Source: UNIVERSITY OF NOTRE DAME submitted to NRP
ANTIMICROBIAL RESISTANCE FATE AND TRANSPORT IN FLOWING WATERS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024735
Grant No.
2021-69015-33500
Cumulative Award Amt.
$995,051.00
Proposal No.
2020-04156
Multistate No.
(N/A)
Project Start Date
Jan 1, 2021
Project End Date
Dec 31, 2024
Grant Year
2021
Program Code
[A1366]- Mitigating Antimicrobial Resistance Across the Food Chain
Recipient Organization
UNIVERSITY OF NOTRE DAME
Galvin Life Sciences Bldg.
NOTRE DAME,IN 46556
Performing Department
Civil & Env Engr & Earth Sci
Non Technical Summary
Agricultural operations represent significant sources of antimicrobial resistance (AMR) to flowing waters, for example via storage and land application of animal manures. Understanding the fate and transport of AMR in flowing waters is critical to inform manure management, land conservation, and monitoring practices. We propose a multiscale experimental approach to quantify degradation, transport, and sorption of AMR in controlled flowing water systems using recirculating mesocosms and experimental streams. This work uses the unique ND-LEEF facility with intermediate sized streams and watersheds to perform controlled fate experiments. Fate and transport experiments will be performed under varying water quality conditions, seasons, and stream substrates; the resulting data will be used to develop a state of the art stochastic transport model. We will measure both culture-based and molecular markers (i.e., antibiotic resistance genes) of AMR to observe differential fate of AMR markers. In addition, we will perform sampling and AMR quantification in natural flowing waters in Pine Creek Watershed, Michigan to both inform and field validate AMR fate and transport predictions. Finally, we will integrate fate and transport models across a gradient of environmental complexity to allow for AMR fate and transport predictions across varying conditions. We will use research activities and findings to develop education and extension modules about AMR, water quality, and conservation practices. Digital education modules will be developed with ND-Learning and implemented through existing ND-LEEF educational activities. Extension modules will be developed and implemented in partnership with the Van Buren Conservation District, the site of Pine Creek Watershed sampling.?
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
7230210202060%
7230210107040%
Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
0210 - Water resources;

Field Of Science
2020 - Engineering; 1070 - Ecology;
Goals / Objectives
There is a clear, critical need to describe offsite antimicrobial resistance (AMR) transport from agricultural operations in flowing waters to inform AMR monitoring and control efforts. Significant unknowns are the persistence and transport of AMR in flowing waters, including the role of environmental drivers such as variation in water quality in receiving waters, seasonality, and the suitability of specific AMR detection methods (e.g., culture versus molecular) and targets (e.g., different antibiotic resistance genes) to represent the wide variety of AMR determinants. Our technical approach will be designed to understand how AMR from multiple agricultural manures is transported and simultaneously degrades in flowing water systems, once in the water column. This will be done using a set of complementary experiments in (1) experimental mesocosms, (2) an outdoor experimental system with linked streams, ponds and wetlands, and (3) field verification in a real-world scenario in the agriculturally-dominated Pine Creek Watershed. Concurrently, we will integrate models with data across a gradient of environmental complexity to understand both culturable and molecular AMR marker degradation and movement in flowing waters. Ultimately, we will build tools and methods for efficient detection and quantification of AMR and construct models to help understand detection in the context of flowing water ecosystems. In addition to technical project tasks, we propose to develop and implement both educational and extension modules relevant to AMR management. Educational and extension modules will be implemented at experimental sites through multiple partners, and based on the best available science to date.Specific project objectives are:Objective 1. Quantify degradation, transport, and sorption of AMR in flowing waters using recirculating mesocosms and replicated experimental streams at the ND-LEEF facility. We will conduct controlled experiments that will test the role of environmental conditions on AMR fate and transport. Both culturable and molecular AMR markers will be quantified to determine agreement between differing AMR quantification approaches.Objective 2. Field validate AMR techniques and detection capabilities in natural stream and ditch ecosystems using the Pine Creek Watershed (PCW) in southwestern Michigan. We will measure parameters derived from experiments from Objective 1 (mesocosms and experimental streams at ND-LEEF) in a variety of natural systems located at PCW to validate our understanding of the interacting role of substrate, stream flow, water quality, biofilms, and seasonality play on AMR dynamics in flowing waters.Objective 3. Integrate empirical data from experiments across a gradient of environmental complexity into a dynamic model to understand and predict AMR transport, degradation, sorption, and movement in flowing waters. This iterative work will continue through all experiments (mesocosms, experimental streams, and field validation in streams and ditches of the PCW). We will use this iterative approach so that empirical results from early experiments inform models and the results of models inform later experiments (Figure 1).Objective 4. Develop education and extension modules about AMR, water quality, and conservation practices. Digital education modules will be developed with ND-Learning and implemented at ND-LEEF (site of Objective 1) and through electronic media allowing widespread dissemination, with target audiences of K-12 through to the general public. Extension modules will be developed in conjunction with our partners at the VBCD (location of Pine Creek, site of Objective 2) and will include extension to local producers/landowners (e.g., conservation meetings, farm field days).
