Progress 01/01/16 to 12/31/19
Outputs
Target Audience:Our target audiences remain the same as described in our initiation project and include state and federal agencies, stakeholders (particularly land and production facility managers), industry (microbiology/antibiotics), trainees, agriculture and environmental science researchers, biomarker developers, and the public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoc and graduate students have had the opportunity to present work at national and international conferences and workshops. Training for data analysis workshops were conducted annually and an analysis was published our final year. How have the results been disseminated to communities of interest?Results have been disseminated through USDA communiciations, social media, and through university communications. We have published in peer-reviewed journals. We have also presented this work at professional conferences. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Accomplishments for Objective 1: We have developed a novel approach to capture ARGs directly using high throughput amplicon sequencing, with multiple amplicons generated in a single sequencing library preparation. For its ability to detect the prevalence and diverse ARGs, we have named this tool Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM). This approach is based on Illumina TruSeq Technology, which has previously been used for detection of biomarkers in clinical samples but is the first demonstration of TruSeq for microbial DNA and for ARGs. The specific highlights of this technology is that it can amplify multiple ARG targets in a single library preparation, detecting hundreds of ARGs simultaneously. We have shown that this technology is quantitatively accurate and specific. Further, we can distinguish resistome profiles from diverse environmental samples. Accomplishments for Objective 2: As agriculture industrializes, concentrated animal feeding operations (CAFOs) are becoming more common. Feces from CAFOs is often used as fertilizer on fields. However, little is known about the effects manure has on the soil microbiome, which is an important aspect of soil health and fertility. In addition, due to the subtherapeutic levels of antibiotics necessary to keep the animals healthy, CAFO manure has elevated levels of antibiotic resistant bacteria. Using 16s rRNA high-throughput sequencing and qPCR, this study sought to determine the impact of swine CAFO manure application on both the soil microbiome and abundance of select antibiotic resistance genes (ARGs) and mobile element genes (erm(B), erm(C), sul1, str(B), intI1, IncW repA) in agricultural soil over the fall and spring seasons. We found the manure community to be distinct from the soil community, with a majority of bacteria belonging to Bacteroidetes and Firmicutes. The soil samples had more diverse communities dominated by Acidobacteria, Actinobacteria, Proteobacteria, Verrucomicrobia, and unclassified bacteria. We observed significant differences in the soil microbiome between all time points, except between the spring samples. These results suggest bacteria in the manure do not survive well in soil and that ARG dynamics in soil following manure application vary by resistance gene. Accomplishments for Objective 3: Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manuretreated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment. Accomplishments for Objective 4: We analyzed our workshops during the extent of this grant and published an analysis of our educational approach and learner evaluations for our EDAMAME data analysis workshop. This will help to provide information to educators and learners.
Publications
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Progress 01/01/19 to 12/31/19
Outputs
Target Audience:Our target audiences remain the same as described in our initiation project and include state and federal agencies, stakeholders (particularly land and production facility managers), industry (microbiology/antibiotics), trainees, agriculture and environmental science researchers, biomarker developers, and the public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?See above accomplishments on Obj 4. How have the results been disseminated to communities of interest?Our training course as been disseminated through USDA communiciations, social media, and through university communications. We have published in peer-reviewed journals. We have also presented this work at professional conferences. What do you plan to do during the next reporting period to accomplish the goals?This is the final year of the funding.
Impacts
What was accomplished under these goals?
