Location: Poultry Microbiological Safety and Processing Research Unit
2025 Annual Report
Objectives
1. Identify and determine the presence and contributing factors for antimicrobial resistant foodborne bacteria in poultry and poultry-associated environments.
1.1. Determine the association of antimicrobial resistance (AR) in foodborne bacteria with resistance to biocides, metals, coccidiostats, and ionophores used in poultry husbandry and processing.
1.2. Evaluate the bacterial metagenome of retail poultry.
1.3. Identify and evaluate markers (resistance genes, genetic elements, virulence genes) to define outbreak and persistent foodborne bacteria in poultry.
1.4. Identify antimicrobial resistance gene cassettes (ARCs) and accumulation on plasmids.
2. Identify and evaluate biological and chemical intervention products and alternatives to antimicrobials to control or reduce foodborne pathogens in poultry.
2.1. Develop, validate, and produce multi-subunit vaccines to control Salmonella and Campylobacter in broiler chickens.
2.2. Develop antimicrobial peptides (AMP) as alternatives to antibiotics to reduce foodborne pathogens associated with poultry.
2.3. Identify and develop broad-spectrum bacteriocins to eliminate foodborne pathogens in poultry.
2.4. Utilize phage isolation, whole-genome sequencing (WGS), and metagenomics to identify lytic phage that target Salmonella and pathogenic Escherichia coli.
Approach
Microbial contamination of food products from poultry continues to be a leading cause of foodborne illness. Antibiotics have been used to treat bacterial infections since the mid-twentieth century. Because of their efficacy in treating and preventing disease, antimicrobials have also been widely used in poultry production contributing to antimicrobial resistance (AR) in foodborne pathogens and commensal bacteria. AR among these bacteria has the potential to compromise therapy and remains a global threat to human health. This research project represents a merger of two teams of scientists to provide solutions to colonization of poultry with human pathogens and AR in foodborne pathogens and commensal bacteria from poultry. Two major approaches will be employed: 1) development of alternatives to antibiotics for use in combating foodborne pathogens, and 2) investigations to accurately understand attributes of antimicrobial resistant foodborne pathogens and commensals. Alternatives to antibiotics include vaccines to control foodborne pathogens in live birds while innovative antimicrobial peptides, bacteriocins, and lytic phage will modulate the poultry microbiome to reduce or eliminate colonization by harmful bacteria from poultry to minimize AR and reduce risk to human health. Data generated on resistance to biocides, metals, coccidiostats, and ionophores used in poultry production and processing is a specific concern to the USDA Food Safety and Inspection Service (FSIS). Research designed to determine ecological niches of foodborne bacteria and identify genetic characteristics facilitating transfer of resistance or a fitness advantage will also benefit FSIS. According to FSIS, increased knowledge of the microbial ecology of antimicrobial resistant pathogens on poultry will result in data that the poultry industry can utilize in development of improved pathogen management strategies. Identification of genetic markers which support survival, persistence, and dissemination of foodborne pathogens, especially those that are resistant to antimicrobials, is critical to this research priority. Data and technology from the proposed research will be used to assist other Federal agencies and the poultry and agricultural biotechnology industry in addressing AR in poultry resulting in safer products for the consumer.
Progress Report
Prediction of co-occurrence of antimicrobial resistance (AMR) using machine learning in Salmonella relating to Sub-objective 1.1 continued and aims to be completed later this year. Data from 10,143 human clinical isolates from the Centers for Disease Control and Prevention (CDC) and 8,028 chicken isolates from the Food Safety and Inspection Service (FSIS) collected from 2016 to 2019 were incorporated into models which considered nine AMR phenotypes (amoxicillin/clavulanic acid, ampicillin, ceftriaxone, cefoxitin, chloramphenicol, tetracycline, gentamicin, streptomycin, and nalidixic acid) as network features. Associations of AMR phenotypes over time were evaluated identifying patterns of re-occurring and transitory associations over the years studied. In addition, network patterns describing isolate populations from human clinical samples were compared against isolate populations from poultry products. Bayesian parameters were used to estimate the conditional probability distributions of the Salmonella network. This allowed for detailed predictions of any dataset feature based on presence or absence of information from any other combination of dataset features. Additional analysis methods to identify co-occurrence patterns included Random Forests for prediction of feature importance and dataset clustering using K-Modes to identify the underlying dataset structure. Across the two populations, patterns of similar co-occurring association were identified along with population unique associations. Consistent identification of AMR co-occurrence across multiple analysis methods demonstrates the accuracy of the results. Models produced from this study will be used to design a web-based tool for prudent selection of antibiotic alternatives for poultry producers.
