Source: UNIVERSITY AT BUFFALO - STATE UNIV OF NEW YORK submitted to
EVALUATING DIFFERENT MANURE MANAGEMENT PRACTICES IN CONTROLLING SPREAD OF ANTIMICROBIAL RESISTANCE FROM DAIRY FARMS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1008068
Grant No.
2016-68003-24601
Project No.
NY.W-2015-07867
Proposal No.
2015-07867
Multistate No.
(N/A)
Program Code
A4171
Project Start Date
Dec 1, 2015
Project End Date
Nov 30, 2018
Grant Year
2016
Project Director
Aga, D. S.
Recipient Organization
UNIVERSITY AT BUFFALO - STATE UNIV OF NEW YORK
(N/A)
BUFFALO,NY 14260
Performing Department
Chemistry
Non Technical Summary
The dairy industry is one of the most important agricultural businesses in the United States and is the number one agricultural business in California, Wisconsin and New York. In modern dairy farms, antimicrobials are commonly used for both therapeutic (disease treatment) and prophylactic (prevention) purposes. However, the use of antimicrobials in animal agriculture has been associated with increased occurrence of antimicrobial resistant bacteria (ARB) and the presence of antimicrobial resistant genes (ARG) in dairy manure, which enter the human food chain when manure is used to fertilize croplands. This proposed integrated research and extension project seeks to evaluate critical control points in dairy farm operations that can prevent the spread of antimicrobial resistance from "farm to fork". Our study is based on 11 participating dairy farms that include small, medium, and large animal feeding operations employing various manure management strategies such as anaerobic digesters (with and without composting or pasteurization), solid-liquid separation, and long-term or short-term lagoon storage systems. The effectiveness of advanced manure treatment systems in reducing the spread of antimicrobial resistance will be evaluated through coordinated bench-scale and pilot-scale experiments to identify important manure characteristics and digester operating parameters for optimum removal of antimicrobials, ARB, and ARG. Plant-uptake experiments will be conducted to determine if certain types of manure treatment systems, combined with appropriate manure application in crop fertilization, can prevent the spread of antimicrobial resistance from "farm to fork". We will also evaluate whether general farm hygienic measures can potentially reduce the use of antimicrobials. Results from the different aspects of this study will be incorporated into a predictive model to simulate the proliferation of ARB and ARG at the watershed-scale.Our research and extension efforts are unique in that they are based on a strong partnership between academic researchers, extension specialists, and commercial dairy operations implementing varying manure treatment systems, which will allow us to directly relate laboratory studies with farm-scale conditions. The knowledge gained from this research will improve the scientific understanding of the role of anaerobic digestion technology and manure composting on mitigating the spread of antimicrobial resistance. Improvements in manure treatment and farming practices (e.g. antimicrobial use, cleanliness of farms, animal health management, manure application) will result from our planned extension and education activities that will include field days and workshops. Ultimately, specific abatement strategies and best management practices (e.g. type of manure treatment and applications, selection of crops grown in specific manure-amended fields) will result in the reduction of antimicrobial use in the animal industry, and ultimately spread of antimicrobial resistance in agricultural environment.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71101991070100%
Goals / Objectives
The major goals of this project are to: (1) evaluate and test conditions for effective and sustainable strategies that mitigate the spread and persistence of ARB and ARG within the agricultural ecosystem, from farm to food crops, and (2) design training, education, and outreach resources (including web-based resources) that can be used by farmers, operators, and policy makers to prevent spread of antimicrobial resistance from farm to fork. The objectives of this project are:Evaluate the fate and removal rates of antimicrobials, ARB, and ARG in conventional and advanced manure treatment systems at 11 dairy farms and in manure-amended soil.Through coordinated bench-scale and pilot-scale AD experiments, identify the driving manure characteristics and AD operating parameters for maximizing removal of antimicrobials, ARB, and ARG from manure.Quantify the amounts of antimicrobials, ARB and ARG that can be transferred from manure-amended soil to human food crops, using different crops and manure treatment.Utilize a watershed-scale hydraulic model to determine antimicrobial transfer from cow to manure to fields, crops and end of field (i.e. streams and ditches) with the three different manure management strategies to ascertain which management strategies have the largest effect and should be the focus of extension activities.Develop accessible extension and education activities to convey the importance of manure management strategies, food system interactions, antimicrobial use, and farm cleanliness in order to mitigate antimicrobial resistance to specific target audiences.