Accomplishments for Objective 1: We have developed anovel approach to capture ARGs directly using high throughput amplicon sequencing, with multiple amplicons generated in a single sequencing library preparation. For its ability to detect the prevalence and diverse ARGs, we have named this tool Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM). This approach is based on Illumina TruSeq Technology, which has previously been used for detection of biomarkers in clinical samples but is the first demonstration of TruSeq for microbial DNA and for ARGs. The specific highlights of this technology is that it can amplify multiple ARG targets in a single library preparation, detecting hundreds of ARGs simultaneously. We have shown that this technology is quantitatively accurate and specific. Further, we can distinguish resistome profiles from diverse environmental samples. Accomplishments for Objective 2:As agriculture industrializes, concentrated animal feeding operations (CAFOs) are becoming more common. Feces from CAFOs is often used as fertilizer on fields. However, little is known about the effects manure has on the soil microbiome, which is an important aspect of soil health and fertility. In addition, due to the subtherapeutic levels of antibiotics necessary to keep the animals healthy, CAFO manure has elevated levels of antibiotic resistant bacteria. Using 16s rRNA high-throughput sequencing and qPCR, this study sought to determine the impact of swine CAFO manure application on both the soil microbiome and abundance of select antibiotic resistance genes (ARGs) and mobile element genes (erm(B), erm(C), sul1, str(B), intI1, IncW repA) in agricultural soil over the fall and spring seasons. We found the manure community to be distinct from the soil community, with a majority of bacteria belonging to Bacteroidetes and Firmicutes. The soil samples had more diverse communities dominated by Acidobacteria, Actinobacteria, Proteobacteria, Verrucomicrobia, and unclassified bacteria. We observed significant differences in the soil microbiome between all time points, except between the spring samples. These results suggest bacteria in the manure do not survive well in soil and that ARG dynamics in soil following manure application vary by resistance gene. Accomplishments for Objective 3:Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment. Accomplishments for Objective 4: We analyzed our workshops during the extent of this grant and published an analysis of our educational approach and learner evaluations for our EDAMAME data analysis workshop. This will help to provide information to educators and learners.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Citation: Smith SD, Colgan P, Yang F, Rieke EL,
Soupir ML, Moorman TB, et al. (2019)
Investigating the dispersal of antibiotic resistance
associated genes from manure application to soil
and drainage waters in simulated agricultural
farmland systems. PLoS ONE 14(9): e0222470.
https://doi.org/10.1371/journal.pone.0222470
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Shade A, Dunivin TK, Choi J, Teal TK, Howe AC. 2019. Strategies for building computing skills to support microbiome analysis: a five-year perspective from the EDAMAME workshop. mSystems 4:e00297-19. https://doi.org/10.1128/mSystems.00297-19.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Citation: Lopatto E, Choi J, Colina A, Ma L, Howe
A, Hinsa-Leasure S (2019) Characterizing the soil
microbiome and quantifying antibiotic resistance
gene dynamics in agricultural soil following swine
CAFO manure application. PLoS ONE 14(8):
e0220770. https://doi.org/10.1371/journal.
pone.0220770
|
Progress 01/01/18 to 12/31/18
Outputs
Target Audience:Our target audiences remain the same as described in our initiation project and include state and federal agencies, stakeholders (particularly land and production facility managers), industry (microbiology/antibiotics), trainees, agriculture and environmental science researchers, biomarker developers, and the public. Changes/Problems:We have had no major problems. Coordinating our probe design with Illumina, a commercial company, has at times been delayed but all groups are enthusiastic and motivated to benchmark and publish this technology. What opportunities for training and professional development has the project provided?We have made all our training for our course openly accessible (www.edamamecourse.org) and students should be able to complete this online material even without attending the physical workshop. All students and postdocs funded by this grant have individualized mentorship plans and regularly meet with our leadership team for professional development. How have the results been disseminated to communities of interest?Our training course as been disseminated through USDA communiciations, social media, and through university communications. We have published in peer-reviewed journals. We have also presented this work at professional conferences (International Society of Microbial Ecology, American Water Resources Association). What do you plan to do during the next reporting period to accomplish the goals?Our final extension year, we hope to finish our publication on the DARTE-QM technology -- we have strong proof of concepts that it is working but have waited for more relevant ARG targets to publish this work for higher impact. We also hope to publish a strong paper helping fill gaps in the persistence of ARGS in our soil column experiments.
Impacts
What was accomplished under these goals?
