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

Goals / 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.

Project Methods
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 05/17/16 to 01/18/21

Outputs
PROGRESS REPORT 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. 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. This is the final report for the project titled ⿿Mitigation Approaches for Foodborne Pathogens in Cattle and Swine for Use During Production and Processing⿝. It has been replaced with 3040-42000-021-00D ⿿Holistic Tactics to Advance the Microbiological Safety and Quality of the Red Meat Continuum⿝. Under Objective 1, studies of novel intervention strategies to reduce or eliminate foodborne pathogens were completed. Two successful interventions were identified, while another was ruled out. A novel aqueous ozone treatment was identified as an efficacious spray chill intervention against Escherichia coli (E. coli) O157:H7 on fresh beef. The last step of beef processing is to rapidly cool the carcass by applying periodic sprays of cold water. Aqueous ozone was found to be 80% more effective than water alone when used for this spray. Ultraviolet Light (UV) light and ozone were used to sanitize food-contact surfaces and to reduce bacteria on food products. Exposing contaminated beef to UV light or UV with gaseous ozone reduced E. coli, Salmonella, and Listeria on the surfaces by 90 to 95%, without impairing the color or taste of the fresh beef. These new proven technologies will allow processors to replace water and energy intensive treatments and produce safer, greener, and more economical beef for consumers. The treatment of cattle hides with a bacteriophage before processing did not improve beef safety. It is established that cattle hides are the main source of beef carcass contamination during processing and that reductions in E. coli O157:H7 on the hides of cattle 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. However, when a commercialized bacteriophage application was sprayed onto cattle hides prior to entering beef processing plants a significant reduction of E. coli O157:H7 on hides or beef carcasses was not found. Objective 2: Develop improved sampling, detection, and tracking technologies to identify points, like biofilms, where pathogens persist and contaminate in the production of red meat, resulted in numerous accomplishments. Novel pre-harvest and post-harvest continuous sampling device (CSD) and manual sampling device (MSD), sample collection methods were established. Rectal mucosal swab (RAM swabs) sampling for detection of pathogenic E. coli in beef cattle pre- or peri-harvest were validated. With RAMS swabs found equal to or better than grab samples for testing of pathogenic E. coli in cattle. Novel sampling methods for beef trim microbiological testing were developed. Traditional methods of beef sampling for pathogen testing examine less than one pound of trimmings from 2000 pounds of beef trimmings destined for ground beef. Two novel sampling technologies: the CSD and the MSD which sample a much greater proportion of the trim and are non-destructive were validated. Results from over 1400 samples 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. A novel approach using surface pH of fresh beef was developed to use as a validation parameter for lactic acid treatment against E. coli O157:H7 and Salmonella. The surface pH of a beef carcass after applying lactic acid was identified as an effective and inexpensive measurement of antimicrobial efficacy and this new practice can improve food safety in the beef industry while reducing costs to beef processors. Improved detection methods of E. coli O157:H7 and non-O157 Shiga Toxin- producing E. coli (STEC) were developed and validated. Many current tests cannot tell E. coli O157:H7 from other E. coli and, thus, produce false positive results. A new real-time Polymerase chain reaction (PCR) linked with melt peak analysis was developed and validated that can more accurately distinguish E. coli O157:H7 from the other E. coli. Two types of STEC are O26 and O111, but there are non-pathogen E. coli of these types that can cause false positive results in current tests. Working with collaborators at Florida State University two new tests that can detect STEC-O26 and STEC-O111 and distinguish them from the non-pathogen O26 and O111 were developed. When in use, these new tests will improve accuracy and implicate less beef products for disposition. Lastly under Objective 2, various E. coli (O157:H7, STEC-O113, and extremely heat resistant E. coli) were characterized to better understand their roles in contamination and disease. E. coli O157:H7 strains isolated during High Event Periods (HEP) of contamination were characterized and results showed that compared to control strains, the HEP strains had a higher biofilm-forming ability and lower sanitizer susceptibility. Moreover, the HEP strains retained higher copy numbers of the pO157 plasmid suggesting that it might be the genetic basis for the HEP strains⿿ enhanced ability to survive in the meat plants and cause contamination. STEC-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 have caused outbreaks of severe disease. In collaboration with the U. S. Food and Drug Administration and the French Agency for Food, Environmental and Occupational Health & Safety, STEC O113 isolated from beef and cattle in the U.S. were compared to the disease-causing strains from other countries. Results showed U.S. strains of STEC O113 form two related groups, with a small portion of one of the groups overlapping with the disease causing STEC O113. However, these had been isolated from imported beef products and are not found in U.S. beef and cattle. Extremely heat resistant (XHR) E. coli can survive in beef patties cooked to 160F and raised concerns regarding food safety. Therefore, the prevalence of XHR E. coli and other heat resistant bacteria present in the feces of U.S. feedlot cattle, cull dairy, and cull beef cows at harvest were examined. Results showed that XHR 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. Objective 3 addressed environmental and management