Project Methods
Our generalized approach. We will take advantage of several novel experimental platforms (i.e., recirculating mesocosms, flow-through experimental streams) to test the role of key environmental variables in flowing waters including variation in stream bottom substrate, water quality, biofilm colonization, and flow rates on AMR detection, degradation and transport. We will measure parameters tested in experiments from Objectives 1 (mesocosms and ND-LEEF streams) and 2 (natural streams and ditches at PCW) during all seasons to explore the role of environmental variability on AMR transport dynamics. These objectives will inform Objective 3 (model development) and Objective 4 (educational outreach and extension activities).AMR CharacterizationCulturable AMR targets will include Gram-negative Carbapenem-resistant Enterobacteriaceae (CRE) and Gram-positive Vancomycin-resistant Enterococcus (VRE) will be evaluated by culturable assays. Molecular AMR targets will include tet(W), tet(O), ermF, sul(I), sul(II), qnrA and int1. For molecular analyses, each sample will be filtered through a 0.45 μm mixed cellulose ester filter and stored at -20C prior to DNA extraction. DNA for ddPCR will be prepared using the Bio-Rad ddPCR supermix, primers, and the fragmented DNA for each quantification target. Data analysis will be conducted with QuantaSoft Software.Water Quality AnalysesWe will measure a variety of characteristics to assess the relationship between stream physical template (i.e., substrate, flow) water chemistry, and stream biology on the subsequent retention and processing of AMR. The day before each experimental release, we will estimate benthic biomass cover. On sampling dates, we will also collect water chemistry samples along with background conductivity, temperature, pH, and dissolved oxygen.Objective 1 - Mesocosm and Stream ExperimentsAMR persistence and transport will be evaluated using animal manure slurries in mesocosms and streams. Mesocosms will be constructed out of 300 L tanks filled with 200 L water and continuous gentle mechanical aeration. Transport will be assessed in experimental streams at ND-LEEF. We will conduct stream sampling in three phases: prior to adding manure slurry, during active addition of manure slurry, and after the manure slurry addition has been stopped. Once manure slurry has been introduced, sampling will occur at fixed sites in each test 'watershed,' including in the pond, along the 50 m of the stream reach, and at multiple sites within the wetland. This will allow us to calculate average AMR transport distances and rates of deposition under our treatment conditions, and these parameters will be used in the modeling efforts. Each AMR target will be calculated independently. Additionally, after active manure slurry addition, each area will then be sampled hourly to measure AMR resuspension after AMR addition has ceased.Objective 2- Field validation in natural flowing waters at PCW (MI).Validation sampling will be conducted in the Pine Creek Watershed (PCW; Van Buren Co., MI).For the proposed research, at each of five sampling sites in the PCW, water samples for AMR will be taken from four different locations within the aquatic habitat: (a) center - surface water, (b) center - near bottom, (c) edge - surface water, and (d) edge - near bottom. Streams and ditches of this size should be well mixed, but we want to confirm this by directly testing this in the field using this "stratified" water column sampling approach. AMR quantification and water quality parameters will be determined as described above.Objective 3- Methods for integrating data with dynamic models.Modeling AMR transport involves multiple levels of complexity and heterogeneity, which makes modeling fate and transport challenging. Potential considerations include 1) variable exchange rates between water channel and sediments 2) heterogeneity in AMR particle association and 3) spatially and temporally heterogeneous degradation due to light, shear forces, and biofilm biogeochemistry, to name a few. In the proposed research, we will focus on models that strike a balance between parsimony, complexity, and fidelity, so as to facilitate accurate interpretation of positive and quantified detections and create a predictive modeling framework applied to AMR transport.The Stochastic Mobile Immobile Model (SMIM) - SMIMs are sophisticated models capable of capturing complex behaviors common in streams. As AMR is not conservative, we will generalize the model to include reactions. Via a hierarchy of experiments, we will measure mechanistic parameters independently and predict transport of AMR in natural streams.Single Upscaled Effective Rate Uptake Length Model - Degradation can often be modeled as a first order process, even in complex settings, resulting in a simple model that for rapid decision making is ideal, but whose parameters are hard to predict without measurements. By working with the SMIM, we will develop a basis to calculate these.Objective 4 - Develop education and extension modules about AMR, water quality, and conservation practicesDigital Module Development: The project will partner with the new ND-Learning to develop>10 short digital learning modules. We will develop and implement a comprehensive education and outreach program focused on the issues associated with AMR, integrating research outcomes resulting from the proposed research.In addition to digital module development, we plan multiple educational outreach activities. In collaboration with Southwest Michigan College Educational Talent Search (ETS), we will develop and implement an AMR field day for participants in the ETS Agricultural Science Camp, which is a career and college prep program. As part of an on-going collaboration with Environmental Science students from John Adams High School (South Bend) and Marion High School (Mishawaka), students will participate in hands-on learning modules to learn about the AMR research being conducted at ND-LEEF. Students will conduct field exercises to measure stream characteristics such as flow, substrate composition and sample for water chemistry, while discussing how these factors may influence AMR transport. Science Sunday is ND-LEEF's annual open house, which usually draws 150-200 visitors each year. ND-LEEF will host a "Science at Sunset" presentation, delivered by the project PI, to discuss AMR and what was learned from experiments at ND-LEEF through the project. A representative from the Notre Dame AMR team will participate at the St. Joseph County and/or Berrien County Fair. At the fair, we will have an information booth about AMR research and display the AMR digital modules using a portable monitor. In addition to in-person implementation, we will also produce and host the AMR digital modules on the ND-LEEF website, and for public view on the touchscreen monitor at the ND-LEEF Education and Outreach Pavilion.Extension efforts will be led through the Van Buren Conservation District. Annual Farm Conference held in March of each project year (120-175 attendees). Annual on-farm field day held in August of the first and last project year (50-100 attendees). Small grower meetings will also be held as needed, and on request (5-20 attendees) with at least three meetings to be held during the project period. Presentations to the Van Buren Tech Center agricultural classes are held annually (25-30 high schoolers per class) on topics including water quality and agricultural best management practices. Annual farm topics newsletter distributed county-wide and regionally, with a mailing list of >1000 addresses. Project updates on the AMR research will be included in ongoing newsletters over the three-year project, and at the end of the project, a newsletter will be specifically focused on connections between water quality, management practices, and AMR.