Accomplishments for Objective 1: In Phase I of DARTE-QM development, we strategically selected a "mock" community containing known genes relevant to antibiotic resistance. Reference genomes were available for the membership of the mock community, allowing us to validate sequenced DARTE-QM amplicons. The results from this effort provided strong support for the success of DARTE-QM: 95% (63 out of 66 ARG) genes from mock community were accurately detected. Comparison of the 16S rRNA gene (DARTE-QM 16S rRNA probe vs. MiSeq 16S rRNA probe) were statistically indistinguishable, suggesting that for this gene, DARTE-QM would provide similar results of community membership. Finally, DARTE-QM estimates of erm gene (associated with macrolide resistance) abundances directly correlate to gene estimates from metagenome sequencing, further support that DARTE-QM can be used for quantitative measurements of ARG genes. Phase II of DARTE-QM expands our Phase I probes for ARGs of increasing environmental relevance. Rather than targeting genes in a mock community, our objective is to broadly target genes relevant for assessing AMR risks in the environment. We have now designed probes for greater than 75% of all ARG genes contained within a curated core database for antibiotic resistant genes, and our in the process of ordering these probes in collaboration with Illumina TruSeq. These probes will be tested soil and water samples with known manure/antibiotic-treatment histories in the next year. This work has supported the publication of one peer-reviewed publications: Choi et al., 2018. We anticipate a publication highlighting this technology this coming year. Accomplishments for Objective 2: As agriculture industrializes, concentrated animal feeding operations (CAFOs) are becoming more common. Feces from CAFOs is often used as fertilizer on fields. However, little is known about the effects manure has on the soil microbiome - an important aspect of soil health and fertility. In addition, due to the subtherapeutic levels of antibiotics necessary to keep the animals healthy, CAFO manure has elevated levels of antibiotic resistant bacteria. Manure application on fields has the potential to disseminate these antibiotic resistance genes (ARGs). The dynamics of ARGs in the soil over time after manure application are also unclear. The results from this objective sought to determine the effects of swine CAFO manure application on both the soil microbiome and abundance of select ARGs and mobile element genes (erm(B), erm(C), sul1, str(B), int1, IncW repA) in agricultural soil over the spring and fall season. We found while the abundance of the main soil phyla does not individually change, the soil microbiome as a whole does significantly change from manure application and does not recover to the same community in pre-manure soil by the spring. Of the measured genes, manure application only significantly increased the abundance of erm(B) and erm(C) which remained elevated in the spring. This work is being submitted as a manuscript to Applied and Environmental Microbiology and is being led by co-PI Hinsa and her student Edward Lopatto. Accomplishments for Objective 3: Using intact soil columns, we simulated an agroecosystem with artificial subsurface drainage, typical of the Upper Midwestern United States. Columns were collected from plots maintained in corn-soybean rotations, with nitrogen application prior to the corn growing season. Nitrogen was applied either in the form of swine manure or urea ammonium nitrate (UAN) and plots were maintained under chisel plow or no-till managements, creating four different overall management practices: manured no-till, non-manured no-till, manured chisel plow and non-manured chisel plow. Manure was applied to columns to simulate manure injection bands. DNA extraction was performed on effluent of six simulated rainfall events, replicating the length of a typical drainage season for the location (108 days). After the second, fourth and sixth rainfall events, subsets of manured columns were destructively sampled from the top 15 cm of soil for DNA extraction. Targeted 16S rRNA gene sequencing was used to characterize and to identify shifts in soil and drainage microbial communities post manure application. These results indicated that soil history was associated with significant differences in community structure, although only 2% of variation was explained by this factor. The orders of bacteria in soil which were stimulated by manure application contained varied responses in drainage waters over the course of the experiment. We also identified a "manure-specific core" of five genera who comprised 13% of manure communities. Results from our study increase our understanding of soils' capacity to attenuate or transport manure associated microbial communities. Identification of manure stimulated and manure derived bacteria in soil and drainage waters provides a holistic approach for tracking manure associated microbial impacts in the environment. Metagenome shotgun sequencing was also used to characterize ARGs in select soil column samples, and we have identified ~50 ARGs that persist in both soil and water samples from the manure in this experiment. These results are currently being integrated into a publication with expected submission in the early Spring. Overall, an important output of this work is a better understanding of what ARGs originate from manure and are distinct from background soil and water resistomes. These genes are being heavily prioritized for development into the DARTE-QM technology and also for probes for high throughput PCR. This work has resulted in one peer-reviewed publications (Rieke et al., 2018) and one in review at Frontiers for Microbiology (Rieke et al, in review). We expect to submit one publication about the persistence of ARGs in the environment this year. Accomplishments for Objective 4: As part of our extension component of this effort, we have used ARG probes to assist in other project to detect resistomes and have collaborated with Diana Aga (University of NY - Buffalo) and Lisa Schulte-Moore (Iowa State University) to detect these genes in manures and soils. EDAMAME, a week long work shop to develop education and training for biologists to use tools developed in this grant, had 103 applicants and we accepted 25 students, prioritizing diverse students locally and internationally. For this year, again, funds from this proposal supported workshop fees for all students. Feedback from the course was extremely positive, with students reporting unanimously that learning objectives were achieved.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Guyer HE, Howe A, Moorman T, Andersen D, Soupir ML. (2018, Nov). Impacts of Manure Application Timing and Tillage Practices on Antibiotic Resistant Bacteria and Genes in Drainage Water from Manured Fields. Presented at the annual meeting of the American Water Resources Association, Baltimore, MD.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Jinlyung Choi, Elizabeth L Rieke, Thomas B Moorman, Michelle L Soupir, Heather K Allen, Schuyler D Smith, Adina Howe; Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance, FEMS Microbiology Ecology, Volume 94, Issue 4, 1 April 2018, fiy006, https://doi-org.proxy.lib.iastate.edu/10.1093/femsec/fiy006
