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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

2020 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, to identify effective control measures to reduce pathogens and in the pre-harvest environment 300 head of feedlot cattle were enrolled in a vaccine trial. The goal of the trial was to determine the efficacy of a novel vaccine targeting factors found in Escherichia coli (E. coli) O157:H7. Cattle have been sampled every three weeks beginning in mid-May and continuing until September. Samples consist of blood and fecal swabs. The fecal swabs have been analyzed for E. coli O157:H7 concentration and prevalence. The blood samples have been used to measure antibody titers. At the end of the trial it will be determined if the vaccine reduced carriage of E. coli O157: H7 compared to controls and if the vaccine impacted cattle production performance. To improve efficacious non-thermal post-harvest interventions, gaseous chlorine dioxide (gCD) was tested for reducing Shiga toxin-producing Escherichia coli (STEC; 15 strains) and Salmonella (12 strains). Inoculated surfaces of fresh beef were subjected to five different gCD treatments for 2 hours at 2 degrees Centigrade. Tissue samples were collected before and after each gCD treatment. The findings indicated that gCD effectively reduces STEC and Salmonella on surfaces of fresh beef during chilling. The reduction of the pathogens was greater as the concentration of gCD increased. The gCD treated tissue samples were sent to the USDA-ARS at Fargo, North Dakota, for residue analyses. To determine if current processing interventions are equally effective on antimicrobial resistance (AMR) bacteria and foodborne pathogens, Salmonella isolates including 35 non-AMR and 33 AMR strains were screened for sensitivity and resistance in fresh beef purge containing half strength of lactic acid (2%), peracetic acid (200 ppm), and cetylpyridinium chloride (0.4%). Tissues were collected before and after spray treatment of each antimicrobial compound. Results indicated that each antimicrobial compound is equally effective in reducing non-AMR and AMR Salmonella when present on the surface of fresh beef. However, lactic acid was the most effective, while peracetic acid and cetylpyridinium chloride had equal effects on reducing Salmonella on fresh beef. In related work under Objective 1, the genetic element of heat resistance (LHR) in four meat-borne E. coli was characterized by using whole genome sequencing. Significant differences in the extent of heat resistance was observed among these E. coli. The LHR is found to be largely conserved, with some degree of variation in both sequence and number. It was observed that a single strain may carry multiple copies and variants of the LHR, and that higher numbers of LHR elements correlate with increased resistance. This genomic data will be further used to investigate how the chromosomal background influences the functions of the LHR. Further, E. coli were screened from various stages of the meat processing continuum and found that the abundance of bacteria possessing the LHR increases at the late and final stages of meat production. Current efforts are focused on evaluating the tolerance of these bacteria to common processing treatments and attempting to track their sources. Under Objective 2, to characterize bacterial and environmental components contributing to E. coli O157:H7 and Salmonella contamination work has been performed with meat industry stakeholders to investigate the contamination mechanism and the potential pathogen sources. Since it is known that multispecies biofilms contribute to sanitizer tolerance of pathogens, and that meat processing plants harbor a wide variety of environmental microorganisms along with pathogens, the environmental microbial community of any mixed biofilm present might enhance pathogen survival. Thus, such protection may increase the pathogen prevalence and the contamination incidence. Environmental samples were collected from various meat processing plants with different Salmonella enterica prevalence histories (sporadic or recurrent). Results showed that certain environmental microbial samples from the plant with recurrent Salmonella history were able to recruit a higher amount of Salmonella cells into their mixed biofilm community, subsequently, after sanitization it was observed that a higher amount of Salmonella cells survived when compared to those in mixed biofilms formed by environmental samples from other plants. Collaborations with Texas A&M University and University of Pennsylvania have been established to further investigate biofilm related protective mechanisms with electron microscope analysis to identify the distribution and location of the pathogen within the mixed biofilm matrix. Further, 16S rRNA sequencing of the biofilm communities are underway to identify the unique bacterial species that might provide protection for the pathogens. Other work under Objective 2 to improve sampling and detections technologies for foodborne pathogens, over 1,650 samples were collected for validation trials of the continuous and manual sampling devices in commercial processing plants (aka the MicroTally swab). To date, more than six commercial beef processing companies have had their employees trained and validated for sample collection using the MicroTally swab. This work has led to widespread implementation of the technology in the beef industry. In addition, work continues to get acceptance criteria approved by the USDA Agricultural Marketing Service (AMS) School Lunch Program in order to allow suppliers of the program to use MicroTally-based sample collection methods. Under Objective 3, to identify practices that influence Salmonella in lymph nodes, cattle feedyards harboring Salmonella posing high risk to human health were identified. Because Salmonella present in ground beef can be due to the incorporation of fat containing lymph nodes, it is important to identify cattle populations that harbor high-risk Salmonella. Over 400 samples were collected across 23 cattle feedlots. A total of 2,055 Salmonella isolates were recovered and presumptive molecular serotypes determined. All Salmonella were further evaluated with a High Pathogenicity Salmonella (HPS) PCR targeting genes associated with Salmonella virulence. For each feedyard these results were used to group the Salmonella isolates. Then for each grouping, at least one strain was arbitrarily identified resulting in a total of 171 representative strains. The serotype and HPS status of each was confirmed, then its susceptibility to 14 antimicrobials determined. In all, 10 Salmonella sub-types posing high risk to human health were identified. Importantly, these high-risk Salmonella serotypes were generally very small sub-populations of the overall Salmonella populations at these feedlots. Additional work is planned to investigate sources of variation in Salmonella among feedyards.