Progress 01/01/21 to 12/31/24

Outputs
Target Audience:Throughout the project, specific target audiences include the broader scientific community with interest in antimicrobial resistance, water quality, and agricultural operations; individuals across multiple age ranges through educational initiatives and digital modules; and regional growers through extension activities. Scientific communication also proceeded through publication and conference presentations. One paper was published in 2024 and we currently have multiple other publications in preparation for submission in the near future and multiple conference presentations/posters. Digital Module Development: The project has partnered with ND-Learning to develop short digital learning modules. We have developed a comprehensive education and outreach program focused on the issues associated with AMR, integrating research outcomes resulting from the proposed research. These modules are freely available online, allowing widespread access and distribution. Additional modules have recently been completed and the web hosting interface will be made public soon.The modules are available here - https://environmentalchange.nd.edu/resources/education-outreach/arg/ Additional educational outreach activities included the 2024 'Science Sunday' at ND LEEF and work with a high school class at ND LEEF. Outreach to growers proceededthrough partners at the Van Buren Conservation District. Changes/Problems:The project was delayed due to COVID-19 but was successfully completed during the No-Cost Extension period. What opportunities for training and professional development has the project provided?The project has provided educational opportunities for one postdoctoral scholar, four graduate students, and four undergraduate students in the project period. This includes field and laboratory experience, data analysis, manuscript preparation, and conference attendance and presentations. How have the results been disseminated to communities of interest?The second manuscript from this work was published in 2024 (Badilla-Aguilar, A., D. M. C. Hallack, O. Ginn, E. Snyder, D. Bolster, J. L. Tank, and K. Bibby. "Streambed immobilization controls the transport of antibiotic resistance genes in flowing water." Water Research 259 (2024): 121833.). Several manuscripts are forthcoming. Team members gave several conference presentations and posters. The educational modules are published and are available: https://environmentalchange.nd.edu/resources/education-outreach/arg/ Van Buren Conservation District worked with growers at several events as detailed above. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Quantify degradation, transport, and sorption of AMR in flowing waters using recirculating mesocosms and replicated experimental streams Manure Management Practices (MMPs) and AMR Transport: Different manure management practices--including fresh manure, lagoon-stored manure with fresh additions, aged lagoon manure, and mixed manure sources--did not significantly alter the in-stream transport behavior of ARGs such as mefA, tetQ, or tetW. While MMPs exhibited variations in ARG concentrations, removal rates were not significantly influenced by manure type. We hypothesize that ARG removal is driven by host-specific factors, physical processes (e.g., substrate interactions, benthic biolayers, and hyporheic exchange), and other potential mechanisms. E. coli Decay and Antibiotic Resistance Profiles: In recirculating mesocosms spiked with different types of cattle manure, viable E. coli exhibited exponential decay across all different treatments. Bacterial isolates showed multi-drug resistance to amikacin, ampicillin, cefotaxime, tetracycline, and sulfonamides, while over 95% remained susceptible to ciprofloxacin. Notably, beta-lactam-resistant E. coli was particularly prevalent in fresh manure sources, most likely linked to cephalosporin use in livestock. These findings highlight the role of agricultural practices in the dissemination of antibiotic resistance. ARG Transport in Experimental Streams: Manure storage appeared to increase the downstream travel distance of certain ARGs compared to fresh manure in experimental freshwater streams. ARGs such as tetQ and tetW exhibited significantly higher removal rates in stored manure additions to the streams. A stochastic mobile immobile model (SMIM) suggested that stored manure had reduced subsurface interactions, potentially allowing ARGs to persist longer in flowing waters. We hypothesize that this differential removal is driven by bacterial adaptations during storage, differences in extracellular vs. intracellular ARGs, or sorption processes at the stream level. Further investigation is needed to elucidate these mechanisms. ARG Interactions with Riverine Biofilms: The interaction of manure-borne ARGs and riverine biofilms was assessed in recirculating mesocosms. ARGs rapidly attached to riverine biofilms upon contact with the water column, with no significant differences observed among tetracycline, beta-lactam, and macrolide resistance genes related to attachment rate. Although biofilms accumulated ARGs over time, detachment of targets began after approximately 12 hours, likely due to biofilm senescence and flow-induced shearing. Objective 2:Field validate AMR techniques and detection capabilities in natural stream and ditch ecosystems We seasonally sampled five points (one above a dairy, four below a dairy) along 2 km of stream in the Pine Creek Watershed in Southwest Michigan on 15 dates spanning all seasons and assessed various models to assess the transport of ARGs from a dairy source. We found that there was a significant increase in ARGs after the dairy. We fitted three different simplified hydrological transport models to four different ARG (ermB, sul1, tetM, and tetW) removal rates and found that of all sampling dates and target instances. We found that 52% exhibited transport behavior that indicated groundwater/runoff contributions may contain ARG concentrations equal to the stream concentration, while 43% exhibited transport behavior that indicated groundwater/runoff