practices that influenced antimicrobial resistance, colonization of lymph nodes, and colonization rates of veal. Antimicrobial use in livestock production is under intense scrutiny in the U.S. due to potential contributions to antimicrobial resistance. Multiple studies were performed and reported that showed 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. Likewise, in-feed tylosin phosphate administration to feedlot cattle was found to have little to no impact on antimicrobial resistance where human health is concerned. Lastly, the occurrence of antimicrobial resistance in beef cows was found not associated with antimicrobial use indicating that other factors more strongly influenced the observed levels of antimicrobial-resistant bacteria in beef cows. It was concluded that, beef cattle production without any antibiotics would not be expected to reduce the amount of antimicrobial resistance (AMR) contributed to the environment compared to conventional production. Meat products are considered by some consumers, regulators, and public health officials to transmit antibiotic resistance from animals to humans. Studies were performed and reported that found pork chops produced from swine ⿿raised without antibiotics⿝ (RWA) had similar levels of antibiotic resistant bacteria and antibiotic resistance genes as pork chops produced from ⿿conventionally⿝ (CONV) raised swine where the animals receive antibiotics. In the case of ground beef, CONV and RWA ground beef products contained similar levels of antimicrobial resistances. This is mounting 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. Beef carcass lymph nodes have been identified as a potential source of human exposure to Salmonella when fat trim containing these nodes is mixed with lean trim and incorporated into ground beef. Salmonella were found in peripheral lymph nodes of healthy cattle at slaughter but the majority were neither resistant to any antimicrobial agents, nor of serotypes commonly identified in human disease. The processing and harvest of bob veal and formula-fed veal calves were studied for E. coli O157:H7, non-O157 STEC, and Salmonella. 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. Further, a greater proportion of bob veal was found to be contaminated by STEC compared to formula-fed veal. In regard to Salmonella, bob veal products were 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, and although formula-fed veal had a lower incidence of Salmonella, the strains were types more often linked to human illnesses. The study was repeated a year later, and processing had improved with less STEC and Salmonella detected on carcasses. Record of Any Impact of Maximized Teleworking Requirement: Some research activities were delayed. Day to day scientist interactions, spontaneous research discussions, and stakeholder interactions are all severely reduced and continue to impact scientific productivity. ACCOMPLISHMENTS 01 Environmental biofilms help Escherichia coli (E. coli) O157:H7 survive sanitation. E.coli O157:H7 is a foodborne pathogen that causes outbreaks of severe disease in humans. Inside meat processing plants a wide variety of bacteria, and occasionally E. coli O157:H7, are found. Floor drains at meat plants catch all of these bacteria, where many survive as a protected community in the form of a biofilm. USDA-ARS scientists in Clay Center, Nebraska, tested biofilm samples from floor drains at two meat plants with different E. coli O157:H7 contamination histories. They found the biofilm forming ability and the bacterial species that made up the biofilms were different between the two plants. Certain bacterial species in the drain biofilms helped E. coli O157:H7 survive and persist by protecting it from sanitation. This new knowledge will inform plant sanitation procedures to disrupt biofilms, making E. coli O157:H7 and other potentially harmful bacteria more susceptible to sanitation and disinfection procedures in the packing plant, which will improve the safety of meat and further protect public health. 02 Shiga toxin-producing Escherichia coli (E. coli) on pork carcasses. Escherichia coli (E. coli) that produce Shiga toxin are pathogenic, making them a food safety threat. Beef processors routinely test for Shiga toxin-producing E. coli, however, the presence of these pathogenic E. coli throughout pork processing is not well known. USDA- ARS scientists in Clay Center, Nebraska, tested pork carcasses each season at two pork processing plants and determined that pathogenic E. coli prevalence was 40 to 100% on pigs coming into the processing plant regardless of season. Although, pork processing steps and antimicrobial interventions reduced pathogenic E. coli levels and prevalence significantly, some types of E. coli capable of causing human illness were identified on the finished pork carcasses. Based on this study, pork processors may need to consider Shiga toxin-producing E. coli a potential contaminate in order to improve pork food safety and to better protect public health. 03 Prevalence of antibiotic resistance in cull cow processing plants. Dairy cows make up about 18% of the cattle harvested in the United States each year. Published studies show antibiotic use has little effect on antibiotic resistance in fed beef cattle and their products, but little data exists regarding culled cows that are typically many years older when harvested for beef. To address this data gap, USDA-ARS scientists in Clay Center, Nebraska, in collaboration with scientists from Colorado State University, Fort Collins, Colorado, tested culled beef cows (conventional beef), culled dairy cows (conventional dairy), and culled organic dairy cows cared for without using antibiotics (organic dairy). Results indicate that all groups had very low levels of antibiotic resistance and that cows managed without antibiotics were not significantly different in measured antimicrobial resistance on their beef products when compared to both conventional beef and dairy cows. These data demonstrate that human exposure to antibiotic resistance through beef is insignificant and not different between conventional and organic production systems.