1. A novel aqueous ozone treatment is an efficacious spray chill intervention against Escherichia coli O157:H7 on fresh beef. The last step of beef processing is to rapidly cool the carcass to 35 degrees Fahrenheit and this is accelerated by applying periodic sprays of cold water. Ozone is a naturally occurring water-soluble gas that is an effective germicide and has been approved as a sanitizer for food-contact surfaces and food products. ARS scientists at Clay Center, Nebraska, evaluated a new nanobubble technology that creates a stable high concentration of aqueous ozone for its effect on pathogenic Escherichia coli that can be present on beef during spray chilling. The results indicated that the novel ozone spray was 80% more effective in reducing the E. coli than water alone. Since carcasses are usually chilled under recurring sprays of water for 6-8 hours, by adding ozone this process can now be a continued antimicrobial step leading to safer beef.

2. Food service pork chops from three U.S. regions harbor similar levels of antimicrobial resistance regardless of antibiotic use claims. Meat products, including pork chops, are considered by some consumers to transmit antibiotic resistance from animals to humans. Pork chops produced from swine "raised without antibiotics" (RWA) are assumed to carry lower levels of antibiotic resistance than pork chops produced from swine raised in a "conventional" (CWA) system where the animals receive antibiotics. This assumption is not based on scientific evidence, therefore ARS scientists at Clay Center, Nebraska, set out to address this data gap. Similar levels of 8 antibiotic resistant bacteria and 10 antibiotic resistance genes were found in CWA and RWA pork chops. The results provide the first comprehensive evidence that antimicrobial use in U.S. swine production does not impact the quality or safety of pork products.

3. In-feed tylosin phosphate administration to feedlot cattle minimally affects antimicrobial resistance. The antibiotic tylosin phosphate is approved for use in cattle feed to improve liver health. Tylosin use in cattle feed has not been linked to any specific antibiotic resistant infection in humans, but concerns persist that tylosin use may increase human exposure to antimicrobial resistance. ARS scientists at Clay Center, Nebraska, compared the presence and levels of antibiotic resistant bacteria and antibiotic resistance genes in cattle and their environment with and without a tylosin feed treatment. Most of the measures of resistance were not different between control and tylosin treatment. However, there were a few small increases in resistance for tylosin treated cattle that were smaller than the natural month-to-month variation measured during the seven-month study. When cattle are fed tylosin there is little to no impact on antimicrobial resistance where human health is concerned.