contributions may not contain ARG concentrations, therefore diluting ARGs as they are removed from the water column. We then analyzed the best model fits according to targets and found that these results mirrored this same pattern. Objective 3:Integrate empirical data from experiments across a gradient of environmental complexity into a dynamic model Proposed Transport Model: Developed a simplified model to represent ARG transport in a real watershed, considering groundwater/runoff contributions and distributed ARG sources along the mainstream. Model Performance: Three models were tested: (A) pure dilution, assuming no ARG removal; (B) removal + dilution, where groundwater dilutes ARGs; and (C) pure removal, assuming groundwater introduces ARGs at stream concentration. Model C was most frequently selected (52%), followed by Model B (43%), with Model A being the least preferred. User-Friendly Tool: An Excel-based tool was created to implement the models, enabling concentration predictions and removal rate optimization analysis with adjustable locations and other input parameters. Objective 4:Develop education and extension modules about AMR, water quality, and conservation practices The project team worked with ND-Learning to publish and finalize the AMR learning modules. At ND-LEEF, the project PI presented a 'Science at Sunset' seminar in August 2024, and in Fall 2024 the experiential learning ARG/water flow program was completed with 3 high school classes (128 students). In 2024, Van Buren Conservation District (VBCD) staff held multiple educational events for farmers in which AMR was a featured topic. These events included: Farming for the Future (3/13/24): this conference was held in Lawrence, MI with 124 farmers, consultants, and agency staff in attendance. Topics included soil fertility, soil health, antimicrobial resistance, no-till, regenerative agriculture, manure management, and more. Grazing/Livestock Farm Field Day (9/19/24): In collaboration with several partner organizations, VBCD held a farm field day in Bloomingdale, MI with 85 farmers, consultants, and agency staff in attendance. This event was targeted to livestock farmers with a goal to provide opportunities to learn from local experts, get hand-on experience with conservation practices, and share knowledge. Discussion topics included best management practices, a naturalist approach to farm planning, pasturing pigs, pasture parasites, cost share opportunities, and AMR. Small Grower Meeting (9/10/24): Staff held a small grower meeting in partnership with Michigan Agriculture Advancement and National Wildlife Federation. This meeting brought together 10 local farmers to discuss and share opinions on new program and policy ideas for supporting conservation agricultural practices in Michigan. The farmers' perspectives will be shared with policy makers and others in the agriculture sector that can help shape conservation programs for farmers. VBCD staff will use farmer opinions to guide programs and outreach in the future. Farmers shared valuable insight into the barriers and challenges they face in implementing on-farm conservation. 4/24/24: Staff presented to 37 high school students in the Van Buren ISD Ag & Natural Resources Program in Lawrence, MI about conservation farming, E. coli, and antimicrobial resistance. 8/1/24: Farming for the Future - Bringing Conservation to the Farm. This newsletter was mailed to 1,163 farmers, partner organizations, and agribusinesses. Articles included information on prairie strips, antimicrobial resistance, MAEAP, pesticide container recycling, and cost share opportunities. Over the course of the project, farmers in the area received direct mailings inviting them to all events, including postcards, brochures, and E-newsletters. Staff also communicated directly with farmers by attending county Farm Bureau board meetings, the Van Buren County Youth Fair, and other community events. Staff also participated in multiple meetings and conference calls with project partners during this project as well as reported progress in quarterly reports. Staff located farms willing to share manure for research purposes and connected farmers with Notre Dame researchers. Staff reviewed four draft educational videos about AMR developed by the University of Notre Dame and provided comments. Additionally, staff continues to work one-on-one with manure-producing farms to help implement conservation practices.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Badilla-Aguilar, A., D. M. C. Hallack, O. Ginn, E. Snyder, D. Bolster, J. L. Tank, and K. Bibby. "Streambed immobilization controls the transport of antibiotic resistance genes in flowing water." Water Research 259 (2024): 121833.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: E.M. Thrift-Cahall, J.L. Tank, K. Bibby, D. Bolster, O. Ginn, M.J. Liddick, U.H. Mahl, A.N. Pruitt. 2024. The impact of manure management on transport dynamics of antibiotic resistance genes in streams. Society for Freshwater Sciences. Philadelphia, PA. (Oral Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: E.M. Thrift-Cahall, J.L. Tank, K. Bibby, D. Bolster, O. Ginn, M.J. Liddick, U.H. Mahl, A.N. Pruitt. 2024. The impact of manure management on transport dynamics of antibiotic resistance genes in streams. Ecological Society of America; Great Lakes Chapter. Kalamazoo, MI. (Oral Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Liddick, M.J., J.L. Tank, E.M. Thrift-Cahall, A.N. Pruitt, E. Snyder, A.E.S. Vincent, D. Bolster, K. Bibby. 2024. The effect of leaf litter decomposition on transport of antimicrobial resistance genes (ARGs) in streams. Freshwater Sciences Meeting. Philadelphia, PA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: E.M. Thrift-Cahall, A. Badilla-Aguilar, M.J. Liddick, K. Bibby. 2024. Antimicrobial Resistance Fate and Transport in Flowing Waters. United States Department of Agriculture (USDA) Project Directors Meeting. Long Beach, CA. (Oral Presentation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Badilla-Aguilar, A. Transport and Fate of Manure-Borne Antibiotic Resistance Genes in Flowing Water. UNC Water and Health Conference. Talk. October 2024. Chapel Hill, North Carolina.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Badilla-Aguilar, A., Hallack, D.M.C., Ginn, O., Snyder, E., Bolster, D., Tank, J., Bibby, K. Transport and fate of Antibiotic Resistance Genes from cattle manure in flowing water. Seventh (7th) Environmental Dimensions of Antibiotic Resistance Conference (EDAR7). Flash talk and poster. May 2024, Montreal Canada