Impacts
(N/A)

Publications

Dass, S.C., Bosilevac, J.M., Weinroth, M., Elowsky, C.G., Zhou, Y., Anandappa, A., Wang, R. 2020. Impact of mixed biofilm formation with environmental microorganisms on E. coli O157:H7 survival against sanitization. NPJ Science of Food. 4:16. https://doi.org/10.1038/s41538- 020-00076-x.
Schmidt, J.W., Vikram, A., Arthur, T.M., Belk, K.E., Morley, P.S., Weinroth, M.D., Wheeler, T.L. 2020. Antimicrobial resistance at two United States cull cow processing establishments. Journal of Food Protection. 83(12):2216-2228. https://doi.org/10.4315/JFP-20-201.
Nastasijevic, I., Schmidt, J.W., Boskovic, M., Glisic, M., Kalchayanand, N. , Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., Bosilevac, J.M. 2020. Seasonal prevalence of Shiga toxin-producing Escherichia coli on pork carcasses for three steps of the harvest process at two commercial processing plants in the United States. Applied and Environmental Microbiology. 87(1):e01711-20. https://doi.org/10.1128/AEM.01711-20.
Schmidt, J.W., Vikram, A., Doster, E., Thomas, K., Weinroth, M.D., Parker, J., Hanes, A., Geornaras, I., Morley, P.S., Belk, K.E., Wheeler, T.L., Arthur, T.M. 2021. Antimicrobial resistance in U.S. retail ground beef with and without label claims regarding antibiotic use. Journal of Food Protection. 84(5):827-842. https://doi.org/10.4315/JFP-20-376.

Progress 10/01/19 to 09/30/20

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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.

Impacts
(N/A)

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.

Progress 10/01/18 to 09/30/19

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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.

