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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** 2022 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**
                                

Under Objective 1, we completed the second feedyard sample collection at the feedlot in Clay Center, Nebraska, and cultured Shiga toxin-containing Escherichia coli O157:H7 (STEC O157:H7) from the samples. The DNA from these strains will be extracted and sequenced during the next fiscal year (FY). The sequencing and analysis were completed from year one feedyard surface sample collection and weaned calf fecal samples. The isolates recovered from feedyard pen surface material before calves were weaned into the feedyard belonged to clade two as designated by our previous study. The prevalence of STEC O157:H7 in calves weaned into the feedyard was 19%, which was greater than expected when compared to previous studies that reported 5% or less. Half of the groups of weaned calves had a prevalence higher than 15%. Time, weather and age didn’t appear to be responsible for the increased prevalence. The increase in prevalence appears to be associated with cows and their calves being moved from pastures into areas where they were fed a premixed ration. Clade 2 and clade 4 STEC O157:H7 strains were cultured from calves weaned into the feedyard plus 2 new strains that we hadn’t previously seen. The two new strains were cultured from two different areas five miles apart where cows and calves were fed before calves were weaned into the feedyard. The two new strains differ in the Shiga toxin genes they carry and by an allele that associates with their ability to cause disease in humans. These two new strains plus strains from the two previously described clades were cultured from pen surface material from the last sampling of the year. From three pens, the new strains were isolated either together or with strains from the previously identified clades. It will be interesting to see the results from sequencing the STEC O157:H7 strains from this year’s feedyard sampling to see if the new strains were able to compete with the previously established strains in the feedyard and establish a niche for themselves. Also of interest, we will be looking at strains that are isolated from the 2022 weaned calves to determine if the new strains are resident in the cattle herd.
Whole genome sequences and antimicrobial susceptibilities were obtained for 190 Escherichia coli (E.coli) isolated from cattle at feedyards shortly before processing and as cattle progressed through processing to final products. Within each of the three lots examined, sequence types and antimicrobial resistances changed as cattle moved through each step of processing. These results suggested that the data gathered from E. coli isolates obtained at a specific point in processing should not be extrapolated forward or backward in processing. The sequencing also revealed that antimicrobial resistant E. coli subpopulations exist in feedyards and in early stages of processing which are unrelated to more abundant antimicrobial susceptible E. coli populations.
Objective 2, progress continues with isolating Salmonella from Food Safety and Inspection Service (FSIS) Salmonella positive enrichments, as part of an incoming Material Transfer Agreement established with FSIS in FY2021. To date, over 1800 Salmonella isolates have been collected, serotyped, and evaluated using the Highly Pathogenic Salmonella (HPS) assay developed at our research lab. Overall, 1,260 enrichments have been received (from testing of chicken, turkey, pork, and beef products) and over 900 have been analyzed. The goal of this project is to further validate the targets of the HPS assay, and to demonstrate to FSIS laboratory personnel the efficacy of the assay for 1) detecting Salmonella strains of greater concern for human health, and 2) decreasing the detection and response time for the presence of HPS strains in food products tested by FSIS. The Salmonella serotypes isolated by our research lab in Clay Center, Nebraska, are being compared to the serotype of the single confirmed FSIS isolate. These comparisons show serotype agreement for 80% of the samples, while multiple serotypes are identified in 20% of samples. Sequence data has been collected for a select set of these Salmonella isolates and will be used as input data for Machine Learning analyses in FY2023.
Complete closed Shiga toxin-containing STEC O157:H7 genomes from two groups of strains that differ in their ability to cause disease in humans were compared using a microbial pan-genome wide association (GWAS) study and a core genome phylogeny study. The core genome study identified 206 single nucleotide polymorphisms (SNPs) that associated with the two STEC O157 groups. Almost twice as many of the SNPs changed the amino acid sequence of the protein as compared to those that didn’t. Typically, the reverse result is observed where there are more SNPs that don’t change the protein’s amino acid sequence. For some proteins, changing the amino acid sequence can alter the function of the protein, which can have a deleterious effect causing the protein to function less efficiently, if at all. There were several SNPs that were located between genes in noncoding regions. These intergenic regions are where the promoter regions for the genes are located so base differences in these regions can influence the transcription of the gene. Lastly, there were seven genes that had SNPs that create a stop mutation for protein synthesis, creating a truncated protein. These SNPs are the most severe as they can cause the loss of function that could disrupt metabolic or catabolic processes. Further experimentation will be needed to determine if any of the SNPs described have a role in determining if a strain can cause disease in humans. The microbial GWAS identified a gene variant in STEC O157:H7 that associated with the ability to cause disease in humans. This gene variant was caused by a 28 base pair (bp) insertion that resulted in a truncated protein. Further investigation of the gene just upstream identified a 10 bp deletion in the gene that also resulted in a truncated protein. This indel also had a high association with the ability of the strain to cause disease in humans. If both genes were intact, the strains were associated with the ability to cause disease in humans. Those strains with one or both genes mutated were associated with the inability to cause disease in humans. The genes are part of the fermentation of lactate to propanoate pathway, therefore, both intact genes are able to ferment lactate to propanoate. Further experimentation is needed to determine how the loss of this pathway reduces STEC O157 strain’s ability to cause disease in humans. 
We have made significant progress towards understanding the impact of environmental microorganisms on pathogen colonization and sanitizer effectiveness. Environmental microbial samples from multiple beef and pork plants have been collected from floor drains and their biofilm-forming ability was examined under processing conditions. Further, the ability of the environmental microorganisms isolated from the different plants to recruit and coexist with foodborne pathogens (E. coli O157:H7 and Salmonella enterica) was investigated. Even though the pathogenic strains were able to effectively form mixed biofilms with most floor drain samples, the presence of certain bacterial species in these complex samples was found to either increase or decrease pathogen colonization within the multispecies community. More importantly, some pathogenic strains isolated from the processing plants exhibited enhanced tolerance against sanitization while forming mixed biofilms with the local environmental microorganisms, which was also related to the community species composition. These findings indicated that multiple factors might contribute to pathogen survival and prevalence at meat plants that need to be taken into consideration while designing sanitization protocols. The high presence of certain environmental species that relates to low pathogen colonization as a result of competitive exclusion, may represent a potential natural alternative strategy for pathogenic biofilm prevention.
Under Objective 3, the Time to Positivity (TTP) for Salmonella detection was evaluated for 96 retail ground meat samples (ground chicken number (n) n=36; ground turkey n=36; ground pork n=12 and ground beef n=12). Chicken products were the most likely to be found contaminated with Salmonella (80.6%), followed by ground turkey (47.2%) and ground pork (41%). Salmonella was not detected in ground beef samples. TTP estimates indicate the majority of Salmonella positive samples were contaminated at levels less than 1cfu/g. **Accomplishments**
                                