Project Methods
The proposed project engages in research and extension activities targeted at likely critical control points for spread of antimicrobial resistance from farm to fork, including: 1) anaerobic digestion, 2) composting, 3) manure application, 4) crop selection, and 5) general farm hygienic measures or management parameters. Specific objectives 1-3 are centered on research specific to each of the critical control points, with a significant extension component (informing extension activities and gathering feedback to inform subsequent research activities). Objectives 4 and 5 are focused on extension.Manure and soil samples will be collected at various locations in eleven participating dairy farms with varying manure management strategies: some with anaerobic digesters (with and without composting or pasteurization), others with solid-liquid separation, and the rest with various forms of lagoon storage (long-term and short-term). The samples collected with be analyzed for veterinary antimicrobials using state-of-the art analytical instruments based on liquid chromatography with tandem mass spectrometry (LC/MS/MS) that can detect very low levels of antimicrobials in highly complex samples, such as manure and soil. For accurate quantification of antimicrobials, we will use isotope dilution mass spectrometry to alleviateproblems associated with matrix effects and low extraction recoveries.The same samples will be analyzed for culturable bacteria from four genera associated with dairy farms and antimicrobial resistance (Enterococcus spp., E. coli, C. perfringens, and P. aeruginosa). We willmonitor for a suite of ARG that induce resistance to antimicrobials commonly applied in dairy cow operations. These genes include a range of resistance mechanisms and also reside on a range of genetic elements including plasmid (tetA, ermB, blaTEM ) or chromosomal (tetM, blaTEM) DNA. Our inclusion of a range of beta lactamase genes (bla), in particular, is due to the fact that beta lactam antibiotics are frequently applied for mastitis, dry cow therapy, and lameness.Resistance genes for macrolides and lincosamides (erm) are also included because macrolides and lincosamides are commonly used for mastitis and pneumonia in calves and these specific genes were detected at high frequencies in manure-amended soils. Wehave chosen to perform quantitative PCR (qPCR) on a focused list of ARG in order to obtain quantitative data so thatdifferent farms with varying manure management practices can be compared, not only based on the presence or absence of resistance genes, but also on the relative abundances of the genes.We will conduct coordinated bench-scale and pilot-scale AD experiments to identify the driving manure characteristics and AD operating parameters for optimum removal of antimicrobials, ARB, and ARG. Pilot-scale AD experiments will be conducted using six plug-flow digesters located at the USDA BARC dairy in Beltsville, MD. The six pilot-scale AD will be operated using: (a) 2 controls (no antimicrobials added), (b) 2 low-level spike antimicrobials, and (c) 2 high-level spike antimicrobials. For the lab-scale AD experiments we will spike 2-L semi-continuous anaerobic bioreactors with antimicrobials and ARG. Tetracycline and β-lactam ARG in intracellular and extracellular DNA will be spiked into the bench-scale reactors and their degradation will be monitored. At least two ARGs will be included in the spikes--tetA and ampC. The genes will be added in several formats, including: (1) purified plasmid solutions, (2) purified chromosomal solutions, (3) purified phage solutions, and (4) purified intact cells with the genes in plasmids or chromosomal DNA. Biomass samples will be collected regularly for ARB and ARG analyses, and general microbial performance will be monitored to evaluate the impact of the spikes on the typical AD microbial community. Multiplexed amplicons will be sequenced by the Host Microbiome Initiative via IlluminaMiSeq using the MiSeq Reagent Kit V4 and sequences will be processed with MOTHUR following the SchlossMiSeq standard operating procedures. Sequences will be classified using the Ribosomal Database Project, and further analysed for operational taxonomic unit (OTU)-based clustering.The efforts that will be used to cause a change in knowledge or conditions will be through field days and workshops presented to dairy farmers and operators. The output(s) will bequantified for its impact based on modeling fate of antimicrobials involving the following factors: feeding rates to animals, processing of the antimicrobials by the animals, transformations of antimicrobials in the waste management system, timeliness of manure application, soil sorption, and plant species-antimicrobial uptake kinetics. Transport of antimicrobials in surface water adds the complexities of soil release of antimicrobials and sediment-antimicrobial kinetics during transport to edge-of-field. The antimicrobials then leave the field either as a component of runoff, bound to sediment in the runoff, or as a component in the harvested plant tissue.Using MACRO 5.1 model, a one-dimensional, process oriented dual-permeability model of water flow and solute transport, will be used to evaluate effects of different manure management regimes on edge-of field solute transport. Rather than create a new hydraulic model, we will use this existing model to compare impacts of manure management. The differences from