NC_old1206: Antimicrobial Resistance

(Multistate Research Project)

Status: Active

NC_old1206: Antimicrobial Resistance

Duration: 10/01/2017 to 09/30/2022

Administrative Advisor(s):

NIFA Reps:

Statement of Issues and Justification

The advent of antibiotics in the 20th century has had a profound effect on both human and animal health, with millions of lives saved annually as a result of these “wonder drugs.” However, over the past several decades, bacterial resistance to antimicrobial drugs has escalated. The Centers for Disease Control and Prevention (CDC),the World Health Organization and more recently President Obama’s President’s Council of Advisors on Science and Technology (PCAST) have identified antibiotic resistance as one of the greatest threats to human health worldwide, economic growth, public health, agriculture, economic security and national security. Furthermore, the CDC estimates that annually 2 million illnesses and 23,000 deaths in the United States alone can be attributed to antibiotic-resistant bacteria.

This serious threat to human, animal and public health has not gone unnoticed. In September 2014, President Obama signed the executive order “Combating Antibiotic-Resistant Bacteria” followed by the “National Action Plan for Combating Antibiotic-Resistant Bacteria.” In response, other government agencies including and CDC and USDA established comprehensive action plans that lay the foundation for research and actions needed to address this critical concern. Additionally, President Obama has proposed to double the budget available for antibiotic resistance research to $1.2 billion in 2016.

The CDC plan focuses on human health, while the USDA action plan focuses primarily on food and animal agriculture. Common themes are the need for the One-Health approach and science-based data to mitigate antibiotic resistance.

The use of antibiotics in animal agriculture is integral to the treatment of and protection against disease. It is imperative that stakeholders in animal agriculture and animal health address the important public health concern of bacterial resistance before antibiotic drugs either become ineffective or are further restricted for use in animals.

The CDC and USDA action plans are comprehensive in their approaches to combat antibiotic resistance. The plans call for applied and basic research to:

  • enhance surveillance and monitoring of antibiotic resistance,
  • better understand the epidemiology and ecology of antibiotic resistance,
  • develop new antibiotics and alternatives to antibiotics,
  • determine the mechanisms involved in resistance and transmission of resistance,
  • determine and/or model patterns and practices that impact the use of antibiotics in animal agriculture, and
  • develop improved diagnostic tests and vaccines.

Achieving these goals require interdisciplinary and transdisciplinary collaborations, which can be greatly facilitated by forming a new NCDC committee.

The 12-state North Central Region has many scientists with expertise that can contribute to the understanding of antibiotic resistance in food animals. The North Central Region also has some of the most concentrated populations of food-producing animals in the United States. These include dairy cattle, beef cattle, swine, laying chickens and turkeys. Because of the wide diversity of food-producing animal populations across the region, we are uniquely positioned to conduct studies directed towards all and specific animal populations relative to broad and specific bacterial species, geographical differences and animal management practices.

Identifying practical and effective intervention strategies to lower antibiotic resistance is only one part of a successful mitigation program. Equally important is the willingness of livestock producers to fully implement specific control measures into production practices. Effective communication is needed between producers and experts about the risks if control measures are not implemented.

The barriers that impact effective communication about antibiotic resistance mitigation have not been studied. Communication barriers and the extent to which each influences the behavior of involved parties should be identified. Potential barriers may include lack of effective communication channels; the relationship between producers and experts; and the impact of ethical, moral, social and economic beliefs on communication and decision-making.

Because the implementation of new mitigation strategies is not trivial and producer attitudes and perceived risk may prevent the adoption of new strategies, it is also important to classify attitudes and risk perceptions about antibiotic mitigation, and develop decision-support communication activities for producers

Antibiotic resistance is a complex issue and addressing it requires working together beyond individual teams and beyond individual animal species. The multistate, multispecies and multidiscipline approach provides an exceptional opportunity to develop collaborative projects in the North Central Region that will allow us to develop and implement comprehensive strategies directed specifically at food-producing animals, establish baseline data to be used as benchmarks over time to ascertain the effectiveness of new research applications and mitigation strategies, and develop innovative tools for combating current and emerging antibiotic resistance threats. Furthermore, such collaborative transdisciplinary and multi-institutional projects will greatly enhance the NCR stakeholder’s position to compete successfully for federal funding on antibiotic resistance.

Related, Current and Previous Work

Emergence, persistence, accumulation and propagation of antibiotic resistance (AMR) is occurring at an alarming rate in animal and human populations1-4.  The Centers for Disease Control and prevention (CDC), the World Health Organization, and President’s Council of Advisors on Science and Technology (PCAST) have identified AMR as one of the greatest threats to human health worldwide, and it is a threat to economic growth, public health, agriculture, economic security, and national security5.  The CDC estimates that more than 23,000 Americans die annually because of infections caused by antimicrobial resistant bacteria1. Furthermore, AMR also threatens many modern medical procedures like cancer chemotherapy, complex surgeries, dialysis for renal disease and organ transplantation5.  The annual domestic impact of antibiotic-resistant infections to the U.S. economy is estimated to be to be in excess of $20-30 billion with an additional $35 billion due to direct health care costs and lost productivity, respectively6,7.  Prophylactic and metaphylatic use of antibiotics in livestock are major concerns considering their possible impact on selection for antibiotic resistance8. Excessive use of antimicrobials stresses the naturally occurring microbiome and allows for resistant bacteria to become dominant9. Therefore, unrestricted use of antibiotics in the livestock industry is likely contributing to the increase of resistant bacteria and emergence of antibiotic resistant strains. Such selection has a direct impact on human health as many of the antibiotics used in animal agriculture are also prescribed for the treatment of diseases in humans (e.g. tetracycline, penicillin, sulfonamides, and 3rd generation cephalosporins)10. Indeed, research has suggested that AMR might spread to humans11 through food products of animal origin12, the environment13, and by direct contact in the case of agricultural workers14. Therefore, the development and evaluation of strategies that can reduce the prophylactic and metaphylatic antimicrobial use in agriculture animals can be pivotal to improving antimicrobial stewardship and containing the AMR threat to global health.

