Location: Meat Safety and Quality
2024 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 fourth 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) 2025. The pens with culturable STEC O157:H7 in 2023 had a prevalence of 33.1% which is in line with the 2021 and 2022 prevalence of 38.3% and 38.9% respectively. The same trend held true with the overall prevalence of STEC O157:H7 in the 2023 at 23.4% when compared to 27% for 2022 and 25% for 2021. Weather conditions play an important part of STEC O157:H7 prevalence. The weather for the last three years has been fairly consistent and unseasonably dry. Consequently, calves have been weaned into the feedlot sooner than intended. This resulted in the last sampling having a higher pen (47%) and sample prevalence (72%). If not for these results, the yearly prevalence average would have been much lower for 2023. 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 2023 was 22.8% which is almost three times the prevalence seen in 2022 and greater than the 16% prevalence seen in 2021. The prevalence from the 8 weaning groups was quite variable with two groups accounting for 85% of the positive samples and two other groups accounting for only one sample. One reason for the dramatic increase in prevalence for the two groups is that the cattle in those groups were supplemented with silage because of the lack of grass due to drought conditions. This created a feedlot-like environment where cattle were in closer confinement than when grazing in a pasture.
The sequencing and analysis were completed for year three pen surface samples and weaned calf fecal samples. A total of 384 strains from the 2023 sampling were sequenced that represent the 324 cultured strains and additional strains picked from the same plate as the original culture strains. The sequencing results from 2023 were quite different than the previous two years with clade 2 strains representing 90% of the sequenced isolates, clade 3 with 6.8% followed by clade 4 with 3.2% of the strains compared to the 2022 sequencing results of 72% for strains that belonged to clade 2 with 19% from clade 3 and 9 % from clade 4. The difference between years is magnified when comparing calf fecal samples. From 2023 samples, 98% of the strains belong to clade 2 whereas only 18% belong to the same clade in 2022. The majority of the 2022 strains belonged to clade 3 (74%) while no clade 3 strains were isolated during the 2023 sampling. Another example of the dominance of clade 2 strains during the 2023 sampling was demonstrated by the lack of clade 3, 4 or 5 strains from the last three samplings. Of the 13 pens with either clade 3 or 4 isolated during the first three samplings, 11 pens subsequently had clade 2 strains isolated from the last 3 sampling dates. Only one pen had a clade 3 strain from the last sampling date compared to the 2022 sampling where 22% of the last pen sampling were clade 4 strains and 8% were clade 3 strains. A virulence gene survey of strains identified a group of clade 2 strains that are missing a Shiga toxin (stx) gene that the other clade 2 strains contain. Also, the clade 3 strains lack stx genes which is different from the reference clade 3 strain. These results demonstrate the complicated feedlot dynamic of E. coli O157:H7 strains and their ability to persist in this environment.
Under Objective 2, collaborative research continued with the research and development team of our research partners on development of a real-time polymerase chain reaction (RT-PCR) assay for the rapid detection of more pathogenic versions of Salmonella (Highly Pathogenic Salmonella or HPS) in meat enrichment samples. As part of this, we shared 129 Salmonella isolates of various serotypes, and 140 BAX (Hygiene, LLC) lysates of corresponding enrichment samples, for alpha testing of the prototype RT-PCR assay they were developing. These initial tests showed vary promising results and we have subsequently received 19 prototype kits (96 tests per kit) to further test with approximately 1200 archived and well characterized enrichment samples, for beta testing and validation testing of the RT assay. If these tests perform as we hope, the assay could be ready for commercial release sometime in FY2025.
We completed sequencing 74 E. coli O26:H11 strains to generate complete closed genomes for subsequent analysis. These 74 strains represent 64 unique genotypes. These strains were picked to represent the known genomic diversity. These strains represent 53 strains isolated from humans and 21 from the environment including 18 bovine strains. There were seven sequence types (ST) identified with the large number belonging to ST21 (number = 37). All but one of these strains contain the virulence gene stx1a while 14 contained stx2a and one strain didn’t contain any stx genes. ST29 has the second most strains (number = 30) with 23 containing the stx2a gene and seven containing no stx genes. ST5172 contained three strains with all three containing the stx2a gene. Analysis is underway to identify potential gene/s that predict the potential of strains to cause human disease.
We have continued making significant progress toward understanding the impact of environmental microbial communities on foodborne pathogen survival and persistence under sanitizer stress. We collaborated with Texas A&M University scientists and characterized the microbiome dynamics in meat facility environments and their response to pathogen recruitment and sanitization. We demonstrated that the relative abundance of the different bacterial genera was affected by both sanitization and pathogen addition. The correlation network analyzing the relationships among the various bacteria showed that Nitrospira had the greatest number of connections in the community network. Since nitrogen residues accumulate in the environment due to repeated use of the sanitizers, and Nitrospira may convert ammonium into nitrites, thus, Nitrospira may potentially function as a keystone species that provides tolerance to pathogen-encompassed mixed biofilms. Further, a novel method using differential staining fluorescence microscopy (DSFM) to measure biovolumes and track pathogen locations in mixed biofilms was developed. Using this method, we showed that the introduction of pathogen strains resulted in diverse alterations of biofilm biovolumes, suggesting distinct impacts on mixed biofilms by different enteric pathogens which were revealed to be located in the upper layer of the mixed biofilms. To further understand the environmental impact on pathogen stress tolerance, we have isolated individual environmental bacterial strains from meat plant floor drain samples and are currently testing the interspecies interactions between pathogens and the natural species using dual-species or triple-species biofilm models.