Under Sub-objective 1.2, work on bacterial metagenomic analysis of conventional (n=80) and no antibiotic ever (NAE, n=80) retail poultry products continued. DNA extractions were completed from whole product rinsates from the two sampling categories. Shotgun sequencing was done followed by metagenomic analysis. Taxonomic comparison of the microbial composition of non-incubated versus incubated samples showed that overnight incubation increased relative abundance of each organism and population diversity to detectable limits while also decreasing abundance of contaminant reads. As non-incubated samples did not provide adequate data for the microbial composition analysis, shotgun sequencing of incubated samples only will be performed going forward. Sequencing of a total of 151 samples (116 incubated samples and 35 unincubated samples) was completed. Antimicrobial, metal, and biocide resistance genes and mobile genetic elements in the samples were also analyzed. Statistical analysis of the metagenomic samples will provide data to the poultry industry on beneficial poultry production practices to decrease AMR.
For Sub-objective 1.3, molecular characterization of Salmonella, Campylobacter, Escherichia coli, Staphylococcus, and Enterococcus from conventional and NAE retail poultry products continued. This research aimed to identify unique attributes of specific pathogen outbreak strains and persistent foodborne bacteria that may increase the probability of foodborne illness, a priority area of FSIS. Whole-genome sequencing (WGS) was completed for all isolates. SeqSero was used to confirm Salmonella serogroup determined from sequencing. Campylobacter isolates (n=36) were identified as either Campylobacter coli or C. jejuni and exhibited resistance against tetracycline with at least four isolates from three samples resistant to two or more of the drugs tested. Staphylococcus aureus was the most common staphylococcal species isolated from both conventional and NAE products. A Staphylococcus lentus isolate from NAE chicken drumsticks contained unusual resistance to antibiotics used in clinical medicine (daptomycin, linezolid, oxacillin, penicillin, quinupristin/dalfopristin, and vancomycin). Enterococcus faecalis was the most identified enterococcal species (n=173) and isolates from both sample types exhibited high-level aminoglycoside resistance (gentamicin, kanamycin, and streptomycin).
The ARS Salmonella Infantis Working Group continued under Sub-objective 1.3 and was transformed into a comprehensive system for responding to all threats to food safety. Previously, the Salmonella Infantis working group was formed in collaboration with the Office of National Programs to address the threat of Salmonella Infantis and pESI to U.S. poultry and present data to the National Chicken Council (NCC) and the National Turkey Federation (NTF). The working group brought together ARS scientists across several locations to work together on this issue. New emerging threats were identified including Salmonella Enteritidis with decreased susceptibility to ciprofloxacin and Salmonella Schwarzengrund in poultry. To address this, the working group was reorganized and named the Emerging Foodborne Pathogens of Concern (EFPOC). The leadership team, including scientists from Athens, Georgia, identified new threats, directed investigations, devised responses, and communicated findings to stakeholders at FSIS, CDC, NCC and NTF. The group now monitors and responds to bacteriological, viral, and parasitic threats to food safety.
For Sub-objective 2.1, 10 recombinant Salmonella sub-unit proteins were tested in chickens to determine if the proteins could induce humoral immune responses in hosts. After administration of the proteins subcutaneously, some clinical signs were observed in chickens from the immunized, but not from un-immunized groups. Utilizing bioinformatic tools, work continued to search Salmonella virulence factors as targets, analyze the immune epitopes with immunoinformatic tools, and assemble and construct multi-epitope targets by joining the amino acid sequences of the adjuvant, the epitopes (including the helper T cell, linear B cell and cytotoxic T cell epitopes), and linkers together. The constructs in a protein format were analyzed with immunoinformatic tools to determine the physiochemical characterization, antigenicity and toxicity. The analysis revealed the constructs were non-toxic and immunogenetic. The constructs were reverse translated into cDNA for gene synthesis. The cDNAs were cloned and expressed in a bacterial expression system. Biological components derived from Salmonella that can induce chicken immune responses and confer protection against colonization and infection in chickens will benefit poultry producers.
