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

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

Location: Meat Safety & Quality Research

2018 Annual Report

1a. Objectives (from AD-416):
Objective 1: Develop and validate novel pre- and post-harvest intervention strategies to reduce or eliminate foodborne pathogen colonization and persistence in the animal and on carcasses and meat products. Sub-objective 1.A: Identify effective control measures to reduce pathogens and in the pre-harvest environment. Sub-objective 1.B: Identify and/or improve efficacious non-thermal post-harvest interventions to reduce contamination of processing plant surfaces, hides, carcasses, and meat products. Sub-objective 1.C: Determine if current processing interventions are equally effective on AMR bacteria and foodborne pathogens. Objective 2: Develop improved sampling, detection, and tracking technologies to identify points, including biofilms, where pathogens persist and contaminate in the production of red meat. Sub-objective 2.A: Characterization of bacterial and environmental components contributing to high event periods (HEP) of E. coli O157:H7 contamination at beef processing plants. Sub-objective 2.B: Identify improved sampling and detections technologies for foodborne pathogens associated with red meat. Sub-objective 2.C: Develop and evaluate indicator organisms as surrogates for tracking pathogens through beef processing. Objective 3: Identify environmental and management practices that influence antimicrobial resistance, colonization of lymph nodes, and colonization rates of cattle, veal, and swine. Sub-objective 3.A: Determine effects of season and production system on occurrence of antimicrobial resistance and foodborne pathogens associated with food animal production. Sub-objective 3.B: Identify environmental and management practices that influence Salmonella in lymph nodes. Sub-objective 3.C: Determine the prevalence of STEC and AMR in veal production systems and identify factors contributing to colonization.

1b. Approach (from AD-416):
Cattle and swine can serve as reservoirs of foodborne pathogens that can spread through the environment or to meat during harvest. Further, pharmacologic antimicrobial use in meat animal production is a concern due to the perceived possibility of emergence and transmission of antimicrobial resistant (AMR) bacteria to the environment and food supply. Research to develop ways to reduce the levels of foodborne pathogens such as Shiga-toxin producing Escherichia coli (STEC) and Salmonella on farms and in foods is important, as is understanding and reducing the risk posed to food safety by AMR bacteria present in the meat production system. To this end, the effects of animal vaccines and direct fed microbial feed additives will be investigated to reduce or eliminate foodborne pathogens in the pre-harvest environment. During the harvest process, chlorine dioxide gas, cold atmospheric plasma, and a unique nano-technology sprayer will be assessed to reduce contamination. Novel methods to detect and track pathogens will be designed and tested including examining processing plants for biofilms and determining their roles during times of widespread pathogen contamination. Environmental and animal management practices that influence antimicrobial resistance and colonization of meat animals by pathogens will be studied, with the goal of identifying management practices that influence Salmonella in beef carcass lymph nodes and the prevalence of STEC in veal production. Successful completion of the project objectives will increase the ability of producers and processors to monitor production and use improved interventions to control contamination and product loss, and clarify the risk of antimicrobial resistance in meat production, while providing meat consumers a decreased risk of foodborne illness.

3. Progress Report:
Under Objective 1, 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.

4. Accomplishments
1. 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.

2. 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.

3. 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.

Review 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.