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

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

Location: Meat Safety & Quality Research

2017 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Under Objective 1, common antimicrobials such as lactic acid and peracetic acid that are commonly used in the beef industry, and silver dihydrogen citrate (a novel antimicrobial compound) were found to form stable micro-nano bubbles (MNB). The application of MNB of these compounds to pathogen contaminated fresh beef will be examined next using a model carcass wash cabinet. Cold atmospheric plasma (CAP) parameters such as application time and distance between surface of fresh beef and plasma source were determined. The application of CAP against E. coli O157:H7 and Salmonella on surfaces of fresh beef reduced these pathogens by more than 90% of these pathogens. However, CAP caused discoloration of fresh beef therefore further studies of CAP will focus on means to reduce discoloration without reducing efficacy against pathogens. The efficacy of radiant catalytic ionization (RCI) on reducing E. coli O157:H7, non-O157 Shiga toxin producing E. coli (STEC), antimicrobial resistance (AMR) and non-AMR Salmonella on surface of fresh beef and on stainless steel knives has been studied. This non-thermal oxidation technology utilizes a combination of ultra violet (UV) light and low-level oxidizers such as ozone, hydroxyl radicals, and hydrogen peroxide to cause antimicrobial action. Our findings indicate that RCI is effective in reducing these pathogens both on surfaces of fresh beef and on surfaces of stainless steel knives, and also is equally effective in reducing non-AMR and AMR Salmonella. Based on the study conditions, RCI did not cause lipid oxidation when applied on surfaces of fresh beef. Further progress under Objective 1 determined if current processing interventions are equally effective on AMR bacteria and foodborne pathogens. Sixty-Eight Salmonella isolates including 35 non-AMR and 33 AMR strains were screened for sensitivity and resistance in fresh beef purge containing half strength lactic acid, peracetic acid, cetylpyridinium chloride, and sodium hydroxide. Sensitivity and resistance of the isolates (non-AMR and AMR) was strain dependent for each antimicrobial compound. Lactic acid was the most effective, while sodium hydroxide was the least effective in reducing Salmonella. Antimicrobials not only inactivated but also inflicted injury to Salmonella strains tested. The findings indicated that antimicrobials were equally effective in reducing non-AMR and AMR Salmonella. This study was a collaboration with the Institute of Agriculture and Natural Resources (IANR), University of Nebraska at Lincoln to determine risk assessment of Salmonella contamination of fresh beef products. Under Objective 2, the development of a novel sampling method for raw beef trim has continued. We have made significant progress towards implementing both the manual and continuous sampling methods in the commercial beef processing industry. A patent covering our invention of novel approaches and equipment for sampling large continuous flow foods or other products for testing is in the final stages of evaluation. Proof of concept data collection has been completed. These data were used in a submission to the USDA Food Safety Inspection Service to achieve a No Objection Letter (NOL) for both methods to be used in validated commercial sampling programs. The NOL was received and disseminated to multiple beef processing companies. Our Cooperative Research and Development Agreement (CRADA) partner is finalizing versions of both manual and continuous sampling methods. Multiple companies have asked for demonstrations in their processing plants. Further validation experiments are planned for sampling versions. Under Objective 3, the impact of nutrient enrichment on the occurrence of antimicrobial resistance was examined. Many of our field trials indicated that a factor other than antimicrobial use in animal production or selective pressure was the major driver for increases in the occurrence of antimicrobial resistance. We have been investigating the effect of nutrient enrichment without antimicrobial selection on the diversity and levels of native antimicrobial resistant populations. The plots received one of three treatments: nutrients (tryptic soy broth), sterile water, or no amendment. Samples were processed for culture-based microbiology, quantitative polymerase chain reaction (qPCR), and metagenomics. Concentrations of antimicrobial resistant bacterial populations and antimicrobial resistance genes were increased through nutrient enrichment to the levels observed in environments directly impacted by human and livestock waste. Preliminary results from this project demonstrate that resistant bacterial populations exist as subpopulations within most if not all environments and nutrient enrichment likely plays a larger role than antimicrobial selection in AMR occurrence and transmission. Additional experiments to verify these results are planned. Further progress under Objective 3 has examined the rates of Salmonella present during the various steps of veal processing. Our recent studies of contamination present in veal processing have allowed us to expand the work and investigate Food Safety and Inspection Service (FSIS) reports that bob veal have a higher Salmonella prevalence rate relative to formula-fed veal and beef. This study measured the rates and concentrations of Salmonella present on veal calf hides, veal pre-evisceration carcasses and final chilled veal carcasses, then characterized the serotypes and antibiotic susceptibility of the Salmonella identified. Finally, under Objective 3, an extremely heat resistant (XHR) non-pathogen strain of E. coli was isolated by Canadian researchers from a meat processing plant. The strain was resistant to 60C (140F) for 60min and survived in ground beef patties cooked to 71C (160F). Analysis of the heat resistant strain identified a Locus of Heat Resistance (LHR) flanked by mobile elements suggesting it was acquired and can be transferred through lateral gene transfer. The group reported that a putative LHR was present in 2% of all reported E. coli genomes. We have initiated studies to examine the threat XHR E. coli may pose in meat processing. A rapid molecular screen has been developed that can accurately identify LHR positive E. coli and has been validated to detect XHR E. coli present in samples of beef and in samples of bovine feces. Investigations are underway to examine our culture collection of bovine, ovine and porcine sourced E. coli. In addition, we have received funding to examine the prevalence of XHR E. coli in feces of feed lot, beef and dairy cattle, as well as determine if the LHR imparts any additional resistance to commonly used processing antimicrobials such as lactic acid or peracetic acid.

