/or on surfaces of fresh beef: (a) micro-nano bubble ozone (MNB_OZ), (b) cold atmospheric plasma (CAP), (c) radiant catalytic ionization (PHI), and (d) dry-ice blasting (DIB). PHI and DIB had equal effects as hot water and reduced more than 99% of E. coli O157:H7 and Salmonella on surface of stainless. CAP reduced the pathogens more than 90%, while MNB_OZ was the least effective on stainless steel surfaces. Fresh beef tissues were contaminated with pathogenic E. coli and Salmonella and treated with CAP or PHI. The findings indicated that CAP and PHI reduced more than 90% of STEC and Salmonella on surface of fresh beef. One large beef cattle processing company is interested in the PHI technology and we will evaluate a custom PHI unit at one of their processing plants on beef trim and variety meats. Studies continued under Objective 1, extreme heat resistant E. coli from red meat were further studied by correlating the amount and portions of the heat resistance genetic material to the degree of heat resistance. The extreme heat resistance of 29 E. coli isolates was confirmed and the rate of transfer of the heat resistance genetic element to pathogenic E. coli was tested. This work directly relates to food safety as heat resistant bacteria can survive processing treatments and the potential for the emergence of heat resistant pathogens such as STEC needs to be identified. Preliminary results suggest the rate of transfer of the resistance genes is very low. Under Objective 2, we continue working with the meat industry to investigate the impact of mixed biofilm formation with environmental microorganisms on sanitizer tolerance of E. coli O157:H7 and Salmonella enterica at meat plants. Since multispecies biofilms possess enhanced tolerance against sanitization, and the meat processing plants harbor a wide variety of microorganisms and occasional foodborne pathogens such as E. coli O157:H7, we investigated the impact of mixed biofilm formation by environmental microorganisms on E. coli O157:H7 survival and prevalence. We collected floor drain samples from three meat processing plants with different E. coli O157:H7 and Salmonella enterica prevalence histories (sporadic or recurrent) and evaluated the protective effects of environmental microorganisms from floor drains on pathogen survival in mixed. The results showed that biofilm forming ability and bacterial species composition varied considerably based on the processing plants and drain locations. E. coli O157:H7 strains obtained significantly greater tolerance to sanitization when they formed mixed biofilms with drain microorganisms from the plant with historic recurrence of this pathogen than those mixed with drain samples from the other plant. A similar result was obtained from the plant with recurrent Salmonella contamination events. Scanning electron microscope analysis indicated that such protective effect was not solely dependent upon mixed biofilm volume. Furthermore, 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. We are currently collaborating with Texas A&M University and Stanford University to further investigate the biofilm protective mechanisms and also to identify the unique bacterial species in the environmental community that might protect the pathogens. Additional studies under Objective 2 have continued to improve detection methods for foodborne pathogens. Specifically evaluating the occurrence of E coli genes espK and espV as markers for non-O157 STEC contamination. The presence of these genes amongst an extensive panel of STEC isolates, and in contaminated beef broth enrichments was examined. Results have shown that nearly 90% of pathogenic STEC possess both genes, and that all pathogenic STEC tested had at least one or the other. The examination of beef enrichment broths showed that their inclusion in a detection assay with other STEC genes (stx and eae) can lead to fewer potential positive broths. Under Objective 3, we have studied seasonal fluctuations in E. coli O157:H7 shedding by cattle. Excretion of E. coli O157:H7 typically peaks in the summer months and is undetectable during the winter. This phenomenon is of particular interest as the feed, water, and internal body temperature are not changed as the cattle transition from winter to summer or summer to winter. The objective of this study was to use immunologic, metabolomic, and metagenomic methods to characterize seasonal shifts in E. coli O157 shedding to determine the mechanism by which E. coli O157:H7 colonization is mitigated. We have completed the second year of sampling. In addition to the summer vs. winter comparison, OMICs analyses will be directed towards samples from animals that transitioned through a super-shedding phase. By comparing samples from the same animal in a longitudinal manner, we hope to minimize the signal-to-noise ratio and identify causal factors responsible for the changes in O157 shedding patterns.

1. Novel continuous and manual sampling methods for beef trim microbiological testing. Beef trim sampling for pathogen testing is one of the final steps in the food safety system beef processors have implemented to keep meat safe and wholesome for consumers. Traditional methods of sampling for pathogen testing examine less than one pound of trimmings from a 2000 pound combo bin of beef trimmings destined for ground beef. ARS scientists in Clay Center, Nebraska, invented, validated, and assisted in commercial adoption through a CRADA partner of two novel sampling technologies: a continuous sampling device (CSD) and a manual sampling device (MSD) which sample a much greater proportion of the trim and are non-destructive. Results from over 1400 samples on numerous days across multiple companies, processing plants, and lean types demonstrated that both the CSD and MSD provide an equal or better level of performance for detecting pathogen contamination in beef trim compared to the existing methods. Implementation of these new trim sampling methods are resulting in improved beef safety with additional benefits in reduced labor and other costs and improved worker safety.