Impacts
(N/A)

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.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report 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. 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. Under Objective 1, studies to determine if current processing interventions are equally effective on antimicrobial resistant (AMR) bacteria and foodborne pathogens have been performed. 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 of lactic acid, peracetic acid, cetylpyridinium chloride, and sodium hydroxide. Twelve Salmonella strains (6 strains of non-AMR and 6 strains of AMR) that were most resistant to 2% lactic acid, 200 ppm peracetic acid, or 0.4% cetylpyridinium chloride were divided into two inocula of 6 strains each, non-AMR inoculum and AMR inoculum. Each inoculum was used to contaminate fresh beef. Contaminated fresh beef tissues were subjected to antimicrobial interventions with 4% lactic acid, 400 ppm peracetic acid, and 0.8% cetylpyridinium chloride. Tissues were collected before and after spray treatment of each antimicrobial compound. Based on this study lactic acid was the most effective, while peracetic acid and cetylpyridinium chloride had equal effects on reducing Salmonella on fresh beef, but less than lactic acid. The findings also indicated that each antimicrobial compound is equally effective in reducing non-AMR and AMR Salmonella on surface of fresh beef. This study project was a collaboration with the University of Nebraska at Lincoln to determine risk assessment of Salmonella contamination of fresh beef products. Further, we have investigated using aqueous ozone as a spray chill intervention to reduce E. coli O157:H7 on surface of fresh beef inside the hot box compared to spray chill with water to keep moisture loss during carcass cooling. Our preliminary results indicated that aqueous ozone spray chill reduced the pathogens on surface of fresh beef more than 90%. Also under Objective 1, the impact of extreme heat resistance among E. coli was examined as it pertains to meat safety. A rapid screening test for the genetic element that provides extreme heat resistance was developed, as was a simple high throughput test to identify and isolate extremely heat resistant bacteria and E. coli. These tools were used to screen feces samples collected from 538 fed, 425 cull dairy, and 475 cull beef cattle arriving at nine beef processing plants located across the U.S. Results showed that the prevalence of extreme heat resistant E. coli is very low in cattle. Further, the USMARC E. coli collection was screened for strains possessing extreme heat resistance. Reinforcing the scarcity of this phenotype, only 22 isolates were found to be extremely heat resistant among 3,076 isolates. None of the extreme heat resistant E. coli were pathogens, and when compared to E. coli O157:H7 and Salmonella exposed to common beef processing interventions (4% lactic acid, 400ppm peroxyacetic acid, or 85C hot water) in a pilot scale carcass wash cabinet, all strains were not significantly different in sensitivity to the interventions. Under Objective 2, we continue to make progress towards understanding the potential impact of environmental components on �High Event Period� (HEP) meat contamination by E. coli O157:H7 at commercial plants. E. coli O157:H7 HEP strains, non-HEP trim isolates and diversity control panel strains were tested and compared for their biofilm forming ability and sanitizer tolerance. More importantly, we investigated the potential impact of environmental microorganisms on colonization and sanitizer tolerance of E. coli O157:H7, which would in turn affect the prevalence rate and meat contamination incident at commercial plants. Since floor drains are an important niche that could harbor a wide variety of environmental microorganisms as a result of bacterial accumulation from wash/rinse of equipment, contact surface and animal hides, we collected samples from floor drains located in cooler and hotbox at two meat processing facilities, which had E. coli O157:H7 prevalence rates at different levels. We phenotypically and genetically characterized and compared these drain samples for their biofilm forming ability using material and temperature commonly encountered in the meat industry under normal operating conditions. Furthermore, the protective effect of the drain microorganisms on E. coli O157:H7 survival capability against sanitization was investigated as well. Overall, a wide diversity of bacterial species was observed in all the samples using 16S rRNA gene sequencing technique, however, there were significant differences in the composition of bacterial species among the various drain samples collected from different locations. Notably, a unique multi-species bacterial community within the cooler drain samples from one particular processing plant, which has had higher O157 prevalence rate, exhibited significant protective effect on E. coli O157:H7 survival by enhancing the tolerance of this pathogen against sanitization. We are currently investigating the mechanism of such protection by isolating individual bacterial species from the mixture and also using scanning electronic microscope to directly visualize the mixed biofilm structure and the distribution of fluorescence-labelled E. coli O157:H7 strain within the mixed biofilm community. Also 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. Our Cooperative Research and Development Agreement (CRADA) partner has finalized commercial versions of both manual and continuous sampling methods. Multiple companies are conducting or have completed validation of the manual method in their processing plants and will begin adoption of the method as their primary beef trim sampling procedure. The continuous method has been trialed in a small number of plants and adoption of the method is expected to follow a similar timeline as the manual method. Finally, under Objective 3, the impact of nutrient enrichment on the occurrence of antimicrobial resistance was examined for a second year. 