Source: UNIVERSITY OF NEBRASKA submitted to NRP
IDENTIFYING AMR GENE RESERVOIRS AND BACTERIAL HOST-AMR GENE ASSOCIATIONS TO IDENTIFY BACTERIAL HOST RANGE OF AMR GENES IN SWINE PRODUCTION SYSTEMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030648
Grant No.
2023-68015-40015
Cumulative Award Amt.
$999,981.00
Proposal No.
2022-08916
Multistate No.
(N/A)
Project Start Date
May 1, 2023
Project End Date
Mar 31, 2027
Grant Year
2023
Program Code
[A1366]- Mitigating Antimicrobial Resistance Across the Food Chain
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
(N/A)
Non Technical Summary
The rise in antibiotic resistance is an emerging global problem affecting human, animal, and environmental health. This increase may be due, in part, to the "industrialization of livestock production and the widespread use of non-therapeutic antimicrobial growth promotants" leading to decreased effectiveness of current antibiotics against human and animal diseases. Understanding the ecology of antibiotic resistance gene pools within livestock production systems and its effect on microbial ecology within host species is critical to better understand the emergence and spread of antibiotic resistance from a One Health perspective. Therefore, a critical gap exists in understanding the ecology, emergence, and transmission of antibiotic resistance in the pork industry, primarily due to the lack of investigations focusing on "plasmids or other integrative and cognitive elements", the hotspots for antimicrobial resistance (AMR) genes that lead to the emergence of AMR among microbial populations. This project, investigates the effects of antibiotic use on the ecology of the microbial community, AMR gene prevalence, enrichment, and transmission by evaluating the mobile genetic elements and the microbiome in the swine production system. Additionally, this study will develop new tools to identify host-plasmid associations and host range of plasmids to predict emerging AMR bacteria. Additionally, this study will evaluate an essential oil identified by this team for nursery pigs and develop science-based intervention strategies to reduce antibiotic use and mitigate emergence of AMR and effect change in management through outreach. This work is expected to translate to the development of management and nutritional strategies that reduce antimicrobial use and establish an outreach network that will improve the capacity nationwide to assess producer and consumer AMR-related food safety needs, and to develop, deliver and evaluate outreach programs that will help producers and consumers to assess and adopt practices that mitigate potential risks from food-borne AMR.
Animal Health Component
20%
Research Effort Categories
Basic
60%
Applied
20%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3073510104060%
3073510110020%
3073510110120%
Knowledge Area
307 - Animal Management Systems;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1040 - Molecular biology; 1101 - Virology; 1100 - Bacteriology;
Goals / Objectives
long-term goal of this project is to determine the ecology and transmission of AMR gene reservoirs in pigs, identify critical control points for AMR throughout the pork production system, and motivate adoption of science-based interventions among industry partners to mitigate emergence of antimicrobial drug resistance in pork production. Research Objective 1: Determine the effects of antimicrobial use on the ecology of microbial community and microbial resistance gene reservoirs in the porcine gut and in wastewater at different production stages using longitudinal sampling.Research Objective 2: Develop neural network models to predict the bacterial host range of raw plasmid metagenomic sequences in complex bacterial communities for rapid risk assessment of emerging multi drug resistant strains using Nanopore sequencing.Research Objective 3: Develop a novel science-based management and nutritional strategy to reduce metaphylactic antibiotic use by utilizing a novel essential oil as an alternative to conventional antibiotics, evaluating the changes in the microbiome due to essential oil feeding and evaluating its applicability as a nutritional strategy to reduce the use of antibiotics.Outreach Objective: Improve knowledge of AMR-related risks and inspire the adoption of practices among food producers and consumers to combat AMR-related health and food safety risks across the food chain using the established, trusted, interdisciplinary iAMResponsibleTM Project platform to disseminate research-based information and best-practice recommendations.
