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





- Bono, James - Jim
- Schmidt, John
- Wang, Rong
- Harhay, Dayna
- 2023
- 2022
- 2021
- Publications
- Food Safety, (Animal and Plant Products) (108)

Research Project: Identification, Genomic Characterization, and Metabolic Modeling of Foodborne Pathogens in the Meat Production Continuum **Location:
                            Meat Safety and Quality** 2021 Annual Report **Objectives**
                                

Objective 1: Evaluate longitudinal ecology of foodborne pathogens in food-animal production continuum. 
Sub-objective 1.A: Determine the population diversity of Shiga toxigenic Escherichia coli in a closed cattle feedlot.
Sub-objective 1.B: Determine the population dynamics of Salmonella at cattle feeding operations.

Objective 2: Application of bioinformatic tools to identify factors that contribute to virulence and persistence in foodborne pathogens. 
Sub-objective 2.A: Development of machine learning approaches for predicting Shiga toxigenic E. coli and Salmonella pathogenicity in humans.
Sub-objective 2.B: In vitro pathogenicity assays and transcriptomic analyses to examine putative virulence factor contribution to Salmonella enterica pathogenicity, in order to increase our understanding of the strains encountered in production agriculture that have the greatest potential impact on human health.
Sub-objective 2.C: Characterization of environmental impacts on pathogen resistance to antimicrobials and sanitizers.

Objective 3: Development and validation of tools that enable regulators, food-animal producers and processors to monitor high-risk foodborne pathogens. **Approach**
                                

Foodborne illness and the resulting loss of productivity in the United States are reportedly greater than $14 billion a year. While research efforts have resulted in significant strides in tracking contamination entry points and identifying mitigation strategies, outlier events continue to occur, and complete prevention of foodborne pathogens entering the food chain remains an elusive goal. Moreover, concerns persist among regulators and health care advocates that antimicrobial use during animal production may impact antimicrobial resistance levels and potential for transfer to foodborne pathogens. Accordingly, the research described here aims to provide new information about these issues by 1) increasing our understanding of both the genomic diversity and persistence of pathogens over space and time in agricultural settings, which will improve foodborne illness traceback investigations 2) improve understanding of the movement of antimicrobial resistance genes among natural reservoirs and foodborne pathogens; 3) using machine learning to identify predictive markers that can be used to rapidly screen samples or isolates for important phenotypic characteristics and further phenotypically characterizing strains predicted to be more pathogenic or persistent in production settings; and 4) developing tools to monitor high-risk foodborne pathogens, including methods for rapidly estimating levels of foodborne pathogens in meat products. The information generated by this research will facilitate the development of solutions to decrease the incidence of pathogen exposure from the meat food chain. The results of this research will be of interest to food regulatory agencies, the pathogen testing industry, livestock and meat processing industries, agricultural and biomedical scientists, and public health professionals. Major beneficiaries of the successful realization and manifestation of the research goals would ultimately be consumers of a safer food supply. **Progress Report**
                                