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Badilla-Aguilar, A., Hallack, D.M.C., Ginn, O., Snyder, E., Bolster, D., Tank, J., Bibby, K. Streambed Immobilizations Drive the Removal of Antibiotic Resistance Genes From Cattle Manure In Streams. May 2024 Midwest Microbiome Symposium. Highlight talk and poster. Purdue University


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Throughout the project, specific target audiences include the broader scientific community with interest in antimicrobial resistance, water quality, and agricultural operations; individuals across multiple age ranges through educational initiatives and digital modules; and regional growers through extension activities. Scientific communication will proceed through publication and conference presentations. One paper was published in 2023 and we currently have one paper under preparation for submission in the near futureand multiple conference presentations/posters scheduled. Digital Module Development: The project has partnered with ND-Learning to develop short digital learning modules. We have developed a comprehensive education and outreach program focused on the issues associated with AMR, integrating research outcomes resulting from the proposed research. These modules will be freely available online, allowing widespread access and distribution. Additional modules have recently been completed and the web hosting interface will be made public soon.The current modules are available here - https://environmentalchange.nd.edu/admin/pages//preview/89ead1971c8e27c3f7cc8ed5ea79b89d Additional educational outreachactivities are proceeding, e.g. the 2023'Science Sunday' at ND LEEF and work with a middle school class at ND LEEF. Outreach to growers is proceeding through partners at the Van Buren Conservation District. Changes/Problems:The project was delayed due to COVID-19 but is on track to be completed during the NCE period of 2024. What opportunities for training and professional development has the project provided?The project has provided educational opportunities for two postdoctoral scholars, four graduate students, and four undergraduate students in the project period. This includes field and laboratory experience, data analysis, manuscript preparation, and conference attendance and presentaitons. How have the results been disseminated to communities of interest?The first manuscript from this work was published in 2023 (Ginn, Olivia, et al. "Persistence of Antibiotic Resistance Genes Varies with Particle Size and Substrate Conditions in Recirculating Streams." Environmental Science & Technology (2023).). Several manuscripts are forthcoming. Team members gave several conference presentations and posters. The educational modules will be published shortly and are available here:https://environmentalchange.nd.edu/admin/pages//preview/89ead1971c8e27c3f7cc8ed5ea79b89d Van Buren Conservation District worked with growers at several events as detailed above. What do you plan to do during the next reporting period to accomplish the goals?Sample analysis for previously completed experiments is ongoing and we expect to submit several manuscripts in 2024. Educational outreach, including module dissemination and ND-LEEF activities are ongoing.

Impacts
What was accomplished under these goals? Team members continued analysis of two field experiments at ND-LEEF (September 2021 and October 2022) and three projects in the experimental mesocosm facility (July 2021, January 2022, Summer 2022). These include molecular analyses and associated modeling. Experiments conducted during 2023 include: The impact of manure management on fate and transport dynamics of ARGs in streams. In July 2023, we used experimental streams, and short-term, steady-state additions of manure slurry, sourced from different stages of management, to examine water column ARG removal paired with biofilm accumulation of two medically-significant ARGs (tetracycline-resistant tetW and tetQ). We collected three manure types: before microbial digestion, from an active digester, and post-digestion from a holding lagoon and screened them for various ARGs and the fecal indicator bacR. Spatiotemporal dynamics of ARGs in agricultural watersheds. From March 2022-2023, weconducted 15 seasonal ARG sampling events across five stream sites in Pine Creek, and one reference cite in Mill Creek (less impacted by agricultural activity) located in the Paw Paw River Watershed (Southwest Michigan) to examine the spatiotemporal dynamics of ARG transport in agricultural watersheds, and areanalyzing these data currently. The effects of leaf litter availability and decomposition on antimicrobial resistance gene (ARG) transport in streams. In June 2023, we finalized results for a study in which we examined the role of organic matter availability on ARG removal rates in recirculating stream mesocosms. To explore this, we used recirculating stream mesocosms amended with uncolonized leaf litter, biofilm colonized leaf litter, or DOC, compared to mesocosms without leaf litter as a control. Assessing antimicrobial resistance gene (ARG) distribution across an agricultural, urban, and forested land use gradient using STARS and SSN spatial modeling framework. In mid October 2023, we conducted a synoptic sampling of streams and rivers (n=105 sites) across 4 major watersheds in Michigan and Indiana (Manistee, Muskegon, St. Joseph, and Tippecanoe) that span an agricultural land use gradient.Our next steps are to extract the DNA and analyze the samples for ARG concentrations using digital PCR. The effect of leaf litter decomposition on transport of antimicrobial resistance genes (ARGs) in streams. In October and