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Rieke, E. L., T. B. Moorman, M. L. Soupir, F. Yang, and A. Howe. 2018. Assessing Pathogen Presence in an Intensively Tile Drained, Agricultural Watershed. J. Environ. Qual. 47:1033-1042. doi:10.2134/jeq2017.12.0500
- Type:
Journal Articles
Status:
Under Review
Year Published:
2018
Citation:
Elizabeth Luby Rieke, Michelle Lynn Soupir, Thomas B Moorman, Fan Yang, and Adina Howe. Temporal dynamics of bacterial communities in soil and leachate water after swine manure application. Frontiers in Microbiology. 2018, in review.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Mobility and persistence of integron-associated antibiotic resistance genes in manure-applied agricultural soil and water. International Society of Microbial Ecology Meeting, Leipzig, Germany, 2018. Phil Colgan, Heather Allen, Thomas Moorman, Michelle Soupir, Elizabeth Rieke, and Adina Howe.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Impacts of Swine Manure Application on Soil Microbial Communities and Associated Drainage Water. International Society of Microbial Ecology Meeting, Leipzig, Germany, 2018. Elizabeth Rieke, Heather Allen, Thomas Moorman, Michelle Soupir, and Adina Howe.
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Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Our target audiences remain the same as described in our initiation project and include state and federal agencies, stakeholders (particularly land and production facility managers), industry (microbiology/antibiotics), trainees, agriculture and environmental science researchers, biomarker developers, and the public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have made all our training for our course openly accessible (www.edamamecourse.org) and students should be able to complete this online material even without attending the physical workshop. All students and postdocs funded by this grant have individualized mentorship plans and regularly meet with our leadership team for professional development. How have the results been disseminated to communities of interest?Our training course as been disseminated through USDA communiciations, social media, and through university communications. We have also presented this work at professional conferences (American Society of Microbiology and American Society of Agricultural and Biosystems Engineers) What do you plan to do during the next reporting period to accomplish the goals?Year 3 will be focused on publishing the DARTE-QM technology to make it widely available to the public. Additionally, we plan to have our results from our soil columns published in a series of 2-3 publications this year.
Impacts
What was accomplished under these goals?
Accomplishments for Objective 1: For our test of the DARTE-QM technology, we designed approximately 150 primers targeting approximately 50 known ARGs, MEs, and bacterial phylogenetic markers (i.e., genes that define which bacteria are present). More than half of these were specifically designed to target ARGs and phylogenetic markers found in a bacterial community containing known members with known genome sequences; this is a so-called mock community (our mock community has been published previously and can be found here: Allen et al. 2016 BMC Res Notes). We took this approach in order to validate how well DARTE-QM did or did not work. That is, if we had tested DARTE-QM exclusively using complex samples from the environment, we wouldn't know what genes to expect from that environment, and so it would have been impossible to detect false-positives or false-negatives. The results of testing DARTE-QM on the mock community showed 44 out of 44 mock community targeted ARGs were detected (100% true positive detection). Further, 70% of the primers amplified to the abundance as expected, with the remaining 30% yielding too many or too few sequences suggesting some range of specificity/sensitivity for our designed primers in the mock community. In environmental samples, we can detect 40 ARGs, including genes previously described as connected to anthropogenic impact. Accomplishments for Objective 2: Sampling from last year is being analyzed for a suite of ARGs with qPCR assays (Grinnell University). Additional sampling is planned for this year. We had delayed field sampling to focus our efforts to ensure the successful development of Objective 1. With positive results now for DARTE-QM, we anticipate thorough sampling and surveillance this year. Accomplishments for Objective 3: We are currently preparing a manuscript to describe microbial community structure change "from rump to runoff" from our soil column experiments and plan to submit this in Spring. Additionally, these samples were included on our DARTE-QM run, where early promising results indicate we have detected anthropogenic ARG genes in these samples effectively with this novel technology. We also have generated metagenomes from these samples and are currently analyzing datasets. Accomplishments for Objective 4: EDAMAME, a week long work shop to develop education and training for biologists to use tools developed in this grant, had 129 applicants and we accepted 25 students, prioritizing diverse students locally and internationally. Funds from this proposal supported workshop fees for all students. Feedback from the course was extremely positive, with students reporting unanimously that learning objectives were achieved. A workshop is planned again this summer, with applications due in February and the workshop in June.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2017
Citation:
Choi, J. +, Rieke, E. +, Moorman, T., Soupir, M., Allen, H.K., Smith, S.D., and Howe, A. Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance. FEMS Microbiology Ecology. Revision submitted Dec, 2017.
