Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Our target audiences for communication of our research include agencies (state and federal), farmers, farmland owners, farm advisors, trainees along with future students through workshops, the public, agricultural and environmental scientists, and environmental health and mitigation specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The described experiments also supported the mentoring and supervision of two undergraduate laboratory assistant students. The graduate student supported by this grant also participated in the Science Olympiad, a state- and national-level competition for public school students. The graduate student acted as facilitator and judge for the middle- and high-school students during the April 2024 competition hosted in Ames, Iowa. Graduate students attended a Microbiome Workshop, hosted by the PI,where they received training on identifying core microbiome between datasets. Presenting this research and generating these datasets have fostered valuable collaborations with fellow researcher and contributed to deeper understanding of strategies for mitigating antimicrobial resistance in agricultural systems. How have the results been disseminated to communities of interest?Graduate students have presented posters, oral presentations, and online webinars about their research. Two manuscripts are under preparation related to this work. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will include finishing up analyses and writing up the papers for submission and responding to comments.
Impacts
What was accomplished under these goals?
Over the past year, the research team has investigated the dynamics of antimicrobial resistance (AMR) in agricultural environments, focusing on the effects of swine manure treatments on antimicrobial resistance genes (ARGs) in soil and leachate. We compared the impacts of untreated and anaerobically digested manure application onto soils with the installation of a conservation practice. Results wer ealso compared to application of a synthetic chemical fertilizer.Results showed that manure runoff containing ARGs primarily infiltrated vertically, with most ARGs retained in surface soil. These results suggest vertical infiltration intosoils may help mitigate AMR dispersal in agroecosystems. Additionally, analysis of plant roots from these columns revealed that exposure to manure did not significantly increase antimicrobial resistance in endophytic bacteria, indicating the minimal impact of manure-based AMR on plant root endophytes. Manure treatment alternatives were compared and evaluated for treatment of AMR risks, including antibiotic residuals, phenotypic resistance, and ARGs. In particular, this year, a comprehensive analysis that combined phenotypic, genotypic, and antibiotic data across various manure treatments was analyzed with the focus on writing a peer-reviewed manuscript. We have explored the effects of chlorination on its action towards the emergence of AMR in manure systems. A graduate student hashave been investigating two interconnected questions: (i) the extent to which chlorine disinfection used as a cleansing agent in animal farms may pose risks related to antimicrobial resistance, and (ii) the degree to which extracellular DNA in manure can contain antibiotic-resistant genes and promote gene transfer based on different manure wash water storage areas. To address the first question, transformation studies have been developed to study the impacts of disinfection concentrations.?
Publications
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Our target audiences for communication of our research include agencies (state and federal), farmers, farmland owners, farm advisors, trainees along with future students through workshops, the public, agricultural and environmental scientists, and environmental health and mitigation specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has afforded numerous professional development opportunities. Presentations have been made to collaborators at various stages of the project. The researchers have presented at national and local meetings. Every student has an individualized mentorship plan and has weekly meetings withPIs. How have the results been disseminated to communities of interest?Findings have been disseminated throughoral and poster presentations at the ASABE Iowa Section meeting and oral presentations at both the ASM Conference at Grinnell College and the ASABE 2023 Annual International Meeting in Omaha, Nebraska. What do you plan to do during the next reporting period to accomplish the goals?We will publish the results from the manure mitigation experiment and the soil column experiment. Experiments to study the effects of selective pressure on AMR in manure are underway.Preliminary experiments to establish Minimum Inhibitory Concentrations (MICs) of antibiotics and heavy metals are underway. These will inform the design of the next experiments to assess resistance responses in swine manure.
Impacts
What was accomplished under these goals?
