IDENTIFICATION AND MANAGEMENT OF CRITICAL CONTROL POINTS IN THE SPREAD OF ANTIBIOTIC RESISTANCE FROM ANIMAL MANURE TO RAW PRODUCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1005148
• Grant No.: 2015-68003-23050
• Project No.: VA-422453
• Proposal No.: 2014-05280
• Program Code: A4171
• Project Start Date: Feb 1, 2015
• Project End Date: Jan 31, 2019
• Grant Year: 2017

Project Director
Pruden, A.

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Civil & Env Engineering

Non Technical Summary
There is growing evidence and concern that agricultural practices, including antibiotic use and manure management, are contributing to the growing antibiotic resistance problem in humans. Simultaneously, there is growing demand for fresh produce, which may carry antibiotic resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) of manure origin. The proposed integrated research, education, and extension program seeks to act proactively by focusing on critical control points that may prevent the spread of antibiotic resistance from farm to fork, including: 1) antibiotic use; 2) manure composting; 3) soil selection; 4) crop selection; and 5) fresh produce post-harvest processing practices. A novel and practical outcome of this project will be examination of whether the proposed FDA FSMA rules for composting manure, which are intended for control of pathogens, will effectively limit the spread of ARGs. Research will focus on tracking the fate of antibiotics, ARBs, and ARGs through each of these critical control points, including lab, greenhouse, and field-scale studies. Methods will include advanced analytical chemical methods, culturing of ARBs, and high-throughput metagenomic and DNA amplicon sequencing. Education activities will include launching an innovative interdisciplinary graduate education program [Agricultural Sources of Antibiotic Resistance (AgSoAR)], hosting undergraduate practicums, and developing and implementing course modules. The extension effort will serve to translate research into practice, using vehicles such as websites, publications, webinars, trainings, and workshops in order to reach a wide array of stakeholders, including producers, consumers, veterinarians, and policy-makers. The proposal addresses Priority Code A4171 Effective Mitigation Strategies for Microbial Resistance.

Animal Health Component

10%

Research Effort Categories

Basic

80%

Applied

15%

Developmental

5%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
711	5370	2000	20%
712	1499	1100	80%

Knowledge Area
711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1499 - Vegetables, general/other; 5370 - Sewage and waste disposal facilities and systems;

Field Of Science
1100 - Bacteriology; 2000 - Chemistry;

Keywords

antibiotic resistance

bovine

compost

extension

manure

mitigation

post-harvest

produce

qpcr

Goals / Objectives
The goals of this integrated research, education, and extension coordinated agricultural program (CAP) are 1) to identify critical control points for the spread of antimicrobial resistance from manure to produce and 2) to engage in education and extension efforts relevant to pre-harvest and post-harvest practices that may mitigate the spread of antibiotic resistance from farm to fork. The specific objectives are to:Determine the effect of common therapeutic and subtherapeutic antibiotic use practices on excretion of antibiotics and ARGs, using dairy and beef cattle as models, in order to inform manure management strategies;Examine the potential for manure composting to degrade antibiotics and attenuate ARBs and ARGs, and recommend appropriate manure treatment practices;Examine the effect of soil type on the persistence of antibiotics, ARBs, and ARGs in compost-amended soils and provide guidance to farmers on implications for field selection;Quantify the transfer of antibiotics, ARBs, and ARGs to various produce grown in soils amended with different compost types and provide guidance to farmers on crop selection;Determine the effect of and develop recommendations for different fresh produce post-harvest practices on survival and re-growth of ARBs and ARGs; andDevelop and implement educational opportunities focused on mitigation of agricultural sources of antibiotic resistance for undergraduate and graduate students.

Project Methods
An integrated research, education, and extension approach will be implemented to achieve the goals and objectives of this CAP.The research effort will employ animal studies (e.g., dosing antibiotics to dairy and beef cattle as models) and both lab- and field-scale studies to evaluate the effects of different composting approaches, soil types, crop types, and post-harvest practices on the potential for antibiotic resistance to spread from farm to fork.The education effort will include lab rotations and specialized coarsework for graduate students selected as AgSOAR trainees. A comprehensive undergraduate field practicum program will also be offered to undergraduate students. Graduates and undergraduates will participate directly in the research.Extension will be integrated throughout, including students and project director/co-directors in disseminating results to producers and growers through eXtension community of practice resources, the Virginia Cooperative ExtensionExtension, a website, blog, facebook page, and webinar.