In the fall of 2015, the White House published its “Action Plan for Combatting Antibiotic Resistant Bacteria”5.  The major goals of the action plan are to 1) Slow the Emergence of Resistant Bacteria and Prevent the Spread of Resistant Infections.  2) Strengthen National One-Health Surveillance Efforts to Combat Resistance. 3) Advance Development and Use of Rapid and Innovative Diagnostic Tests for Identification and Characterization of Resistant Bacteria., 4) Accelerate Basic and Applied Research and Development for New Antibiotics, Other Therapeutics, and Vaccines, and 5) Improve International Collaboration and Capacities for Antibiotic-resistance Prevention, Surveillance, Control, and Antibiotic Research and Development.  Participants on this multi-state proposal have the extensive research, education, extension and outreach experiences needed to thoroughly address the major themes presented in the National Plan to Combat Antibiotic-Resistant Bacteria.

Current and previous work across participating organizations on surveillance and monitoring of antibiotic resistance has centered on phenotypic surveys of Campylobacter spp., Listeria monocytogenes, Avian Pathogenic E. coli (APEC), Salmonella, Staphylococcus aureus, and Clostridium perfringens in various livestock species. At NCSU, PCR is routinely used to detect specific genes coding for resistance to tetracycline, erythromycin and gentamicin.  Since 2014, phenotypic surveillance has revealed a trend towards occasional turkey flocks colonized with erythromycin-resistant Campylobacter jejuni, a phenotype that had not been encountered among C. jejuni from turkeys in North Carolina over the previous 14 years. Any identified erythromycin-resistant C. jejuni are analyzed by PCR and subsequent sequencing of a fragment of the 23S rRNA gene to determine whether they harbor the A2075G or other mutations previously found to be associated with macrolide resistance in Campylobacter. To date all screened macrolide-resistance C. jejuni isolates harbor the A2075G mutation.  Investigators at Iowa State University (ISU) are involved in a range of projects pertaining to AMR including studies of antimicrobial resistance associated with poultry production.  Specifically those studies focus on APEC and commensals bacteria from poultry; E. coli, Campylobacter, and Salmonella. In addition, collaborators at ISU investigate AMR associated resistance in S. aureus and MRSA of swine while exploring the emergence of novel resistance genes linked with methicillin resistance, and the resistances associated with Campylobacter15.

Investigators at South Dakota State University (SDSU) are using whole genome sequencing based surveillance of antibiotic resistance genes in food animal pathogens. Next generation sequencing (NGS) based whole genome sequencing (WGS) currently has become an affordable and much more accurate method for antibiotic resistance gene detection 16. NGS is being used on pathogens such as Salmonella enterica and E. coli.

Investigators at Cornell University (CU) are investigating antimicrobial resistant organisms in goose production.  Salmonella enterica serovar Typhimurium strains of phage types DT104 and U302 are often resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (the ACSSuT resistance type) and are major zoonotic pathogens. Increased consumption of goose meat may enhance the risk of transferring S. enterica serovar to humans. The CU group is characterizing S. enterica serovar Typhimurium strains isolated from four goose farms and one hatchery farm to determine the epidemic and genetic differences among them. Antibiotic susceptibility tests and multiplex PCR are used to detect ACSSuT strains.  CU is also investigating the transmission of antimicrobial resistance genes from resistant bacteria to non-resistant strains, including multiple resistance gene transfer between Escherichia coli and Salmonella in the natural setting. Microarray analysis revealed that E. coli isolated from different farms were closer than the profile of E. coli and Salmonella isolated from the same farm. Evidence suggests that the transfer of multiple resistance genes between these species is unlikely.

Clostridium spp. has rapidly reemerged as a human and animal pathogen. The detection and identification of pathogenic Clostridium spp. is therefore critical for clinical diagnosis and antimicrobial therapy. Traditional diagnostic techniques for clostridia are laborious, are time consuming, and may adversely affect the therapeutic outcome. The CU group is investigating an oligonucleotide diagnostic microarray for detection of pathogenic Clostridium spp. This approach demonstrates a high-throughput detection and identification of Clostridium spp, and it provides several advantages over traditional methods and proved a practical method to determine patterns of virulence and resistance genes.

Current and previous work regarding the ecology and transmission of resistant bacteria and antimicrobial resistance determinants of collaborators at cooperative institutions has investigated the ecology, transmission, and mechanisms of antimicrobial resistance (AMR) in multiple bacterial species in multiple food animal species. In respect to Campylobacter, efforts at NDSU and ISU have been directed at identifying chromosome-encoded and plasmid carried mechanisms mediating resistance to fluoroquinolones, macrolides, tetracyclines, and aminoglycosides17-23; evaluating the impact of antibiotic resistance on Campylobacter fitness and pathogenesis24,25; and monitoring the emergence of new AMR determinants, such as erm(B), cfr(C) and ‘super” efflux pumps22,26. The Campylobacter work has covered multiple animal species including chicken, turkey, sheep, cattle, swine, and wildlife. For Listeria, NDSU is analyzing its resistance to quaternary ammonium disinfectants, heavy metals (cadmium, arsenic) and antibiotics27. For E. coli, researchers at ISU has been focusing on AMR in avian pathogenic E. coli (APEC) and the influence of antibiotics on the selection of these organisms. Additionally, work is ongoing at ISU to assess the presence of the mcr-1 gene in isolates of APEC and avian E. coli from around the world and understand the global distribution of mcr-1 in collaboration with investigators in South America. For MRSA, collaborators at ISU are studying its prevalence and ecology in swine production. Furthermore, collaborators at ISU are conducting PK/PD analysis of antibiotics in sheep as it relates to sheep abortions and the urinary tract and IV sepsis model work in sheep. Work at Illinois focuses on AMR ecology in animal manure and previous studies showed that the predominant culturable microorganisms from stored swine manure were obligate anaerobic, low mol% G + C Gram-positive bacteria (Firmicutes) comprised of members of Clostridial, Eubacterial, and Lactobacillus/Streptococcus phylogenetic groups28.