Objective 1. Characterize AMR response to manures under varying antibiotic usage and pressures. Objective 2. Identify and compare the underlying mechanisms of action of emerging AMR in organisms under varying antibiotic and metabolite pressures. An experiment was completed to investigate the effect of different tetracycline concentrations on the bacteria populations within manure. This experiment is associated with both Objective 1 and 2. Two manure types were used in this experiment, untreated (NT) manure and anaerobically digested (AD) manure. Three tetracycline treatments were studied, 0 mg tetracycline/kg manure, 1.6 mg/kg and 16 mg/kg. Sampling from the jars took place on days 0, 3, 7, 14, 28, and 35. Samples were taken from homogenized manure in jars for plating, DNA extraction, and antibiotic analysis. More antibiotic resistance, overall, was seen in the NT manure compared to the AD manure. In the NT manure, significant differences were seen between tetracycline treatments at days 14 and 28, however, no differences were seen in the AD manure. There were significant differences within time and manure treatment variables. All ARGs had significant differences between at least two time points, and there were significant differences between tetracycline rates only in tetX and sul1. Many ARGs had a relative decrease in quantity over time while others increased or were stable. Proliferation trends were similar between manure treatments. Looking at the microbial community composition, both the alpha and beta diversity is distinctly different between untreated and anaerobically digested manure. Coordinate analysis shows some separation based on time but no real differences between tetracycline rates. In culturable bacteria counts, untreated manure contained more bacteria while anaerobically digested manure started with much fewer. When looking at their populations using molecular techniques, we were able to see a higher abundance of bacteria in anaerobically digested manure. This is likely due to an increase in anaerobic bacteria during the anaerobic digestion process, however, these bacteria are unlikely to thrive in the aerobic culturing conditions used in this experiment. Anaerobic digestion of manure had a far greater effect on antibiotic resistance in manure than tetracycline rate. Statistical differences were observed between manure treatments in all data. Anaerobically digested manure had more copies of bacterial genes then untreated manure, suggesting that it contained more bacteria. This may be partially accounted for by bacteria that proliferated during anaerobic digestion. When looking at ARG copy numbers normalized to 16S copy numbers to control for the changes in ARGs as the number of bacteria changes, the benefit of anaerobic digestion is less apparent. Results are mixed with some genes having untreated manure with more normalized ARGs than anaerobically digested manure, some with less, and some that change depending on the time point or rate. There are many directions this project could go in the future. Repeating this experiment with untreated manure and more tetracycline rates, especially on the lower end, may help us tease apart more details on how ARGs and tetracycline interact in that environment. It would also be interesting to incorporate an investigation into the role in extracellular ARGs in the buildup of bacteria resistance in manure. Extracellular ARGs have been shown to be a reservoir or resistance in soil but very few studies have been published on their role in manure. Objective 3. Evaluate AMR mitigation effectiveness of manure treatment alternatives. An experiment investigated the temporal effects of various treatments (heat, aeration, anaerobic digestion, liming, and solid-liquid separation) on tetracycline-resistant bacteria populations. Comparative phenotypic analysis has indicated the effectiveness of all five treatments in the reduction of these bacteria. Notably, liming proved to be the most efficient, achieving a significant decrease within the first 24 hours and peaking in reduction by day five. A publication is in draft form and will be submitted next year on the microbial data associated with this experiment. Antibiotic data is also available and will be considered as a stand alone publication. In another experiment, samples were obtained from soil columns treated with no manure, undigested swine manure, or mesophilic-anaerobically digested swine manure (digestate). DNA was extracted from soil and water samples and analyzed for antimicrobial resistance genes (ARGs), specifically tetM, tetO, tetQ, and ermF in soil, and additional genes tetM, tetO, tetQ, ermF, ermB, sul1, and sul2 in leachate. The study compared ARG absolute and relative abundance in samples treated with digestate or undigested manure, or no manure. Abundances were assessed at different depths and time points. Undigested swine manure-treated columns showed the highest ARG abundances in soil and water, notably on days 7, 21, and 56 for soil and days 7-56 for leachate. However, leachate samples on day 0 exhibited higher ARG abundances in columns treated with anaerobically digested swine manure. ARGs did not increase in soil or leachate samples from soil columns which did not receive a manure or digestate treatment. For manure- or digestate-treated columns, ARGs peaked at 1-7 days post treatment and decreased by day 56. At this point, ARGs in soil from manure and digestate columns exceeded those with no manure, though differences were not significant in soil samples. Leachate samples at 56 days showed no elevation in ARGs in manure- or digestate-treated columns. Analysis indicated that the majority (75-95%) of ARGs in manure and digestate treatment solution were retained in the soil column profile, rather than passing out with leachate. These findings suggest that swine manure or digested swine manure runoff elevates ARGs in soil and leachate for several weeks post-manuring, with a significant retention of runoff ARGs in soil profiles. This retention may reduce ARG transport in leachate. Additionally, ARGs in soil and leachate samples from columns which received anaerobically digested swine manure were generally reduced compared to ARGs from manure treated columns. These results indicate that mesophilic anaerobic digestion of swine manure may change the transport and persistence of ARGs in soil and leachate. Objective 4. Assess indicators (antibiotics, ARGs, and ARBs) of AMR in diverse manures. This work has been published now and is reported as part of this annual report.