4. Accomplishments
1. Characterization of pathogenic E. coli on veal hides and carcasses. Beginning in 2012 the USDA Food Safety Inspection Service increased scrutiny of bob veal (calves less than 3 weeks old) and formula-fed veal (calves 20 weeks in age) when a higher percentage of positive tests for pathogenic Shiga toxin-producing Escherichia coli (STEC) were found in veal compared to beef. To investigate this problem, ARS scientists in Clay Center, Nebraska measured the levels and prevalence of E. coli O157:H7 and non-O157 STEC on veal hides and carcasses just after the hide was removed and before any antimicrobial interventions were applied at five veal processors. A year later follow-up samples were collected at three of the processors. Significantly more non-O157 STEC were found on veal hides and carcasses than E. coli O157:H7, as compared to beef where the opposite has been reported. The follow-up samples showed that processing had improved and less STEC was detected on carcasses. In addition, a greater proportion of bob veal was found to be contaminated by STEC compared to formula-fed veal. Changes in processing have improved the safety of veal.

2. Effects on animal health and antimicrobial resistance after feeding cattle preventative antimicrobials. Antimicrobial use in livestock production is under intense scrutiny in the U.S. due to potential contributions to antimicrobial resistance. ARS scientists in Clay Center, Nebraska evaluated the effect of a one-time, five-day in-feed chlortetracycline regimen as preventative treatment for bovine respiratory disease. Chlortetracycline is an antimicrobial that is not considered critically important for human medicine. Over 25% of the animals that did not receive the in-feed treatment developed illnesses requiring therapeutic treatment with antimicrobials critically important to human medicine. None of the cattle that received the in-feed treatment developed pneumonia during the outbreak period. No differences in antimicrobial resistance were observed between treated and non-treated in-feed animals. The U.S. Food and Drug Administration has taken an approach to maximize therapeutic efficacy and minimize selection of resistant microorganisms through judicious use of antimicrobials. This study demonstrated that prophylactic in-feed treatment of chlortetracycline administered for five days to calves entering feedlots is judicious as this therapy reduced animal illnesses, reduced the use of antimicrobials more critical to human health, and had no long-term impact on the occurrence of antimicrobial resistance.