Also under Sub-objective 2.1, a collaboration with Emory University, Atlanta, Georgia, continued to develop and produce an mRNA-based vaccine against Salmonella Infantis for use in poultry. First, in vitro tests in chicken macrophage cells were performed. The results showed the mRNA could express in the cells within 24 hours after transfection. Second, the mRNA was administered via amniotic fluid or embryo to determine whether mRNA could express in chickens. Analysis of the results showed: (1) mRNA was expressed in chickens administered via amniotic delivery, but not via embryo, and (2) mRNA was expressed in intestine, gizzard, liver and heart. The results indicate that egg injection is a viable method to inoculate the chicken with an mRNA vaccine. The mRNA for the vaccine was designed and will be tested in eggs as soon as it is synthesized. This will yield a promising new vaccine for reducing Salmonella in chickens.
Under Sub-objective 2.3, work continued to identify bacteriocins using genome mining of whole-genome sequences from multidrug resistant Staphylococcus isolated from poultry. DNA was prepared from the isolates and used to determine the genome sequences. The WGS data were analyzed using antiSMASH which performs automated genome-wide identification, annotation, and analysis of secondary metabolite biosynthesis gene clusters in bacterial genomes. Work has also begun to extract the bacteriocins from cultures of the isolates so the bacteriocins can be biologically tested and biochemically characterized. This will identify bacteriocins for use in poultry feed to reduce or eliminate Salmonella and Campylobacter.
For Sub-objective 2.4, bacteriophage that kill S. Infantis-pESI were isolated from local surface water determined to be contaminated with Salmonella. The phages were further characterized for the ability to form plaques and kill the top 20 Salmonella serotypes that cause human infections. Salmonella Typhimurium, Enteritidis, Infantis and Heidelberg were used as initial targets. Currently the phages are being characterized for activity at a range of temperatures (4oC to 70oC) and a range of pH (pH 4 to pH 10). WGS analysis has shown the phages are closely related to known phages that can be selected for evaluation as an intervention to kill Salmonella on processing plant surfaces, retail meats, and in poultry.
Relating to Sub-objective 2.4, development of Bdellovibrio as a probiotic against Salmonella continued as a complementary project to the bacteriophage. These predatory bacteria kill bacteria including Salmonella. Previously, 20 new Bdellovibrio were isolated from local soil and water samples collected in the Athens, Georgia area. The isolates were found to kill the top 10 Salmonella serotypes that infect humans, including S. Infantis with the pESI plasmid. WGS data from the Bdellovibrio was subjected to phylogenetic analysis determining the strains were highly divergent and are likely different species. Microscopic analysis found two of the isolates were periplasmic predators that invade the prey cell, while another was an exovorus strain that attaches to the outside of the Salmonella prey cell. Predation efficiency assays are being used to determine which type of predation is most likely to work as an intervention to eliminate Salmonella from poultry.
Accomplishments
1. Improved detection and estimation of Salmonella in poultry rinse. Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can only detect Salmonella and cannot quantify or estimate the Salmonella load. ARS researchers in Athens, Georgia, and Clay Center, Nebraska, in collaboration with Florida State University standardized and validated a digital PCR (dPCR) assay to detect and estimate Salmonella levels in chicken rinse samples and a novel varying amplification efficiency real-time PCR assay for broad Salmonella contamination levels. The dPCR assay was highly specific with a low limit of detection (0.001 ng/µL) and quantification (0.01 ng/µL) and detected all cold-stressed Salmonella in inoculated samples following a 5-hour enrichment and accurately estimated the inoculated Salmonella levels. The assay generated reproducible results with minimal sample-to-sample variations, was highly resistant to PCR inhibitors, and showed high DNA tolerance. The standardized multiplex PCR assay was validated with 131 pure culture strains and 260 laboratory-inoculated chicken-rinse samples. The multiplex assay specifically identified all Salmonella strains, detected Salmonella in all inoculated samples also following a 5-hour enrichment, and was able to discriminate the high and low levels in most of the samples. These approaches can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination as well as enable the food industry to detect and identify high-risk samples contaminated at higher levels.
