Progress 11/01/23 to 10/31/24
Outputs
Target Audience:In the past year, we have targeted audiences in the following groups: - Undergraduate students (including those from underrepresented groups): we continued recruitingundergraduate student researchers through the RISE (Research In Science and Engineering) program at UCR to provide undergraduate students research experience related to this project's topics. - Scientific communities: we presented our research findings as oral and poster presentations at student symposiumsandinvited talks at other universities. - Farmers and wastewater engineers: we conmunicated with local farmers and wastewater engineers about our projects and obtained treated wastewater and manure samples for our large-pot and lysimeterexperiments to investigate the effect oftreatedwastewater and manure on the spread of antimicrobial resistance during agricultural activities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Seven undergraduate students from University of California - Riverside were hired as part of UCR's RISE (Research in Science and Engineering) program from June 2024 to September 2024 (10-week program) in which the students were supported using a stipend funded by this NIFA grant. Within RISE, we previously established a program named "AMR in AGRI" https://rise.ucr.edu/usda-amr-in-agri to which UCR students could apply. The students assisted in research supporting the NIFA project and were given mentoring and training in research methods by the PD and co-PDs. At the end of the program, the students presented research findings at a RISE symposium held at UCR (poster/talk titles are listed below). Five of the students were subsequently retained after RISE (~10 hours per week for 10 weeks/quarter), for which they are receiving quarterly stipends funded by this NIFA grant. How have the results been disseminated to communities of interest?Besides publications in high-impact journals, we have disseminated our findings to communities of interest, as follows. - Oral and poster presentationsby undergraduate students at the 2024 RISE/MARC Summer Symposium on August 28/2024 at UCR: Evelyn Dibos (Advisor: co-PDJorge Ferreira), Duc Phan, Ananda S. Bhattacharjee, Abasiofiok M. Ibekwe, Daniel Ashworth, Yujie, Men, Michael Schmidt, Jorge F.S. Ferreira. Biomass accumulation and nutritional value of vegetables irrigated with wastewater. 2024 RISE/MARC Summer Symposium, Poster Session 1. August 28, 2024. UC Riverside, Riverside, California. Ty Kannangara (Advisor co-PDAshworth) at the 2024 RISE/MARC Summer Symposium 08/24/2024. Effect of biochar on uptake of emerging contaminants by various crops. Anthony Baxa (Advisor co-PDAshworth) at the 2024 RISE/MARC Summer Symposium 08/24/2024. Sand/Biochar filtration for antibiotic and DNA removal from treated wastewater. Adam Izaguirre (Advisor, co-PDMark Ibekwe)Desmond Hanan, Duc Phan, Daniel Ashworth, Yujie Men, Michael Schmidt, Jorge F. S. Ferreira, Abasiofiok M. Ibekwe.Impact of Treated Municipal Wastewater on Antimicrobial Development in E. coli Recovered from Agricultural Production Environment. 2024 RISE/MARC Summer Symposium, Poster Session 1. August 28, 2024. UC Riverside, Riverside, California. 2024 RISE/MARC Summer Symposium, Poster Session 1. August 28, 2024. UC Riverside, Riverside, California. Justin Vinuya (Advisor, co-PDMark Ibekwe), Desmond Hanan, Duc Phan, Daniel Ashworth, Yujie Men, Michael Schmidt, Jorge F. S. Ferreira, Abasiofiok M. Ibekwe. Impact of treated municipal wastewater on antimicrobial development in Enterococcus. 2024 RISE/MARC Summer Symposium, Poster Session 1. August 28, 2024. UC Riverside, Riverside, California. Yosef Soliman (Advisor, Michael Schmidt), Duch Phan, Daniel Ashworth, Yujie Men, Jorge F. S. Ferreira, Abasiofiok M. Ibekwe. Post-modification of Biochar for adsorption of contaminants in Wastewater. 2024 RISE/MARC Summer Symposium, Oral Session 3. August 28, 2024. UC Riverside, Riverside, California. Leena Nimir (Advisor, PD Yujie Men), Transmission of Antibiotic Resistance in the Soil-Plant System Under Reuse of Treated Wastewater. 2024 RISE/MARC Summer Symposium, poster presentation. August 28, 2024. UC Riverside, Riverside, California. - Invited talks Ashworth, D. 2024. "Biochar based removal of antibiotics from recycled water used for agricultural irrigation".US Forest Service. Men, Y. 2024. "Effects of non-antibiotic and antibiotic co-exposure on the development and propagation of antibiotic resistance in E. coli populations". University of Calgary, Canada. What do you plan to do during the next reporting period to accomplish the goals?Data from the lysimeter experiment are currently being processed for biomass, antioxidant and total phenolic analysis. Plant and soil materials from this experiment are also currently being analyzed for antibiotic residues to determine uptake of trimethoprim, erythromycin, and sulfamethoxazole from TMW into spinach, lettuce, and radish. This work is scheduled for completion by April 2025. The microbiological analysis of samples from this experiment (e.g., ARGs, ARB) is also currently being undertaken. Once all the experimental data are compiled, Task 3 will be completed to assess the risk associated with antibiotic resistance dissemination through the food chain.