Plasmids are a major driving force in the dissemination of antibiotic resistance in Enterobacteriaceae. University of Minnesota (UMN) has focused on understanding the core genome structures of these plasmids, and their basic biological mechanisms enabling success. One such example is the IncA/C plasmid type, which has emerged as a major plasmid type in the spread of MDR Enterobacteriaceae among humans and animals. Researchers at UMN examined multiple IncA/C plasmid sequences from production animals to define their core and accessory regions29. Several studies at UMN also characterized the fitness costs30 and regulon of IncA/C plasmids31,32. Collaborators at UMN has also studied the plasmids of the emergent Extraintestinal Pathogenic E. coli sequence type ST13133,34, a MDR pathogen of global concern. 

Several non-antibiotic interventions to reduce antimicrobial resistance in food production systems are being developed by investigators at the participating institutions. A potential source of non-antibiotics is phytochemicals35. At the University of Illinois (UI), research is directed toward determining the cellular, biochemical and molecular mechanisms underlying the antibacterial effect of phytochemicals. Use of phytochemicals to control multidrug resistant Salmonella Enteritidis in chicken is one of the interventions tested by groups at UMN36. It was shown, that enhancing host gut health, by determining how dietary composition alters mucosal immunity, consequently reducing pathogen colonization37. Identifying beneficial species from healthy gut microbiota that produce beneficial metabolites or enzymes is another potential intervention that has been attempted by the UI group38. A healthy gut microbiota is composed of several species that might suppress the colonization of animal gut by pathogens such as drug resistant Escherichia coli, Salmonella enterica and pathogenic Clostridia. Researchers at SDSU are developing gut microbiota strain libraries using the recently developed culturomics method39. Collaborators at SDSU will continue these studies to identify strains that could inhibit Salmonella and Clostridium difficile in co-cultures. Once characterized, such isolates could be formulated as competitive exclusion products to suppress drug resistant bacterial colonization of swine and poultry40. Several probiotic strains are known to suppress the growth of antibiotic resistant pathogens or modulate their virulence. The NCSU group has shown that probiotic supplementation of swine and poultry improves the host health41.

Studies conducted by ISU have shown that Peptide nucleic acids (PNAs) sensitize Campylobacter jejuni to antibiotics42. Therefore, PNAs in combination with other interventions have the potenial to control C. jejuni infections. Recombinant subunit vaccines have been used by the group at CU to reduce Leptospira infection43.

Work at MSU is investigating the use of pan-susceptible and non-virulent E. coli as competitive exclusion cultures given immediately after birth in dairy calves, and its effect on reducing colonization with resistant coliform bacteria which reaches concentration of up to 109 within 48 hours postpartum. 

Investigators at University of Illinois (UI) have shown that immunization of dairy cows using inactivated bacterial components prevents puerperal metritis. These results shows that vaccinating animals effectively controls bacterial infections and therby indirectly reducing the need for antibiotic treatment.

Because of the established links between antibiotic use and selection of plasmids described earlier, alternative approaches have been sought as a means to reduce or eliminate the use of antibiotics in poultry. One approach at UMN has involved first defining the baseline microbiome of commercial turkeys44, then understanding how antibiotics such as penicillin, virginiamycin, tylosin, and monensin modulate the turkey microbiome45 and/or broiler microbiome46. These baseline studies pave the way for development and assessment of alternative products.  In a recent study at MSU, probiotics were investigated as a means to reduce AMR in pre-weaned calves. However, the probiotic significantly increased cephalosporin resistance in coliform bacteria as compared to calves not fed the probiotic47. This study stresses the need to determine the impacts on AMR for any intervention to reduce AMR and for investigation of alternatives to antibiotics.

In summary, use of probiotics, prebiotics, competetive exclusion cultures, phytochemicals, PNAs, and vaccines are some of the potenital non-antibiotic interventions that this multistate project will develop and evaluate.

Measures to reduce the risks associated with antibiotic use in livestock include the identification of critical points of control, the quantification of animal health in food production systems, the implementation of technological solutions that can prevent contamination with antimicrobial resistant bacteria and genes, and the development of reliable surveillance and risk assessment procedures. Without a better understanding of the complex epidemiology of resistant bacteria and genes, as well as adequate risk-benefit assessments of a variety of antimicrobial intervention scenarios, the effectiveness of antimicrobial use policies could be weakened in reducing the risks posed by antimicrobial resistance that occur in the intermingling interface between humans, animals and the environment48. Research addressing those measures will inform sound decision making on antimicrobial use policies in food animals based on a careful weighing between the expected beneficial and potential detrimental impacts on animal, human health, environmental sustainability and associated social-economic effects.

NCSU groups are investigating whether Campylobacter strains with certain resistance profiles differ in their virulence in animal models and also whether they differ in their capacity to survive on poultry products and in water. The latter attributes would be important in determining risk for transmission to humans. They are also assessing whether Campylobacter strains with certain antimicrobial profiles have greater propensity to be encountered in houseflies from commercial turkey houses. Current data suggest that strains with certain antimicrobial susceptibility profiles indeed have greater virulence than genotypically similar strains (same sequence type based on Multiple Locus Sequence Typing) with different antimicrobial susceptibility profiles. Data also suggest that houseflies are an exquisite sampling device for diverse strains of Campylobacter, including those with multidrug resistance.  