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 continued investigating the effect of nutrient enrichment without antimicrobial selection on the diversity and levels of native antimicrobial resistant populations. All Phase II plots received nutrients as opposed to only one set of plots in Phase I. Samples were processed for culture-based microbiology, quantitative polymerase chain reaction (qPCR), and metagenomics. Concentrations of antimicrobial resistant bacterial populations and antimicrobial resistance genes again increased through nutrient enrichment to the levels equal to or exceeding those 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. Accomplishments 01 Impact of raising beef cattle without antibiotics on the occurrences of antimicrobial resistance. There is a significant societal concern that traditional antimicrobial use patterns for food-animal production have contributed to the occurrence of antimicrobial resistance (AMR) in human infections. In response to this concern, ARS researchers at Clay Center, Nebraska compared fecal AMR levels between U.S. beef cattle produced conventionally, with no restrictions on antibiotic use other than regulatory compliance, and U.S. beef cattle raised without antibiotics. Fifty of 67 individual microbial AMR levels were not different between production systems, while 17 of 67 levels exhibited significant increases in conventional animals. However, while statistically significant, these increases in AMR were so small they are likely biologically insignificant. More importantly, cattle raised without antibiotics typically grow slower, so they must be fed 50 days longer and thus, produce about 2500 pounds more manure. Therefore, the 31% increase in amount of manure from cattle raised without antibiotics more than offsets the small reduction in a few resistances and may actually increase total AMR in the environment. Thus, beef cattle production without any antibiotics would not be expected to reduce the amount of AMR contributed to the environment compared to conventional production. 02 Salmonella in peripheral lymph nodes of healthy cattle at slaughter. Beef carcass lymph nodes have been identified as a potential source of human exposure to Salmonella when fat trim containing these nodes is mixed with lean trim and incorporated into ground beef. To more fully characterize this possible contamination route, ARS researchers at Clay Center, Nebraska, in collaboration with researchers from Texas Tech University in Lubbock, Texas, and commercial industry partners, collected beef cattle peripheral lymph nodes from healthy feedlot cattle at slaughter, and from healthy cattle culled from breeding herds. Salmonella was recovered from 5.6% of all cattle lymph nodes across all cattle sources, with 2.9% of all lymph nodes having a high enough level to be quantifiable. The majority (80.6%) of the Salmonella were neither resistant to any antimicrobial agents, nor of serotypes commonly reported by the CDC in human disease. The results of this study increase our understanding of the sources of Salmonella contamination of beef products and shed light on transmission dynamics that may be useful in targeting interventions to prevent foodborne illness resulting from contaminated beef. 03 Surface pH of fresh beef as a parameter to validate effectiveness of lactic acid treatment against Escherichia O157:H7 and Salmonella. The food safety system implemented by beef processors includes use of antimicrobials such as lactic acid sprayed on beef carcasses to mitigate bacterial contamination. Antimicrobial interventions used by beef processors are required to be validated under the actual conditions used, however antimicrobial intervention sprays are applied under many different parameters (concentration, spray volume and pressure, etc.) making validation studies for commercial beef processors cumbersome and expensive. ARS researchers at Clay Center, Nebraska, conducted a study to determine if surface pH of a beef carcass after applying lactic acid could be used as an effective and inexpensive measurement of antimicrobial efficacy and reduction of pathogenic bacteria if present. Results indicated that carcass surface pH was very effective in validating reductions of both E. coli O157:H7 and Salmonella on beef carcasses. Therefore, surface pH can now be used by the beef processing industry to efficiently validate the effectiveness of lactic acid intervention for pathogen reduction in beef. This new practice can improve food safety in the beef industry while reducing costs to beef processors.

