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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** 2023 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 third sample collection at the feed yard in Clay Center, Nebraska, and cultured Shiga toxin-containing Escherichia coli (E. coli) O157:H7 (STEC O157:H7) from the samples. The DNA from these strains will be extracted and sequenced during fiscal year (FY) 2024. The pens with culturable STEC O157:H7 in 2022 had a prevalence of 38.9% which is comparable to the 2021 prevalence of 38.3%. The percentage of samples with STEC O157:H7 in 2022 was 27% compared to 25% in 2021. While the prevalence of STEC O157:H7 in the pens and percentage of samples were similar, this trend is not expected for the remaining years. Weather conditions play an important part of STEC O157:H7 prevalence. The weather for the last 2 years has been fairly consistent and unseasonably dry leading to similar results. It remains to be seen if this weather pattern will hold or if there will be a change in temperature or moisture. The prevalence of STEC O157:H7 in calves weaned into the feed yard in 2022 was 8%, which was half as much as seen in year 2021 and more in line with previous studies that reported 5% or less. One reason for the decline could be the mass treatment with antibiotics for respiratory disease in the calves during the summer. 
Sequencing and analysis were completed for year 2 pen surface samples and weaned calf fecal samples. The goal of the sequencing objective was to sequence 480 strains cultured from the 2 samplings. Since only 189 strains were cultured, multiple picks from the same plate were used to increase the number to 480. Similar to the sequencing results from 2021, clade 2, 4 and one of the previously unidentified strains (now called clade 5) identified in the 2021 sampling were identified in the 2022 strains. However, strains from clade 3 were also identified in the 2022 results. The weaned calf fecal STEC O157 isolate clades associated with the pasture where the calves were raised. From the weaned calf fecal samples, clade 3 had the highest prevalence with 74% while clade 2 was 17% and clade 4 was 9%. The last pen surface sampling after the calves were weaned had clade 2 with the highest prevalence at 70% followed by clade 4 with 22% and clade 3 with 8%. This indicates that there were calves not sampled that had a high prevalence of clade 4 and that clade 3 strains didn’t survive well as the prevalence in the pen surface material dropped significantly. From the 88 positive pen surface samples before weaned calves were put in the pen, clade 2 had the highest positive rate at 86% followed by clade 5 with 13% and clade 4 with 1%. When comparing these positive rates to the sampling of last year the highest positive rate was clade 2 with 68% followed by clade 5 with 29% and clade 4 with 3%. These results would suggest that clade 4 and clade 5 were able to maintain their prevalence in the feed yard, although the positive rate for clade 4 did drop from 29% to 13% over winter. These results indicate that currently there are three STEC O157:H7 strains that reside in the feed yard, with clade 2 being the predominate strain followed by clade 5 and clade 4. This year was the first time that clade 3 strains were isolated in calves and in the pens. It will be interesting to see if this clade is able to maintain itself in the pens after being reintroduced this year. 
Under Objective 2, complete closed genome sequences from 130 Salmonella enterica strains representing 51 serotypes were either sequenced or downloaded from the National Center for Biotechnology Information (NCBI) database. These isolates were placed into two groups according to their designation as a high pathogenicity Salmonella (HPS) or not. The HPS assay is a recently developed molecular screening method for classifying Salmonella isolates for their ability to cause disease in humans. Fifty-four HPS isolates and 76 non-HPS strains were analyzed using a microbial pan-genome wide association (GWAS) study to identify genes that associate with the HPS genotype as well as effector genes. Effector proteins are important in the pathogenicity of bacteria as they promote colonization of and persistence in the host. There were 2,617 core genes and 15,138 accessory genes identified from the 130 Salmonella strains, with 35 being identified as effector proteins. From those, the GWAS study identified 207 gene clusters that are significantly associated with the HPS genotype (Bonferroni p=<0.05) with six identified as effector proteins. Corroborating the observed utility of the HPS assay, six of the seven chromosomal encoded molecular targets for the assay were identified in the list of 207 significant genes. There wasn’t a gene cluster that had a 100% association with the HPS genotype. A hypothetical gene next to the long polar fimbriae operon had the greatest association with the HPS genotype with a sensitivity of 98.1 %, specificity of 90.8% and an odds ratio of 522.4. The effector protein with the greatest association with the HPS genotype was sspH2 with 90.7% specificity, 89.5% sensitivity and an odds ratio of 57.9. In addition, these same genome sequences were analyzed using a Salmonella virulence gene finder developed by the Federal Drug Administration (FDA). This analysis revealed six additional gene targets that were more commonly present in HPS genomes, and less frequently observed in non-HPS genomes. These gene targets will be further examined to determine the products they encode and what possible role they may play in increasing Salmonella fitness as human pathogens. Overall, the results of these computational analyses suggest that there isn’t one particular virulence factor or effector protein that is responsible for causing disease, rather it is likely a combination of factors within each HPS serovar that contribute to the success of that pathogen and an increased ability to cause illness 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 processing plants have been collected from floor drains and their biofilm-forming ability was examined under processing conditions. E. coli O157:H7 and Salmonella enterica were evaluated for their tolerance in mixed biofilms with pre-exposures to reagents at various concentrations. Environmental microbial samples from several meat processing plants were also collected from floor drains at multiple locations and used to form mixed biofilms on different types of contact surfaces with pathogen co-inoculation under processing conditions. Pre-exposure to traditional sanitizers such as quaternary ammonium compounds (QAC) showed an impact on Salmonella biofilm formation and stress tolerance that was serovar/strain dependent. However, such impact was not observed in most examined E. coli O157:H7 strains.