The NCSU groups are also exploring approaches to quantify animal health. Inflammatory response status and redox status have been measured in the intestine of newly weaned pigs with various dietary and environmental challenges. It seems that inflammatory response status and redox status in the intestine are closely related to growth performance of pigs after weaning. It is hypothesized that inflammatory response status and redox status of the intestinal mucosa can be used to quantify intestinal health of newly weaned pigs related to their growth performance. 

The UMN groups are investigating the potential of alternatives to antimicrobial interventions (probiotics, prebiotics, phytobiotics and other emerging agents) to reduce multidrug-resistant pathogen colonization in poultry and mitigate antimicrobial resistance development in MDR zoonotic pathogens. Efficacy of a few Generally Recognized As Safe (GRAS) status probiotic bacteria and phytobiotics against major multidrug-resistant Salmonella serotypes have been evaluated. Findings suggest that the tested probiotic organisms and phytobiotics were able to reduce/inactivate multidrug-resistant Salmonella in in vitro models. The in vitro findings were validated by using challenge models in turkeys and broilers, showing that the selected probiotic bacteria were able to reduce multidrug-resistant pathogens. Similarly, results indicate the potential of phytobiotics to mitigate the development of antibiotic resistance (effects on plasmid- or chromosomally-encoded MDR determinants) in major foodborne pathogens.

The University of Nebraska, Lincoln (UNL) groups developed a relative exposure assessment model to address the relative contributions of environmental pathways compared to the food consumption on the human exposures to a variety of antimicrobial resistance pathogens associated with the antimicrobial use in cattle primary production. The major outputs from this project were the identification of data gaps that provide the research directions for other collaborators to further collect data to reduce the model output uncertainty.  The development of a risk assessment framework to assess the risk of human exposure to antimicrobial resistance bacteria and/or genes by incorporating environmental transmission pathways will be improved through an iterative process.

The CU groups are investigating the mechanistic pathways by which antibiotic contamination of freshwater ecosystems may enrich resistant bacteria and impact on pathogen transmission among farmed fish via modulation of their microbiome and other mechanisms; and assess the likelihood of each pathway, severity of the consequences and associated uncertainty.

Implementation of new mitigation strategies is not trivial and producer attitudes and perceived risk and barriers may prevent the adoption of new strategies, hence the MSU group will work to classify attitudes and risk perceptions about AMR mitigation, and develop decision-support communication activities for livestock producers.

The UI group is investigating whether selective dry cow therapy can be used strategically to reduce antimicrobial use and what is its impact on the dissemination of AMR. A series of studies from Europe and Canada have suggested that combined use of teat sealant and on-farm culture might allow targeting the use of antimicrobial on selective dry cow therapy only for the high-risk group of cows. However, the impact of these strategies might vary considerably from different regions, and its impact on the milk microbiome has not been evaluated yet.

Purdue University is investigating alternatives to feed antibiotics, including the potential for bacteriophage additives or treatments that include a cocktail of phage types to control pathogenic bacteria including Salmonella and E. coli O157:H7 in both live animals and food matrices49,50.

Ongoing research, at Purdue University, is directly tied to strong Extension programming to facilitate the rapid transfer of the findings into educational programming and application. Purdue University has launched an online course, Diversity in Veterinary Medicine, to students and faculty at veterinary colleges across the United States as well as to veterinarians in private practice. While the topic is different, the foundation of this online course could be used to create courses in which to educate and certify people who work in the food production industry on the important topic of AMR, combining data from studies such as the one on selective dry cow therapy currently in progress at the University of Illinois and antimicrobial alternatives research at Purdue.

Collaborators at UC Davis Vet Med Extension is evaluating how antibiotics are used in California dairies. On most large California dairies, hired labor of Hispanic ethnicity is ultimately responsible for sick cow identification, disease diagnosis, and treatment administration. Bi-lingual researchers have visited 45 dairies throughout the state of California to meet with dairy employees while treating cows during postpartum cow checks and during milking of cows with mastitis. Information on signs of health disorders leading to diagnosis and treatment have been recently summarized51. The type of drugs, the dose and the treatment duration of postpartum cows and mastitic cows is currently being summarized.


Proposed multi-state activities related to other projects in NIMSS and CRIS bases

To identify related and potentially duplicative research, the NIMSS ( ) and CRIS ( ) databases were searched with the keywords: ‘antimicrobial’ and ‘resistance’.   

The NIMSS database produced 119 projects in which one or both of these keywords were present.  The majority of these projects were related to plant and crop breeding and production. Fewer project were related to disease resistance in animals. The following projects in NIMSS also address antimicrobial resistance in bacteria of food animal origin:

NE1048: Mastitis Resistance to Enhance Dairy Food Safety

            This project focuses on development and surveillance of antimicrobial resistance in mastitis pathogens, antimicrobial resistance and it’s impact on treatment success, and alternatives to current mastitis therapeutics.

NC1202: Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety

            The long term goal of this project is to develop strategies to prevent and control enteric diseases of cattle, swine, and poultry, ultimately to decrease the incidence of enteric.  The project also addresses antimicrobial resistance, however, we did not find direct duplication with research proposed in NC_temp 1206.

NC1192: An integrated approach to control of bovine respiratory diseases (NC-1027)

            This project focuses on respiratory pathogens.  More notably it focuses on resistance in respiratory pathogens, to develop alternatives to antimicrobials for BRD, and evaluating the efficacy of antimicrobials in treatment of BRD.