Progress 02/01/15 to 01/31/19

Outputs
Target Audience:We aimed to comprehensively reach multiple target audiences with this effort. We judge that we were successful in reaching all target audiences outlined in our stakeholder diagram at some level, including policy makers, extension agents, veterinarians, producers, dairy farmers, growers, consumers, students, and academics. Within academia, results were presented at conferences geared towards animal scientists, food scientists, environmental engineers, watershed scientists, soil scientists, microbiologists, water engineers, agricultural scientists and scientific communities focused on addressing the spread of antibiotic resistance. In particular, we emphasized a comprehensive "One Health" perspective in disseminating this work, emphasizing the environmental domain of One Health in particular. Invited presentations were delivered to the Presidential Advisory Council on Combatting Antibiotic Resistance (PACCARB), the New York Academy of Sciences, and the One Health and Food Safety Congress in Bonn, Germany, as well as invited keynotes at the Virginia Tech/Maryland College of Veterinary Medicine Annual Symposium and the University of Minnesota School of Veterinary Medicine. A webinar was also delivered to the Center for Infectious Disease Research and Policy (CIDRAP) community to reach doctors and veterinarians and others in the scientific and practitioner community dealing with policy to prevent the spread of antibiotic resistance. Within the educational domain, as part of our Agricultural Sources of Antibiotic Resistance (AgSOAR) program, we served a total of 22 undergraduate, 16 Masters and PhD students, and four post-docs through direct, integrated team-science-based contributions to the research portion of this project. This is in addition to numerous students that enrolled and completed the 12 courses that contributed to the AgSOAR curriculum. Within the extension and outreach domain, we maintained a website and Facebook page to bring awareness to the general public about the project. We also developed and published 7 Fact Sheets about agricultural sources of antibiotic resistance, geared to help extension agents engage with and educate producers and growers about antibiotic resistance. We also developed an online educational animation posted to YouTube which summarizes the basic take-home messages derived from our research in terms of practical advice for producers, growers, and dairy farmers. Changes/Problems:All changes were noted in previous Progress Reports, there are no new changes or problems to report in our Final Report. In summary from previous Progress Reports, we changed our model root vegetable from carrots to radishes in the greenhouse and field experiments because radishes are more amenable to growth in clay soils. We also were not able to successfully grow broccoli to a crowning stage in the greenhouse study and therefore focused on just the lettuce and radish in the greenhouse. In the composting experiment, the static condition achieved the FSMA time-temperature requirements, but the turned condition did not. We repeated with external heating and at full scale and were able to successfully achieve the time-temperature requirements. We concluded that small-scale composting is more vulnerable to heat-loss, and focused more on the static-compost condition in subsequent experiments, since time-temperature requirements were achieved for that condition. What opportunities for training and professional development has the project provided?Twenty-two undergraduate students, sixteen graduate students, and four post-doctoral researchers directly participated in the AgSOAR program and contributed to the research products. These students had the opportunity to learn experimental design and execution of research as well as the preparation of manuscripts and presentations. All graduate students and post-doctorates had the opportunity to present their work at professional conferences and some undergraduates also had the opportunity to present posters. Two prior post-docs working on this project are now faculty members at other universities, one now works for a USDA ARS laboratory in Wyndmoor, PA, and the fourth is a Research Scientist at Virginia Tech. Among the masters students that participated, many continued on for a PhD or pursue a Veterinary Medicine degree, while others chose to work in environmental consulting, in the food safety industry, or in environmental research. Training opportunites were made available to the broader community of Virginia Tech students across several disciplines through the development of curricula and course modules derived from this project. The following is a list of specific courses that contributed to the AgSOAR program: DASC 4984: Antibiotic Resistance from Farm to Fork Capstone. Readings and seminar-based course targeting undergraduates on spread of antibiotic resistance from Farm to Fork led by K.F. Knowlton Fall 2018. GRAD 5134. Developed an instructional module and guided two student-led team research projects related to environmental routes of antibiotic resistance dissemination and agricultural/aquacultural practices for GRAD 5134 Interdisciplinary Research, Spring 2016 (16 students). FST/BIOL 3604. Developed a course module on rise of antibiotic resistance in the food system and strategies to prevent spread in junior level Food Microbiology course in Spring 2017, 2018. (240 students) FST 4634/5634. Developed instructional module on antibiotic resistance for course in Food Science and Technology and delivered to FST 4634/5634g Epidemiology of Food and Waterborne Disease in Spring 2015, 2017 (40 students). FST 4524. Developed and delivered 3 modules on antimicrobials and antimicrobial resistance for Food Science and Technology FST 4524 Food Quality Assurance, Spring 2016 (60 students). CSES/ENSC 4314 Water Quality. Developed and taught an instructional module on antibiotics and pharmaceutical contamination in water resources for Crop Soil and Environmental Sciences/ Environmental Sciences CSES/ENSC 4314 Water Quality (50 students) ENSC 4414. Taught an instructional module on analyzing and tracking antibiotics in the environment for Environmental Sciences ENSC 4414, Monitoring and Analysis of the Environment, 2016 (15 students). BSE 2004. Developed instructional module on antibiotic resistance for course in Biological Systems Engineering (BSE) department, vBSE 2004 Intro to Biosystems Engineering. Students interviewed experts in the field and produced video on antibiotic resistance genes, 2016 (ARGs). BSE 2004. Module developed for Biological Systems Engineering, including student projects focused on ethical consideration of antibiotic use in agriculture, BSE 2004 Introduction to Biological Systems Engineering 2016 (55 students) BSE 3334. Module developed for Biological Systems Engineering BSE 3334 Non-point Source Assessment and Control, 2016 (15 students). BSE 5404. Module developed for online course, Biological Systems Engineering BSE 5400 Agricultural Non-point Source pollution, 2016 (20 students), 2018 (15 students). CEE 5194. Environmental Engineering Microbiology. Module developed for environmental sources of antibiotic resistance. Students learn about methods for monitoring antibiotic resistance in the environment. Student-led research projects developed on this topic and shared with the class, 2025, 2017, 2018 (24-36 students) How have the results been disseminated to communities of interest?Policy Makers- Keynotes and Invited talks at policy-oriented national and international symposia (e.g., Presidential Advisory Council on Combatting Antbiotic Resistant Bacteria (PACCARB), NY Academy of Sciences/Sackler Institute, Bonn Germany One Health Symposium). A webinar was also delivered to the Center for Infectious Disease Research and Policy (CIDRAP) community, which is largely represented by veterinarians and medical doctors interested in public health and One Health. Academics- 15 journal articles in print by the end of this report, plus several others expected to come out in the next year. Thirteen invited talks and keynotes were delivered to major disciplinary and interdisciplinary conferences, featuring the research and education program here and its findings. Twenty-nine oral or poaster presentations were delivered at conferenes. Co-PIs representing Environmental Engineering, Food Science, Soil and Crop Sciences, Dairy Sciences, Agricultural Engineering, and Biological Systems Engineering contributed to this effort and helped to disseminate the work to relevant communites. Extension Agents, Veterinarians, Producers, Growers, and Dairy Farmers. The main findings and recommendations of this research were boiled down into practical terms in an educational animation made available on YouTube https://youtu.be/ob4NmLmhFTE . We published seven extension fact sheets on agricultural sources of antibiotic resistance as a resource to these communities. We also had a local dairy farmer and a grower participate on the advisory board for this project. Students- Through the AgSOAR educational program, several undergraduate, graduate, and post-doctoral researchers participated directly in this effort, in addition to many more students that enrolled in and completed the classes. The course material dveloped will continue to be taught in the future. Advisory Board- Our advisory board served not only in the role of evaluation, assessment, and feedback, but also helped to disseminate the work broadly to multiple communities nationally and internationally. Our advisory board included leading scientists at USDA ARS labs, the World Health Organization, Food Scientists, Veterinarians, and Policy/Consultants. We held two on-site face-to-face advisory board meetings and a final meeting via Zoom online conferencing. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Summary of Key Findings (Chambers et al. 2015)-Direct effects of antibiotic administration, in this case ceftiofur- a 3rd gen cephalosporin, on manure resistome are subtle, but measurable. Effects on "stress response" genes most apparent. It may take time for resistome to attenuate even in manure of livestock not receiving antibiotics because of the legacy of antibiotic use and persistence of resistant bacteria. (Ray et al. 2017)- Composting helps enhance dissipation of antibiotics (chlortetracycline, sulfamethazine, tylosin, pirlimycin, and cephapirin) in manure. Tylosin was the most persistent. (Keenum, Williams et al., in preparation)-Composting reduces total ARGs and tet(W). However, sul1 and intI1 increase during composting. Total ARGs (measured by antibiotics) were most recalcitrant in the dairy with antibiotics condition. (Chen et al. 2018a)- Composting before soil application reduces antibiotic inputs to soil, compared to raw manure containing antibiotics. Dissipation of antibiotics was bi-phasic in soil over a 120 day monitoring period. For the first phase, the slowest to fastest ranked dissipation rates for the different antibiotics in soil was: chlortetracycline, sulfamethazine, tylosin, pirlimycin, cephapirin. There was no effect of soil type on the rate of antibiotic dissipation. (Chen, Pankow et al. 2019)- Addition of raw manure initially elevated total ARGs and "resistome risk" (Oh et al. 2018) to a greater extent than addition of compost to soil. The attenuation of total ARGs occurred in soil amended with dairy manure containing antibiotics. All conditions resembled background soil after 120 days. (Guron et al., 2019/Fogler et al. 2019/Guron et al. in prep)-Radish and lettuce carried different "resistomes" relative to each other and also depending on whether grown in sandy loam or silty clay loam. Both lettuce and radish carried more antibiotic resistance genes when soil was amended with manure or compost versus chemical fertilizer. Lettuce were more affected by amendments than radish, although radish carried more total antibiotic resistance genes than lettuce. Effects of collecting manure during antibiotic use was measureable in lettuce resistomes. Markers of resistance carried by radishes (sul1 and intI1) where highest when raw manure containing antibiotics were amended to the sandy loam, suggesting synergistic manure-soil type effect. (Chen et al. 2018b)- Dissipation of some antibiotics was enhanced in the rhizosphere. There was no apparent difference in tet(W), intI1, or ermB in the soil in the immediate root zone (i.e., rhizosphere) versus the rest of the soil. (Wind et al. 2018)-Antibiotic-resistant fecal coliforms were elevated in soil initially after amending with manure or compost versus chemical fertilizer. The magnitude of