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Neher, T., Soupir, M., Andersen, D, ONeill, M., and Howe, A. Comparison of antibiotic resistance genes in swine manure storage pits of Iowa, USA. Frontiers of Antibiotics. (2023). https://doi.org/10.3389/frabi.2023.1116785
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Our target audiences for communication of our researchinclude agencies (state and federal), farmers, farmland owners, farm advisors, trainees along with future students through workshops, the public, agricultural and environmental scientists, and environmental health and mitigation specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students have presented research in posters at theAmerican Society of Agricultural and Biosystem Engineering Iowa section meeting and oral presentation during American Society of Microbiology North Central Branch conference. Weekly meetings are used to discuss research and professional goals. Team meetings are once a semester to update on research progress and promote collaborations. How have the results been disseminated to communities of interest?Results have been communicated in posters, oral presentations, and extension documents to communities of interest. What do you plan to do during the next reporting period to accomplish the goals?We plan to mentor graduate students to prepare manuscripts about their research to submit to peer-reviewed publications.
Impacts
What was accomplished under these goals?
Objective 1. Characterize AMR response to manures under varying antibiotic usage and pressures. Objective 2. Identify and compare the underlying mechanisms of action of emerging AMR in organisms under varying antibiotic and metabolite pressures. To study the time-based effect of antibiotic resistance in bacteria populations within swine manure at various rates of tetracycline, both anaerobically digested and untreated swine manure were spiked with three different tetracycline concentrations (0 µg/kg, 1600 µg/kg, and 16000 µg/kg). Untreated manure was spiked and incubated at room temperature while manure from the sample collection bucket was anaerobically digested before it was spiked. Samples were collected for chemical analysis, genotypic analysis, and phenotypic analysis at six different time points over 35 days. Bacteria counts performed for phenotypic analysis at each time point showed a rate response in the untreated manure with more resistance seen at higher tetracycline concentrations by day 7, a trend that continued through day 28, peaking at day 14. After the peak, bacteria counts overall decreased. No significant differences in resistance were seen between spiked tetracycline concentrations in anaerobically digested manure. DNA has been extracted from all samples for genotypic analysis and quantification of antibiotic resistance genes is in progress. Samples for tetracycline quantification (chemical analysis) have been freeze dried in preparation for analysis. Objective 3. Evaluate AMR mitigation effectiveness of manure treatment alternatives. Objective 4. Assess indicators (antibiotics, ARGs, and ARBs) of AMR in diverse manures. The effect of manure treatment strategies on AMR indicators was compared. Treatments included heat, aeration, anaerobic digestion, liming and solid-liquid separation. Assessment of AMR risks focused on impacts on tetracycline resistant bacteria. To date, our comparative phenotypic results showed that each of the five treatments was effective in reducing TET-resistantbacteria. Liming was found to be most effective among all treatments, suggesting it as practical and rapid treatment to reduce tetracycline resistant bacteria within 24 hours. It showed its maximum reduction during day 5. Gene counts are currently being estimated also for ARGs in these manure treatments.Manures have been sent to our collaborators for antibiotic charcterization and future opportunities to access antibiotics, ARGs, and ARBs together. UBuffalo activities: Freeze-dried swine manure samples shipped from Iowa State University were received in sets of 16. The first set of samples were received on June 6, 2022. All samples were stored at -20 °C prior to analysis. Samples were analyzed for the following veterinary antimicrobials that belong to 5 major classes of interest: sulfonamides [sulfachloropyridazine (SCP), sulfadimethoxine (SDM), sulfamethazine (SMI), sulfamerazine (SMR), sulfameter (SMT), sulfamethoxazole (SMX), sulfamethizole (SMZ), sulfadiazine (SPD), sulfathiazole (STZ)], tetracyclines [anhydrochlorotetracycline (ACTC), anhydrotetracycline (ATC), chlorotetracycline (CTC), 4-epichlortetracycline (ECTC), 4-epitetracycline (ETC), ocytetracycline (OTC), tetracycline (TC)], macrolides [azithromycin (AZI), clarithromycin (CLA), erythromycin (ERY), roxithromycin ROX, spiramycin 2 SPI2, spiramycin 3 SPI3, tylosin (TY)L, tilmicosin (TIL)], pleuromutilin [tiamulin (TIA)], and ionophores [monensin (MON), lasalocid (LAS), nigericin (NIG), salinomycin (SAL), nonactin (NON), maduramycin (MAD) and narasin (NAR)]. Antimicrobial analysis was completed by liquid chromatography with tandem mass spectrometry (LC-MS/MS).