S1056: Enhancing Microbial Food Safety by Risk Analysis

            The goal of this project is the establishment of a multi-disciplinary network of scientists that perform comprehensive and integrated risk-based research and outreach to improve the safety of food from farm to fork.  Specifically, the project aims to understand the prevalence and frequencies of pathogens and antimicrobial resistance within the environment, food products and food production processing, distributions and consumer systems.

Forty projects were identified in the CRIS system.  Of these, approximately 24 specifically mention antimicrobial/antibiotic or antimicrobial/antibiotic resistance in the title.  Six projects are pilot testing for NARMS sampling and there was one conference grant.  There does not appear to be substantial overlap between these projects and NC_temp 1206. Further, our project in unique it terms of it’s comparative and comprehensive focus encompassing multiple pathogens linked to AMR, multiple animal species susceptible to AMR microorganisms and its integrative approach to the problem with efforts focused on interconnected  foundational objectives outlined in Figure 1.


  1. Enhance surveillance and monitoring of antibiotic resistance and develop improved diagnostic tests.
  2. Determine the ecology and mechanisms involved in resistance and transmission of resistance.
  3. Develop and evaluate interventions (including alternatives to antibiotics) that reduce antimicrobial resistance in food production systems.
  4. Quantify animal health, public health, social, economic, and environmental impacts of antimicrobial interventions in food production systems.
  5. Create and deliver programs on antibiotic stewardship in food production systems through education and outreach.


Objective 1:  The goal of this objective is to continue and enhance our current surveillance methods for detection of antimicrobial resistance in a range of pathogens and commensals associated with food animals. Researchers focus on methods to enhance detection of resistance as well as design of new methods to detect new resistance traits identified through phenotypical and genotypic analysis and to address the use of newer technologies (e.g. MALDI-TOF) that can be investigated and implemented to identify new and novel resistance traits. 

Investigators at NCSU will use phenotypic assessments to survey Campylobacter spp. and Listeria monocytogenes for antimicrobial resistance (AMR) profiles. For specific resistance determinants, PCR will be employed to detect the corresponding genes. Such applications are routinely done for resistance to tetracycline, erythromycin and gentamicin. For gentamicin resistance, PCR will be employed to survey for presence of aph(2”)-If or aph(2”)-Ig. In addition, WGS18 will be used to examine selected strains of Campylobacter and Listeria. Any identified erythromycin-resistant C. jejuni will be analyzed by PCR and sequencing of a fragment of the 23S rRNA gene to determine whether they harbor the A2075G or other mutations earlier found to be associated with macrolide resistance in Campylobacter.

Investigators at ISU will study the antimicrobial resistance in pathogens of poultry (APEC), and those of commensals associated with poultry including avian fecal E. coli, Campylobacter and Salmonella. A new project will focus on understanding the role of the poultry environment in the selection of pathogens, and they will be assessing the entire microbiome as well as the genomic traits (resistance genes) of the environment using a metagenomics based approach. They will use agar, broth and disc assays to phenotypically screen for resistance as well as novel designed multiplex PCR panels to rapidly screen for a range of resistance associated genes for use in standard PCR and real time assays52.  Additionally, collaborators at ISU will investigate AMR in S. aureus and MRSA of swine and explore the emergence of novel resistance genes linked with methicillin resistance, and the resistances associated with Campylobacter15. Other researchers at ISU will work on a method to enrich metagenomic samples for targets of interest, in this case the targets would be AMR genes. These will then be combined with long read sequencing and will be used to look at horizontal gene transfer (HGT) events in the resistome between different species.

Investigators at SDSU are using whole genome sequencing based surveillance of antibiotic resistance genes in food animal pathogen. Next generation sequencing based WGS currently has become an affordable and much more accurate method for antibiotic resistance gene detection53. We will use NGS based WGS of pathogens such as Salmonella enterica, and E. coli for the surveillance of antibiotic resistance gene prevalence and transmission in agricultural food systems. Custom bioinformatics pipelines will be developed for extracting the resistance gene sequences from whole genome data. This approach can capture more than 3000 resistance genes based on comparisons against AMR gene databases54,55.

Collaborating faculty on this objective will share samples, bacterial isolates and microbial laboratory techniques. Additionally, faculty from various universities working on this objective will provide isolates from their states to the Scaria lab for genome sequencing and analysis. This will enable antibiotic resistance gene surveillance from most regions of the United States.

To develop NGS based surveillance methods, whole genomes of S. enterica and E. coli isolated from animal or food sources will be sequenced. Sequencing reactions will be carried out using Illumina 2x250 cycles paired end sequencing chemistry56. Mapping of the resistance genes from each sample will be carried out using a custom bioinformatics pipeline.


Objective 2:  A functional metagenomic approach will be employed to identify antimicrobial resistance genes (ARGs) in reservoirs relevant to the turkey production ecosystem. Samples will be from three types of commercial turkey farms: 1) confined with antibiotic use (conventional, CONV); 2) confined and antibiotic-free (C-ABF); 3) free-range and antibiotic-free (FR-ABF). History and current use data for antibiotics, copper sulfate and anti-coccidials will be obtained from the company, and the same number of farms of each type will participate. For CONV and C-ABF comparisons, host genetics and environmental issues will be pre-empted by use of birds of the same commercial breed, and under control of the same company in the same region. Samples will include litter collected during the downtime between flocks to maximize our ability to assess litter’s potential as an ARG reservoir; house flies and darkling beetles will be collected at the same time as the litter samples. Flies will be collected and darkling beetles will be manually retrieved from the litter. To assess resilience of ARG types and diversity in the poultry GI tract, cecal samples will be obtained twice from the same flock, once at week 1-2 and once at the processing plant. A composite sample of 10 ceca will be used for DNA extractions. Total genomic DNA will be extracted using a FastDNA® SPIN kit. DNA will be sheared to a) 20 kb and b) 2-3 kb fragments, cloned into the expression vector pZE21-MCS, and transformed into E. coli electrocompetent cells in order to construct a library for each sample. The goal is to generate a total size of ~ 109 bp per library. To select antibiotic-resistant clones, each library will be spread-plated on Luria broth (LB) agar containing various antibiotics, incl. tetracycline, chloramphenicol, spectinomycin, ciprofloxacin, kanamycin and gentamicin. Up to 5 colonies from each plate will be randomly selected, re-grown in LB with the corresponding antibiotic and stored at -80oC. Inserts conferring AMR will be sequenced using primers pZE-F and pZE-R (196) by conventional PCR (small fragments) and by Illumina sequencing (large fragments). The sequences will be edited, annotated, and analyzed as described previously. Special attention will be paid to signatures for possible mobile elements (integrases and insertion sequences, known for capacity to assemble resistance islands, phage, plasmids) in the vicinity of the ARGs. Sequences flanking the ARGs will be analyzed for tentative identification of the microorganism harboring the ARGs. Shannon’s index of Diversity will be used to compare ARG diversity from the different farm types and samples. BLASTn will be used to in-silico interrogate the Campylobacter WGS databases for presence of the identified ARGs in campylobacters from diverse sources.