the model output under the various manure management scenarios will provide an estimate of the pollution control value of each management regime.These model outputs will be conveyed to farmers using clear graphics and easy-to-understand language in order to convey to farmers the effect of manure management on antimicrobial resistance. The model results will be tied to nutrient management application rates, as nutrient management regimes control the quantity of manure added to each field, in order to provide the information in a farmer-based platform that will be meaningful to their farm.The extension/outreach component of this project will utilize the time-proven approach developed and implemented by the Cornell PRO-DAIRY Program and UMD's Cooperative Extension Service. Cornell will develop and distribute announcements on the many list serves used to keep the dairy industry informed of environmental topics in NY, PA and MD. Cornell's website is tied in with the eXtension program's web site allowing for ease of material findings at the national level. Two types of outreach activities will be conducted. In beginning of Year 2, a field workshop will be conducted to demonstrate and raise awareness about advanced manure management technologies at the USDA Beltsville Agricultural Research Center, and Cornell University will prepare awareness focused materials and deliver them as part of annual extension/outreach meetings. In Year 3, we will conduct one farm tour in MD and PA and two in NY, which involve visits on cooperators' farms so that farmers can talk to farmers. This will allow tour participants to evaluate and compare different farm-based manure management technologies and talk directly to farmers about their experiences with AD systems.We will conduct field days and/or strategically partner with other extension programs to engage specific target audiences (dairy farmers and their advisors, policy makers, government agency personnel, and NGOs) to disseminate the lessons learned. We will develop and deliver oral presentation materials at dairy industry meetings, conferences, and other related events in the Northeast as opportunities become available. All materials will be posted on the Cornell University's PRO-DAIRY Program's Dairy Environmental Systems web site (www.manuremanagement.cornell.edu).

Progress 12/01/16 to 11/30/17

Outputs
Target Audience: Scientists, Researchers & Extension/Outreach professionals were reached through national and international professional meetings (Waste-to-Worth, ASABE, AEES, EDAR), seminars (Cornell Dairy Center of Excellence), classroom trainings (Cornell Vet School), and dairy manure extension events (Maryland Dairy Farmer Field Day). Industry professionals were reached through professional meetings (Biocycle East and Maryland Dairy Farmer Field Day) and direct correspondences. Farmers were reached through on-farm discussions and field day events, aided by the development of Extension Fact Sheets and other outreach materials. High School and undergraduate students were reached through internship opportunities that included laboratory instruction and experiential fieldwork learning opportunities. Graduate Students and faculty at Scientific conferences such as the American Chemical Society (Washington DC) and EDAR (Michigan State University), and at the Antibiotic Resistance Conference at the Nanjing University, China Changes/Problems: The antibiotics data collected from the 11 farms in June and August 2016 were removed from the average results due to a change in the LC/MS/MS method of analyzing the antibiotics after August 2016; The modification in the method was to improve detection limit and reproducibility of analysis which turned out to be very challenging due to the wide ranging nature of manure samples from various farms. The complexity of each manure sample varied from farm to farm, therefore we had to optimize the method after observing the results from June and August 2016 were not reproducible due to matrix effects. What opportunities for training and professional development has the project provided?Graduate Student Emily Crossette attended a 9-day workshop at the Woods Hole Marine Microbiology Laboratory to study computational microbial ecology methods. How have the results been disseminated to communities of interest? 1. University at Buffalo: We presented data at the following conferences: Dr. Aga presented as invited speaker at the Symposium for Antibiotics and Antibiotics Resistance Genes in the Environment, Nanjing University, Nanjing, China, June 14-16, 2017 Three posters were presented at the4th International Symposium on the Environmental Dimension of Antibiotic Resistance (EDAR) Conference, Lansing, Michigan.August 13-17, 2017. American Chemical Society, Washington, D.C. August 20-24, 2017. Oral Presentation titled: "Assessing Dairy Manure Management Strategies for the Removal of Antimicrobials and Spread of Antimicrobial Resistant Genes". 