4. Ultraviolet Light (UV) light and ozone for use to sanitize food-contact surfaces and to reduce bacteria on food products. Ultraviolet (UV) light and the combination of UV+Ozone are natural resource conserving interventions and are approved for use to sanitize food-contact surfaces and to reduce bacteria on food products. Both UV light and ozone are effective in reducing microorganisms and do not leave any chemical residues after treatment. ARS scientists at Clay Center, Nebraska, exposed fresh beef to UV light or UV in combination with gaseous ozone to evaluate their effect on reducing pathogenic bacteria. The results indicated that UV light or UV + ozone reduced pathogenic Escherichia coli, Salmonella, and Listeria on the surfaces of the beef by 90 to 95%, without impairing the color or taste of the fresh beef. UV light and UV + ozone are now proven technologies that processors can start using to replace water and energy intense treatments that produce safer, greener, and more economical beef for consumers.

5. Extreme heat resistant gram-negative bacteria carried by U.S. cattle at harvest are very low in prevalence. An extremely heat resistant Escherichia coli was reported to have been isolated from a beef processing plant environment. The extremely heat resistant E. coli survived in beef patties cooked well done (160F) and raised concerns regarding food safety. Since cattle are the source of Escherichia coli entering beef processing plants, ARS scientists at Clay Center, Nebraska, investigated the prevalence of extremely heat resistant E. coli and other heat resistant bacteria present in the feces of U.S. feed lot cattle, and cull dairy and cull beef cows at harvest. Results showed that extremely heat resistant bacteria and their respective genes are present in feces of all cattle types and in all geographical regions across the U.S. The prevalence is, however, very low and none of the heat resistant bacteria were identified as food-borne pathogens so they do not currently pose any food safety risk.

6. High-resolution PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes. Shiga toxin-producing Escherichia coli (STEC) are dangerous foodborne pathogens that contaminate beef and fresh produce. For that reason, sensitive and accurate tests are needed to detect them in food. Two types of STEC are O26 and O111, but there are safe non-pathogen Escherichia coli of these types that can cause false positive results in current tests. Therefore, ARS scientists at Clay Center, Nebraska, designed two new tests with collaborators at Florida State University in Tallahassee, Florida, that can detect STEC-O26 and STEC-O111 and distinguish them from the non-pathogen O26 and O111. The two tests were shown to work in beef and spinach that was inoculated with different STEC and non-STEC. Use of these two tests will improve testing accuracy resulting in greater food safety and reduced response times during foodborne illness outbreaks.

Review Publications
Kalchayanand, N., Worlie, D., Wheeler, T. 2019. A novel aqueous ozone treatment as a spray chill intervention against Escherichia coli O157:H7 on surfaces of fresh beef. Journal of Food Protection. 82(11):1874-1878.
Vikram, A., Miller, E., Arthur, T.M., Bosilevac, J.M., Wheeler, T.L., Schmidt, J.W. 2019. Food service pork chops from three U.S. regions harbor similar levels of antimicrobial resistance regardless of antibiotic use claims. Journal of Food Protection. 82(10):1667-1676.
Schmidt, J.W., Vikram, A., Miller, E., Jones, S., Arthur, T.M. 2020. In-feed tylosin phosphate administration to feedlot cattle minimally affects antimicrobial resistance. Journal of Food Protection. 83(2):350-364.
Guragain, M., Smith, G.E., King, D.A., Bosilevac, J.M. 2020. Prevalence of extreme heat-resistant gram-negative bacteria carried by U.S. cattle at harvest. Journal of Food Protection. 83(8):1438-1443.
Singh, P., Cubillos, G., Kirshteyn, G., Bosilevac, J.M. 2020. High-resolution melting real-time PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes. LWT - Food Science and Technology. 131:109785.