2. Antimicrobial resistance is similar in food-service ground beef and pork regardless of antibiotic use claims. Antibiotic use during food-animal production is theorized to contribute significantly to antimicrobial resistance in humans. United States beef and pork products produced from cattle and swine “raised without antibiotics” (RWA) are assumed to harbor lower levels of antibiotic resistance than “conventionally” (CONV) raised animals that may have received antibiotics. ARS scientists in Clay Center, Nebraska, found that CONV and RWA ground beef products contained similar levels of 13 antimicrobial resistances, with one antibiotic resistance level higher in CONV ground beef and two antibiotic resistance levels higher in RWA ground beef. For CONV and RWA pork chops, similar levels of all 16 antimicrobial resistances assessed were found. These results are consistent with prior research and provide further evidence that antimicrobial uses in U.S. cattle and swine production do not significantly impact the antibiotic resistance present in beef and pork products; and that claims of detrimental impacts of antibiotic use during cattle and swine production on human health from eating beef or pork are without merit.

3. In-feed chlortetracycline treatment in beef cattle does not impact antimicrobial resistance gene levels. The Food and Drug Administration has recently implemented significant restrictions on use of antimicrobials for growth enhancement in food animals. However, concern remains about the impact of in-feed antimicrobials on antimicrobial resistance. Chlortetracycline is an antimicrobial commonly fed to calves for five days shortly after entry into feedlots to prevent bovine respiratory disease. ARS scientists in Clay Center, Nebraska, found no differences in the levels of 10 antimicrobial resistance genes between chlortetracycline and control groups at any time from 5 to 117 days following a 5-day in-feed chlortetracycline regimen and concluded this treatment enhances animal welfare, but does not increase antimicrobial resistance levels.

4. Prevalence and characterization of Salmonella present during veal harvest. Veal products can be contaminated by Salmonella, a bacteria that causes gastroenteritis. There are two commonly harvested types of veal. Bob veal, calves that are harvested at a few days old; and formula-fed veal are calves raised on a milk replacer formula for about 20 weeks. The Food Safety Inspection Service reported that they find Salmonella more often in bob veal than formula fed veal. ARS scientists in Clay Center, Nebraska, collected data from five veal processing plants and found that bob veal products are at higher risk of Salmonella contamination than formula fed veal. However, the strains of Salmonella from bob veal were types rarely seen in human illness, although formula-fed veal had a lower incidence of Salmonella, the strains were more often linked to human illness. These results indicate further efforts to control Salmonella are necessary from both bob and formula-fed veal processors.

5. Evaluation of real-time PCR linked with melt peak analysis for the detection of Escherichia coli O157:H7 in beef products. Escherichia coli O157:H7 can cause very severe disease and be transmitted through contaminated beef. For this reason beef trim and ground beef are routinely tested for these bacteria. Many current tests cannot tell E. coli O157:H7 from other E. coli and, thus, produce false positive results. ARS scientists in Clay Center, Nebraska, participated in the development and validation of a new commercial test kit that can more accurately distinguish E. coli O157:H7 from the other E. coli than other commonly used assays. When in use by beef processors for E. coli O157:H7 testing the new method will not produce the false positive results often seen with other test methods, and will implicate less beef products are adulterated requiring disposition. Thus the new method stands to save wholesome beef products from destruction.

Review Publications
Miller, E.W., Vikram, A., Agga, G.E., Arthur, T.M., Schmidt, J.W. 2018. Effects of in-feed Chlortetracycline prophylaxis in beef cattle on antimicrobial resistance genes. Foodborne Pathogens and Disease. 15(1):689-697.
Bosilevac, J.M., Dwivedi, H.P., Chablain, P., Ullery, M., Bailey, J.S., Dutta, V. 2019. Comparative performance evaluation of real-time PCR and dual labeled fluorescence resonance energy transfer probe-based melt peak analysis for the detection of Escherichia coli O157:H7 in beef products. Journal of Food Protection. 82(3):507-512.
Wang, R. 2019. Biofilms and meat safety: A mini-review. Journal of Food Protection. 82(1):120-127.
Bosilevac, J.M., Zhilyaev, S., Wang, R., Luedtke, B., Wheeler, T.L., Koohmaraie, M. 2019. Prevalence and characterization of Salmonella present during veal harvest. Journal of Food Protection. 82(5):775-784.
Rejeski, W.J., Rushing, J., Guralnik, J.M., Ip, E.H., King, A.C., Manini, T.M., Marsh, A.P., McDermott, M.M., Fielding, R.A., Newman, A.B., Tudor-Locke, C., Gill, T.M. 2015. The MAT-sf: identifying risk for major mobility disability. Journal of Gerontology Medical Science. 70(5):641-646.
Wheeler, T.L., Arthur, T.M. 2018. Novel continuous and manual sampling methods for beef trim microbiological testing. Journal of Food Protection. 81(10):1605-1613.
Vikram, A., Miller, E., Arthur, T.M., Bosilevac, J.M., Wheeler, T.L., Schmidt, J.W. 2018. Similar levels of antimicrobial resistance in U.S. food service ground beef products with and without a "Raised Without Antibiotics" claim. Journal of Food Protection. 81(12):2007-2018.