Project Methods
Research Objective 1- Determine the effects of antimicrobial use on the ecology of microbial community and microbial resistance gene reservoirs in the porcine gut and wastewater (to evaluate downstream effects) at different production stages using longitudinal sampling. Specifically, we will evaluate the changes in the microbial community composition and microbial resistance gene reservoirs in the gastrointestinal tract of antimicrobial treated (55 mg/Kg carbadox and 250 mg/Kg copper sulfate in (AB1) and chlortetracycline/tiamulin hydrogen formate (AB2) and untreated animals (CONTROL) at different stages of the production cycle (day 28, 56, 91, and 126 covering weaning, nursery, growing and finishing phase of the production cycle) and in wastewater by sequencing the virome, plasmidome, microbial community and associated microbial resistance genes. The study will be carried out at the pork production facility at U.S. Meat Animal Research Center (USMARC) using 12 pens with 8 head per pen for each rep resulting in a total of 192 pigs with 64 pigs per treatment.Research Objective 2 - Develop neural network models to predict the bacterial host range of raw plasmid metagenomic sequences in complex bacterial communities for rapid risk assessment of emerging multi-drug resistant strains using Nanopore sequencing.We will train a language model with the fastai deep learning library using dereplicated plasmid genomes (RefSeq) split into 5,000 base pair segments with a 100 base pair overlap. A subword tokenization algorithm will be used to tokenize the genomic segments. The validation set for the language model will be genomic segments from 10% of the dereplicated genomes, rather than 10% of all genomic segments, to avoid data leakage between the training and validation datasets. We will train a four-layer quasi-recurrent neural network (QRNN) based on the concepts of masked language modeling (15% of tokens masked). Transfer learning involves further training (i.e., fine-tuning) a model to be more specific to a downstream task, which improves model performance. Nanopore sequences have unique error-profiles which can be simulated with NanoSim. We will fine-tune the pretrained plasmid language model using simulated Nanopore metagenomic sequences generated from dereplicated plasmid reference genomes. The training approach will be identical to pretraining, yielding a plasmid language model fine-tuned to the characteristics of Nanopore sequencing data. The fine-tuned plasmid language model will be used to develop a classifier to assign Nanopore metagenomic sequences to previously described and novel PTUs derived from the plasmid enriched metagenomic data in Objective 1 or an 'Other' class (bacteria, viruses, etc.). Nanopore plasmid metagenomic sequences for training the classifier will be simulated with NanoSim from a dereplicated set of RefSeq genomes (bacterial, viral, and plasmids). The classifier will be evaluated based on the accuracy of the test data, the accuracy on simulated and real metagenomic datasets, and the accuracy of newly sequenced plasmids not included in the training data. The plasmid enriched metagenomes from Objective 1 will be used to evaluate the classifier by comparing the assigned bacterial taxa obtained from methylation assisted binning and the plasmid language model classifier. The classifier accuracy will be compared to baseline tools, minimap2, a metagenomic mapping tool for long-reads, and Kraken. The developed model will be applied to the plasmid enriched metagenomic data generated in Objective 1 to better understand the host-range of plasmid-borne AMR genes throughout different production stages. Nanopore metagenomic reads will be fed into the model and sequences classified to PTUs and will be screened for AMR genes with DeepARG. Using the PTU network, we can assess the if the potential mobility of plasmid-borne AMR genes into foodborne pathogens is higher at different stages of production. For instance, the model will allow us to assess the potential host-range of plasmids with AMR genes early in production that may transfer to Escherichia coli and verify if those transfer events occurred via isolate screening included in Objective 1.Research Objective 3 - Develop a novel science-based management and nutritional strategy to reduce metaphylactic antibiotic use by utilizing a novel essential oil as an alternative to conventional antibiotics, evaluating the changes in the microbiome due to utilizing the essential oil as a feed additive and evaluating its applicability as a nutritional strategy to reduce the use of antibiotics. Currently, antibiotics are