2. Chicken immune responses to Salmonella fimbriae and flagellar proteins. Salmonella is the leading bacterial cause of human foodborne illnesses worldwide. The major source of this bacteria for human infection is from consumption of contaminated poultry products. Implementation of a vaccination program is one of the most effective means to control infectious diseases during poultry production. Although lived attenuated vaccine are available, there are problems with these vaccines, such as persistence and shedding of Salmonella in and from the vaccinated animals. ARS researchers in Athens, Georgia, and Raleigh, North Carolina, produced and tested two flagellar and two fimbrial surface-exposed recombinant proteins from Salmonella Heidelberg for their antigenicity in chickens. The recombinant flagellar proteins triggered high levels of immune responses in vaccinated birds, but not the unvaccinated group, indicating the antigenicity of the recombinant proteins. The recombinant fimbrial proteins barely induced antibody responses in vaccinated chickens. These antibody studies suggest that recombinant flagellar proteins have potential as targets for vaccine development in chickens.
3. Antigen determinant mapping of therapeutic targets in Salmonella. Salmonella is the leading bacterial pathogen linked to human acute gastroenteritis worldwide. Outbreaks of human salmonellosis have often been associated with consumption of contaminated poultry products. Various strategies have been explored to control this microorganism during poultry production and processing. Vaccination is regarded as one of the effective means to control Salmonella in poultry production. ARS researchers in Athens, Georgia, compared two methods (in silico prediction and in vivo using mass spectrometry with immunoprecipitation proteomics) for antigenic determinant identification in the Salmonella Heidelberg flagellar protein. The protein contains 553 amino acids with a molecular mass of 61 kDa and is conserved among S. Heidelberg isolates. Both approaches identified three common shared consensus peptide antigenic sequences in the protein suggesting that these shared linear antigens may be targets for therapeutic development to reduce Salmonella infection in poultry.
4. Genes specific for survival in cattle identified in Salmonella Dublin. There are over 2000 Salmonella serovars that vary in their ability to cause disease in specific host animals. Determining the biology behind these differences will aide in understanding host specificity of Salmonella and enable identification of better methods to block these host specific serovars from infecting cattle. Salmonella Dublin (S. Dublin) and Salmonella Typhimurium (S. Typhimurium) are commonly linked to infections in cattle. S. Dublin is considered a bovine-adapted serovar for primarily infecting and thriving in cattle. S. Typhimurium is a generalist serovar and can infect many hosts. ARS researchers in Athens, Georgia, in collaboration with Tuskegee University compared S. Typhimurium to S. Dublin to identify unique genetic factors contributing to S. Dublin’s adaptation in U.S. cattle. Minimal genomic variation among S. Dublin isolates was observed and S. Dublin carried more antimicrobial resistance genes against key antimicrobials, including aminoglycosides, beta-lactams, tetracyclines, and sulfonamides, commonly used in U.S. cattle production. Additionally, Type VI secretion system virulence genes used in colonization were found exclusively in S. Dublin isolates and over 50% of these isolates also possessed genes conferring resistance to heavy metal stressors, like mercury. These findings suggest that S. Dublin’s adaptation to bovine hosts in the U.S. is supported by a conserved genetic makeup enriched with antimicrobial resistance genes, virulence factors, and stress-related genes, enabling it to colonize and persist in the bovine gut. With this information, the Type VI secretion system can be used as a target with interventions to prevent colonization of cattle.