November of 2023, we explored the effects of leaf litter decomposition and biofilm colonization on ARG fate and transport, we conducted five short-term, steady-state additions of cow manure over a 21-day period (Fall 2023) in four experimental streams. Analysis is ongoing. Educational activities during 2023 included: Team membershosted two groups of Environmental Science classes from Marian HS on October 16 (55 students) and October 30 (33 students). They learned about AMR and how to sample and plate environmental water samples. We incubated the samples and then sent them images of the plates for them to quantify and interpret. Team members worked with ND Learning to develop educational outreach modules. Outreach activities during 2023 included: In 2023, Van Buren Conservation District (VBCD) staff held multiple educational events for farmers in which AMR was a featured topic. These events included: Farming for the Future: this regional farm conference was held in Lawrence, MI on 3/7/23 with over 160 farmers, consultants, and agency staff in attendance. Farm Field Day: held on 6/14/23 in Bangor, MI with 79 farmers, consultants, and agency staff in attendance. This event was targeted to livestock producers with a goal to provide opportunities to learn from local experts and share knowledge. Farm Field Day: held on 8/17/23 in Bangor, MI with 65 farmers, consultants, and agency staff iin attendance. This event was targeted to farmers with a goal to provide opportunities to learn from local experts, see conservation practices in situ, and share knowledge amongst all attendees. Small Grower Meeting: held on 12/20/23 in Lawrence, MI with 8 farmers and 4 agency staff in attendance. This event was targeted to a small group with a goal to provide an intimate setting for farmers to share their opinions about and experiences with conservation practices. VBCD staff also facilitated a discussion about antimicrobial resistance to assess farmer understanding and opinions. Staff also distributed a farm newsletter on 8/1/2023 to 1,081 farmers, partner organizations, and agribusinesses that shared information about AMR. Additionally, staff continues to work one- on-one with manure-producing farms to help implement conservation practices.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Ginn, O., Tank, J.L., Badilla-Aguilar, A., Snyder, E., Brand�o-Dias, P.F., Thrift, E., Bolster, D. and Bibby, K., 2023. Persistence of Antibiotic Resistance Genes Varies with Particle Size and Substrate Conditions in Recirculating Streams. Environmental Science & Technology.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Liddick, M.J., J.L. Tank, O. Ginn, A. Badilla-Aguilar, E.D. Snyder, E.M. Thrift, K. Bibby, D. Bolster. 2023. Organic matter additions differentially impact antimicrobial resistance gene (ARG) removal from flowing waters. Freshwater Sciences Meeting (oral). Brisbane, QLD, Australia
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Liddick, M.J., J.L. Tank, E.M. Thrift-Cahall, A.N. Pruitt, E.D. Snyder, A.E.S. Vincent, C. Dahn, K. Bibby, D. Bolster. 2023. The effects of leaf litter availability and decomposition on antimicrobial resistance gene transport in streams. Colleges of Science and Engineering Research Horizons Symposium, University of Notre Dame. Notre Dame, IN
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: E.M. Thrift-Cahall, J.L. Tank, O. Ginn, E.D. Snyder, U.H. Mahl, A. Badilla-Aguilar, M.J. Liddick, D. Bolster, K. Bibby. Society for Freshwater Sciences, Brisbane, Australia. Nutrients, light, and biofilms interact to influence removal rates of antimicrobial resistance genes (ARGs) in mesocosms. 2023.


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Throughout the project, specific target audiences include the broader scientific community with interest in antimicrobial resistance, water quality, and agricultural operations; individuals across multiple age ranges through educational initiatives and digital modules; and regional growers through extension activities. Scientific communication will proceed through publication and conference presentations. We currently have one paper under review and multiple conference presentations/posters scheduled for Summer 2023. Digital Module Development: The project haspartneredwith ND-Learning to develop short digital learning modules. We are developing and implementing a comprehensive education and outreach program focused on the issues associated with AMR, integrating research outcomes resulting from the proposed research. These modules will be freely available online, allowing widespread access and distribution. The current modules are available here -https://www.youtube.com/playlist?list=PLuk3dGfI10k2CpRN_azwjngd-V2cqTSUv Additional educational outread activities are proceeding, e.g. the 2022'Science Sunday' at ND LEEF. Changes/Problems:Overall experimental progress was slightly delayed by challenges associated with COVID-19. We anticipate requesting a one-year no cost extension to facilitate project completion. What opportunities for training and professional development has the project provided?The project contributed to the training of one postdoctoral scholar (Dr. Olivia Ginn), sixPhD students (Andrei Badilla, Emma Thrift, Daniel Hallack, Sabrina Volponi, Mitch Liddick, and Bethany Oceguera), and two undergraduate studentsto date. How have the results been disseminated to communities of interest?Student presentation (Andrei Badilla, ~15 minutes) at Notre Dame COSE-JAM meeting. Manuscript submitted. Outreach activities detailed above. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Additional experimentation in mesocosm (1 experiment) and experimental streams (2 experiments) evaluating the role of biofilm colonization and activity on ARG transport. Objective 2. Continued monthly sample collection and analysis from PawPaw River basin. Objective 3. Continued modeling of experimental data from Objective 1, and initial coupling of experimental data in Objective 1 to data from Objective 2. Objective 4. Additional digital module development with ND Learning. Continued outreach activities.