Both conventional and advanced methods will be used to study ecology and mechanisms of AMR. These include, but are not limited to, antimicrobial susceptibility tests, molecular typing (such as PFGE and MLST analysis), PCR amplification, gene transformation and cloning, genomics, transcriptomics, and whole genome sequence analysis20,22.  Animal models will be used to assess the development, persistence, and fitness of AMR bacteria; evaluate the pathogen response, transcriptome and resistome in the face of PK/PD data analysis; and determine the role of dose, dosing interval, and concurrent disease in impacting the rate of resistance development and expression. Additionally, new tools will be developed for monitoring the resistome that allow for more in depth evaluation of horizontal gene transfer (HGT) and drivers of AMR HGT in complex microbial communities.

Investigators at CU will investigate antimicrobial resistance in goose production, specifically focusing on Salmonella enterica serovar Typhimurium strains of phage types DT104 and U302 with the ACSSuT resistance type. S. enterica serovar Typhimurium strains isolated from four goose farms and one hatchery farm  will be investigated to determine the epidemic and genetic differences among them. Antibiotic susceptibility tests and multiplex PCR will be used to detect ACSSuT strains isolated from these farms. Work will focus on specific farms where the organism is prevalent and a hatchery supplying the farms as potential sources of the resistant organism. A second project will focus on Clostridium spp. that have rapidly reemerged as human and animal pathogens. The detection and identification of pathogenic Clostridium spp. is therefore critical for clinical diagnosis and antimicrobial therapy. The CU group will use an oligonucleotide diagnostic microarray for detection of pathogenic Clostridium spp. In addition, the pattern of virulence and antibiotic resistance genes of tested strains will be determined through the microarrays. Another project will investigate the transmission of antimicrobial resistance from resistant bacteria to non-resistant strains. In this study, the possibility of multiple resistance gene transfer among E. coli and Salmonella in the natural setting will be investigated.

Plasmids are a major driving force in the dissemination of antibiotic resistance in Enterobacteriaceae. At UMN, the plasmid structure in relation to MDR phenotypes in Escherichia coli of poultry in Minnesota will be assessed.  Researchers at UMN will determine the propensity of basic plasmid types to acquire, retain, and disseminate MDR phenotypes.

Collaborating faculty on objective 2 use similar approaches to determine antimicrobial ecology based on applied intervention and experimental studies as well as observational studies.  Faculty on this objective will work closely to share and integrate samples and methodologies.  Some results from these studies will be directly translatable for producers and veterinarians after development by our extension and education teams.

Objective 3:  Investigators from UI and NCSU will test several phytochemicals for antimicrobial activity. Phytochemicals showing positive activity will be further investigated to determine the cellular, biochemical and molecular mechanisms underlying the inhibitory effect. CU investigators will attempt to develop recombinant subunit vaccines to control Salmonella Dublin and Leptospira sp. Researchers at ISU will explore the development of peptide nucleic acids as treatment for infections caused by Campylobacter jejuni. Researchers at UMN will investigate the use of probiotics and prebiotics to modulate mucosal immunity with the goal of improving overall gut health. Culturomics39 methods will be used at SDSU to identify beneficial bacteria from healthy animals. Researchers at SDSU have setup facilities for high-throughput screening of gut bacteria. Culturomics will be directed towards isolating bacteria from healthy microbiota that provide colonization resistance to pathogens. Main criteria for selecting such species will be the production of beneficial metabolites such as butyrate and propionate. Strains will be isolated using strict anaerobic culture methods and will be identified using MALDI-TOF. Inhibition of these strains against drug resistant bacteria will be then identified using co-culture assays.

Investigators at CU have found a potentially important innate immunity protein(s)/peptide(s) that stems from a plant. This plant protein/peptide shows strong bactericidal activities to Salmonella and E. coli, and its activity is optimized in acidic pH, the condition that intracellular pathogenic bacteria such as Salmonella and E. coli live within human and other animal hosts. We aim to identify and characterize the nature of this bactericidal activity, by identifying the protein/peptide behind the bactericidal activity via size-exclusion chromatography and Mass Spectrometry, followed by defining their mechanisms of action via multidisciplinary approaches involving biochemical and cell biological analysis. We anticipate that this plant protein/peptide is new from any known molecules of antibacterial activities, because of its unique adaptation to acidic pH.  This implies that this molecule could be extremely useful to treat antimicrobial-resistant intracellular pathogens that live in the acidic pH environment in humans and animals.

At UMN, investigators will assess turkey-specific and broiler-specific probiotic blends for their ability to enhance performance and modulate the microbiome of the bird. 