Impacts
What was accomplished under these goals?
Under Task 1.3, we used a large-scale outdoor lysimeter system to evaluate the effect of recycled waters on antibiotic uptake, fresh biomass accumulation, and nutritional values, antimicrobial resistance, and antibiotic residue accumulations on spinach, radish, and lettuce. The dissemination of antibiotic resistance in the systems was also studied. Spinach and radish were directly seeded in all sand tanks while lettuce was seeded in control tanks and transplanted to tanks irrigated with treated municipal wastewater (TMW) on May 29, 2024. All irrigation waters were adjusted for macronutrients and micronutrients equivalent to a half-strength Hoagland's nutrient solution to ensure balanced nutrition for the crops. Control (no antibiotics) and spiked (10 and 100 ppb trimethoprim, sulfamethoxazole, and erythromycin) treatments were established. Crops were harvested on July 8, 2024. Radish tubers and leaves, spinach, and lettuce leaves were evaluated for fresh biomass accumulation as well antibiotic resistance gene and antibiotic-resistant bacteria on the edible portions of plants. Fresh weight was recorded on July 8, 2024. Oven-dried leaves are being analyzed for macronutrients (N, P, K, Ca, Mg, and S), micronutrients (Mo, Cu, Mn, Fe, and Zn), and the salt minerals sodium and chloride. A subset of these samples was frozen in liquid nitrogen, and freeze-dried and is currently being processed for the analyses of antioxidant and total phenolics. Edible portion of plants as well as soil and rhizosphere soils were used for DNA extraction and isolation of E. coli and Enterococcus. DNA samples have been sent out for 16S and shotgun sequencing. Isolates will be sent out for whole genome sequencing after sensitive analysis for resistant isolates. Additionally, antibiotic-resistant bacteria belonging to specific genera (i.e., Escherichia spp., Salmonella spp., Pseudomonas spp.) were isolated frombulk soil samples taken from the large pot experiment and the lysimeter samples, with a total of about 1000. Minimal inhibition concentrations (MICs) of cephalexin, a commonly used antibiotic, were determined for all isolates, according to which they were binned into four categories: susceptible, mild resistance (~10-fold increase in MIC compared to that of the susceptible strain), medium resistance (~ 50-fold increase in MIC), and strong resistance (> 50 fold increase in MIC). Genomic DNA of select isolates in each bin is being extracted and will be sent to the sequencing facility at UC, Davis for whole-genome sequencing analysis. Comparative genomics will be conducted to identify the signature genetic mutations conferring the increase in antibiotic resistance.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Duc Phan, Ananda S. Bhattacharjee, Desmond Hanan, Stanley Park, Daisy Herrera, Daniel Ashworth, Michael Schmidt, Yujie Men, Jorge F.S. Ferreira, Abasiofiok M. Ibekwe. 2024. Dissemination of antimicrobial resistance in agricultural ecosystems following irrigation with treated municipal wastewater. Science of The Total Environment, Volume 934. DOI: 10.1016/j.scitotenv.2024.173288
- Type:
Other
Status:
Other
Year Published:
2024
Citation:
Ashworth, D. 2024. Biochar based removal of antibiotics from recycled water used for agricultural irrigation US Forest Service (Invited talk)
- Type:
Other
Status:
Other
Year Published:
2024
Citation:
Effects of non-antibiotic and antibiotic co-exposure on the development and propagation of antibiotic resistance in E. coli populations. University of Calgary, Canada. (Invited Talk)
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Progress 11/01/22 to 10/31/23
Outputs
Target Audience:In the past year, we have targeted audiences in the following groups: - Undergraduate students (including those from underrepresented groups): we recruited undergraduate student researchersthrough the RISE (Research In Science and Engineering) program at UCR to provide undergraduate students research experience related to this project's topics. - Scientific communities: we presented our research findings to researchers from other department on campus, at professional conferences, and a broader audience at other universities. - Farmers and wastewater engineers: we conmunicated with local farmers and wastewater engineers about our projects and obtained manure samples for our green-house experiments to investigate the effect of manure and treated wastewater on the spread of antimicrobial resistance during agricultural activities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The PD and Co-PDs supervised Dr. Ananda S Bhattacharjee (assistant project scientist) and Dr. Duc Phan (postdoctoral research scholar). Dr. Bhattacharjee and Dr. Phan analyzed the development and mitigation of antibiotic resistance across the objectives including investigation of the fate of ARBs and ARGs in treated municipal wastewater, soil, and vegetables, and the implementation of appropriate QA/QC procedures. Dr. Bhattacharjee and Dr. Phan presented their findings at conferences (list below), invited talks at the Department of Environmental Sciences, University of California, and USDA-ARS monthly seminar series. Dr. Bhattacharjee and Dr. Phan also supervised and mentored graduate and undergraduate students. Seven undergraduate students from University of California - Riverside were hired as part of UCR's RISE (Research in Science and Engineering) program from June 2023 to September 2023 (10 week program) in which the students were supported using a stipend funded by this NIFA grant. Within RISE, we established a program named "AMR in AGRI" https://rise.ucr.edu/usda-amr-in-agri to which UCR students could apply. The students assisted in research supporting the NIFA project and were given mentoring and training in research methods by the PD and co-PDs. At the end of the program, the students presented research findings at a RISE symposium held at UCR (poster titles are enlisted under publication). Four of the students were subsequently retained after RISE (~10 hours per week for 10 weeks/quarter), for which they are receiving quarterly stipends funded by this NIFA grant. How have the results been disseminated to communities of interest?The team is continuing to disseminate the finds on 'AMR in agriculture' to member of the academic and scientific community by training (undergraduates, graduates, and postdoctoral research associates), presenting at conferences and publishing in peer reviewed journals. Dissemination of results to the scientific community via publication in a high-impact journal: Bhattacharjee A. S., Phan D., Zheng, C., Ashworth, D., Schmidt M., Men, Y., Ferreira, J. F. S., Muir, G., Hasan, N. A., Ibekwe, A. M., Dissemination of antibiotic resistance genes through soil-plant-earthworm continuum in the food production environment. Environment International, 183, 108374. https://doi.org/10.1016/j.envint.2023.108374 Soil Science Society of America News Article: https://www.soils.org/news/science-news/using-biochar-remove-antibiotics-wastewater/ Presentation of research data at scientific meetings:American Society for Microbiology, June 2023; American Chemical Society Meeting, August 2023, and UCR RISE Symposium for undergraduates. We also presented our results at seminars inthe Department ofEnvironmental Sciences, as well as webinars organized by other universities. See below: Invited Talks: Bhattacharjee A. S., Phan D., Zheng, C., Ashworth, D., Schmidt M., Men, Y., Ferreira, J. F. S., Muir, G., Hasan, N. A., Ibekwe, A. M., Dissemination and Risk of Anthropogenically Induced Antimicrobial Resistance in the Food Production Environment. Department of Environmental Science, University of California-Riverside. March 1st 2023. Ashworth, D. J., Phan D., Bhattacharjee A. S., Zheng, C., Schmidt M., Ibekwe, A. M, Ferreira, J. F. S., Men, Y. Fate transport and mitigation of antibiotics in irrigated agriculture. Department of Environmental Sciences, University of California-Riverside. May 2023. Men, Y., and Xing, Y.Effects of non-antibiotic and antibiotic co-exposure on the development and propagation of antibiotic resistance in E. coli populations. Webinar. Harbin Institute of Technology, China. What do you plan to do during the next reporting period to accomplish the goals?Under Objective 1, large-scale sand lysimeter studies will be conducted using different as-collected treated municipal wastewaters as the source of irrigation. For these experiments, we will grow spinach, lettuce, radish, and carrot in the lysimeter plots. Several permutations can be made in the experimental protocols to allow study of other plants and mixtures of wastewater effluent from different locations and compositions. Irrigation water will be pumped from water reservoirs housed underneath the lysimeter facility to the plots above, completely saturating and leaching the culture medium. In each lysimeter, the excess irrigation solution is returned to the reservoir after each irrigation (via a subsurface drainage system at the bottom of the tanks). Using sand as the culture medium will remove solid phase interactions and allow greater focus on plant root interactions with the antibiotic compounds and antibiotic resistance determinants in the solution phase.