widely used to reduce pathogen colonization associated with weaning stress and this objective will help develop dietary intervention strategies using alternatives to antibiotics to reduce antibiotics in the swine industry.Utilizing the pork production facility at USMARC, the feeding experiments will be performed over the whole production cycle from farrowing to finish. Two replicates of 16 sows per farrowing group will be utilized that will contain n=4 litters/replicate. Litters will be standardized to a difference of 1 pig or less (10 or 11, 11 or 12, etc.), depending on number of pigs per sow, and cross-fostering will be performed in the first week of life. Pigs from the 32 litters (8 total litters per treatment) will be fed one of 4 treatments (1) Control, 2) Control + essential oil (EO, 2.5% inclusion rate), 3) Control + carbadox/copper sulfate (AB1, 55 mg/kg and 250mg/kg diet, respectively), or 4) Control + chlortetracycline/tiamulin hydrogen formate (AB2, 55 mg/kg and 1.65 g/kg, respectively) as creep feed during lactation starting at 7 d prior to weaning and throughout the nursery phase (28d; 35 d total). All pigs will be fed common diets in the growing and finishing phases of production. The pigs will have free access to each diet and diets will be formulated to meet or exceed NRC nutrient recommendations (1). At weaning, treatments will be moved into the nursery together and be group-housed and blocked by litter. Body weight will be measured gravimetrically starting on days 0, 10, 28, 56, 91 and 126 of life. Weekly pen feed disappearance will be measured in the nursery (d28 to 56). Individual weights and feed intakes will be measured in the USMARC Feed efficiency Barn (described in Obj. 1). At days 0, 28, 56, 91 and 126 a fecal grab sample will be collected from each animal. Subsamples of fecal grabs will be pooled by pen and be snap frozen and stored at -80°C for evaluating microbial community compositional changes.Outreach Activity #1: Deliver relevant outcomes generated by achievement of the project's research objectives through the iAMResponsibleTM Project and the eXtension Livestock and Poultry Environmental Learning Community (LPELC).Outreach Activity #2: Deliver project outcomes through the existing iAMResponsibleTM Project-led university graduate-level course, AMR from a One Health Perspective.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:Target audiance include extension educators, scientists, researches, livestock producers, consumers and policymakers. Changes/Problems:We are sorry for the delay in submitting this report. This is due to having an incorrect email for the PI in the REEport system. This has been fixed and future reports will be submitted in a timely manner. What opportunities for training and professional development has the project provided?During the reporting period 1 PhD student, 1 master's student, and 2 undergraduate students developed or refined STEM communication skills by contributing to the development and distribution of educational materials for the iAMResponsible networks. The continued expansion of media development by the iAMResponsible team has also required several team members to develop new expertise in video production, audio production, and graphic design, utilizing programs such as Canva, and the Adobe Creative Suite, and database management capacity in Airtable. Additionally, 1 PhD student was recruited and has been learning techniques in microbial ecology and molecular biology. Additionally, This student is also engaged in multidisciplinary research and is learning bioinformatic analysis methods. How have the results been disseminated to communities of interest?Primary dissemination for the iAMResponsible team has been via social media, and other online publications, in course materials, and at professional, educational, and community events. Aditionally, research findings have been dessiminated through poster and oral presentations at conferences and peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the iAMResponsible team will continue to expand their web resources to incorporate and make accessible research findings from a wider selection of contributors and expand into to new media types and delivery methods. The team is also eager to expand on this year's success in partnership with student-led outreach and plans to continue to work with existing groups as well as to seek out new student and community partners with similar needs. Additionally, we plan to complete animal studies and begin sequencing the microbiome, plasmidome and virome to understand the effect of antimicrobials in the ecology and emergence of AMR.