5. Distinct differences found between strong and weak colonizing Campylobacter jejuni. Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of poultry, including chickens, and consumption of chicken and poultry products is identified as a common route of transmission. ARS researchers in Athens, Georgia, collaborated with the U.S. Food and Drug Administration to analyze and compare a strong versus a weak colonizing C. jejuni strain. The two strains were genotypically similar; however, extensive differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and cellular transport were found. Carbon metabolism and motility proteins were distinctively overexpressed in the robust colonizing strain. The robust colonizer also exhibited significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. These findings demonstrate that chick colonization of C. jejuni is not a simple process involving just a few colonization factors, but one incorporating many colonization factors, which together may contribute to commensalism in the chicken. Identification of colonization factors in chickens will aide in development of effective strategies to mitigate C. jejuni contamination in poultry flocks, an important goal in public health.
6. A new multidrug-resistant Escherichia coli control strain for use in environmental and agricultural studies. Control strains with known characteristics ensure consistency and reproducibility of assays across laboratories and are an important part of quality control in the microbiology laboratory. Ideally, control strains should be representative of the assay’s target and be widely available from reputable sources. However, for work involving antibiotic resistance, most controls come from human and veterinary clinical sources and are not optimized for work in agriculturally impacted environments or are not widely available. ARS researchers in Athens, Georgia; Clay Center and Lincoln, Nebraska; Riverside, California; Beltsville, Maryland; and Maricopa, Arizona, collaborated to identify and make available two Escherichia coli isolates sourced from agricultural production settings that could be used as external controls supporting method development, research, and environmental monitoring for extended spectrum beta-lactamase producing (ESBL) and tetracycline resistant E. coli. The positive control strain, ARS-C301, was ESBL positive and contained the CTX-M-55 and tet(A) genes, and the negative control strain, ARS-C101, was negative for both targets. These two agriculturally sourced, fully characterized, and genetically sequenced control strains are now available via publicly accessible culture collections and commercially as a quantitative pellet.
7. Plasmids contribute to antibiotic resistance and virulence in Staphylococcus aureus from retail chicken meat. Staphylococcus aureus is a bacterium that is commonly found on the skin or in the nasal passages of humans and animals. Staphylococcal food poisoning, caused by enterotoxins and characterized by vomiting and diarrhea, is a leading cause of foodborne illness in the U.S. As food sources of S. aureus include retail meat, ARS researchers in Athens, Georgia, in collaboration with Tuskegee University, investigated prevalence, toxin gene, and antibiotic resistance profile of S. aureus recovered from retail poultry meat samples. Of 200 samples, 16% were positive for S. aureus recovered from the thigh, wings, gizzards, and livers. Antibiotic resistance was low; only one isolate was resistant to more than two classes of antibiotics. Four isolates were positive for the mecA gene associated with Methicillin-Resistant S. aureus (MRSA). One MRSA isolate contained a large plasmid which was the source of resistance to several antibiotics including penicillin, ampicillin, erythromycin, and oxacillin. Plasmids harbored most antimicrobial resistance genes and staphylococcal enterotoxin genes identified among the isolates. This study raises awareness of the continuous circulation of pathogenic microbes like S. aureus in retail poultry meat. This is especially useful for scientists as they develop prevention and control strategies to protect consumers and personnel who handle raw meat.
8. Probiotic discovery in novel bacteria from poultry. Bird species have been reported to harbor communities of microorganisms in their gastrointestinal tract that play crucial roles in providing the host with nutrition and protection from pathogens. Although most uses for bacterial species in the genus Sporosarcina are industrial, three members of the genus have been reported to possess properties that make them potential poultry probiotics. ARS researchers in Athens, Georgia, collaborated with Oregon State University and Western University of Health Sciences to isolate new probiotic bacteria from poultry origins. Two bacteria of interest were isolated from feces of Canada geese, one which was able to grow in high salt levels and exhibited lipase activity, while the other could not grow under these conditions. Both isolates were positive for starch hydrolysis and inhibited the growth of Staphylococcus aureus. Phenotypic and phylogenetic data indicate these bacteria are two novel isolates of the Sporosarcina genus. Both strains contain urease genes and a fibronectin-binding protein gene indicating that these bacteria may bind to eukaryotic cells in host gastrointestinal tracts. These new species of bacteria will be beneficial to the poultry industry as novel bacteria with probiotic characteristics which could be introduced into poultry feed.