MSU will determine the impact of competitive exclusion cultures of pan-susceptible non-pathogenic E. coli on reduction of colonization of newborn calves with resistant coliforms. 

Overall, this objective aims at determining alternatives therapies to current antimicrobial therapies and to reduce the use of current antimicrobials by improving animals’ immunity and health.  Faculty working on this objective will work closely to share ideas and methods for determining the most effective study designs to answer the research questions and work with faculty participating on other objectives sharing study designs and microbiological methods.

Objective 4:  Researchers at NSCU will investigate the persistence and/or virulence of animal-originated pathogens/commensal bacteria on food products and in a variety of environmental niches through molecular genetics techniques, such as DNA fingerprinting approaches. NSCU collaborators will also focus on studies to quantify animal health in response to removal of antimicrobial growth promoters in animal feed. In pig production, weaning is done at an early age of pigs’ life (between 2 and 4 weeks of age) and newly weaned piglets often encounter intestinal stress including inflammatory response, oxidative stress, pathogenic infection, and physical damages causing diarrhea followed by dehydration, weight loss, and even death. Quantification of intestinal health in newly weaned pigs will be the target measurement when antimicrobial growth promoters are removed from nursery diets.

UMN researchers will investigate the potential alternatives to antimicrobial interventions, including probiotics, prebiotics, phytobiotics and other emerging agents, to evaluate their efficacy in reducing the development of multidrug-resistant zoonotic pathogens. Efficacies will be evaluated through in vitro models using co-culture, cecal multiplication, motility-, adhesion- and invasion assays and validated in in vivo models, i.e., challenge trials in food animals. UNL collaborators will conduct field trials to investigate the development, persistence and transfer of antimicrobial resistant genes in several chained environmental niches due to the land application of cattle manure collected from beef cattle raised in the feedlot with and without antimicrobial uses. The temporal and spatial changes in resistome will be investigated through metagenomics analysis. Cultural-based approaches will also be used to investigate e changes in prevalence and concentration in resistant bacteria in environmental samples, which will then be used to incorporate into and improve the established relative exposure assessment model.

CU researchers will conduct a qualitative risk assessment. The hazards will be defined as 1) an increase in antibiotic resistance genes in freshwater ecosystems above the baseline (defined as the expected level with negligible antimicrobial pollution); and 2) an increase in the transmission of fish pathogens above baseline. Specific resistance genes and pathogens will be prioritized for study according to likelihood of occurrence and severity of consequences, as part of the risk assessment process.

Faculty participating in this objective will collaborate close with faculty on other objectives in sharing effective study designs and microbiological methods. 


Objective 5:  University of Illinois researchers will investigate the effect of selective dry cow therapy in US farms and its effects on milk microbiome and milk resistome to determine downstream impacts on dissemination of AMR. The study will compare decision-making strategies based on on-farm culture, somatic count cells, and history to determine which approach translates into a better outcome for milk quality and containment of mastitis. Furthermore, an economic analysis will be conducted to evaluate the feasibility of implementing these strategies for the food supply chain. Finally, an evaluation of the impact of these strategies on AMR based on the characterization of milk microbiome and resistome will be performed.

Online courses will be developed at Purdue University (PU) to communicate findings from AMR studies to people working in food production systems. A current online course at PU will be used as the foundation for formatting and logistics. The course will be divided into modules based on production type. For example, one module will focus on the milk industry and include the findings from the current study on selective dry cow thereapy at the University of Illinois.

PU Extension will expand upon the Purdue Food Animal Education Network program, which uses web-based media and experiential learning to provide research-based information to consumers who have little to no connection to livestock production. Information regarding AMR will be a key element to this informational resource. To date, the anchor website ( has over 22 informational pages and the site has been visited over 200,000 times with over 75,000 unique visitors. Educational videos are hosted on its corresponding YouTube channel (viewed over 6,000 times). Much of the information is a direct extension of Purdue Extension faculty research with topics including the role of antibiotics in livestock production, foodborne illness trends, emerging diseases, and environmental impact of livestock production.

Modules for veterinary students regarding prudent use of antibiotics in food animals will be developed for the underclassman and 4th year levels.

Based on the knowledge gained on how antibiotics are currently being used on large CA dairies, UCD researchers will develop specific training materials and educational strategies to promote judicious use of antibiotics on dairies. Our target clientele will be individuals treating cows, mostly of Hispanic ethnicity, with little to no formal training on animal husbandry.

Agricultural and risk managers need improved ways to communicate with the public about AMR risks to a) promote attitude and behavior change (i.e., the adoption of AMR reduction methods or support for specific AMR policies) and b) facilitate decision-making. To effectively communicate, managers need information about how stakeholders search for and engage with information about AMR risks and how this information, once received and interpreted, influences risk-related attitudes and behaviors. Such information is currently absent, and the MSU group will work toward elucidating gaps in this knowledge.

We anticipate the results from applied studies from objectives 1, 2, 3, 4 and 5 easily can be prepared for sharing through Purdue and UC Davis existing education and extension pipelines.  Additionally, information gained in extension and education efforts will inform the other objectives of new needs.