observed coliform counts differed by soil amendment type and type of antibiotic resistance assayed. Compost-amended soils had the highest levels of cephalosporin- resistant fecal coliforms, regardless of whether the cows from which the manure was derived were administered antibiotics. Samples from control plots or those treated with inorganic fertilizer trended toward lower counts of resistant coliforms, although these differences were not statistically significant. No statistical differences were observed between soils that grew leafy (lettuce) versus rooted (radish) crops. (Jacobs et al. in revision)- Runoff from all soil plots, including controls that were not amended with manure or compost, yielded ARGs in associated runoff. Compost and manure-amended soils resulted in higher levels of target ARGs (sul1 and ermB) in runoff. Vegetable type influenced sediment concentrations in runoff, but not ARGs, in runoff. (Dharmarha et al. 2019a) Including sanitizer in wash water suppresses total bacterial numbers, including resistant E. coli and Pseudomonas aeruginosa that are inoculated. Storage temperature not sanitizers resulted in the most changes to the carrot microbiota. Storage at 10oC was associated with growth of bacteria resistant to cefotaxime on carrots. Storage at 2oC prevents growth of spiked antibiotic-resistant Pseudomonas sp. and E.coli O157 on carrots. (Dharmarha et al. 2019b)- Post-harvest interventions are needed to mitigate pathogens, but also ARB, and ARGs on vegetables. Washing followed by irradiation resulted in a different resistome chiefly due to reductions in ARG classes that confer resistance to: multidrug, triclosan, polymyxin, β-lactam, and quinolones. Results of this study indicate that washing, irradiation and storage of modified atmosphere packaged lettuce at 4ºC are effective strategies to reduce antibiotic-resistant E. coli O157:H7 and P. aeruginosa and some other ARBs and ARGs. Specific Recommendations Multiple Farm-to-Fork Management Practices/Barriers for Reducing Opportunity for Antibiotic Resistance to Spread. We recommend multiple barriers as a means to minimize the chances of antibiotic resistance spreading through the food chain. Because of the uncertainties associated with the developing science of risk assessment and identifying monitoring targets, we focus here on management practices that are currently recommended for reducing other biological and chemical contaminants, fairly straightforward to implement, and are known to have other benefits. These are summarized in the YouTube Animation and below. Use antibiotics judiciously. When possible, segregate manure from livestock undergoing antibiotic treatment so that the manure can be treated and handled separately. Treat manure (e.g., by composting) before land-applying, which can also provide other benefits to the soil. Consider soil type- more research needs to be done, but we noticed instances where the soil was a key factor shaping the kinds of ARGs carried by the vegetables and that adding manure-based amendments to the sandy soil that we studied resulted in elevated numbers of ARGs. Consider crop type and soil amendments. Root vegetables have more direct contact with soil and soil amendments than leafy vegetables. Here we found radishes carried greater total numbers of ARGs than lettuce, but lettuce was more measurably affected by the amendment type, including whether antibiotics had been administered during manure collection. Impose time intervales between aplication of manure-derived amendments and planting or harvest. We focused on 120 days, based on guidelines for organic produce, and noticed that markers of antibiotic resistance markedly declined with time, though some differences were still apparent. Impose vigilant post-harvest processing and storage conditions, including post-harvest washing, addition of sanitizers, and storage at 4 degrees C limits ability of all bacteria to re-grow, including resistant forms.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Wind, L., Jacobs, K., Krometis, L., Hession, C., and A. Pruden. Environmental dissemination of antibiotic resistance genes from vegetable production. American Ecological Engineering Society Annual Meeting, June 7-9, Knoxville, TN (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Oral Presentation by Le, H., R. O. Maguire, and K. Xia. Effects of Manure Land Application Technologies and Timing on Environmental Fate of Four Antibiotics Commonly Used in Dairy Production. ASA-CSSA-SSSA International Annual Meetings, November 6-9, Phoenix, AZ (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Oral Presentation by Chen, C., Ray, P., Pruden, A., Knowlton, K.F., and Xia, K. Fate and Transformation of Manure-borne Veterinary Antibiotics in Soils. 252nd American Chemical Society National Meeting. August 21-25, Philadelphia, PA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Chen, C., Guron, G., Pruden, A., Ponder, M., and Xia, K. Antibiotics and ARGs in manure-amended soils growing vegetables. 254nd American Chemical Society National Meeting. August 20-24, Washington, D.C. (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Guron, G., Ray, P., Ponder, M., and Pruden, A. Assessing the potential for antibiotic resistant bacteria and resistance genes to carry over from soil amendments to vegetable surfaces: A greenhouse study. International Association for Food Protection (IAFP), July 31-Aug 2, St. Louis, MO (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pulido, N., Dharmarha, V., Ponder, M., Pruden, A., and Boyer, R. Effect of Sanitizers on the Survival of Antibiotic-resistant Bacteria Applied to Raw Carrots through Contaminated Compost. International Association for Food Protection (IAFP), July 31-Aug 2, St. Louis, MO (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Chen, C. and Xia, K. Effect of Earthworm Activity on the Fate of Antibiotics, ARB, and ARGs in a Compost Amended Soil. August 20-24, Washington, D.C. (2017).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Jacobs, K.; Krometis, L-A.; Hession, W.C.; Wind, L., Pruden, A. Recovery of Antibiotic Resistance Genes in Storm Runoff from Manure and Compost-Amended Vegetable Fields. (Revision submitted to Journal of Environmental Quality, March 2019).
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Guron, G., Chen, C.; Du, P.; Pruden, A.; Ponder, M. Manure-based amendments influence surface-associated bacteria and markers of antibiotic resistance on radishes grown in different soil types. Applied and Environmental Microbiology (Revision and Resubmission in Preparation)
• Type: Journal Articles Status: Other Year Published: 2019 Citation: Keenum, I.M.; Williams, R.K.; Ray, P.; Garner, E.D.; Knowlton, K.F.; Chen, C.; Xia, K.; Pruden, A. Does Composting Alter Effects of Prior Antibiotic Use on Cattle Manure-borne Microbiota and Resistomes? (In preparation for submission to ISME Journal)
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Pulido, N. 2016. EFFECT OF STANDARD POST-HARVEST INTERVENTIONS ON THE SURVIVAL AND RE-GROWTH OF ANTIBIOTIC-RESISTANT BACTERIA ON FRESH PRODUCE. Masters Thesis. Food Science and Technology. Virginia Tech
• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Williams, Robert K. 2016. EFFECT OF COMPOSTING ON THE PREVALENCE OF ANTIBIOTIC RESISTANT BACTERIA AND RESISTANCE GENES IN CATTLE MANURE. Masters Thesis. Civil Engineering. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Pankow, Christine A. 2017 Effect of Soil Type, Composting, and Antibiotic Use on Fate of Antibiotic Resistance Genes and Microbial Community Composition in Dairy and Beef Manure Applied Soils. Masters Thesis. Civil Engineering. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Wind, Lauren. 2017. PERSISTENCE OF CULTURABLE ANTIBIOTIC RESISTANT FECAL COLIFORMS IN MANURE AMENDED VEGETABLE FIELDS. Masters Thesis. Biological Systems Engineering. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Fogler, Kendall W. 2017. Effect of Soil Amendments from Antibiotic Treated Cows on Antibiotic Resistant Bacteria & Genes Recovered from the Surfaces of Lettuce and Radishes: Field Study. Masters Thesis. Food Science and Technology. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Dharmarha, V. 2018. EFFECT OF STANDARD POST-HARVEST INTERVENTIONS OF FRESH VEGETABLES ON BACTERIAL COMMUNITY DYNAMICS, PATHOGEN SURVIVAL, AND ANTIBIOTIC RESISTANCE. Doctoral Dissertation. Food Science and Technology. Virginia Tech.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Oh, M.; Pruden, A.; Chen, C.; Heath, L.; Xia, K.; Zhang, L. (2018). MetaCompare: A computational pipeline for prioritizing environmental resistome risk. FEMS Microbiology Ecology. 94(7): fiy079, https://doi.org/10.1093/femsec/fiy079.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: B�rgmann, H.; Frigon, D.; Gaze, W.; Manaia, C.; Pruden, A.; Singer, A.C.; Smets, B.; Zhang, T. (2018). Water & Sanitation: An Essential Battlefront in the War on Antimicrobial Resistance. FEMS Microbiology Ecology. 95(9): fiy101. https://doi.org/10.1093/femsec/fiy101.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Jacobs, Kyle B. 2017. RECOVERY OF ANTIBIOTIC RESISTANCE GENES FROM AGRICULTURAL RUNOFF. Masters Thesis. Biological Systems Engineering. Virginia Tech.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Chen, C.; Pankow, C.; Oh, M.; Heath, L.; Zhang, L.; Du., P.; Xia, K.; Pruden, A. (2019). Effect of antibiotic use and composting on antibiotic resistance gene abundance and resistome risks of soils receiving manure-derived amendments. Environment International (accepted April 18, 2019).
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Guron, G., Arango-Argoty, G.; Zhang, L., Ponder, M.; Pruden, A. (2019). Effect of Dairy Manure-Based Amendments and Soil Texture on Lettuce and Radish Associated Microbiota and Resistomes. mSphere DOI:10.1128/mSphere.00239-19
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dharmarha, V.; Guron, G., Boyer, R.R.; Niemara, B., Pruden, A.; Strawn, L.K., Ponder, M.A. (2019). Gamma irradiation influences the survival and regrowth of antibiotic resistant bacteria and antibiotic resistant genes on romaine lettuce. Frontiers in Microbiology. 10: 710. doi: 10.3389/fmicb.2019.00710
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Fogler, K.; Guron, G.K.; Wind, L.; Hession, W.C.; Krometis, L-A, Strawn, L.K.; Pruden, A.; Ponder, M.A. Profiling the Resistomes of Bacterial Communities of Field-Grown Lettuce Leaves and Radish Taproots Grown in Soils Receiving Dairy Manure-Based Amendments. Frontiers in Sustainable Agriculture. 3:22. https://doi.org/10.3389/fsufs.2019.00022
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dharmarha, V.; Pulido, N., Boyer, R.R.; Pruden, A.; Strawn, L.K., Ponder, M.A. (2019). Effect of Post-harvest Interventions on Surficial Carrot Bacterial Community Dynamics, Pathogen survival, and Antibiotic Resistance. International Journal of Food Microbiology 291:25-34. doi: 10.1016/j.ijfoodmicro.2018.11.006.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wind, L.; Krometis, L-A; Hession, W.C.; Chen, C.; Du, P.; Jacobs, K.; Xia, K.; Pruden, A. (2018). Fate of Pirlimycin and Antibiotic-Resistant Fecal Coliforms in Field Plots Amended with Dairy Manure or Compost during Vegetable Cultivation. Journal of Environmental Quality. 47(3): 436-444. doi:10.2134/jeq2017.12.0491
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Chen, C.; Guron, G.K.*; Pruden, A.; Ponder, M.; Du, P.; Xia, K. (2018). Antibiotics and antibiotic resistance genes in bulk and rhizosphere soils subject to manure amendment and vegetable cultivation. Journal of Environmental Quality. doi: 10.2134/jeq2018.02.0078.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Chen, C.; Ray, P.*; Knowlton, K.F.; Pruden, A.; Xia, K. (2018). Effect of composting and soil type on dissipation of veterinary antibiotics in land-applied manures. Chemosphere. 196:270-279. doi.org/10.1016/j.chemosphere.2017.12.161.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Le, H.T.V.; Maguire, R.O.; Xia, K. (2018). Method of Dairy Manure Application and Time before Rainfall Affect Antibiotics in Surface Runoff. Journal of Environmental Quality. doi:10.2134/jeq2018.02.0086
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen, C.; Xia, K. (2017). Fate of Land Applied Emerging Organic Contaminants in Waste Materials. Current Pollution Review. 3:38-54.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ray, P., Chen, C., Knowlton, K.F., Pruden, A., and Xia, K. (2017). Fate and effect of antibiotics in beef and dairy manure during static and turned composting. Journal of Environmental Quality. 46(1):45-54. DOI: 10.2134/jeq2016.07.0269