1. Antibiotic resistance transfer from non-pathogens to pathogens not observed in cattle feedyards. It has been theorized that Salmonella present at cattle feedyards may become resistant to the primary antibiotic used to treat serious human Salmonella infections by acquiring the genes responsible for resistance from non-pathogenic resistant Escherichia coli (E.coli). This theory has not been tested because although this gene transfer can be shown in artificial laboratory conditions, it has not been conclusively demonstrated in real world conditions. USDA-ARS scientists at Clay Center, Nebraska, sequenced over 200 each of both E. coli and Salmonella isolated from a beef cattle feedyard. The primary resistance gene in resistant Salmonella was present in only 37.9% of the resistant E. coli. Likewise, the primary resistance gene in most resistant E. coli was present at 0% in resistant Salmonella. These results demonstrated that resistant Salmonella at this feedyard were primarily due the persistence of a well-adapted Salmonella sub-population with very minimal or no contribution of resistance genes from the resistant E. coli. Available data do not support widespread transfer of resistance from non-pathogens to pathogens under commercial cattle feeding conditions. **Review Publications**
                            *Amadio, A., Bono, J.L., Irazoqui, M., Larzabal, M., Marques Da Siliva, W., Eberhardt, M.F., Riviere, N.A., Gally, D., Manning, S.D., Cataldi, A. 2021. Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle. PLoS ONE. 16(10). Article e0258753. https://doi.org/10.1371/journal.pone.0258753.*
                                *Weinroth, M.D., Clawson, M.L., Arthur, T.M., Wells, J., Harhay, D.M., Strachan, N., Bono, J.L. 2022. Rates of evolutionary change of resident Escherichia coli O157:H7 differ within the same ecological niche. BMC Genomics. 23. Article 275. https://doi.org/10.1186/s12864-022-08497-6.*
                                *Schmidt, J.W., Murray, S.A., Dickey, A.M., Wheeler, T.L., Harhay, D.M., Arthur, T.M. 2022. Twenty-four-month longitudinal study suggests little to no horizontal gene transfer in situ between third-generation cephalosporin-resistant Salmonella and third-generation cephalosporin-resistant Escherichia coli in a beef cattle feedyard. Journal of Food Protection. 85(2):323-335. https://doi.org/10.4315/JFP-21-371.*
                                *Macori, G., Nguyen, S.V., Naithani, A., Hurley, D., Bai, L., El Garach, F., Woehrlé, F., Miossec, C., Roques, B., O'Gaora, P., Bono, J.L., Fanning, S. 2021. Characterisation of early positive mcr-1 resistance gene and plasmidome in Escherichia coli pathogenic strains associated with variable phylogroups under colistin selection. Antibiotics. 10. Article 1041. https://doi.org/10.3390/antibiotics10091041.*
                                *Fitzgerald, S., Lupolova, N., Shaaban, S., Dallman, T.J., Greig, D.Q., Allison, L., Tongue, S.C., Evans, J., Henry, M.K., McNeilly, T., Bono, J.L., Gally, D.L. 2021. Genome structural variation in Escherichia coli O157:H7. Microbial Genomics. 7(11). Article 000682. https://doi.org/10.1099/mgen.0.000682.*


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