Impacts
What was accomplished under these goals? Objective 1. Three mesocosm experiments - Evaluating the persistence of ARGs (1) with the addition of leaf litter simulating a natural stream (2) withmanure sources of various ages and (3) with the addition of varying nutrient levels. Experimental stream - transport of ARGs from manure sources of various ages. Objective 2. Monthly sampling for ARGs within the Paw Paw River basin. Objective 3. Persistence and transport modeling of prior year experimental data. Objective 4. Developed digital educational modules with ND Learning (available here:https://www.youtube.com/playlist?list=PLuk3dGfI10k2CpRN_azwjngd-V2cqTSUv ) Outreach activities included (note: activities noted below not exclusive to this project but included project portions): Postcard "Farming for the Future" sent 1/11/22 to 1,084 farmers Brochure "Farming for the Future" flier & agenda sent 2/8/22 to 1084 recipients Conference "Farming for the Future Farmers" held 3/8/2022 102 (93 attendees in person, 9 via Zoom) Presentation "Conservation farming presentations "to Van Buren ISD classes Students 4/20/22 to 35 participants Field Day "MAEAP Field Day" held 8/10/22 for 42 farmers. Discussed and demonstrated cover crop use in perennial crop row middles. Also shared information about AMR. Postcard "Invitation to Fall Field Day" mailed 8/15/22 to 1085 farmers Newsletter "Farming for the Future - Bringing Conservation to the Farm" mailed 8/25/22 to 1,085 farmers. Field Day Fall Field Day - "Nutrient Efficiency Farmers" Held 9/7/22 75 attendees. Small Grower Meeting "Small Grower Meeting" held 12/15/22 for 25 farmer attendees.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Olivia Ginn, Jennifer L. Tank, Andrei Badilla-Aguilar, Elise Snyder, Pedro FP Brand�o-Dias, Emma Thrift, Diogo Bolster, Kyle Bibby. Persistence of antibiotic resistance genes varies with particle size and substrate conditions in recirculating streams. Submitted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Transport and removal of ARGs in freshwater experimental streams.


Progress 01/01/21 to 12/31/21

Outputs
Target Audience:Through this project period, project results have largely not yet been communicated with target audiences; below is a description of the overall intended audience for the work completed during this project period. Throughout the project, specific target audiences include the broader scientific community with interest in antimicrobial resistance, water quality, and agricultural operations; individuals across multiple age ranges through educational initiatives and digital modules; and regional growers through extension activities. Scientific communication will proceed through publication and conference presentations. Digital Module Development: The project is partnering with the new ND-Learning to develop short digital learning modules. We are developing and implementing a comprehensive education and outreach program focused on the issues associated with AMR, integrating research outcomes resulting from the proposed research. These modules will be freely available online, allowing widespread access and distribution. Additional educational outread activities are proceeding, e.g. the 2021 'Science Sunday' at ND LEEF. We expect these activiteis to ramp up in the coming project year with reduced COVID-19 conerns and limitations. Extension activities were limited by COVID-19 concerns in the past project period. The plan for upcoming project extension activities is detailed below. Annual regional farm conference (120-175 attendees) March 2021: CANCELED March 8, 2022 focus Soil Health o Sessions: Ecosystem management strategies for improving soil health, Measuring on-farm success for soil health, Farmer perspective: a systems approach to achieving soil health on the farm, Corn Tar Spot Update, Intro to Antimicrobial Resistance March 2023 TBD o Tentative breakout sessions: - Better manure management options to improve soil health and water quality - A case on cover crops: how they are the ultimate tool to keeping farm inputs on the farm and out of the water Annual on-farm field day (50-100 attendees) - August 31, 2022: Soil Health - August 2023: Manure Bus Tour (tentative) Small grower meetings (5-20 attendees) (as needed, at least three meetings) - VBCD holds meetings for growers participating in VBCD projects o Opportunities to discuss research and BMPs and why farmers should care - VBCD will hold a focused meeting for growers who have manure to manage o Presenter: Farmer with manure doing practices to improve WQ o University Speaker: Research on ARG and E. coli and why farmers should care o Cover Crops and soil health - Gauge grower interest in participating in future projects to monitor farm runoff and evaluate BMP effectiveness at pathogen and nutrient runoff reduction Presentations to the Van Buren Tech Center agricultural classes (25-30 high schoolers) - Annual presentation including topics of BMPs, E. coli and ARG Annual farm topics newsletter (county-wide + additional growers on mailing list @ 1000 addresses) - Annual newsletter on farm projects in the county, farmer highlights, BMP updates o 2021: Building Resilient Farms o 2022: TBD (Manure/alternative nutrients inputs focus) o 2023: TBD Direct mailings to targeted farmers in Van Buren County - Targeted letters to farmers about project/cost-share opportunities - Focused letter with information on nutrients, E. coli, ARG and water quality - Invitations to events Changes/Problems:Overall project initiation was delayed by COVID-19 limitations and concerns; however, progress in all areas has been strong and we expect to return to schedule. Extension activities were limited by COVID-19 limitations and concerns; however, we have a plan in place to ramp up these activities in the coming project period. What opportunities for training and professional development has the project provided?The project contributed to the training of one postdoctoral scholar (Dr. Olivia Ginn) and four PhD students (Andrei Badilla, Emma Thrift, Daniel Hallack, and Sabrina Volponi) to date. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We have already run one mesocosm experiment during this reporting period as described below (January/February 2022). We expect additional mesocosm and LEEF stream experiments during this period as well, as described below. We conducted an additional mesocosm experiment assessing the effect of leaf litter in various states on the removal of ARGs from the water column of recirculating streams. Treatments included 100 g/m2 of uncolonized leaves, 100 g/m2 of biofilm colonized leaves, dissolved organic carbon (leaf leachate), and no treatment. We collected 4 replicate samples for each treatment at the following time points: pre-spike and at 0, 4, 12, 24, 48, 72 hours and 1 and 2 weeks post spike. Samples have been stored and analysis will be conducted within this reporting period. Future plans for this reporting period include the following: Mesocosm experiment assessing the effects of solar irradiation on ARG removal from the water column of recirculating streams. Mesocosm experiment assessing ARGs in manure at different stages: fresh manure, multiple time lengths of aged manure, and land-applied manure. ND LEEF experiment assessing the effect of underlying stream substrate and biofilm colonization on ARG removal. Objective 2. As noted above we expect to initiate sample collection and analysis. Objective 3. We plan on coupling the models developed for conservayive transport in streams to reactive transport model that are being developed to interpret mesocosm experimental data. Objective 4. Educational module development is ongoing. Annual regional farm conference (120-175 attendees) March 2021: CANCELED March 8, 2022 focus Soil Health o Sessions: Ecosystem management strategies for improving soil health, Measuring on-farm success for soil health, Farmer perspective: a systems approach to achieving soil health on the farm, Corn Tar Spot Update, Intro to Antimicrobial Resistance March 2023 TBD o Tentative breakout sessions: - Better manure management options to improve soil health and water quality - A case on cover crops: how they are the ultimate tool to keeping farm inputs on the farm and out of the water Annual on-farm field day (50-100 attendees) - August 31, 2022: Soil Health - August 2023: Manure Bus Tour (tentative) Small grower meetings (5-20 attendees) (as needed, at least three meetings) - VBCD holds meetings for growers participating in VBCD projects o Opportunities to discuss research and BMPs and why farmers should care - VBCD will hold a focused meeting for growers who have manure to manage o Presenter: Farmer with manure doing practices to improve WQ o University Speaker: Research on ARG and E. coli and why farmers should care o Cover Crops and soil health - Gauge grower interest in participating in future projects to monitor farm runoff and evaluate BMP effectiveness at pathogen and nutrient runoff reduction Presentations to the Van Buren Tech Center agricultural classes (25-30 high schoolers) - Annual presentation including topics of BMPs, E. coli and ARG Annual farm topics newsletter (county-wide + additional growers on mailing list @ 1000 addresses) - Annual newsletter on farm projects in the county, farmer highlights, BMP updates o 2021: Building Resilient Farms o 2022: TBD (Manure/alternative nutrients inputs focus) o 2023: TBD Direct mailings to targeted farmers in Van Buren County - Targeted letters to farmers about project/cost-share opportunities - Focused letter with information on nutrients, E. coli, ARG and water quality - Invitations to events

Impacts
What was accomplished under these goals? Under objective 1, initial experiments were completed in both recirculating mesocosms and experimental streams at ND LEEF. Brief descriptions of the individual experiments are noted below. We conducted an initial experiment in recirculating mesocosms in July 2021. We assessed water column removal of antibiotic resistance genes (ARGs) originating from cow manure slurry collected from a dairy farm. We assessed the effect of three underlying substrate variations (pea gravel, pea gravel with fine particulate organic matter, and no substrate) on removal rates by collecting sequential water samples over time: pre-spike, and at 0, 4, 16, 24, 48, 72 hours, 1 week and 2 weeks after spiking with manure slurry. Analysis is ongoing, though we have determined removal rates across the first 4 time points for two ARG targets: tetracycline resistance encoding gene, tetW and erythromycin resistance encoding gene, ermB. Our results indicate that ARGs may persist in the water column of flowing waters for >24 hours, providing ample opportunity for dispersal via bacterial horizontal gene transfer. Additionally, we conducted an initial experiment at ND LEEF in September of 2021. As with the mesocosm experiment, we assessed water column removal of ARGs originating from cow manure slurry collected at the same dairy farm. We collected samples at 6 points along a 100 m stream with mixed substrate. We spiked the stream with manure slurry at a constant drip rate of X, with an overall stream flow rate of Y. We measured conductivity throughout the stream to determine the point at which the stream reached steady state. We collected 3 replicate samples at each of the 6 points and repeated this process on four sequential days. Sample extraction and analysis is ongoing. Under objective 2, sampling plans for PCW have been established and we expect to initiate sampling in this project year. Under objective 3, we have implemented and tested transport models for conservative tracer tests at the ND LEEF streams. Under objective 4, we have begun working with ND Learning to develop educational outreach modules and expect to have the first modules completed in the next project period. Outreach activities have also been conducted through ND LEEF (e.g., Science Sunday). Extension efforts were delayed due to COVID-19 concerns and grower's reluctance to meet virtually; these activities are expected to significantly ramp up in the next project period.

Publications