Publications
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Progress 01/01/21 to 12/31/21
Outputs
Target Audience:During this Year 1 reporting period, our efforts focused on recruiting and training students and communicating the rationale and scope of our resesarch to agencies (state and federal), farmers, farmland owners, farm advisors, trainees along with future students through workshops, the public, agricultural and environmental scientists, and environmental health and mitigation specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?All students and staff participated in an AMR workshop, including encouragement to participate in extension material development, in December 2021. Diana Aga from UBuffalo traveled to ISU to discuss best practices for shipping manure and also plans for developing new experiments around antibiotic degradation and metabolites. Students and postdocs meet weekly with PI Howe to discuss research progress and individual mentorship plans. How have the results been disseminated to communities of interest?We have published one review article discusssing the importance of AMR in manure applications. What do you plan to do during the next reporting period to accomplish the goals?We need to recruit a full time student to this project and that has been challenging due to both COVID and student engagement. Our plans our to work more closely to link genes, phenotypes, and antibiotics in repeated experiments in the next year.
Impacts
What was accomplished under these goals?
In Year 1, we focused our efforts on recruiting students to this work, which was more challenging due to COVID interruptions of student engagement and also the numbers of individuals allowed in the research laboratory simultaneously. Despite a slower initiation, we accomplished several accomplishments towards mainly our first and third objectives. Objective 1: We studied thetime-based effect of antibiotic resistance in bacteria isolated from erythromycin spiked pig manure. We expected that antibiotic resistance in the spiked manure sample would decrease over time, likely due to the antibiotic degradation. Also, we expected that the addition of antibiotics wouldincrease the number of antibiotic resistant bacteria (ARBs) isolated and the range of antibiotics the manure bacterial community is resistant to. After spiking swine manure with three different concentrations of erythromycin (0 ug/kg, 10 ug/kg, and 3000 ug/kg), we observed that the greater concentrations of erythromycin resulted inhigher numbers of erythromycin resistant isolates from the manure. Additionally, performing phenotypic testing on manure isolates showed that they were multi-drug resistant even prior to spiking the manure with erythromycin. Objective 3:A preliminary experiment consisting of four different short-term manure treatments was performed to compared to determine their relative suppressive effect against bacteria resistant to tetracycline. These four treatments were: aeration, heat treatment, liming, and solid-liquid separation. Liming was the most effective treatment with significantly fewer tetracycline resistant bacteria present in samples taken after liming than before. Heat treatment and solid-liquid separation also saw a decrease in the presence of tetracycline resistant bacteria. Conversely, there was an increase in the number of tetracycline resistant bacteria in the aeration treatment, possibly due to favorable conditions increasing bacterial populations overall. These treatments were performed as a preliminary experiment; a larger-scale experiment including both a composting treatment and an anaerobic digestion treatment is recommended. Objective 4: We are developing standard operating protocols for phenotypic resistance plating and a list of indicators for our swine manures that are represenetative with antibiotic resistance, espeicially to tetracyline and erythromycin. These genes will be further developed in the future for more specificity to detect bacterial hosts.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Howe, A. C., & Soupir, M. L. (2021). Antimicrobial resistance in integrated agroecosystems: State of the science and future opportunities. J. Environ. Qual. 50: 1255 1265. https://doi.org/10.1002/jeq2.20289
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