2. University of Michigan: Preliminary metagenomic work was shared at the UNC Water Microbiology/IWA Health-Related Water Microbiology joint conference in Chapel Hill, NC. The bioinformatic methods for the metagenomic study was presented in a poster presentation entitled "Quantitative metagenomic approach to monitor the dynamics of antimicrobial resistance gene (ARG) profiles through engineered treatment systems" at the "Environmental Dimension of Antimicrobial Resistance" Symposium in Lansing, Michigan. Intracellular/Extracellular DNA extraction methods were presented at a poster session at the Borchardt Conference in Ann Arbor, Michigan. The poster was entitled "Extra- and intracellular antimicrobial resistance genes (ARGs) and their fate in dairy cow manure treatment structures." Preliminary findings from the virus DNA extraction methods were shared in poster presentations entitled "Quantitative Analysis of dsDNA Phage Through Wastewater Treatment Plants" at the "Environmental Dimension of Antimicrobial Resistance" Symposium in Lansing, Michigan and at the "Association of Environmental Engineering and Science Professors" Annual Conference in Ann Arbor, Michigan. 3. Cornell University and Maryland (Extension Part) Professional meetings, seminars, and teleconferences have been used to disseminate preliminary phase I results to Scientists, Researchers, Extension personnel, Veterinaries, Agricultural Engineers, and Dairy Industry professionals Fact sheets have been prepared to disseminate information on antimicrobial resistance as it relates to dairy production to farmers and their stakeholders Currently, reports that summarize Phase I efforts are being prepared for the collaborating farms in advance of sit-down meetings to discuss the project's Phase I findings What do you plan to do during the next reporting period to accomplish the goals? Phase II sampling focusing in on one New York farm and one Maryland farm to conduct a more intensive exploration of specific treatment processes including bedding recovery and composting and the effect of time and temperature on antibiotic degradation. Bench and pilot-scale experiments will be conducted to focus in and explore the specific contribution of anaerobic digestion to antimicrobial degradation and resistant gene and bacteria generation. Phase I results will be used to calibrate and create a model to track antimicrobial passage through the different farm manure systems. Further analyze antibiotic data for eight sampling events for correlations to antibiotic usage and possible removal across manure management systems. Extract and analyze for antibiotics in focused sampling on bedding recovery and anaerobic digestion units to assess absolute removal. Continue investigation of degradation of beta-lactam antibiotics in dairy manure by analyzing degradation exposure samples. Investigate possible transformation products of parent beta-lactams by high resolution mass spectrometry. Complete second crop uptake exposure experiments for antibiotic and ARG's. Prepare a manuscript for publication describing uptake of antibiotics and presence of ARG's in maize when grown with manure amended soil. Sample collection is being planned to target specific processes on dairy farms participating in the study. Samples from this round of collection will be analyzed for both antimicrobials and the int1, sul1, tetO and beta-lactam associated resistance genes. Intracellular DNA (iDNA) and extracellular DNA (eDNA) co-extraction processes will be finalized. This will involve the quantification of contaminating DNA fractions using internal standard DNA with qPCR. These internal standards include a virus, bacteria, and an extracellular plasmid. The final bioinformatic processes will be completed for themetagenomic ARG quantification method,which includes quantification of the internal standard genes recovered through sequencing and the identification of ARG on assembled DNA sequences. Additionally, the quantification method will be compared to qPCR, a common approach for gene quantification. PrepareManuscripts for publications in scientific journals

Impacts
What was accomplished under these goals? Objective 1.Evaluate the fate of AMR During Phase I sampling, samples were taken every six weeks over a year long period from participating farms in Maryland (MD1, MD2, MD3), New York (NY1, NY2, NY3, NY4, NY5, NY6), and Pennsylvania (PA1, PA2) and analyzed to determine the fate and transport of antimicrobials, ARB and ARG. Each farm utilizes a different manure management system, but the manure was sampled at each point in their management practice (i.e. inception, before and after separation, compost or digestion, before lagoon storage and prior to field application). Manure samples were freeze-dried and homogenized; 100 mg of dried manure solids were analyzed using LC/MS/MS.Antimicrobial analysis of tetracyclines, sulfonamides, and macrolides revealed detection of sulfonamides in 6% of the total samples. Tetracyclines were detected with a higher frequency of 86%, suggesting higher environmental stability and use of tetracyclines on dairy farms compared to sulfonamides. Macrolides were not detected in any manure samples, which reflects the low reported average antibiotic usage of macrolides per month (i.e. 1-5 cows are treated with erythromycin per month). Screening for the presence of beta-lactam antibiotics was performed using a commercial ELISA kit. Screening efforts using ELISA for the analysis of ceftiofur and penicillin G yielded no detection for manure samples which were chosen to screen based on antibiotic usage data from six of the NY farms. These farms reported high usage of beta-lactam antibiotics, however, no parent compounds of the beta-lactam molecules were detected. In addition to antimicrobials, ARB and ARG analyses, the following analyses were conducted on the samples to determine if correlations exist (and can be modeled) between