Impacts
What was accomplished under these goals? As for research objectives; Under Objective 1, the animal trials are underway. We hope to have the animal trials completed by summer of 2025. As for objective 2, we have not started objective 2 as we are awaiting data from objective 1 to start objective 2. For objective 3, we have planned the essential oil feeding trial. The trial will start end of October 2024 and will be completed by spring of 2025. As for outreach objectives, core to the iAMResponsible team's mission is a train-the-trainer approach to equipping current and future one-health professionals to educate and empower their communities to address the AMR-One-Health challenge. This year, the team partnered with student groups based in Monrovia, Liberia and Lincoln, NE (USA) to plan, promote, and conduct three community outreach events to introduce AMR health risks and potential interventions to youth audiences (ages 4 to 18). The iAMResponsible Team's role in these events was to develop educational materials and provide guidance and training to the student-leaders both about AMR and in delivering STEM youth programming. More than 330 community members participated in one or more of these student-led events. Participants indicated a high level of new understanding of the AMR problem and intention to take individual action to combat the crisis. 92% of participants said they improve their awareness of AMR to a great or very great extent, and 84% said they planned to engage in research, policy development, or advocacy to address AMR in future. Student-leaders were also positively impacted by the programs as teaching further improve their own understanding of AMR and deepened their commitment on the issue. 11% of student-leaders said they learned something new about AMR and 35% intended to expand their awareness efforts in their personal networks.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: WA Tom, JV Judy, PJ Kononoff, SC Fernando. Influence of empirically derived filtering parameters, ASV, and OTU Accession No. 1029171 Project No. NEB-26-261 pipelines on assessing rumen microbial doi: 10.3168/jds.2023-24479.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Alison C Neujahr, Duan S Loy, John Dustin Loy, Bruce W Brodersen, Samodha C Fernando. Rapid detection of high consequence and emerging viral pathogens in pigs. Frontiers in Veterinary Science. Volume 11 - 2024 | https://doi.org/10.3389/fvets.2024.1341783
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Tien Doan, Alison C Neujahr, Samodha C. Fernando. Development of genetically engineered microbes as probiotics to enhance ruminant productivity [Poster Presentation]. Midwest Animal Science Meetings, Wisconsin Madison, March 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Seidu Adams, Matthew L Spangler, Samodha Fernando. Oral and rumen microbiome characterization to predict host phenotypes[Poster Presentation]. Midwest Animal Science Meetings, Wisconsin Madison, March 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 1. Lubna, Nafisa, Burbach, Mark, Zelt, Mara, Schmidt, Amy. 2024-05-20. Integrating Natural Resource Management Strategies in Antimicrobial Resistance Education and Prevention [Poster Presentation]. 2024 NIAMRRE Annual Conference. Columbus, OH.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 2. Palala-Martinez, Hector, Zelt, Mara, Zelt, Beth, Oviedo-Ventura, Maria Jose, and Schmidt, Amy. 2024-06-20. Empowering Youth to Create a Healthier Future Through STEM Education About Antimicrobial Resistance. [Proceedings Paper]. 2024 ASEE Annual Conference & Exposition. Portland, OR. https://nemo.asee.org/public/conferences/344/papers/44540
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 3. Lubna, Nafisa, Burbach, Mark, Zelt, Mara, Schmidt, Amy. 2024-06-18. Integrating Natural Resource Management Strategies in Antimicrobial Resistance Education and Prevention [Poster Presentation]. 2024 Farm Foundation Roundtable Meetings. Denver, CO.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 4. Lubna, Nafisa, Burbach, Mark, Zelt, Mara, Schmidt, Amy. 2024-07-29. Integrating Natural Resource Management Strategies in Antimicrobial Resistance Education and Prevention [Oral Presentation]. 2024 Annual ASABE International Conference. Anaheim, CA.