Impacts
(N/A)

Publications

Vikram, A., Rovira, P., Agga, G.E., Arthur, T.M., Bosilevac, J.M., Wheeler, T.L., Morley, P., Belk, K., Schmidt, J.W. 2017. Impact of "raised without antibiotics" beef cattle production practices on occurrences of antimicrobial resistance. Applied and Environmental Microbiology. 83:e01682-17.
Webb, H.E., Harhay, D.M., Brashers, M.M., Nightengale, K.K., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Schmidt, J.W., Wang, R., Granier, S.A., Brown, T.R., Edrington, T.S., Shackelford, S.D., Wheeler, T.L., Loneragan, G.H. 2017. Salmonella in peripheral lymph nodes of healthy cattle at slaughter. Frontiers in Microbiology. 8:2214.
Kalchayanand, N., Arthur, T.M., Bosilevac, J.M., Schmidt, J.W., Shackelford, S.D., Brown, T., Wheeler, T.L. 2018. Surface pH of fresh beef as a parameter to validate effectiveness of lactic acid treatment against Escherichia O157:H7 and Salmonella. Journal of Food Protection. 81(7):1126- 1133.
Vikram, A., Schmidt, J.W. 2018. Functional blaKPC-2 sequences are present in U.S. beef cattle feces regardless of antibiotic use. Foodborne Pathogens and Disease. 15(7):444-448.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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.

Impacts
(N/A)