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Arango-Argoty, G., Singh, G., Heath, L.S., Pruden, A., Xiao, W., and Zhang, L. MetaStorm: A Public Resource for Customizable Metagenomics Annotation. (2016). PLoS One. doi.org/10.1371/journal.pone.0162442.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chambers, L.R., Yang, Y., Littier, H., Ray, P., Zhang, T., Pruden, A., Strickland, M. and K.F. Knowlton. (2015). Metagenomic Analysis of Antibiotic Resistance Genes in Dairy Cow Feces following Therapeutic Administration of Third Generation Cephalosporin. PLoS One. 10(8): e0133764.
• Type: Other Status: Published Year Published: 2017 Citation: Claire Gleason, Ellen Maurer, Katharine Knowlton, and Amber Vallotton. 2017. Battling Resistance: Judicious Antibiotic Use in Beef and Dairy Cattle. Extension Fact Sheet DASC-106NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Katharine Knowlton, Amy Pruden, and Amber Vallotton. 2017. What is the Veterinary Feed Directive? Extension Fact Sheet HORT-262NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. The Phenomenon of Antimicrobial Resistance: A One-Health Issue. Extension Fact Sheet. HORT-259NP
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. How Do We Measure Antimicrobial Resistance? Extension Fact Sheet HORT-258NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. Antimicrobial Resistance: What is it and Whats at Stake? Extension Fact Sheet HORT-257NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. The Journey of Antibiotics from Farm to Fork. Extension Fact Sheet HORT-261NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. Understanding the Public Health Risks of Antimicrobial Resistance. Extension Fact Sheet HORT-260NP.
• Type: Websites Status: Published Year Published: 2015 Citation: Reducing Antibiotic Resistance from Farm to Fork. https://www.args.hort.vt.edu/
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Plenary Keynote Lecture by Pruden, A. Metagenomic Insight into Preventing the Spread of Antibiotic Resistance from Farm to Fork. One Health & Food Safety Congress 2018. Bonn, Germany, Sept. 19 (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Plenary Keynote Lecture by Pruden, A. Critical Control Points for Preventing the Spread of Antibiotic Resistance from Cattle Manure to Fresh Produce. World Congress on Animal Production. Vancouver, BC, Canada, July 5-12 (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Plenary Lecture by Pruden, A. Antibiotic Resistance in the Water Environment: What Do We Monitor? What Do We Mitigate? Gordon Research Conference. Environmental Science: Water. Holderness, NH, June 24-29 (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Plenary Keynote by Pruden, A. ARGs as Contaminants of Emerging Concern: Towards Actionable Policy. International Water Association (IWA) Leading Edge Technology (LET) Conference, May 27-30, Nanjing, China (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Symposium Keynote by Pruden, A. Critical Control Points for AMR from Farm to Fork. 29th Annual Virginia-Maryland Veterinary College of Medicine Research Symposium, March 15 (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Invited Symposium Speaker, Pruden, A. Tracking AMR from agroecosystems to fresh produce. Symposium on Minimizing the Risk of Antimicrobial Resistance from Food Animal Production, New York Academy of Sciences (NYAS), Sept 28, New York, NY (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Invited Symposium Speaker, Pruden, A. Environmental assessment and analysis for control of antibiotic resistance in the environment. Presidential Advisory Council on Combating Antibiotic Resistant Bacteria (PACCARB) Public Meeting, Sept 27, Columbus, OH (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Invited Symposium Speaker, Ponder, M. A Metagenomic Odyssey to Assess Contributions of Antibiotic-resistance Genes from Biological Soil Amendments to Fresh Produce. Symposium on Understanding Antibiotic Resistance from an Environmental Perspective, International Association for Food Protection General Meeting 2018, July 11, Salt Lake City, UT (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Invited Seminar by Pruden, A. Critical Control Points for AMR from Farm to Fork. University of Minnesota Vet School, March 8 (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Invited Seminar by Pruden, A. Critical Control Points for AMR from Farm to Fork. Workshop on Management and Control of Antibiotics and Antibiotic Resistance in Environment. Beijing, China Dec. 4-6 (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Keynote Lecture by Pruden, A. Antimicrobial Resistance in Wastewater Treatment Plants: A Call to Action. International Water Association Specialty Group on Microbial Ecology and Water Engineering. Sept. 4-6, Copenhagen, Denmark (2016)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Invited Speaker, Guron, G. Metagenomic Profiling of Antibiotic Resistance Genes Associated with Lettuce Leaf Surfaces Grown in Soils Receiving Cattle Manure-Based Amendments International Association for Food Protection General Meeting 2018, July 9, Salt Lake City, UT (2018)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Invited Plenary Lecture by Pruden, A. Synergizing Water Sustainability and Public Health: Antibiotic Resistance and Opportunistic Pathogens in Recycled Water. International Water Association Leading Edge Technology Conference (LET 2016), June, Jerez de la Frontera, Spain (June 13-16, 2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Poster Presentation by Wind, L., Krometis, L.A., Hession, W.C., Chen, C., Jacobs, K., Du, P., and Pruden, A. Tracking antibiotic resistance along the Farm to Fork agricultural production chain. November 10-14, 2018. American Public Health Association Annual Meeting. San Diego, CA. (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Poster Presentation by Dharmarha, V., Trimble, K., Guron, G., Niemera, BK, Pruden, A., Boyer, R.R., Strawn, L.K and Ponder, M.A. Gamma Irradiation Reduces the Survival and Regrowth of Inoculated Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes on Romaine Lettuce. International Association for Food Protection General Meeting 2018, July 11, Salt Lake City, UT (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Short Talk and Poster Presentation by Wind, L., Krometis, L.A., Hession, W.C., Chen, C., Jacobs, K., Du, P., Xia, K., and Pruden, A. Parallels among culturable antibiotic-resistant fecal coliforms and resistance genes from soils amended with dairy manure or compost during vegetable cultivation. June 7-11, 2018. American Society for Microbiology Annual Meeting. Atlanta, Georgia (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Poster Presentation by Fogler, K., Guron, G., Wind, L, Krometis, LA, Hession, WC, Pruden, A., and Ponder, M.A. Antibiotic Resistance Genes on Lettuce and Radishes Field-Grown in Soils Amended with Manure or Compost from Antibiotic-Treated Cattle. International Association for Food Protection General Meeting 2018, July 10, Salt Lake City, UT (2018).
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Oral Presentation by Keenum, I.*,Williams,R., Ray P., Guron, G. ,Garner,E., Knowlton,K. and Pruden,A. Metagenomic and ARG Shifts During Composting of Antibiotic Treated Beef and Dairy Cattle Manure. American Chemical Society General Meeting 2018, March 18, New Orleans, LA (2018)
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Wind, L., Krometis, L.A., Hession, W.C., Chen, C., Jacobs, K., Du, P., Xia, K., and Pruden, A. Environmental dissemination of antibiotic resistance in manure-amended agricultural fields. August 13- 17, 2017. 4th International Symposium on the Environmental Dimension of Antibiotic Resistance. Lansing, Michigan (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Wickware, C., Garner, E., Guron, G., Williams, R., Krometis, L. A., Ponder, M., Pruden, A. Phenotypic, phylogenetic, and functional shifts in antibiotic resistant Gammaproteobacteria following composting of cattle manure. International Symposium on the Environmental Dimension of Antibiotic Resistance. August 13-17, 2017, Lansing, MI.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Guron, G.*, Ponder, M.A., Pruden, A. Quantifying sulfonamide resistance genes on lettuce grown in soil amended with manure and compost from antibiotic-treated cattle. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Guron, G.*, Ponder, M.A., Pruden, A. Metagenomic analysis of antibiotic resistance genes and mobile genetic elements found on radish taproots grown in soils amended with manure and compost from cattle. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Fogler, K., Guron, G.*, Ponder, M., and Pruden, A. Microbial Community Analysis of Field Grown Produce in Soil Amended with Manure in Compost from Antibiotic Treated Cattle. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Dharmarha, V., Pulido, N.A., Guron, G., Ponder, M.A., Pruden, A., Boyer, R.R., Strawn, L.K. Storage temperature and sanitizer washing influences bacterial community dynamics on carrots. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Wind, L., Jacobs, K., Cully, W.C., Krometis, L.A., Du, P., Chen, C., Pruden, A. Persistence of culturable antibiotic-resistant fecal coliforms from manure-amended vegetable fields: One of the Puzzle Pieces Connecting Antibiotic Resistance from Farm to Fork. 17th Annual Meeting of the American Ecological Engineering Society "Ecological Engineering for Adaption in the Anthropocene". May 23-25, Athens, GA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Jacobs, K., Wind, L, Krometis, LA, Hession W.C., Pruden, A. Indicator bacteria, sediment, and antibiotic-resistance gene transport of via runoff from vegetable plots amended with manure. 17th Annual Meeting of the American Ecological Engineering Society "Ecological Engineering for Adaption in the Anthropocene". May 23-25, Athens, GA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Guron, G., Ponder, M., and Pruden, A. Characterizing the communities found from radishes grown in manure-based soil amendments from cattle administered antibiotics. ASM Conference on Innovative Microbial Ecology for Mitigation of Antibiotic Resistance and Bacterial Diseases, March 22-25, Crystal City, VA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Pankow, C., Chen, C., Williams, R., Guron, G., Xia, K., Ponder, M., and Pruden, A. Effect of soil type, composting, and antibiotic use on fate of antibiotic resistance genes in dairy manure applied soil. ASM Conference on Innovative Microbial Ecology for Mitigation of Antibiotic Resistance and Bacterial Diseases, March 22-25, Crystal City, VA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pankow, C., Guron, G., Williams, R., Chen, C., Pruden, A., and Ponder, M. Assessing the potential for the spread of antibiotic resistance genes from cattle manure-based soil amendments to fresh radishes. American Society for Microbiology- Virginia Branch Annual Meeting, November 6-7, Richmond, VA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Guron, G., Chen, C., Ray, P., Pruden, A., and Ponder, M. Quantifying antibiotic resistance genes associated with compost-amended greenhouse radish taproots. American Society for Microbiology- Virginia Branch Annual Meeting, November 6-7, Richmond, VA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Williams, R., Ray, P., Knowlton, K.F., Krometis, L-A, Ponder, M. and Pruden, A. Effect of Composting Method and Antibiotic Residue in Manure on the Fate of E. coli and Antibiotic Resistance Genes in Cattle Manures. American Society for Microbiology Microbe 2016, June 16-18, Boston, MA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pankow, C., Chen, C., Williams, R., Xia, K., Ponder, M. and Pruden, A. Effect of soil type, composting, and antibiotic use on fate of antibiotic resistant bacteria and resistance genes in dairy and beef manure applied soil. American Society for Microbiology Microbe 2016, June 16-18, Boston, MA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pankow, C., Guron, G., Williams, R., Chen, C., Pruden, A., and Ponder, M. Assessing the potential for the spread of antibiotic resistance genes from cattle manure-based soil amendments to fresh radishes. American Society for Microbiology- Virginia Branch Annual Meeting, November 607, Richmond, VA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Guron, G., Chen, C., Ray, P., Pruden, A., and Ponder, M. Quantifying antibiotic resistance genes associated with compost-amended greenhouse radish taproots. American Society for Microbiology- Virginia Branch Annual Meeting, November 607, Richmond, VA (2016).