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Our target audiences remain the same as described in our initiation project and include state and federal agencies, stakeholders (particularly land and production facility managers), industry (microbiology/antibiotics), trainees, agriculture and environmental science researchers, biomarker developers, and the public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have made all our training for our course openly accessible (www.edamamecourse.org) and students should be able to complete this online material even without attending the physical workshop. All students and postdocs funded by this grant have individualized mentorship plans and regularly meet with our leadership team for professional development. How have the results been disseminated to communities of interest?Our training course as been disseminated through USDA communiciations, social media, and through university communications. We have also presented this work at professional conferences (American Society of Microbiology and American Society of Agricultural and Biosystems Engineers). What do you plan to do during the next reporting period to accomplish the goals?As year 1 has been a ramp up year with experimental setup and sample collection, year 2 will be a year of data analysis and refinement for our objectives. Our team regularly meets (every other week) to discuss our goals and to remove any challenges for accomplishing objectives. Next year, we hope to have peer-reviewed publications to communicate our results.
Impacts
What was accomplished under these goals?
Objective 1 and 2: We proposed adapting Illumina's TruSeq technology to the sequencing of hundreds of antibiotic resistance genes in parallel. While one team developed the bioinformatic pipeline for primer design, another team executed the TruSeq technology using existing qPCR primers for resistance genes (Looft et al. 2012 PNAS). We sent the resistance gene primer sequences to Illumina, they generated TruSeq probes, and we tested them in the laboratory. The results showed failure to amplify antibiotic resistance genes in all samples tested (manure, soil, and an artificially assembled community of 19 known bacteria (mock community). Troubleshooting suggested that the amplicons generated by the qPCR primers were too small to be effective in the TruSeq protocol. Therefore, we turned to the primer design team to generate new primer sets for antibiotic resistance genes that would amplify targets appropriately sized for TruSeq. We have now tested two versions of primers for TruSeq that were designed by our bioinformatic pipeline. We chose to test them via qPCR prior to ordering them as TruSeq probes from Illumina. Ten primer pairs (forward and reverse) were randomly chosen from the nearly 200 that we generated. The ten pairs were tested via qPCR assay both in individual reactions and pooled (i.e., all ten primer sets in one PCR), and the template DNA tested was either a single genome (multi-drug resistant Salmonella enterica serovar Typhimurium) or a pool of known genomes (the mock community). The first version of primers failed to amplify resistance genes and were shown to be too specific. The second version of primers were met with much greater success: 7/10 primer sets had ideal efficiency, and although 3/10 showed slightly lower efficiency they nonetheless amplified the desired product. Prior to proceeding with this version of the primers, we discovered that the window of PCR products was too large. We need to generate amplicons of a specific size range, and our products were exceeding that range. Therefore, we have once again redesigned the primers with the exact amplicon size specified by Illumina for their TruSeq technology, and we are currently testing them in qPCR assay prior to ordering them as probes for another TruSeq attempt. We have collected manure, soil, and water samples and this DNA has been extracted and ready for testing with DARTE-QM as it develops. Objective 3: Soil Column Experiments Soil columns were extracted from four plots at Iowa State's Northeast Research and Demonstration Farm, near Nashua, IA (43.0° N, 92.5° W). Soils at the farm consist of moderately well to poorly drained Kenyon silty-clay loam, Floyd loam, and Readlyn loam overtop of glacial till, with 1 to 3% slopes (Luby et al. 2016). Plots were maintained in corn soybean rotations, with nitrogen application prior to the corn growing season. Nitrogen was applied either in the form of swine manure (one chisel plow and one no-till) or urea ammonium nitrate (UAN) (one chisel plow and one no-till). Manure