Under Objective 3, research was conducted in collaboration with Food Safety Inspection Service (FSIS) and University of Georgia, Athens (UGA), collaborators examining enrichment media biases that could impact the ability to detect more pathogenic versions of Salmonella when present. Research included Deep Serotyping (DST) analysis of 1244 Salmonella positive enrichment samples that had been sent by FSIS for serotype and HPS analysis. For this, Salmonella was isolated from Rappaport-Vassiliadis Soya Peptone broth secondary enrichment samples by centrifugation, from which total DNA was isolated. DNA samples were sent to UGA collaborators for DST analysis. Additionally, 33 primary enrichment samples (13 Chicken, 10 Pork, 6 Beef and 4 Turkey) were subjected to three different secondary enrichment methods (one traditionally used by ARS in Clay Center, Nebraska, and two that are routinely used at FSIS) and plated onto three different selective and differential agar media. Up to eight colonies were selected from each plate, resulting in a total of 2,376 Salmonella colonies that were isolated and subsequently characterized for serotype and HPS genotype. Secondary enrichments were centrifuged to isolate Salmonella populations present, total DNA extracted, and DST performed. A subset of these samples was analyzed to completion and demonstrated enrichment media biases in the Salmonella serotypes that were isolated. These investigations into the effects of enrichment media biases, using traditional culture methods and novel molecular methods have high potential for improving the methods used to monitor for Salmonella, especially those noted for an increased ability to cause human illness. Analysis of the remainder of the data collected will continue in FY2024.
Accomplishments
1. Genomic variation in Escherichia coli O157:H7 does not completely explain disease phenotype in humans. Over time, changes at the genome level can provide valuable information about a bacteria’s abilities to survive or adapt to different environments. One such change in a virulence factor of Escherichia coli (E. coli) O157:H7 associates with a strain's ability to cause disease in humans. Previous studies to address this association have used draft genomes instead of complete closed genomes. USDA-ARS scientists at Clay Center, Nebraska, generated complete closed genomes from 48 strains along with another ten complete closed genomes from an international depository to compare the genome change that is associated with a strain’s ability to cause disease in humans. While genomic differences exist between the two groups, none on their own completely explain why one group has a greater likelihood for causing disease in humans. Further characterization of these bacteria may provide a better explanation for this association. Another added value is the release of accurate complete close genomes that doubled the number of E. coli O157:H7 available to the research community.
2. Growth assessment of Salmonella enterica multi-serovar populations with commonly used enrichment and plating media. Isolation of Salmonella from food or environmental samples is a complicated process. Numerous factors impact which strains grow best and are subsequently identified from a given sample. A recently developed approach for identifying all the Salmonella strains from a sample (Deep Serotyping) is providing new insight into how these factors impact the strains identified, when different selective enrichment media are used. ARS scientists in Clay Center, Nebraska, and collaborators used two selective growth media and found that sometimes the Salmonella strain present at lower concentration was incorrectly identified from the sample instead of the more numerous strain. These data indicate specific media combinations must be used to identify Salmonella strains of greater concern for human health.
Review Publications
Weinroth, M.D., Clawson, M.L., Harhay, G.P., Eppinger, M., Harhay, D.M., Smith, T.P.L., Bono, J.L. 2023. Escherichia coli O157:H7 tir 255 T > A allele strains differ in chromosomal and plasmid composition. Frontiers in Microbiology. 14. Article 1303387. https://doi.org/10.3389/fmicb.2023.1303387.
Katz, T.S., Harhay, D.M., Schmidt, J.W., Wheeler, T.L. 2024. Identifying a list of Salmonella serotypes of concern to target for reducing risk of salmonellosis. Frontiers in Microbiology. 15. Article 1307563. https://doi.org/10.3389/fmicb.2024.1307563.
Kalalah, A.A., Koenig, S.S.K., Bono, J.L., Bosilevac, J.M., Eppinger, M. 2024. Pathogenomes and virulence profiles of representative big six non-O157 serogroup Shiga toxin-producing Escherichia coli. Frontiers in Microbiology. 15. Article 1364026. https://doi.org/10.3389/fmicb.2024.1364026.
Carter, M.Q., Quinones, B., He, X., Pham, A.C., Carychao, D.K., Cooley, M., Lo, C., Chain, P.S., Lindsey, R.L., Bono, J.L. 2023. Genomic and phenotypic characterization of Shiga toxin-producing Escherichia albertii strains isolated from wild birds in a major agricultural region in California. Microorganisms. 11(11). Article 2803. https://doi.org/10.3390/microorganisms11112803.
Gorski, L.A., Shariat, N., Richards, A.K., Siceloff, A.T., Aviles Noriega, A., Harhay, D.M. 2023. Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media. Food Microbiology. 119. Article 104431. https://doi.org/10.1016/j.fm.2023.104431.
Dickey, A.M., Schmidt, J.W., Bono, J.L., Guragain, M. 2024. The GEA pipeline for characterizing Escherichia coli and Salmonella genomes. Scientific Reports. 14. Article 13257. https://doi.org/10.1038/s41598-024-63832-z.
Kalalah, A.A., Koenig, S.S., Feng, P.C., Bosilevac, J.M., Bono, J.L., Eppinger, M. 2024. Pathogenomes of Shiga toxin positive and negative Escherichia coli O157:H7 strains TT12A and TT12B: Comprehensive phylogenomic analysis using closed genomes. Microorganisms. 12(4). Article 699. https://doi.org/10.3390/microorganisms12040699.