Progress 02/01/17 to 01/31/18

Outputs
Target Audience:Target audiences reached this reporting period included extension agents, students, and various scientific communities. Extension agents and the growers and producers that they serve were reached through publication of 7 fact sheets providing educational material related to agricultural sources of antibiotic resistance and the role of pre- and post-harvest management practices. Twenty undergraduates, twelve graduate students, and two post-doctoral researchers contributed to this effort during this period. Four graduate students completed their Masters degrees and published their theses, one in Food Science & Technology, one in Environmental Engineering, and two in Biological Systems Engineering. Additionally CEE 5194 Environmental Engineering Microbiology, FST 4634/5634g Epidemiology of Food and Waterborne Disease, FST/BIOL 3604 Food Microbiology were taught during this reporting period and included modules related to agricultural sources of antibiotic resistance. Within the scientific community, presentations were delivered to the American Chemical Society, The 4th International Symposium on the Environmental Dimension of Antibiotic Resistance, the International Association for Food Protection, and a Workshop on Management and Control of Antibiotics and Antibiotic Resistance in Environment. Beijing, China Changes/Problems:We will require and request a one year no cost extension to complete analysis and publication of the key findings of this study. What opportunities for training and professional development has the project provided?Four Masters students completed and published their theses during this reporting period. Two decided to continue for a PhD, one took a job in environmental consulting, and the fourth is now working in food safety for a major US corporation. One post-doc completed his project and took a position as a professor in Dairy Science at the University of Reading in the U.K. Several of the undergraduates have continued to contribute to the research, many of them gaining their first lab experience, and also have contributed to the preparation of education, outreach, and extension materials by assisting with videos and PI and student interviews. How have the results been disseminated to communities of interest?• Academics- Two key journal articles were published during this reporting period. One was focused on the fate of antibiotics during the controlled composting study. The second was a review articla on the fate of chemical contminants, including antibiotics, in land-applied wastes and residuals. One major invited talk was delivered at an antibiotic resistance symposium in China. Ten oral or poaster presentations were delivered at conferenes, including the International Association for Food Protection, the 4th International Environmental Dimension of Antibiotic Resistance Conference, the Ecological Engineering for Adaption in the Anthropocene, and the ASM Conference on Innovative Microbial Ecology for Mitigation of Antibiotic Resistance and Bacterial Diseases. • Extension Agents, Veterinarians, Producers, Growers, and Dairymen. We published seven extension fact sheets on agricultural sources of antibiotic resistance as a resource to these communities. • Students- Through the AgSOAR educational program, several undergraduate, graduate, and post-doctoral researchers participated directly in this effort, in addition to many more students that enrolled in and completed the classes. Four Masters students graduated and published their Masters' thesis during this reporting period. Three of the courses were taught during this period. What do you plan to do during the next reporting period to accomplish the goals?We have nearly completed the experiments at this stage of the effort, except for experiments related to gamma irradiation of lettuce. These experiments will be completed within the next six months. However, substantial effort is needed to appropriately analyze the data and write up manuscripts. We will request a one year no cost extension in order to make sure that the main findings of this project are published or submitted for publication by the end of this project. We will also devote substantial effort towards the teaching of the capstone AgSOAR Farm to Fork AMR class and to extension and education via preparation of an educational animation.

Impacts
What was accomplished under these goals? Composting experiments were complete and the results of the fate of antibiotics were published (Ray et al. 2017). Composting was found to enhance dissipation of antibiotics (chlortetracycline, sulfamethazine, tylosin, pirlimycin, and cephapirin) in manure. Tylosin was the most persistent, and appeared to revert from a metabolite back to the parent compound during the initial phase of composting. Analysis of metagenomic and qPCR data is underway to understand the fate of the ARGs during these experiments and is presented in the Masters Thesis of Robert Williams. The field study was completed in 2016 and some of the data was analyzed and published during this reporting period in 2017. Studies focused on the fate of the antibiotic resistant bacteria in soil were published in Lauren Wind's Masters thesis and in a corresponding journal article (Wind et al. 2018). In brief, the findings were that antibiotic-resistant fecal coliforms were elevated in soil initially after amending with manure or compost versus chemical fertilizer. The magnitude of observed coliform counts differed by soil amendment type and type of antibiotic resistance assayed. Compost-amended soils had the highest levels of cephalosporin- resistant fecal coliforms, regardless of whether the cows from which the manure was derived were administered antibiotics. Samples from control plots or those treated with inorganic fertilizer trended toward lower counts of resistant coliforms, although these differences were not statistically significant. No statistical differences were observed between soils that grew leafy (lettuce) versus rooted (radish) crops. A second Masters thesis from the field study focused on the fate of antibiotic resistant bacteria and ARGs in runoff. Runoff from all soil plots, including controls that were not amended with manure or compost, yielded ARGs in associated runoff. Compost and manure-amended soils resulted in higher levels of target ARGs (sul1 and ermB) in runoff. Vegetable type influenced sediment concentrations in runoff, but not ARGs, in runoff. Further detail may be found in Kyle Jacob's Masters thesis. Analysis of the antibiotic resistant bacteria and ARGs associated with surfaces of vegetables harvested in the field study is presented in the Masters thesis of Kendall Fogler. In brief, it was found that both lettuce and radish carried more ARGs when soil was amended with manure or compost versus chemical fertilizer. Lettuce were more affected by amendments than radish, although radish carried more total antibiotic resistance genes than lettuce. Effects of collecting manure during antibiotic use was measureable in lettuce resistomes. Other major accomplishments in terms of extension and education included the publication of seven new fact sheets focused helping extension agents educate their constituents about agricultural sources of antibiotic resistance and also the teaching of three of the core classes in the AgSOAR curriculum.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Pankow, Christine A. 2017 Effect of Soil Type, Composting, and Antibiotic Use on Fate of Antibiotic Resistance Genes and Microbial Community Composition in Dairy and Beef Manure Applied Soils. Masters Thesis. Civil Engineering. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Wind, Lauren. 2017. PERSISTENCE OF CULTURABLE ANTIBIOTIC RESISTANT FECAL COLIFORMS IN MANURE AMENDED VEGETABLE FIELDS. Masters Thesis. Biological Systems Engineering. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Fogler, Kendall W. 2017. Effect of Soil Amendments from Antibiotic Treated Cows on Antibiotic Resistant Bacteria & Genes Recovered from the Surfaces of Lettuce and Radishes: Field Study. Masters Thesis. Food Science and Technology. Virginia Tech.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Jacobs, Kyle B. 2017. RECOVERY OF ANTIBIOTIC RESISTANCE GENES FROM AGRICULTURAL RUNOFF. Masters Thesis. Biological Systems Engineering. Virginia Tech.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen, C.; Xia, K. (2017). Fate of Land Applied Emerging Organic Contaminants in Waste Materials. Current Pollution Review. 3:38-54.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ray, P., Chen, C., Knowlton, K.F., Pruden, A., and Xia, K. (2017). Fate and effect of antibiotics in beef and dairy manure during static and turned composting. Journal of Environmental Quality. 46(1):45-54. DOI: 10.2134/jeq2016.07.0269
• Type: Other Status: Published Year Published: 2017 Citation: Claire Gleason, Ellen Maurer, Katharine Knowlton, and Amber Vallotton. 2017. Battling Resistance: Judicious Antibiotic Use in Beef and Dairy Cattle. Extension Fact Sheet DASC-106NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Katharine Knowlton, Amy Pruden, and Amber Vallotton. 2017. What is the Veterinary Feed Directive? Extension Fact Sheet HORT-262NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. The Phenomenon of Antimicrobial Resistance: A One-Health Issue. Extension Fact Sheet. HORT-259NP
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. How Do We Measure Antimicrobial Resistance? Extension Fact Sheet HORT-258NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. Antimicrobial Resistance: What is it and Whats at Stake? Extension Fact Sheet HORT-257NP.
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. The Journey of Antibiotics from Farm to Fork. Extension Fact Sheet HORT-261NP.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Guron, G.*, Ponder, M.A., Pruden, A. Quantifying sulfonamide resistance genes on lettuce grown in soil amended with manure and compost from antibiotic-treated cattle. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Guron, G., Ponder, M.A., Pruden, A. Metagenomic analysis of antibiotic resistance genes and mobile genetic elements found on radish taproots grown in soils amended with manure and compost from cattle. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Fogler, K., Guron, G.*, Ponder, M., and Pruden, A. Microbial Community Analysis of Field Grown Produce in Soil Amended with Manure in Compost from Antibiotic Treated Cattle. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Dharmarha, V., Pulido, N.A., Guron, G., Ponder, M.A., Pruden, A., Boyer, R.R., Strawn, L.K. Storage temperature and sanitizer washing influences bacterial community dynamics on carrots. International Association for Food Protection (IAFP), July 9-12, Tampa, FL (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Wind, L., Jacobs, K., Cully, W.C., Krometis, L.A., Du, P., Chen, C., Pruden, A. Persistence of culturable antibiotic-resistant fecal coliforms from manure-amended vegetable fields: One of the Puzzle Pieces Connecting Antibiotic Resistance from Farm to Fork. 17th Annual Meeting of the American Ecological Engineering Society "Ecological Engineering for Adaption in the Anthropocene". May 23-25, Athens, GA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Jacobs, K., Wind, L, Krometis, LA, Hession W.C., Pruden, A. Indicator bacteria, sediment, and antibiotic-resistance gene transport of via runoff from vegetable plots amended with manure. 17th Annual Meeting of the American Ecological Engineering Society "Ecological Engineering for Adaption in the Anthropocene". May 23-25, Athens, GA (2017).
• Type: Other Status: Published Year Published: 2017 Citation: Ellen Maurer, Amber Vallotton, and Amy Pruden. 2017. Understanding the Public Health Risks of Antimicrobial Resistance. Extension Fact Sheet HORT-260NP.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Invited Seminar by Pruden, A. Critical Control Points for AMR from Farm to Fork. Workshop on Management and Control of Antibiotics and Antibiotic Resistance in Environment. Beijing, China Dec. 4-6 (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Wind, L., Krometis, L.A., Hession, W.C., Chen, C., Jacobs, K., Du, P., Xia, K., and Pruden, A. Environmental dissemination of antibiotic resistance in manure-amended agricultural fields. August 13- 17, 2017. 4th International Symposium on the Environmental Dimension of Antibiotic Resistance. Lansing, Michigan (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Wickware, C., Garner, E., Guron, G., Williams, R., Krometis, L. A., Ponder, M., Pruden, A. Phenotypic, phylogenetic, and functional shifts in antibiotic resistant Gammaproteobacteria following composting of cattle manure. International Symposium on the Environmental Dimension of Antibiotic Resistance. August 13-17, 2017, Lansing, MI.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Guron, G., Ponder, M., and Pruden, A. Characterizing the communities found from radishes grown in manure-based soil amendments from cattle administered antibiotics. ASM Conference on Innovative Microbial Ecology for Mitigation of Antibiotic Resistance and Bacterial Diseases, March 22-25, Crystal City, VA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poster Presentation by Pankow, C., Chen, C., Williams, R., Guron, G., Xia, K., Ponder, M., and Pruden, A. Effect of soil type, composting, and antibiotic use on fate of antibiotic resistance genes in dairy manure applied soil. ASM Conference on Innovative Microbial Ecology for Mitigation of Antibiotic Resistance and Bacterial Diseases, March 22-25, Crystal City, VA (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Chen, C., Guron, G., Pruden, A., Ponder, M., and Xia, K. Antibiotics and ARGs in manure-amended soils growing vegetables. 254nd American Chemical Society National Meeting. August 20-24, Washington, D.C. (2017).
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Oral Presentation by Chen, C. and Xia, K. Effect of Earthworm Activity on the Fate of Antibiotics, ARB, and ARGs in a Compost Amended Soil. August 20-24, Washington, D.C. (2017).