Review Publications
Yeh, H., Read, Q.D. 2024. Immune responses of chickens against recombinant Salmonella enterica serotype Heidelberg FimA and FimW fimbriae and FliD and FlgK flagellar proteins. Veterinary Immunology and Immunopathology. 280:2025(e110870). https://doi.org/10.1016/j.vetimm.2024.110870.
Bentum, K., Kuufire, E., Nyarku, R., Osei, V., Price, S., Bourassa, D., Samuel, T., Jackson, C.R., Abebe, W. 2025. Salmonellosis in cattle: sources and risk of infection, control and prevention. Zoonotic Diseases. 2025, 5, 4. https://doi.org/10.3390/zoonoticdis5010004.
Faraj, R., Ramadan, H., Bentum, K., Alkaraghulli, B., Woube, Y., Hassan, Z., Samuel, T., Adeslyun, A., Jackson, C.R., Abebe, W. 2025. Prevalence, antimicrobial resistance, and virulence gene profiles of Staphylococcus aureus isolated from retail chicken meat. Pathogens. 14, 107. https://doi.org/10.3390/pathogens14020107.
Bentum, K., Kuufire, E., Nyarku, R., Osei, V., Adu-Addai, B., Frye, J.G., Jackson, C.R., Samuel, T., Abebe, W. 2025. Comparative genomic profiles of Salmonella Typhimurium and Salmonella Dublin bovine isolates from the U.S. indicate possible factors associated with host adaptation of Salmonella Dublin in the region. Microorganisms. 13(4):886. https://doi.org/10.3390/microorganisms13040886.
Cosby, D.E., Berrang, M.E., Frye, J.G., Hinton Jr, A. 2023. Filter sterilized carcass rinsate for recovery of Salmonella species in various concentrations of cetylpyridinium chloride. Food Science and Nutrition. 11(8):4353-4866. https://doi.org/10.1002/fsn3.3463.
Kandula, N., Velez, F.J., Jackson, C.R., Bosilevac, J.M., Singh, P. 2024. Varying amplification efficiency Real-time PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse. Food Bioscience. https://doi.org/10.1016/j.fbio.2024.104777.
Kehri, J., Smith, K.M., Svedsen, M.K., Keillor, H.R., Moss, M.L., Jordan, H.J., Larkin, A.M., Garrish, J.K., Line, E., Bali, P.N., Oakley, B.B., Seal, B.S. 2023. Phenotypic characterization and draft genome sequence analyses of two novel endospore-forming Sporosarcina spp. isolated from Canada Goose (Branta canadensis) feces. Microorganisms. 12:70. https://doi.org/10.3390/microorganisms12010070.
Sung, K., Gao, Y., Yu, L., Chon, J., Hiett, K.L., Line, E.J., Kweon, O., Park, M., Khan, S.A. 2024. Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.. Microbial Pathogenesis. 193: 102766. https://doi.org/10.1016/j.micpath.2024.106766.
Velez, F.J., Kandula, N., Blech-Hermoni, Y., Jackson, C.R., Bosilevac, J.M., Singh, P. 2024. Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse. Food Control. https://doi.org/10.1016/j.crfs.2024.100807.
Wells, J., Durso, L.M., Ibekwe, A.M., Frye, J.G., Sharma, M., Williams, C.F., Shamimuzzaman, M. 2025. Agriculturally sourced multidrug-resistant Escherichia coli for use as control strains. Pathogens. 14. Article 14050417. https://doi.org/10.3390/pathogens14050417.
Bentum, K., Kuufire, E., Nyarku, R., Samuel, T., Jackson, C.R., Abebe, W. 2025. Hydrogen sulfide negative Salmonella and their implication for standard culture-based identification: looking at the other side of the coin. Journal of Food Protection. https://doi.org/10.1016/j.jfp.2025.100549.
Yeh, H. 2025. Epitope mapping of recombinant Salmonella enterica serotype Heidelberg flagellar hook-associated protein by in silico and in vivo approaches. BMC Veterinary Research. 21(54). https://doi.org/10.1186/s12917-025-04479-4.