manure characteristics and antimicrobial transformations: pH, moisture content, total and volatile solids (TV, VS), nutrients (TKN, TKP, NH4, PO4), and volatile fatty acids (VFAs). Manure system performance data was collected from each farm (e.g. manure produced, manure digested, AD temperature, biogas production, rotary drum composter temperature) as well as drug treatment records were collected from each farm and along with several meetings with farm herdsman daily drug use is being summarized for each farm. Phase I sampling, manure system monitoring, and antibiotic usage monitoring was completed in September 2017, samples have been analyzed, and the team is moving forward with statistical analysis of the collected data. 2016 antibiotic usage has been summarized for NYS, PA and MD farms, with summary of all data on-going. Herd health and antibiotic administration on farms is variable. Administration data is being compared to antibiotic residual data to see if there are correlations between administration spikes and concentrations in the sampled manure. Under the first goal, ARGs were identified and quantified from metagenomic data. The data will reveal most abundant ARG in the samples and samples containing the most ARGs. This will inform ARGs to track throughout treatment. Do date, the metagenomes have been assembled to compare both ARG annotation with short metagenomic reads and annotation with assembled genes. Read-mapping to the CARD database was completed and an algorithm is being developed to convert relative abundances of reads to copies of an ARG/volume of sample. ARG presence in untreated and storage lagoon manure samples for each dairy farm and each collection date (Sept'16, Oct'16, Dec'16, March'17, June'17, and Aug'17) was evaluated using quantitative polymerase chain reaction (qPCR) and customized Qiagen Microbial DNA qPCR Arrays. The samples analyzed thus far represent ARG concentrations prior to treatment and/or field application.Temporal and location-based variability among each farm and the collection dates reveal no trends. However, the temporal variability observed may be related to climatic conditions and further data analysis is underway. No correlation was observed between int1 normalized copies per gram manure and total antibiotic concentration (Spearman correlation, p=0.082). In addition to qPCR, a Customized Qiagen Microbial DNA qPCR Arrays were used to evaluate the presence of nine ARGs simultaneously. Nine total genes were targeted (ermB, mefA, ereB, tetA, OXA-2, CTX-M-1, VEB-1, aada1, and aadac-6-lb) which represent resistance mechanisms to the following antimicrobial classes: macrolides, tetracyclines, beta-lactams, and aminoglycosides. DNA extracted from untreated manure from each of the 11 farms over six collection dates (Sept'16, Oct'16, Dec'16, March'17, June'17, and Aug'17) and storage drawdown samples were analyzed for the nine ARGs. Objective 2. Bench-scale and pilot-scale Anaerobic Digestion experiments A face-to-face project collaborators meeting was hosted at the EDAR meeting in Lansing, MI to discuss Phase I project status and future directions for Phase II of the project. In addition, another face-to-face meeting between the Cornell and UB teams occurred in Buffalo in September to discuss the best approach for Phase I data analysis. Phase II sampling strategies are being planned, with farmer meetings conducted and scheduled to work out the logistics. Experimental design for bench and pilot scale experiments is being finalized. The bench-scale experiment will be conducted over the winter with the AD pilot scale experiment occurring in Spring 2018. To help meet Goal 2, extraction methods for different forms of ARGs have been investigated. ARGs in the intracellular or extracellular fractions may behave differently in manure which could cause one fraction to persist longer than another. So far, DNA internal standards have been developed to assess recovery of DNA in each fraction and contamination. Additionally, methods to minimize the virus DNA contamination from iDNA/eDNA fractions have also been evaluated. Another graduate student, Kathryn Langenfeld, is developing DNA and RNA extraction methods for viruses from manure. Two foreign phage are used as internal standards to determine the absolute recovery of viral nucleic acid extraction methods. Objective 3. Crop uptake experiments Field soils and long-term storage manures were collected from a NY farm on August 2017 for crop uptake experiment Objective 4. Hydraulic modelling effort Phase I sampling information will be used for model calibration. This will begin in 2018. Objective 5. Extension and education activities A five-part Fact Sheet series on "Antibiotic Residues in Dairy Manure" is near completion. The series includes: Part 1: Critically important antimicrobials labeled for dairy use, Part 2: Sampling dairy manure for antibiotic detection, Part 3: Laboratory methods for extracting antibiotic residues from dairy manure, Part 4: Laboratory methods for analyzing antibiotic residues extracted from dairy manure, Part 5: What is known about antibiotic residues in dairy manure? Cornell spoke to a class of 4th and 5th year veterinary students in May 2017 on the topic of antimicrobial resistance and dairy manure systems in a course titled "Veterinary Perspectives on Pathogen Control in Animal Manure" In October 2017 a Field Day and Tour was hosted by UMD at the Central Maryland Research and Education Center-Clarksville Facility (Ellicott City, MD). The event including four talks from various research experts and extension/outreach professionals on manure management. A farm tour was given where two posters were presented at various locations talking about antibiotic administration on farm and antibiotic detection levels throughout the manure trail (Figure 4). A manure injection demonstration was also provided. The event included 59 participants.