Progress 02/01/16 to 01/31/17

Outputs
Target Audience:During the second year of the project, we began to execute our communications and marketing plan that we developed in the first year, including building and strengthening the networks of stakeholders that will allow for effective translational research. As a core aspect of this effort, we constructed a stakeholder map to define the target audiences and guide direction of dissemination of results as the project progresses. The stakeholder map incorporates the schemata of the project logic models and includes: formal educational institutions, non-formal educational trainers, dairy and livestock producers, produce growers, consumers, industry stakeholders, and policy-makers at state, regional, and national levels) for our various efforts. Specific accomplishments include: • Maintaining an up-to-date website to provide the latest project information and resources to a wide variety of stakeholders and the general public: http://www.hort.vt.edu/args/index.html • Launched the Agricultural SOurces of Antibiotic Resistance (AgSoAR) program. • Undergraduate research practicums for an additional 12 undergraduate students. • Graduate research practicums for an additional 12 graduate students to date, with two MS and one MPH students completing to date. • Carried out survey of antibiotic use practices among livestock operators in Virginia. • Developed instructional modules on antibiotic resistance for ten courses to date, serving a total of 306 students to date. • Student projects focused on ethics of antibiotic use in livestock and tracking antibiotic resistance in the environment • On-site advisory board meeting held in October 2016. Included employees of USDA ARS, a progressive Virginia Dairy Farmer, an area extension specialist on horticulture, small fruit, and specialty crops, Associate-Director of the University of Arizona Water Resources Research Center, and a Research Scientist at AgriFood Canada. Our Stakeholder map has a comprehensive summary of our target audiences: Target audiences included: Students in multiple disciplines (e.g., Environmental Engineering, Biological Systems Engineering, Animal Sciences, Soil Science, Food Science & Technology, Horticulture, Education, Public Health, and Extension). Scientists in multiple disciplines (e.g., Environmental Engineering, Biological Systems Engineering, Animal Sciences, Soil Science, Food Science & Technology, Horticulture, Education, and Extension). Veterinarians Growers Dairy Farmers Cattlemen General public Changes/Problems:As the reviewers of the original proposal noted that the proposed project was highly ambitious, we have been able to achieve ~85% of the objectives as originally proposed. Nonetheless, a few unexpected results and obstacles have been encountered, which have required us to repeat and/or redesign experiments or eliminate some experimental conditions. Broccoli has been particularly problematic, as it did not grow well in the greenhouse. After two attempts, we were unable to stimulate the broccoli to crown. As a compromise, the aerial surfaces of the broccoli were sampled instead of the crown. Correspondingly, broccoli was dropped from the post-harvest washing study because the number of experimental conditions was unmanageable and for concordance with the greenhouse study. The upcoming irradiation study will focus on lettuce because most work on pathogens to date has been done doing lettuce, which provides a point of reference in extending this knowledge towards understanding effects on antibiotic resistant bacteria. Broccoli was also eliminated from the field study, for concordance with the greenhouse and post-harvest study, with the design simplified to focus on lettuce and radishes. Elimination of the broccoli condition made it possible to add control plots without planting, including plots with fertilizer only and plots with no amendment or fertilizer (background). What opportunities for training and professional development has the project provided?This project has served as an ideal interdisciplinary training ground for undergraduate students (n=24 to date), graduate students (n=12 to date), and post-doctoral researchers (n=3 to date). The students and post-docs work along with the PD and Co-PDs as part of an integrated team. The full project team meets on a monthly basis, with the individual objective teams meeting regularly between full project team meetings. As part of the Agricultural Sources of Antibiotic Resistance (AgSoAR) program, undergraduate students gained hands-on research experience, with the graduate students and postdoctoral researchers gaining mentoring experience. New course modules on antibiotic resistance were developed for eleven courses across six departments to date. Together the courses associated with this project have served 75 students in AY 2015 and 231 students in AY 2016. Students are co-authors on papers and presentations and presented at four different conferences, one winning a best student presentation award. The students also had the opportunity to meet and interact with the advisory board, which consists of employees of USDA ARS, faculty at other universities, a member of the World Health Organization, dairy farmers, and growers. How have the results been disseminated to communities of interest?Our extension team has developed a stakeholder map to guide dissemination and impact of this project. The stakeholder map includes formal educational institutions, non-formal educational trainers, dairy and livestock producers, produce growers, consumers, industry stakeholders, and policy-makers at state, regional, and national levels. We launched our website to reach a wide variety of audiences with the latest information and happenings about the project: http://www.hort.vt.edu/args/index.html. Our advisory board includes a dairy farmer and a grower, and we regularly interact with them to share information and also request input to the project. We have also published two peer-reviewed articles related to this work. The first is focused on excretion of ARGs by dairy cows administered antibiotics (Chambers et al., PLoS ONE, 2015), the second is focused on the fate of antibiotics during composting (Ray et al., JEQ, 2016), and the third examines the fate of antibiotics in manure applied to soil (Kulesza et al. JEQ, 2016). To date, the PD delivered four keynotes and other invited lectures and other members of the team delivered a total of nine conference presentations. Two on-site meetings have been held with the advisory board, where results were further discussed and disseminated. We have also maintained a website with up to date project information and have maintained a presence on Facebook and Instagram, to build awareness about the project. What do you plan to do during the next reporting period to accomplish the goals?1. Objective 1: Prepare manuscripts related to excretion of antibiotics in beef cattle. Present at conference and provide input to education and extension materials. 2. Objective 2: Prepare manuscript describing the effect of prior antibiotic dosing and composting on the fate of ARBs and ARGs in dairy and beef manure. Present at conference and provide input to education and extension materials. 3. Objective 3: Prepare two manuscript describing the effect of soil type on the fate of ARBs and ARGs in dairy and beef manure and compost. Prepare a manuscript describing the effect of soil type on the fate of antibiotics in dairy and beef manure and compost. Present at conference and provide input to education and extension materials. 4. Objective 4: Complete analysis of greenhouse examining the interactive effects of the different types of compost on two vegetables (lettuce and radish) grown in two types of soil (sand and loam). Complete analysis of field study examining the effects of prior antibiotic dosing and composting of dairy manure on the fate of ARBs and ARGs in soil and on vegetable surfaces (lettuce and radish). Prepare manuscripts, present at conference and provide input to education and extension materials. 5. Objective 5: Carry out lettuce irradiation study. Complete analysis of stored lettuce samples in terms of microbiome analysis, metagenomics, and qPCR. Prepare and submit manuscripts for each vegetable type. 6. Objective 6: Continue to teach the core classes and modules developed here. Mentor undergraduate and graduate training practicums via AgSOAR. Evaluate the effectiveness of student training program. 7. Objective 7: As research phase is winding down and more results become available, disseminate findings to scientific and agricultural community via extension. We will finalize online video content and operate booths at local fairs and other events. Evaluate the effectiveness of extension program.