Measurement of Progress and Results


  • A comprehensive catalogue of antibiotic resistance genes and resistance-gene variants in pathogens of importance in agricultural productions systems. Report on progress and collaborations. Comments: ISU, NCSU, SDSU, and CU will report research findings on a yearly basis through the NCDC 230 program lifetime.
  • Evidence on how treatment with antimicrobial drugs impacts the host/pathogen interface and how it affects the resistome and selection and dissemination of AMR. Report on progress and collaborations. Comments: NCSU, UMN, SDSU, CU, and IU will report research findings on a yearly basis through the NCDC 230 program lifetime.
  • Direct fed microbials, dietary formulations, phytochemicals, new vaccines and novel therapeutic agents to improve gut health and reduce pathogen colonization and antimicrobial resistance. Report on progress and collaborations. Comments: UCD, NCSU, UI, UMN, SDSU, ISU, MSU, and CU will report research findings on a yearly basis through the NCDC 230 program lifetime.
  • Biological indicators, assays and models to evaluate the impact of different antimicrobial use scenarios in food-producing animals on animal, human health, environmental protection and related social-economic effects. Report on progress and collaborations. Comments: NCSU, UMN, UNL, and CU will report research findings on a yearly basis through the NCDC 230 program lifetime.
  • Population studies evaluating the impact of different antimicrobial use strategies in food-producing animals and their results on AMR dissemination through the food supply chain. Report on progress and collaborations. Comments: UI, PU, UCD, ISU and MSU will report research findings on a yearly basis through the NCDC 230 program lifetime.

Outcomes or Projected Impacts

  • Improved tracking and identification of current and newly identified resistance genes and variants will result in better antibiotic stewardship methods.
  • Development of a new platform for studying the resistome in animals will facilitate monitoring of AMR, the development of practical means to reduce AMR and transmission between livestock, the environment and humans.
  • Novel non-antibiotics developed through our collaborative efforts will lower the amount of antibiotics used in food productions systems and hence reduce the development of drug resistance in enteric bacteria.
  • Provide scientific evidence to food animal producers, extension educators and policy makers to maximize the judicious use of antimicrobials in food-producing animals by weighing benefits and risks of their uses in animal, human health and environmental sustainability.
  • Deliver scientifically sound evidence to all parties of the food supply chain to maximize the judicious use of antimicrobial drugs and reduce AMR in food-producing animals.


(2022):The agricultural systems in which antimicrobial resistance emerge, evolve, accumulate and disseminate are exceedingly complex. Hence, these problems dictate a trans-institutional and interconnected trans-disciplinary approach creating collaborations between people with different and overlapping areas of expertise. We have brought together a group of investigators under the umbrella of antimicrobial resistance and with noticeably diverse areas of expertise ranging from molecular biology/microbiology, modeling, epidemiology, statistics, infectious diseases, antimicrobial resistance mitigation, applied research and education and extension/outreach. Although some participants in this project have collaborated previously on research, extension and education projects, prior history of significant interaction and collaboration amongst members of the group is limited. The research activity described under above described objectives represents a foundational starting point and extensive inter, and intra-disciplinary collaboration amongst and between objectives will evolve as the multistate effort matures. Hence, the specific experiments, interventions, surveillance, and outreach efforts as described in this proposal are the foundational basis for further collaborations and identification of new needs and efforts. We will also design the agenda of group meetings to help further promote integrative and new collaborations within and across objectives of the project. We believe that the group meeting will evolve the project and objectives naturally, and furthermore foster new and innovative collaborations within current and future new institutions not yet included in this project. Specifically, the first meeting of our group will focus on synergizing and completely integrating all objectives and sub-objectives (Figure 1. ) Another goal of the first meeting will be to further outline protocols for delivery of findings to our audiences, as well as approaches and solutions for how to take advantage of the broad diversity of expertise, methods, materials and conditions within the project group. This will include sharing of most cost-effective and innovative project designs, many varieties of samples and bacterial isolates, as well as methods which can be used across laboratories and investigators. At the end of the first meeting we will have outlined specific standard operating procedures for these topics. To avoid that our meetings will focus exclusively on current efforts and achieved results and outcomes, presentation of data/new results will be limited to 5 minutes per station. A large amount of time at the first meeting and subsequent meetings will be used in breakout groups to further discuss and synergize projects and participants’ contributions. Focus groups, based on for example animal and/or microorganism of interest with overarching questions to be addressed, will present participants the opportunity to comprehensively brainstorm together and develop action items relative to the current project across objectives. Groups will then report ideas to the entire group which will help to identify new and emerging needs not currently identified and addressed and also potentially lead to new outputs and impacts. Progress in meeting our objectives will be measured on an annual basis in terms of current and new collaborations within and across objectives, work completed; publications (peer reviewed and otherwise); outputs in terms of students graduated; students mentored; development of outreach materials, presentations of research at professional and other meetings; and additional funding secured on the basis of the research and extension activities generated.

Projected Participation

View Appendix E: Participation

Outreach Plan

Outreach and education are arguably critical elements in efforts to control and to mitigate AMR on livestock premises, and they are advocated for by national and international organizations.  The stakeholders, in respect to AMR in livestock, are many and diverse with different perspectives and often conflicting agendas, therefore our basic message needs to be clear and consistent with room for customization to each unique audience.  This calls for a coordinated effort in creating communication and appropriate messages for all involved parties.  We will develop an outreach and extension program that will engage students, veterinarians, extension personnel, producers and their employees across outcomes form different objectives in the understanding of the importance of antibiotic management decisions necessary to implement antibiotic stewardship and AMR mitigation programs.  Our messages will be delivered to audiences through a previously established media platform containing informational pages and educational videos.  Educational courses and materials will be tailored to weigh benefits and risks of their applications in animal and human health and for environmental sustainability.  Additionally, findings from studies conducted as part of the NDCD230 antimicrobial resistance program will be presented at national scientific and producer organization meetings and workshops and through undergraduate and graduate-level courses at participating universities.  We will document all activities that the multistate NCDC230 Antimicrobial Resistance studies impact, and we will measure where capacity to improve antimicrobial stewardship and antimicrobial resistance control has been strengthened.


The recommended Standard Governance for multistate research activities include the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for at least two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a NIFA Representative.

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Land Grant Participating States/Institutions


Non Land Grant Participating States/Institutions

National Animal Disease Center, University of California, Davis
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