This report documents progress from Project Number 3040-42000-020-00D, which started on January 2021 and continues research from Project Number 3040-42000-017-00D, entitled “Genomic and Metagenomic Differences in Foodborne Pathogens and Determination of Ecological Niches and Reservoirs.”  Progress was made on all three objectives.  
Under Objective 1, completed the first sample collections and culture of Shiga toxin-containing Escherichia coli O157:H7 (STEC O157:H7) and antimicrobial resistant (AMR) Salmonella and E. coli. First, STEC O157:H7 was collected from a cattle feedlot to study the ecology and rate of genomic change of over time. Second, AMR Salmonella and E. coli were completed for a project focused on the antimicrobial resistance impact of in-feed administration of tylosin phosphate to feedlot cattle. Third, sample collection and culture were completed for a project with the goal of determining the impacts of sampling method (feces, boot socks, and ropes) and characterization of multiple isolates per sample on antimicrobial resistant Salmonella and E. coli detection rates. Fourth, whole genome sequencing was completed for 450 antimicrobial resistant Salmonella and E. coli obtained over two years at a cattle feedlot. Furthermore, analyses of evolutionary relationships, plasmids, and antimicrobial resistance genes for these 450 isolates are nearing completion but indicate that a single cephalosporin resistant Salmonella sub-type persisted over the two years and that horizontal gene transfer from cephalosporin resistant E. coli populations did not contribute significantly to this persistence. Lastly, research was conducted in a collaborative project between ARS scientists at Clay Center, Nebraska, and researchers at Kansas State University, to evaluate the efficacy of a direct fed microbial for decreasing bovine lymph node contamination with Salmonella. The environmental prevalence of Salmonella in pen surface material and water trough samples was found to be 98.6% and 50%, respectively. Salmonella concentration was low over all (13.8%) and was not found to differ significantly in the mesenteric lymph node (MLN) samples collected from control and treated cattle. While Salmonella was consistently detected in pen surface material, enumerable levels were moderate ranging from 200-5200 colony forming units per gram. This may have contributed to the low overall prevalence of Salmonella isolated from MLNs in this study. Salmonella prevalence in water trough samples was observed to be relatively low, however the subtypes identified matched the dominant subtypes isolated from the yard, and pens with enumerable levels in surface material were more likely to have Salmonella isolated from the corresponding water troughs. Salmonella contamination of bovine lymph nodes continues to be a mechanism by which Salmonella contamination can enter the ground beef production chain and preharvest mitigation methods are needed to control this source of contamination. The results of this project further our understanding of Salmonella transmission dynamics in fed cattle and their environments and provide a valuable source of isolates to examine the population dynamics of Salmonella in cattle feeding operations. Further characterization of the Salmonella isolates is underway.
Under Objective 2, substantial progress has been made comparing genomes of STEC O157:H7 strains and defining various phenotypes associated with specific genotypes. Currently, there are 119 STEC O157:H7 genomes ready to start the comparison analysis. We have made significant progress towards understanding the mechanisms and genetic basis for biofilm formation and sanitizer tolerance by common foodborne pathogens as well as the biofilm impact on meat safety at commercial plants. Environmental microbial samples from multiple meat processing plants have been collected from floor drains located at hot box, cooler, and fabrication, including samples from trim storage and grind rooms. All samples have been examined for general bacterial counts (aerobic plate count [APC] mesophiles, psychrophiles, etc.), pathogen isolation (STEC O157:H7 and Salmonella enterica), and biofilm forming ability. Our results showed that bacterial cell density and biofilm forming ability of the different environmental microbial communities varied considerably based on the plants and drain locations. Importantly, most pathogenic strains were isolated from the environment at processing plants with the history of the pathogen recurrence, supporting the hypothesis that pathogen persistence in the environment could be a contamination source at commercial establishments. All floor drain samples have been properly stored as experimental materials for further investigation. 
Additionally, an incoming Material Transfer Agreement was established with Food Safety Inspection Service (FSIS), whereby they will send approximately 1200 Salmonella positive enrichment broths (poultry, pork, and beef) over the course of one year. The goal of this project is to further validate the targets of the highly pathogenic strains (HPS) assay, and to demonstrate to FSIS laboratory personnel the efficacy of the assay for 1) detecting HPS of Salmonella, and 2) decreasing the detection and response time to the presence of HPS strains in food products tested by FSIS. The Salmonella serotypes obtained from the submitted samples will be compared to the single serotype obtained from the single confirmed isolated colony by FSIS laboratories.  
Under Objective 3, progress was made towards evaluating differences in pathogen growth dynamics in different food matrices and different enrichment media. A total of 36 beef, pork, and poultry samples (325 g to 975 ml of enrichment medium for beef and pork incubated at 42 degrees C; 325 g to 1675 ml of medium for poultry, incubated at 35 degrees C) were evaluated using different media. Beef and pork inoculation studies were conducted using Salmonella Montevideo and Typhimurium in the Mesenteric lymph node (MLG) standard enrichment medium (mTSB) as well as in modified enterohemorrhagic Escherichia coli, (mEHEC) medium and showed that Salmonella growth rates were negatively impacted by growth in mEHEC, with an approximate 3 min increase in growth rate (average 23 min doubling time in mEHEC as opposed to the average 20 min doubling time in mTSB). As a result, it was decided to proceed with the FSIS MLG standard method using mTSB for beef and pork in the remainder of the experiments to be conducted in Objective 3. Poultry samples were evaluated for isolation of Salmonella, APC, and Enterobacteriaceae (EB) using the standard buffered peptone water (BPW), in comparison with mTSB. As anticipated, APC and EB levels were enhanced by growth in mTSB in comparison with BPW, due to the increased nutrient content of this medium in comparison with BPW. Salmonella was readily isolated from enrichments using either growth medium, so it was decided to proceed with the FSIS MLG standard method using BPW for chicken and turkey enrichments for the remaining experiments to be conducted under Objective 3. **Accomplishments**
                                

1. Rapid identification of highly pathogenic Salmonella. Current technology only allows detection of all Salmonella strains, but some strains are much more pathogenic and more likely to cause illness. Rapid methods are needed to detect Salmonella that specifically pose a greater risk to human health in food products. USDA-ARS scientists at Clay Center, Nebraska, developed a novel assay for the detection of highly pathogenic Salmonella strains in food products. A food safety testing company has partnered with ARS to develop the assay into a rapid test kit for use in industry settings. This test will enable the food processing industries to clearly identify and proactively remove food products that are contaminated with pathogenic Salmonella strains most likely to cause human illness. This test will greatly improve food safety and public health, while reducing product condemnation. **Review Publications**
                            *Guragain, M., Brichta-Harhay, D.M., Bono, J.L., Bosilevac, J.M. 2021. Locus of Heat Resistance (LHR) in meat-borne Escherichia coli: Screening and genetic characterization. Applied and Environmental Microbiology. 87:e02343-20. https://doi.org/10.1128/AEM.02343-20.*


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