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 1. Arikan, O., Rice, C., Mulbry, W., Lansing, S., 2017. The fate and effect of lasalocid during anaerobic digestion of dairy manure. Progress in Biogas IV International Conference. Stuttgart, Germany. March 9, 2017. 2. Oliver JP, Gooch CA. Effectiveness of dairy manure management practices at controlling antimicrobial resistance. Biocycle East Coast. Baltimore, MD. Apr. 4-7, 2017. 3. Oliver JP, Gooch C. 2017. Effectiveness of different dairy manure management practices in controlling the spread of antibiotics and antibiotic resistance. Waste-to-Worth Annual Conference, Cary, NC. Apr. 18-21, 2017. 4. Schueler J, Lansing S. Fate and Transport of Antibiotics and Nutrients in Manure Management Systems. American Ecological Engineering Society. Athens, GA. May 22-25, 2017. 5. Oliver JP, Schueler J, Hurst J, Crossette E, Langenfeld K, Gooch C, Lansing S, Felton G, Wigginton K, Raskin L, Sassoubre L, Aga D. 2017. Antibiotics and antibiotic resistant bacteria and genes in northeastern dairy manure management systems  Project overview and preliminary findings from an 11 farm case study. ASABE International Meeting, Spokane, Washington. July 16-19, 2017. 6. Lansing, S., Oliver JP, Schueler J, Gooch C, Felton G, Raskin L, Wigginton K, Crossette E, Langenfeld K, Hurst J Sassoubre L, Aga D. 2017. The Effect of Manure Collection and Inherent Variability on Antibiotic Testing of Dairy Manure Systems (poster). 4th International Symposium on the Environmental Dimensions of Antibiotic Resistance. Lansing, MI. August 13-17, 2017. 7. Jerod Hurst, Jason Oliver, Jenna Schueler, Stephanie Lansing, Lauren Sassoubre, Diana Aga. Assessing Dairy Manure Management Strategies for Removal of Antimicrobials, (poster). 4th International Symposium on the Environmental Dimensions of Antibiotic Resistance. Lansing, MI. August 13-17, 2017. 8. Jerod J. Hurst, Lauren Sassoubre, and Diana S. Aga. "Assessing Dairy Manure Management Strategies for Removal of Antimicrobials and Spread of Antimicrobial Resistant Genes", (oral), 254th ACS National Meeting, Washington, DC, August 20-24, 2017.


Progress 12/01/15 to 11/30/16

Outputs
Target Audience:Chemists, Agricultural Engineers, Farmers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The University of Maryland participants had involved two African American high school interns on this project, two college undergraduate students, and one Master Students who have assisted with and trained under this project. The University at Buffalo housed Benjamin Davis as part of the Research for Undergraduate Education (REU) program (Summer 2016). The student assisted with research for the USDA project. Through this process, the student learned how we collect samples at the dairy farms, how manure is analyzed for antibiotics, and how manure is analyzed for ARBs and ARGs. His REU experience culminated with a poster presentation. The University of Michigan sent Emily Crossette to the Gordon Research Conference: Environmental Sciences Water, which promoted networking between scientists evaluating antimicrobial resistance gene dissemination in the environment. Additionally, the grant supported a student's attendance of an R-workshop which enhance skills in data processing and coding relevant for microbial community analysis. The workshop also facilitated networking between professionals and researchers in the field of microbiology. The Cornell efforts are being led by a postdoctoral associate. This individual has worked with farm personal and veterinarians to understand the dynamics of farm antibiotic usage and record keeping. Under the supervision of Curt Gooch and with farm personal this individual has sharpened their understanding of the various manure treatment systems and their operational parameters. Additionally, this induvial has attended several professional engineering trainings, outreach/extension events, and is gaining project management skills. Throughout the 2016 sampling season Benjamin Davis, Mitchell Mayville, and Jerod Hurst regularly shadowed Jason Oliver at participating farms during sampling to improve their knowledge of the dynamic manure treatment systems, the sampling process and its subtleties. Approximately 30 private and public NYS professional engineers working in the areas of livestock nutrient management and manure treatment systems were updated on the issue of antibiotic resistance in dairy systems at the May 2016 training meeting. A meeting is being planned for spring 2017 with collaborating NYS dairies their operators and herdsmen to discuss different approached to improve antibiotic treatment recording. How have the results been disseminated to communities of interest? A kickoff meeting was conducted with each participating farmer, followed by several one-on-one meetings with our participant farmers throughout the past seven months to discuss their manure management systems and antibiotic usage and transformations on-farm. In