Impacts
What was accomplished under these goals? 1. Objective 1 experiments are complete. Both dairy and beef cattle antibiotic dosing experiments are concluded. Antibiotic analysis of the manure and urine is complete and a manuscript is under preparation. Fecal samples from the beef dosing study have been preserved for later ARG analysis. Results of ARG analysis of dairy manure during antibiotic dosing has been published in PLoS ONE. Results of beef manure excretion are in preparation for publication. 2. Objective 2 experiments are complete. A study was conducted comparing static versus turned composting of beef manure, with and without antibiotic (sulfamethazine, tylosin, chlortetracycline, and tetracycline) administration, and dairy manure, with and without antibiotic (pirlimycin and cephapirin) administration. This resulted in an experimental matrix of 8 conditions carried out in triplicate, accounting for 24 compost piles. The large number of conditions necessitated small-scale composters, which were insulated and equipped with temperature sensors and subject to manual turning twice a day for 42 days. A second run of composting was conducted for the dairy manure, with and without antibiotics, under static conditions only and manually forced to maintain >55ºC for 14 days followed by an ambient curation stage out to 42 days. Finally, a third field-scale trial was carried out with static dairy manure compost, with and without prior antibiotic administration, versus just stockpiling dairy manure with prior antibiotic treatment. Antibiotic analysis has been completed now on all compost samples. E. coli was enumerated and a sub-set was assayed for resistance to the classes of antibiotics administered using Kirby-Bauer. These isolates have been submitted for whole-genome sequencing. In the mid-and field-scale compost, enterics and heterotrophs were directly enumerated on plates with and without antibiotics amended. Metagenomic DNA sequencing and 16S rRNA gene amplicon sequencing is now complete for all compost samples, as is qPCR of tet(W) and sul1. One manuscript about the fate of antibiotics during composting has been accepted for publication in Journal of Environmental Quality and one MS thesis has been completed on the fate of ARBs and ARGs. 3. Objective 3 experiments are complete. Microcosms were set up with three kinds of soils (sandy, loamy, and clayey) and amended with all 8 types of compost produced in objective 2 plus the four raw manures, for a total of 36 conditions. Microcosms were set up in small jars for sacrificial sampling of triplicate samples over a 120 day period (for consistency with latest FSMA guidelines regarding land-application), with moisture maintained at 50% field capacity. Samples have been collected and preserved. Antibiotic analysis is complete, as is metagenomics analysis and qPCR of 16S rRNA genes, tet(W) and sul1. One manuscript has been published in Journal of Environmental Quality and a second manuscript is in preparation regarding the fate of the antibiotics. 4. Objective 4 experiments are complete. The second full-scale greenhouse experiment successfully produced radishes and lettuce, although the broccoli never crowned. Two types of soil amended with the same 8 types of compost and four manure types were investigated, for a total of 24 conditions for each vegetable in the greenhouse. In the field, radishes and lettuce were grown on plots amended with dairy compost with and without a prior history of antibiotic use and compared to amendment with stockpiled dairy manure with prior history of antibiotics (positive control). Plots without planting, with only fertilizer (fertilizer control) or no amendment (no amendment control) were also monitored. Radishes and lettuce were harvested in summer 2016. Plate counting from vegetable washes and soil are complete for both the greenhouse and field study. qPCR for tet(W) is complete for the greenhouse study and vegetable wash samples have been submitted for metagenomics sequencing. qPCR analysis of tet(W) and sul1 is just getting underway for the field study. Antibiotic analysis is also complete, but antibiotics were only generally detectable in the soil on day zero. A manuscript is currently in preparation about the radishes. 5. Objective 5 experiments are about 80% complete. A study was carried out by inoculating lettuce and carrots from the retail store with diluted compost and Pseudomonas spiked in. Different post-harvest washing practices were tested, including washing in water dosed with chlorine or peracetic acid, versus tap water. The carrot study is complete and a manuscript is currently under preparation. The lettuce samples remain to be analyzed. A lettuce irradiation experiment is pending. 6. Objective 6 is integrated throughout the project, with major milestones being new modules taught in five courses across five departments and 12 additional undergraduate practicums for 2016. 7. Objective 7 is integrated throughout the project, with major milestones being development of the Stakeholder Map, the launching of the website, production of YouTube videos, and a field study of antibiotic use practices in Virginia. A second on-site advisory board meeting was held in October 2016 and input was provided in evaluation of the project.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chambers, L.R., Yang, Y., Littier, H., Ray, P., Zhang, T., Pruden, A., Strickland, M. and K.F. Knowlton. (2015). Metagenomic Analysis of Antibiotic Resistance Genes in Dairy Cow Feces following Therapeutic Administration of Third Generation Cephalosporin. PLoS One. 10(8): e0133764.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kulesza, S. B, J. Cushman, K. Knowlton, R. O. Maguire , P. Ray, and K. Xia. 2016. Manure injection impacts fate of pirlimycin in surface runoff and soil. J. Environ. Qual. 45: 511-518.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Williams, R., Ray, P., Knowlton, K.F., Krometis, L-A, Ponder, M. and Pruden, A. Effect of Composting Method and Antibiotic Residue in Manure on the Fate of E. coli and Antibiotic Resistance Genes in Cattle Manures. American Society for Microbiology Microbe2016, June 16-18, Boston, MA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pankow, C., Chen, C., Williams, R., Xia, K., Ponder, M. and Pruden, A. Effect of soil type, composting, and antibiotic use on fate of antibiotic resistant bacteria and resistance genes in dairy and beef manure applied soil. American Society for Microbiology Microbe2016, June 16-18, Boston, MA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pankow, C., Guron, G., Williams, R., Chen, C., Pruden, A., and Ponder, M. Assessing the potential for the spread of antibiotic resistance genes from cattle manure-based soil amendments to fresh radishes. American Society for Microbiology- Virginia Branch Annual Meeting, November 607, Richmond, VA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Guron, G., Chen, C., Ray, P., Pruden, A., and Ponder, M. Quantifying antibiotic resistance genes associated with compost-amended greenhouse radish taproots. American Society for Microbiology- Virginia Branch Annual Meeting, November 607, Richmond, VA (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Guron, G., Ray, P., Ponder, M., and Pruden, A. Assessing the potential for antibiotic resistant bacteria and resistance genes to carry over from soil amendments to vegetable surfaces: A greenhouse study. International Association for Food Protection (IAFP), July 31-Aug 2, St. Louis, MO (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Pulido, N., Dharmarha, V., Ponder, M., Pruden, A., and Boyer, R. Effect of Sanitizers on the Survival of Antibiotic-resistant Bacteria Applied to Raw Carrots through Contaminated Compost. International Association for Food Protection (IAFP), July 31-Aug 2, St. Louis, MO (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poster Presentation by Wind, L., Jacobs, K., Krometis, L., Hession, C., and A. Pruden. Environmental dissemination of antibiotic resistance genes from vegetable production. American Ecological Engineering Society Annual Meeting, June 7-9, Knoxville, TN (2016).
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ray, P., Chen, C., Knowlton, K.F., Pruden, A., and Xia, K. (2017). Fate and effect of antibiotics in beef and dairy manure during static and turned composting. Journal of Environmental Quality. 46(1):45-54. DOI: 10.2134/jeq2016.07.0269
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Invited Plenary by Pruden, A. Mitigation Strategies for Environmental Sources of Antibiotic Resistance. Environmental Dimensions of Antibiotic Resistance (EDAR). May 17-21, Wernigerode, Germany (2015).
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Invited Plenary Lecture by Pruden, A. How do We Combat Antibiotic Resistance? Human Progress and the Laws of Nature. Fate of the Earth Symposium, Michigan State University, Lansing, MI. April 1-3 (2015)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Keynote Lecture by Pruden, A. Antimicrobial Resistance in Wastewater Treatment Plants: A Call to Action. International Water Association Specialty Group on Microbial Ecology and Water Engineering. Sept. 4-6, Copenhagen, Denmark (2016)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Invited Plenary Lecture by Pruden, A. Synergizing Water Sustainability and Public Health: Antibiotic Resistance and Opportunistic Pathogens in Recycled Water. International Water Association Leading Edge Technology Conference (LET 2016), June, Jerez de la Frontera, Spain, June 13-16, (2016).
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Oral Presentation by Le, H., R. O. Maguire, and K. Xia. Effects of Manure Land Application Technologies and Timing on Environmental Fate of Four Antibiotics Commonly Used in Dairy Production. ASA-CSSA-SSSA International Annual Meetings, November 6-9, Phoenix, AZ (2016). (Best Student Paper Award)
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Oral Presentation by Chen, C. Q., P. Ray, K. Xia, K. F. Knowlton, and A. Prudent. Fate and Transformation of Manure-borne Veterinary Antibiotics in Soils. 252nd American Chemical Society National Meeting. August 21-25, Philadelphia, PA (2016).
• Type: Websites Status: Published Year Published: 2015 Citation: http://www.args.hort.vt.edu/resources/

Progress 02/01/15 to 01/31/16

Outputs
Target Audience:During the first year of the project, we focused on developing a communications and marketing plan, including building and strengthening the networks of stakeholders that will allow for effective translational research. As a core aspect of this effort, we constructed a stakeholder map to define the target audiences and guide direction of dissemination of results as the project progresses. The stakeholder map incorporates the schemata of the project logic models and includes: formal educational institutions, non-formal educational trainers, dairy and livestock producers, produce growers, consumers, industry stakeholders, and policy-makers at state, regional, and national levels) for our various efforts. Specific accomplishments include: Launching the website to provide the latest project information and resources to a wide variety of stakeholders and the general public: http://www.hort.vt.edu/args/index.html Launched the Agricultural SOurces of Antibiotic Resistance (AgSoAR) program. Undergraduate research practicums underway for 12 undergraduate and 7 graduate students. Developed instructional module on antibiotic resistance for course in Food Science and Technology (FST) department and delivered to FST 4634/5634g Epidemiology of Food and Waterborne Disease in Spring 2015. 20 students were enrolled in the class, including graduate and undergraduate students at Virginia Tech. Developed instructional module on antibiotic resistance for course in Biological Systems Engineering (BSE) department, BSE 2004 Intro to Biosystems Engineering. Students interviewed experts in the field and produced video on antibiotic resistance genes (ARGs). Delivered to 55 BSE undergraduate students at Virginia Tech. On-site advisory board meeting held in July 2015. Included a member of the World Health Organization, employees of USDA ARS, a progressive Virginia Dairy Farmer, an area extension specialist on horticulture, small fruit, and specialty crops, Associate-Director of the University of Arizona Water Resources Research Center, and a Research Scientist at AgriFood Canada. Changes/Problems:There are two changes to the experimental design as described in the proposal. These are not expected to influence the project schedule, but could impact the rate of expenditure, particularly for the metagenomic DNA sequencing analysis. We determined that the local Virginia soil is not amenable to growing carrots. We have selected radishes as an alternative root vegetable that will allow us to address the same experimental question (differences in root, leafy, and cruciferous vegetables). Carrots will still be used in the post-harvest practice portion of the experiment, using carrots purchased from retail. Despite three different trials with the compost experiments, E. coli remained detectable in the finished compost at 42 days. FSMA time x temperature requirements were achieved for the static compost (3 days >/= 55ºC), but not for the turned compost (15 days >/= 55ºC, we achieved 3 days > 55ºC). We determined that the difficulty in maintaining the higher temperature for the turned compost was likely because of the small compost piles, which was necessary because of the large experimental matrix. We proceeded with greenhouse and soil microcosm studies with these different composts, as they will still be valuable from a research standpoint to see if the different management practices matter for downstream persistence of ARBs and ARGs. We also conducted a follow-up study of just dairy manure, with and without antibiotics, under static conditions- in which we externally forced the temperature to > 55ºC for 15 days. This compost will be applied in a small sub-set of experiments for comparison. We do not anticipate that there will be a problem achieving the FSMA time x temperature guidelines in the large-scale composting that will be getting underway for the field study in Spring 2016. What opportunities for training and professional development has the project provided?This project has served as an ideal interdisciplinary training ground for undergraduate students (n=12), graduate students (n=7), and post-doctoral researchers (n=3). The students and post-docs work along with the PD and Co-PDs as part of an integrated team. The full project team meets on a monthly basis, with the individual objective teams meeting regularly between full project team meetings. As part of the Agricultural Sources of Antibiotic Resistance (AgSoAR) program, undergraduate students gained hands-on research experience, with the graduate students and postdoctoral researchers gaining mentoring experience. New course modules on antibiotic resistance were developed for two courses, FST 4634/5634g Epidemiology of Food and Waterborne Disease and BSE 2004 Intro to Biosystems Engineering. Together these two courses served 75 students in AY 2015. Students are co-authors on papers and presentations and as research progresses will attend and present at conferences in the coming year. The students also had the opportunity to meet and interact with the advisory board, which consists of employees of USDA ARS, faculty at other universities, a member of the World Health Organization, dairy farmers, and growers. How have the results been disseminated to communities of interest?Our extension team has developed a stakeholder map to guide dissemination and impact of this project. The stakeholder map includes formal educational institutions, non-formal educational trainers, dairy and livestock producers, produce growers, consumers, industry stakeholders, and policy-makers at state, regional, and national levels. We launched our website to reach a wide variety of audiences with the latest information and happenings about the project: http://www.hort.vt.edu/args/index.html. Our advisory board includes a dairy farmer and a grower, and we regularly interact with them to share information and also request input to the project. We also published our first peer-reviewed article related to this work, focused on excretion of ARGs by dairy cows administerd antibiotics (Chambers et al., PLoS ONE, 2015). The PD delievered invited conference presentations at the Fate of the Earth Symposium at Michigan State University (April 2015), the Environmental Dimensions of Antibiotic Resistance conference in Germany (May 2015), and the Project Directors' Meeting in Portland OR (July 2015). We are currently in the progress of creating YouTube videos to post to the website. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will focus on: Objective 1: Analysis of ARGs in beef manure collected during dosing study, analysis and publication of results. Objective 2: Analysis of ARGs in compost samples, analysis and publication of results Objective 3: Completion of soil microcosm experiments, analysis of samples for antibiotics and ARGs. Objective 4: Completion of greenhouse studies examining the effect of the different types of compost on three vegetables (lettuce, broccoli, and radish) grown in two types of soil (sand and loam). We will also carry out the field portion of the project in Spring 2016. Objective 5: Now that methods are established, carry out and complete the produce washing experiments. Objective 6: Develop modules for other courses and teach the core class for the program, GRAD 5134 Interdisciplinary Research, and recruit and train new students. Complete and post-YouTube videos to the website. As research results become available, continue to populate content for extension materials.