  • Type: Websites Status: Published Year Published: 2023 Citation: 1. Zelt, Mara and Johnson, Leslie [Hosts]. Improving disease identification, treatment, & antibiotic stewardship in livestock production. [Webinar] August 25, 2023. Part of eXensions Livestock and Poultry Environmental Learning Community Webinar Series. https://lpelc.org/improving-disease-identification-treatment-antibiotic-stewardship-in-livestock-production/
  • Type: Other Status: Published Year Published: 2023 Citation: Zelt, Beth, Zelt, Mara. 2023-10-17. Post-episode discussion of: More drug-resistant bacteria in recycled wastewater or natural water? Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/13680143-post-episode-discussion-of-more-drug-resistant-bacteria-in-recycled-wastewater-or-natural-water Zelt, Beth, Zelt, Mara. 2023-10-11. More drug-resistant bacteria in recycled wastewater or natural water? It's not as easy an answer as you think! Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/episodes/13680129-more-drug-resistant-bacteria-in-recycled-wastewater-or-natural-water-it-s-not-as-easy-an-answer-as-you-think Zelt, Beth, Zelt, Mara. 2023-09-26. Ok, you rule the world. Now solve antimicrobial resistance! What would YOU do first?. Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/episodes/13254074-ok-you-rule-the-world-now-solve-antimicrobial-resistance-what-would-you-do-first Lanwermann, Saskia, Zelt, Mara, Zelt, Beth. 2023-11-21. Antibiotic Resistance: Challenges for the Development of New Antimicrobials. Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/13766556-antibiotic-resistance-challenges-for-the-development-of-new-antimicrobials Zelt, Beth, Zelt, Mara. 2023-12-05. Artificial Intelligence can be applied to AMR. Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/14003924-artificial-intelligence-can-be-applied-to-amr Zelt, Beth, Zelt, Mara. 2023-12-19. Talking about AMR, what words should we use? Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/episodes/14174084 Zelt, Beth, Zelt, Mara. 2023-09-12. Hospitals -a danger zone? Drug-resistance in the ICU. Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/13253960 Zelt, Beth, Zelt, Mara. 2023-11-07. The one about smog. Tales of the Resistance [Podcast episode]. iAMResponsible. https://www.buzzsprout.com/1877103/13629592-the-one-about-smog
  • Type: Other Status: Published Year Published: 2023 Citation: Zelt, Beth. [iAMResponsible]. 2023-10-11. Recycled Wastewater and Antibiotic Resistance -Should we be concerned? [Video]. YouTube. https://www.youtube.com/watch?v=y6b_ToXz5Us Zelt, Beth. [iAMResponsible]. 2023-11-14. Antibiotic Resistance: Should You Be Concerned? [Video]. YouTube. https://youtu.be/392rhDDDKGQ Vogel, Amber, Ortiz Balsero, Andrew. [iAMResponsible]. 2023-11-15. Antibiotic Resistance is found all over the world: Latin America [Video]. YouTube. https://youtu.be/q2ecWt9D97Y Zelt, Beth. [iAMResponsible]. 2024-05-17. Rising Through the Ranks: A Young Fungus Lieutenant's Journey in the War on Humans [Video]. YouTube. https://youtu.be/y3Nr6TyioV8 Zelt, Beth. [iAMResponsible]. 2024-06-19. Meet E. Coli: The Bacteria Living Lavishly in Your Gut [Video]. YouTube. https://youtu.be/qloPZlJjIrs Zelt, Beth. [iAMResponsible]. 2023-11-28. Extension Outreach: The Steps to Success [Video]. YouTube. https://youtu.be/G75o8omq1xQ Lanwermann, Saskia. [iAMResponsible]. 2023-11-29. AMR is a Global Threat [Video]. YouTube. https://youtube.com/shorts/oD_y83j8VCo?si=g7ZA16AyzG-oXv_7 Lanwermann, Saskia. [iAMResponsible]. 2024-06-19. Stop Superbugs 4: Ways to Prevent Antibiotic Resistance [Video]. YouTube. https://youtube.com/shorts/ScENO1CLObw Zelt, Beth. [iAMResponsible]. 2024-03-04. Beyond Borders: Liberia's Antimicrobial Resistance Challenge [Video]. YouTube. https://youtu.be/u9pu6GxZh-U Zelt, Beth, Lubna, Nafisa. [iAMResponsible]. 2024-03-08. Inside the Lab: What We Learned About Drug-Resistance This Year [Video]. YouTube. https://youtu.be/K1ju5mRt-9E Zelt, Beth. [iAMResponsible]. 2024-07-31. Tuberculosis Tales: A Chat on Survival and Resistance [Video]. YouTube. https://youtu.be/iMZ7yct4CAk