addition, see presentations given in Year 1 and events planned for Year 2 in Goal 5 section above Preliminary work was shared with the scientific community through a poster presentation at the Gordon Research Conference, "The forms and persistence of antimicrobial resistance genes (ARG) in dairy farm manure treatment processes". What do you plan to do during the next reporting period to accomplish the goals?A. University of Maryland Finalize bench-scale and pilot-scale design and implement the experiments. Implement one large field day and several smaller county-extension based field days on the participating farms. Begin calibrating the hydraulic model to each farm using data collected in Years 1 and 2. University of Michigan During the next reporting period, the metagenomic data will be processed and relative and absolute abundance of resistance genes will be compared from both metagenomic data and qPCR assays. Additionally, intracellular DNA (iDNA) and extracellular DNA (eDNA) extraction processes will be optimized. Similar to the bulk DNA extraction kit comparison, the iDNA/eDNA extraction methods will be compared on the basis of yield, absolute abundance of spiked intracellular or extracellular DNA, and purity. Additionally, contamination of intracellular DNA into the extracellular fraction and vice-versa will be quantified and minimized. A viral DNA extraction method will be established and used to quantify the fraction of resistance genes in the viral community of the sample. C. Cornell University Integrate all field data to evaluate the effect of the various manure treatment systems on antibiotic residues. Complete the study investigating the effect of bedding recovery systems on pathogenic ARB. Launch a webpage for the project. In coordination with the UMD team, execute the on-farm meetings and various extension events that are planned and are in planning. Complete and publish the review of the impacts of dairy production on antibiotic resistance D. Univeristy at Buffalo Begin to extract DNA from the manure collected by the field teams. Based on the results from collaborators at University of Michigan, we have decided on an extraction kit for the entire project to be consistent across laboratories. We are in the process of deciding what assays to use for the antimicrobial resistance genes. Complete our laboratory experiments investigating the effect of lime on ARBs, ARGs and antibiotics. We plan to prepare a manuscript with this data. Continue experiments investigating which antimicrobial resistance genes are carried by which bacteria cultured on AccuMast plates. The development of new methods to analyze compounds of interest that have been identified by farm drug usage. The target antibiotics of these new methods include: ampicillin, ceftiofurpenicillin G procaineenrofloxacin, hetacillin potassium, and Amoxicilin Trihydrate . The majority of these target compounds of interest are beta-lactams, which are known to be highly unstable in the environment, degradation and transformation products of these antibiotics will be investigated using high resolution mass spectrometry if parent compounds are not seen in manure samples.

Impacts
What was accomplished under these goals? Major Goal 1 Samples were taken every six weeks over the past seven months from participating farms in Maryland (MD1, MD2, MD3), New York (NY1, NY2, NY3, NY4, NY5, NY6), and Pennsylvania (PA1, PA2) and analyzed to determine the fate and transport of antimicrobials, ARB and ARG. Each farm utilizes a different manure management system, but the manure was sampled at each point in their management practice (i.e. inception, before and after separation, compost or digestion, before lagoon storage and prior to field application). Initially, project personnel met with the farmers to better understand their manure management practices (lagoon storage time, solid separation, digester operation, etc.), their antibiotic usage (type, dosage rate, number of cows treated per day/month), and manure field application practices. Manure samples were freeze-dried and homogenized. 100 mg of dried manure solids were added to polypropylene centrifuge tubes. Each sample was spiked with 50mL of surrogate solution containing d4-sulfa-methoxazole, phenyl-13C6-sulfamethazine, demeclocycline, and13C-erythromycin (500 ng mL-1) and allowed to equilibrate for 30 minutes. Solids were suspended with 5 mL of 20:30:50 acetonitrile-methanol-0.1 M EDTA-McIlvaine buffer (pH = 4; v/v/v), vortexed for 30 seconds, ultrasonicated (40 kHz, 120 W) for 10 minutes and centrifuged at approximately 4000gfor 10 min.The supernatant was decanted into a 500-mL HDPE bottle and the solids were extracted twice more. Extracts were diluted with 400 mL of NANOpure water to reduce percentage organic content (≤4.0%) and the pH was adjusted to 4.0±0.2 with H3PO4. Diluted extracts were subjected to SPE with tandem NH2-HLB cartridges. Loaded cartridges were washed with 10 mL of water/methanol (95:5, v/v), the NH2 cartridges were removed, and the HLB cartridges were dried under vacuum.The HLB cartridges were eluted with 10 mL of methanol and evaporated to 200mL under N2at 30°C. Extracts were reconstituted to 1 mL with water/methanol (95:5, v/v) plus 0.1% acetic acid solution, and the mixture was vortexed. A 200-mL aliquot of the extract was placed into a vial insert and spiked with 10mL of 500 ng mL−1spiking solution and ISTD. A second aliquot of 200mL was spiked only