3. Characterization and virulence potential of serogroup O113 Shiga toxin-producing Escherichia coli strains isolated from beef and cattle in the United States. Shiga toxin-producing Escherichia coli (STEC) can cause very severe disease. STEC of serogroup O113 are commonly found in U.S. beef but little disease is reported in the U.S. for this STEC. In other countries STEC O113 has caused severe disease, therefore ARS scientists in Clay Center, Nebraska in collaboration with scientists at the Food and Drug Administration, Center for Food Safety and Applied Nutrition in College Park, Maryland and scientists at the French Agency for Food, Environmental and Occupational Health & Safety in Maisons-Alfort, France examined STEC O113 isolated from beef and cattle in the U.S. and compared them to the disease causing strains from other countries. U.S. strains of STEC O113 were from two related groups. A small portion of one of the groups had overlapping profiles with the disease causing STEC O113, but these came from imported beef products and are not found in U.S. beef and cattle. Therefore, imported ground beef manufacturing components may require monitoring for STEC-O113 while domestic components do not.

4. Antimicrobial resistance in mature beef cows is not correlated to antimicrobial use. There is a growing concern that antimicrobial use in food animals increases antimicrobial resistance in bacteria. Previous studies have shown that following antimicrobial treatment in feedlot cattle there is an increase in the antimicrobial-resistant bacterial population, which then returns to pre-treatment levels approximately 14 to 36 days after treatment. Due to their longevity, beef cows are more likely to receive antimicrobial treatments than feedlot cattle, albeit spread over a longer period of time. ARS scientists in Clay Center, Nebraska compared the occurrences of resistance to antimicrobials in bacteria from beef cows for which complete antimicrobial treatment records were available. Approximately half of the cows sampled for this study were treated with antimicrobials for the treatment of disease, while the other half did not receive any antimicrobial treatments over their lifetime. The occurrence of antimicrobial resistant bacteria was not associated with prior history of antimicrobial treatments or duration of time since the last antimicrobial treatment. The occurrence of antimicrobial resistance in beef cows was not associated with antimicrobial use indicating that other factors more strongly influenced the observed levels of antimicrobial-resistant bacteria in beef cows.

5. High Event Period (HEP) Escherichia coli O157:H7 have strong biofilm-forming ability and resist sanitizers. A HEP is defined as a time period when meat plants experience an increased occurrence of product contamination by E. coli O157:H7. The contamination mechanism and pathogen source responsible for HEPs is currently unknown, so ARS scientists at Clay Center Nebraska characterized E. coli O157:H7 strains isolated during HEPs for their biofilm forming ability, sanitizer resistance, and the genetic basis for these traits. Results showed that compared to the control panel strains, the HEP strains had a higher biofilm-forming ability and lower sanitizer susceptibility. Moreover, the HEP strains retained significantly higher copy numbers of the pO157 plasmid which was positively correlated to their strong biofilm formation and low sanitizer susceptibility, suggesting that it might be the genetic basis for the HEP strains’ enhanced ability to survive in the meat plants and cause contamination. This study highlights the potential role of biofilm formation and sanitizer resistance in HEP contamination of beef by E. coli O157:H7 and reveals a potential molecular mechanism for HEP strain's enhanced survival.

6. Treatment of cattle hides with a bacteriophage before processing may not improve beef safety. Escherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies have provided evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing facilities will lead to reductions in carcass contamination. Bacteriophage, viruses capable of killing bacteria, have been proposed as a novel technology to reduce the levels of E. coli O157:H7 on cattle hides. ARS scientists in Clay Center, Nebraska evaluated a commercialized bacteriophage application sprayed onto cattle hides prior to entering beef processing plants for the ability to reduce E. coli O157:H7 contamination of cattle hides and carcasses. The results demonstrated that the treatment of cattle hides with bacteriophage prior to processing did not produce a significant reduction of E. coli O157:H7 on hides or beef carcasses during processing. Therefore, treatments using bacteriophage before processing may not improve beef safety.