was last injected as 10-15 cm deep bands on November 6, 2014. Manure has not been applied to control (non-manured plots) since 1978, while manured plots application rates have varied since 1993. Chisel plow tillage and no-till management were chosen due to their prevalence in Iowa agriculture. Agricultural fields under no-till management form preferential flow paths, or macropores. Chisel plow tillage disrupts macropores and aerates soil, while leaving crop residue on top, limiting erosion potential.? Manure application and Pre Manure Soil Sampling and Manure Application Simulated manure injection zones were incorporated in manure treated columns by removing a 10.16 cm diameter, 15.24 cm deep section of soil from each column. Removed soil was subsampled in 10-20 g aliquots. The tops of non-manured control columns were also sampled at the same time by scraping off 10-20 g off the top of the column and frozen at -20 ?C. Manured columns were treated with 750 mL of liquid swine manure to approximate a 7.8 cm wide, 15.24 cm deep manure injection band. Simulated Rainfall and Effluent capture Six simulated rainfall events were conducted 7, 21, 35, 49, 77 and 107 days after manure application. Single column rainfall simulators were constructed by drilling holes in 15.24 cm diameter PVC pipe end caps to insert X needles. Simulators were placed on top of each column and filled with 1 L of DI water. Leachate was collected in 1 L sterile Nalgene bottles located on racks below the columns and processed 48 hours after the beginning of each event. In order to maintain soil moisture contents representative of field conditions columns received additional wettings in between rainfall events. A 1 L pan of DI water was placed on the top of the column rack during the experiment. 48 hours prior to rainfall events the amount evaporated from the pan was recorded. DI water was added at a rate of amount evaporated plus an additional 100 mL to ensure column water contents reached field capacity. Additionally, this process was repeated once a week throughout the experiment during weeks without simulated rain events. DNA Extraction and 16S rRNA Sequencing Column effluent was filtered through 0.22 um sterile filters and frozen at -20 ?C until DNA extractions were performed using Mo Bio Power Water DNA kits. Three 50 mL aliquots were filtered from manure treated effluent. Two 250 mL aliquots and remaining effluent was filtered from non-manured control columns. Greater volumes of water were filtered from non-manured columns to ensure enough DNA was recovered for downstream analysis. Soil DNA was extracted using PowerSoil 96 Well Soil DNA Isolation kits. Soil aliquots for each well were weighed and recorded. DNA samples above 10 ug ml-1 were normalized to 10 ug ml-1. The V4 region of bacterial 16S rRNA was amplified using methods previously by Kozich et al. (2013). Sequencing of bacterial amplicons was performed on Illumina Miseq with Miseq Reagent Kit v2. Mitigation strategies We have set up the experiment to evaluate the potential of manure management strategies: anaerobic digestion, ionophore treatment, two-phase manure storage, liquid solid segregation, and centrifugation manure treatment systems for reduction of ARB (entoeroccci, TET resistant enterococci, and TYL resistant enterococci) and ARGs (ermB, ermF, ermC, msrA, tetW, tetM and tetQ). Information on reductions of ARB and ARGs by manure management practices is needed as a strategy to mitigate the spread of antibiotic resistance through agroecosystems. We are currently analyzing manure for antibiotics. Total enterococci concentrations, TYL-resistant concentrations, and TET-resistant concentrations are determined by spread plate techniques on mENT agar (U.S. EPA 2000) and TYL and TET infused mENT agar at a concentration of 35 and 16 mg/L, respectively (Benning and Mather, 1999; CLSI, 2010; Kaukas et all, 1988). This DNA will be screened for antibiotic resistance genes with qPCR techniques and also with DARTE-QM as it continues to develop. Objective 4: We have piloted our bioinformatic workshop (edamamecourse.org) and developed online training material as well as deployed and evaluated it in a one week workshop. Assessment on this course is currently undergoing. Applications for this year's course are already open and we are anticipating diverse applicants. We are collaborating with partners to gather environmental samples to screen with DARTE-QM, prioritizing experiments with high statistical power to inform ARGs in the environment or are related to mitigation technologies.
Publications
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