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.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report 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. 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. This is the annual report for the project titled Mitigation Approaches for Foodborne Pathogens in Cattle and Swine for Use During Production and Processing (3040-42000-018-00D) that has replaced the project titled Pathogen Mitigation In Livestock And Red Meat Production (3040-42000-014- 00D) progress has been made on all three objectives. Under 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, two studies have been completed. First, current antimicrobials such as lactic acid, peracetic acid, cetylpyridinium chloride, and sodium hydroxide have been used in the meat industry to reduce pathogenic bacteria. However, effectiveness of these antimicrobials on non- antimicrobial resistance (non-AMR) and antimicrobial resistance (AMR) bacteria has not been determined. A study was performed using beef purge containing either non-AMR or AMR Salmonella and subjected to the antimicrobials. Our findings indicate that lactic acid, Peracetic acid, and cetylpyridinium chloride are equally effective in reducing non-AMR and AMR Salmonella. Second, there has been increased interest in the application of antimicrobial spray treatments to beef trimmings prior to grinding and to beef subprimals before packaging for the reduction of microbial contamination during processing. However, the Food safety and Inspection Service (FSIS) only allows for a maximum of 0.5% of liquid gained after spray application of antimicrobials. A study was performed in which beef trimmings and subprimals were surface inoculated with a ten-strain cocktail mixture of E. coli O157:H7 and Salmonella and subjected to water and four different concentrations of peracetic acid (130, 150, 200, and 400 ppm). Results indicated that all concentrations of peracetic acid significantly reduced E. coli O157:H7 and Salmonella on beef trimmings and subprimals compared to water and can be used during beef processing to improve the safety of beef trimmings and subprimals when weight gain is limited to <= 0.5% to meet regulatory requirements. Under 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, we have been making significant progress towards determining specific bacterial and environmental components that might contribute to �High Event Period� (HEP) trim contamination by E. coli O157:H7 at beef processing plants. We have phenotypically and genetically characterized and compared the E. coli O157:H7 HEP strains and control panel strains for their biofilm forming ability and sanitizer resistance. Our results showed that compared to the control panel strains, E. coli O157:H7 strains isolated from HEP events had stronger biofilm forming ability on materials commonly used in the meat industry as well as lower susceptibility to common sanitizers. The HEP strains also harbored a higher copy number of the pO157 plasmid, which was positively correlated to the stronger potency of �mature� biofilm formation and higher survival /recovery growth capability after sanitization. Taken together, these results suggest that the pO157 plasmid might play important roles in the phenotypes of biofilm formation and sanitizer resistance by certain E. coli O157:H7 strains that pose higher potential of causing HEP contamination in the meat plants. This study may help answer the important question for the industry if environmental colonization of certain E. coli O157:H7 strains contribute to HEP contamination at the processing plants. Under Objective 3: Identify environmental and management practices that influence antimicrobial resistance, colonization of lymph nodes, and colonization rates of cattle, veal and swine, we investigated the relationship between antibiotic usage in livestock and development of antimicrobial resistance in associated bacterial populations. Breeding beef cows (hereafter referred to as beef cows), typically pasture raised, make up one third of the cattle inventory in the United States, but there is a paucity of information regarding antimicrobial resistance in beef cows. We compared the occurrences of resistance to specific classes of antimicrobials in bacteria from the fecal samples of beef cows more than 8 years old 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 prevalences of antimicrobial resistant bacteria were not significantly (P > 0.05) associated with prior history of antimicrobial treatments or duration of time between last antimicrobial treatment and sampling. In conclusion, occurrences of antimicrobial resistance in comingled beef cows were not associated with antimicrobial use indicating that other factors more strongly influenced the observed levels of antimicrobial-resistant bacteria in feces of beef cows. We conducted a study to follow cattle that had experienced a salmonellosis outbreak from the feedlot through harvest and determine if salmonellosis, resolved through antimicrobial therapy, results in persistent colonization of bovine peripheral lymph nodes. Based on the results of the study it is clear that salmonellosis outbreaks in cattle do not result in long-term carriage of the outbreak strain. It was unknown prior to this study if bovine peripheral lymph nodes from cattle that had salmonellosis infections would be persistently infected leading to increased food safety risk at harvest. The findings of this study indicate that cattle that are treated for salmonellosis provide no increased risk of lymph node colonization by Salmonella at harvest. We have begun to determine the effects of season and production system (conventional and raised without antimicrobials) on occurrence of antimicrobial resistance and foodborne pathogens associated with food animal production. To date, 720 fecal samples have been collected. Half from cattle produced conventionally (with antimicrobials) and half from cattle raised without antimicrobials. Antimicrobial resistant bacteria have been cultured, and genomic DNA has been isolated from these samples. Accomplishments 01 Antimicrobial-resistant bacterial populations and antimicrobial resistance genes obtained from environments impacted by livestock and municipal waste. The impact of potential antimicrobial resistant bacteria in livestock waste runoff has been a growing topic of public concern. ARS scientists at Clay Center, Nebraska, compared the populations of antimicrobial-resistant bacteria and the presence of antimicrobial resistance genes within samples of livestock and municipal waste streams discharged from municipal wastewater treatment facilities, cattle feedlot runoff catchment ponds, swine waste lagoons and environments considered low impact (a municipal lake and a prairie). The results showed prevalences and concentrations of antimicrobial- resistant bacteria were similar among the livestock and municipal sample sources, but there were differences among the antimicrobial resistance genes found in agricultural, environmental, and municipal samples, with municipal samples harboring the highest number of antimicrobial resistance genes. It was concluded that antimicrobial resistance is a very widespread phenomenon where antimicrobial resistance can be found in cattle, swine, and human waste streams, but the higher diversity of antimicrobial resistance can be found in human waste streams.

Impacts
(N/A)

Publications