As per request from major meat industry processors, we further tested novel biofilm pretreatment reagents (enzyme-based detergents) at either low or high concentrations, followed by traditional sanitizers of oxidizing agents peracetic acid (PAA) or QAC. Pathogens in mixed biofilms showed enhanced tolerance against detergent treatment and enumerable viable cells were observed at various levels depending upon the different drain samples. Even though follow-up treatments using traditional sanitizers at recommended lethal concentrations were able to reduce viable pathogens to non-enumerable levels, the pathogens in most treated mixed biofilms exhibited positive prevalence after enrichment. However, detergents applied at high concentrations increased pretreatment efficiency and reduced pathogen tolerance in mixed biofilms as most enriched samples exhibited negative pathogen prevalence after QAC or PAA follow-up treatments. Scanning electron microscopy analysis confirmed that such pretreatment reduced the mixed biofilm matrix and weakened the extracellular polymeric substances (EPS) connection with the contact surfaces. Biofilm morphology was altered after the treatment as in most cases EPS structures were dissolved and intact mature biofilm architecture was not found. Instead, scattered clusters of bacterial aggregates or individual cells were observed. Our results showed novel mitigation strategies such as enzyme-based detergents applied at low concentrations were not efficient in eliminating pathogens as they likely obtain increased tolerance in environmental mixed biofilms. Detergent pretreatment should be applied at sufficient concentration to destroy mixed biofilm 3D structure which provides protection to the harbored pathogens in order for the follow-up sanitization to minimize potential pathogen prevalence. Studies are ongoing using pretreatment with additional products followed by treatment with novel multicomponent sanitizers. 
Under Objective 3, the Time to Positivity (TTP) method for Salmonella detection was used previously to evaluate 96 retail ground meat samples. Chicken products were most frequently 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 indicated that the majority of Salmonella positive samples were contaminated at levels less than 1 colony forming units per gram (cfu/g) of ground beef. The TTP method was further evaluated along with novel molecular quantification methods (BioMerieux GeneUP Salmonella quant and Hygiena SalQuant methods) as well as with the traditional Most Probable Number (MPN) method, with 12 inoculated poultry rinse samples. This evaluation provided further evidence that the TTP method allows for the rapid and economical identification of meat samples contaminated at the 5 cfu/g or greater range. Continued evaluation with other meat commodities will be undertaken in FY2024. **Accomplishments** **Review Publications**
                            *Weinroth, M.D., Bono, J.L. 2022. Comparative genomics of Escherichia coli serotype O55:H7 using complete closed genomes. Microorganisms. 10(8): Article 1545. https://doi.org/10.3390/microorganisms10081545.*
                                *Marques Da Silva, W., Larzabal, M., Figueira Aburjaile, F., Rivieri, N., Martorelli, L., Bono, J.L., Amadio, A., Cataldi, A. 2022. Whole-genome sequencing analysis of Shiga toxin-producing Escherichia coli O22:H8 isolated from cattle prediction pathogenesis and colonization factors and position in STEC universe phylogeny. Journal of Microbiology. 60:689-704. https://doi.org/10.1007/s12275-022-1616-z.*
                                *Wynn, E.L., Hille, M.M., Loy, J.D., Schuller, G., Kuhn, K.L., Dickey, A.M., Bono, J.L., Clawson, M.L. 2022. Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants. BMC Microbiology. 22(1). Article 258. https://doi.org/10.1186/s12866-022-02670-3.*


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