7. Evaluation of rectal mucosal swab sampling for detection of pathogenic E. coli in beef cattle. Studies of pathogenic E. coli in cattle require large numbers of fecal samples to be collected and analyzed. Feces is usually collected by the grab method which is slow and cumbersome. An alternate method uses large foam tipped swabs at the rectum (RAM swabs). RAM swab samples are rapid and simple to collect and target the location of pathogenic E. coli. ARS scientists in Clay Center, Nebraska compared the presence of pathogenic E. coli in feces collected by grab sample and RAM swabs and found that RAMS swabs are equal to or better than grab samples for testing of pathogenic E. coli in cattle. These results validate RAM swab sample collection and will facilitate studies of cattle to identify ways to control pathogenic E. coli and improve beef safety.

Review Publications
Bosilevac, J.M., Wang, R., Luedtke, B.E., Hinkley, S., Wheeler, T.L., Koohmaraie, M. 2017. Characterization of enterohemorrhagic Escherichia coli on veal hides and carcasses. Journal of Food Protection. 80(1):136-145. doi:10.4315/0362.028X.JFP-16-247.

Agga, G.E., Schmidt, J.W., Arthur, T.M. 2016. Effects of in-feed chlortetracycline prophylaxis of beef cattle on animal health and antimicrobial-resistant Escherichia coli. Applied and Environmental Microbiology. 82(24):7197-7204. doi:10.1128/AEM.01928-16.

Feng, P., Delannoy, S., Lacher, D., Bosilevac, J.M., Fach, P. 2017. Characterization and virulence potential of serogroup O113 Shiga toxin-producing Escherichia coli strains isolated from beef and cattle in the United States. Journal of Food Protection. 80(3):383-391. doi:10.4315/0362-028X.JFP-16-325.

Agga, G.E., Arthur, T.M., Hinkley, S., Bosilevac, J.M. 2017. Evaluation of rectoanal mucosal swab sampling for molecular detection of Enterohemorrhagic Escherichia coli in beef cattle. Journal of Food Protection. 80(4):661-667. doi:10.4315/0362-028X.JFP-16-435.

Agga, G.E., Schmidt, J.W., Arthur, T.M. 2016. Antimicrobial-resistant fecal bacteria from ceftiofur-treated and nonantimicrobial-treated comingled beef cows at a cow-calf operation. Microbial Drug Resistance. 22(7):598-608. doi:10.1089/mdr.2015.0259.

Arthur, T.M., Kalchayanand, N., Agga, G.E., Wheeler, T.L., Koohmaraie, M. 2017. Evaluation of bacteriophage application to cattle in lairage at beef processing plants to reduce Escherichia coli O157:H7 prevalence on hides and carcasses. Foodborne Pathogens and Disease. 14(1):17-22. doi:10.1089/fpd.2016.2189.

Wang, R., Luedtke, B.E., Bosilevac, J.M., Schmidt, J.W., Kalchayanand, N., Arthur, T.M. 2016. Escherichia coli O157:H7 strains isolated from High Event Period beef contamination have strong biofilm-forming ability and low sanitizer susceptibility, which are associated with high pO157 plasmid copy number. Journal of Food Protection. 79(11):1875-1883. doi:10.4315/0362-028X.JFP-16-113.

Fratamico, P.M., Bosilevac, J.M., Schmidt, J.W. 2017. Methods for detecting pathogens in the beef food chain: an overview. In: Acuff, G., Dickson, J. Ensuring safety and quality in the production of beef. Volume 1: Safety. Cambridge, UK: Burleigh Dobbs Science. p.35-51.

Fratamico, P.M., Bosilevac, J.M., Schmidt, J.W. 2017. Methods for detecting pathogens in the beef food chain: detecting particular pathogens. In: Acuff, G., Dickson, J. Ensuring safety and quality in the production of beef. Volume 1: Safety. Cambridge, UK: Burleigh Dobbs Science. p.59-72.