with 10mL of 500 ng mL−1ISTD, d10-carbamazepine, and minocycline. Both aliquots were centrifuged at 7000gfor 5 min to remove anyfine particles from thefinal extract before analysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS). In addition to antimicrobials, ARB and ARG analyses, the following analyses were conducted on the samples in order to determine if correlations exist (and can be modeled) between manure characteristics and antimicrobial transformations: pH, moisture content, total and volatile solids (TV, VS), nutrients (TKN, TKP, NH4, PO4), and volatile fatty acids (VFAs). The following data has been produced over the last 7 months and will continue to be produced for the next year: 1) antibiotics usage and total antibiotic load averaged on a monthly basis, 2) nutrients, solids, pH and VFAs, 3) antibiotic. Manure system performance data was collected from each farm (e.g. manure produced, manure digested, AD temperature, biogas production, rotary drum composter temperature) as well as drug treatment records were collected from each farm and along with several meetings with farm herdsman daily drug use is being summarized for each farm. Initial analysis of preliminary antibiotic mitigation data is underway. Progress towards the first project goal includes the submission of manure samples for complete genome sequencing. This first task required optimizing the DNA extraction techniques in order to best obtain a high-quality and representative sample of DNA for sequencing and other downstream analysis. The absolute yield, DNA purity, and absolute abundance of spiked bacteria were compared from samples of dairy manure using two different commercial extraction kits. Major Goal 2 Development of the experimental design for the bench-scale and pilot-scale anaerobic digestion experiments begun based on our antibiotic usage data from the farms participating in this study. Major Goal 3 Sassoubre and Oliver are collaborating with Dr. Rodrigo Bicalho to investigate the antimicrobial resistance genes carried by different species and types of bacteria in manure used for bedding. Bicalho is an associate professor at Cornell University and started FERA Animal Health which makes AccuMast plates, a quick, culture-based method to diagnose mastitis on dairy farms. The research will give a better understanding of which bacteria carry ARGs and which ARGs which will inform treatment strategies for inactivating and removing ARBs and ARGs. Major Goal 4 The model framework and data for model calibration is being collected, and the model platform will be built in year 2. Major Goal 5 We will offer programming to help dairy farmers understand the new FDA regulations instated on January 1, 2017 that eliminate the growth promotion use of human medically important antibiotics and expanding the list of feed-grade antibiotics classified as Veterinary Feed Directive (VFD) drugs. Antibiotic resistance and dairy production Fact Sheets were developed for public and farm audiences and will be published soon to a page on the topic at the Cornell Dairy Env. Systems webpage. The extension programming has begun, with a large field-day planned for Spring 2017 in Maryland, and additional smaller programming in the county offices in MD, PA and NY. We also plan to conduct events in conjunction with PA dairy farm events that will occur in Summer 2017, and we will be part of the Vermont Digester Operator's Group event in December 2017. March 23rd, 2016. Presentation titled "Emerging Manure to Energy Technologies - Are Cost Effective Small Scale Digesters Possible" at the USDA Climate Hub Greenhouse Gas Mitigation Workshop by S. Lansing. This included information on manure management styles and how digesters fit into manure management strategies for dairy farmers. This presentation was followed by a live and archived one-hour webinar available for USDA CEU credit (52 participants for credit, 146 live participants total) give on June 1, 2016. Available at: http://www.climatewebinars.net/ webinars/emerging-manure-to-energy-technologies-are-cost-effective-small-scale-digesters-possible Two more presentations on dairy manure technologies were given entitled, "Emerging manure to energy technologies." The first presentation (May 26, 2016) was sponsored by the World Bank in order to share experiences of good management practices in terms of integrated nutrient pollution control and water quality issues to a Romanian delegation. The second presentation was given to the University of Maryland Agriculture Faculty at the Emeritus Recognition Luncheon. May 2016. Presentation titled "Manure treatment and antibacterial impacts" was developed and presented to Applied Agricultural Engineering Continuing Education Series Session No. 14. Nov. 2016. Presentation titled "Antimicrobial resistance from dairy farms: Evaluating the potential of manure management practices to control its spread" was presented at the Agriculture, Food & Environmental Systems In-Service Meeting, and Extension professionals training held annually at Cornell. Abstracts have been submitted to three professional meetings in 2017 (Waste-to-Worth, Biocycle East, and American Society of Agricultural and Biological Engineering). These meeting are typically attended by industry, Extension and academic audiences that specialize in animal agriculture, waste management, composting and anaerobic digestion systems.

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