Impacts
What was accomplished under these goals? Antibiotic resistance is a serious and growing human health problem. Antibiotics are losing effectiveness, which limits means to prevent and treat bacterial infections in humans. There is an urgent need to understand why and how antibiotic resistance is spreading in order to develop strategies to slow the spread of resistance. In particular, there is concern that antibiotic use in agriculture results in higher levels of resistant bacteria in their manure, which could contribute to elevated levels of antibiotic resistant bacteria (ARBs) in soil and ultimatey on the surfaces of vegetables. In addition to ARBs, the antibiotic resistance genes (ARGs) that they carry are a concern because they are known to sometimes spread among different kinds of bacteria. The overall goal of the project is to provide an integrated understanding of the potential for ARBs and ARGs to spread from farm to fork, i.e., from manure to raw produce. A novelty of the proposed work is the incorporation of advanced metagenomic techniques to broadly probe and track ARGs through each critical control point: 1) type of antibiotic used; 2) method of composting; 3) type of soil selected for land application; 4) type of vegetable selected; and 5) and post-harvest produce washing practices. Where possible, the experimental parameters are benchmarked against Food Safety and Modernization Act (FSMA) guidelines for limiting the spread of pathogens, such as E. coli and Salmonella. The overarching research question is whether the same barriers for limiting the spread of bacterial pathogens also limit the spread of ARGs. This is an integrated project, involving research, education, and extension. Faculty, students, and post-doctoral scientists across various programs at Virginia Tech (Environmental Engineering, Biological Systems Engineering, Dairy Science, Food Science and Technology, Horticulture, Agricultural Science, and Microbiology) have teamed together to carry out the research. Extension is integrated and operates from the stakeholder map developed in the first year. Education is also integrated, including the Agricultural Sources of Antibiotic Resistance (AgSoAR) program, which consistes of research practicums for undergraduate students and new course modules on antibiotic resistance for graduate and undergraduate courses. While we are only 10 months into the project, significant progress has been made on all of the objectives. Most progress has been made through objectives 1 and 2, where we examined excretion of antibiotics and ARGs in manure and their fate during composting. Preliminary results at this point suggest that the static versus turned composting regimes had little to no effect on the fate of the various antibiotics, and that the presence of antibiotics in the manure did not influence the progress of composting according to standard physicochemical measurements (e.g., Temperature). The main differences observed were among the antibiotics themselves, in which cephapirin appeared to degrade very quickly and was not detected beyond day zero; pirlimycin degradation followed 1st order kinetics with 90% removal by day 14 and 99% removal by 42 days; sulfamethazine followed a similar pattern as pirlimycin but was 95% removed by 42 days; chlortetracycline and tetracycline were subject to 72-76% removal over 42 days; and tylosin was the least effectively removed, with 60-80% removal by 42 days and some evidence that turning the compost offered some benefit. E. coli persistence also generally did not appear to be affected by the different composting conditions. This project offers practical value, already yielding insight into how different composting practices may affect residual antibiotics in manure. Ideally, limiting antibiotic residues in manures will limit the ability of antibiotics to select for resistant bacteria in the compost, soil and on vegetables. This will be subject to further investigation as the project progresses and guide formulation of mitgation strategies to present multiple barriers to the spread of antibiotic resistance from farm to fork. Objective 1 experiments are complete. Both dairy and beef cattle antibiotic dosing experiments are concluded. Antibiotic analysis of the manure and urine is complete and a manuscript is under preparation. Fecal samples from the beef dosing study have been preserved for later ARG analysis. Results of ARG analysis of dairy manure during antibiotic dosing has been published in PLoS ONE. Objective 2 experiments are complete. A study was conducted comparing static versus turned composting of beef manure, with and without antibiotic (sulfamethazine, tylosin, chlortetracycline, and tetracycline) administration, and dairy manure, with and without antibiotic (pirlimycin and cephapirin) administration. This resulted in an experimental matrix of 8 conditions carried out in triplicate, accounting for 24 compost piles. The large number of conditions necessitated small-scale composters, which were insulated and equipped with temperature sensors and subject to manual turning twice a day for 42 days. A second run of composting was conducted for the dairy manure, with and without antibiotics, under static conditions only and manually forced to maintain >55ºC for 14 days followed by an ambient curation stage out to 42 days. Antibiotic analysis has been completed on the first run of composting and is under way for the second run. E. coli was enumerated and a sub-set was assayed for resistance to the classes of antibiotics administered. Objective 3 experiments are about 50% complete. Microcosms have been set up with three kinds of soils (sandy, loamy, and clayey) and amended with all 8 types of compost produced in objective 2 plus the four raw manures, for a total of 36 conditions. Microcosms were set up in small jars for sacrifical sampling of triplicate samples over a 120 day period (for consistency with latest FSMA guidelines regarding land-application), with moisture maintained at 50% field capacity. Samples are being collected and preserved for later antibiotic and ARG analysis. Monitoring of E. coli at 2 weeks into the study indicates that they are persisting in soils where untreated manures were applied, and sporadically where compost was applied. Objective 4 experiments are about 20% complete. We have carried out pilot studies of broccoli, lettuce, and carrots in the greenhouse in two types of soil amended with the same 8 types of compost and four manure types, for a total of 24 conditions for each vegetable. Objective 5 experiments are about 20% complete. A pilot study was carried out by inoculating lettuce, broccoli, and carrots from the grocery store with diluted compost and Pseudomonas. Different post-harvest washing practices are being tested, including washing in water dosed with chlorine or peracetic acid or irradiation. Methodology has been developed for quantifying antibiotic resistant heterotrophic bacteria from the edible portions of the produce on R2A media. Objective 6 is integrated throughout the project, with major milestones being the new modules taught in the two courses, the undergraduate practicums, launching of the website, the 1st on-site advisory board meeting, and development of the stakeholder map.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Chambers, L.R., Yang, Y., Littier, H., Ray, P., Zhang, T., Pruden, A., Strickland, M. and K.F. Knowlton. (2015). Metagenomic Analysis of Antibiotic Resistance Genes in Dairy Cow Feces following Therapeutic Administration of Third Generation Cephalosporin. PLoS One. 10(8): e0133764.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pruden, A. Mitigation Strategies for Environmental Sources of Antibiotic Resistance. Environmental Dimensions of Antibiotic Resistance (EDAR). May 17-21, Wernigerode, Germany (2015).
• Type: Websites Status: Published Year Published: 2015 Citation: http://www.hort.vt.edu/args/index.html
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Invited Plenary Lecture by Pruden, A. How do We Combat Antibiotic Resistance? Human Progress and the Laws of Nature. Fate of the Earth Symposium, Michigan State University, Lansing, MI. April 1-3 (2015)
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Archibald, T., Hession, W.C., Krometis, L-A., Xia, K., Knowlton, K., Ponder, M., Vallotton, A., Ray, P., Guron, G., Chen, C., Pulido, N., Williams, R., Fogler, K., Pruden, A. Identification and Management of Critical Control Points in the Spread of Antibiotic Resistance from Animal Manure to Raw Produce. USDA NIFA Institute of Food Safety and Nutrition Project Directors Meeting, Portland, OR, July 24, 2015