Impacts
What was accomplished under these goals?
Under Objective 1, (Processes and Dissemination), A greenhouse pot experiment was conducted to assess the role by soil-plant, and plant-animal (earthworm) in the dissemination of antimicrobial resistance in the agriculture. Spinach and Radish plants (representing vegetables with above and below ground edible portions, that are typically eaten raw) were grown and irrigated with deionized water (with and without antibiotics) and treated municipal wastewater (with and without antibiotics) in the greenhouse. The antibiotics used in the study were trimethoprim (TMP), sulfamethoxazole (SMZ), and sulfapyridine (SPD) The harvested plant material was then fed to earthworms (Eisenia fetida). Genomic DNA (gDNA) was extracted from water, bulk soil, plant phyllosphere, plant endosphere, and earthworm feces to elucidate the microbial composition, antibiotic resistance determinants (ARDs, such as antibiotic-resistance genes (ARGs), virulence factor (VF) genes antibiotic-resistant bacteria (ARBs), and mobile genetic elements (MGEs)). The gDNA was analyzed for ARDs by, a) sequencing (16SrRNA amplicon and shotgun), and b) high throughput-qPCR (HT-qPCR). Recently, we published the role of vegetable plants, earthworms, and their endophytic microbes in the dissemination of antimicrobial resistance (AMR) in the agricultural environment/food chains (DOI: https://doi.org/10.1016/j.envint.2023.108374). Briefly, our results showed that antibiotic resistance genes (ARGs) were enriched in the soil-plant-earthworm microbiomes irrigated with TMW and TMW spiked with higher concentrations of antibiotics. The number of ARGs and antibiotic resistant bacteria (ARB) enrichment varied with plant type, with spinach harboring a significantly higher amount of ARGs and ARB compared to radish. Our data showed that bulk and rhizosphere soils of spinach and radish plants irrigated with MilliQ water, TMW, TMW10, or TMW100 had significant differences in bacterial community (p < 0.001), ARG (p < 0.001), and virulence factor gene (VFG) (p < 0.001) diversities. The abundance of ARGs significantly decreased from bulk soil to rhizosphere to phyllosphere and endosphere. Using metagenome assembled genomes (MAGs), we recovered many bacterial MAGs and a near complete genome (>90 %) of bacterial MAG of genus Leclercia adecarboxylata B from the fecal microbiome of earthworm that was fed harvested radish tubers and spinach leaves grown on TMW10 irrigated waters, and this bacterium has been shown to be an emerging pathogen causing infection in immunocompromised patients that may lead to health complications and death. Therefore, crops irrigated with TMW containing residual antibiotics and ARGs may lead to increased incidences of enrichment of ARB in the soil-plant-earthworm continuum. Currently we in the process of submitting a manuscript on ARDs detected with HT-qPCR in water-soil-plant-earthworm continuum. The study provides us with the role of vegetable plants, earthworms, and their endophytic microbes in the dissemination of AMR in the agricultural environment/food chains. Briefly, the 16S data indicated that the rhizosphere microbial ecology structure was more resilient to antibiotic residuals in the irrigated water, with radish showing less susceptibility than spinach. The HT-qPCR analysis revealed that a total of 271 ARGs (out of 285) and 9 mobile genetic elements (MGEs) (out of 10) were detected in all samples. Higher diversity and abundance of ARGs were observed for samples irrigated with higher concentrations of antibiotics both spinach and radish treatments. However, compared to radishes, spinaches showed more susceptibility to changing the ARG dynamics in the soil biome. For individual ARG classes, the multi-drug resistance (MDR) class was altered significantly by the presence of antibiotics in irrigation water. Earthworm fecal samples and their corresponding soil environments showed different ARG dynamics structures, suggesting that earthworms could play a role in reducing ARG dissemination in the soil environments. These findings will not only provide an insight of the dissemination of ARGs in agricultural environments due to antibiotic residuals in irrigated water but could be further helpful in understanding potential human health risks. Also under Objective 1, an intermediate-scale outdoor pot experiment (70 L pots) was conducted to study the dissemination of antibiotic resistance determinants (antibiotic compounds, antibiotic resistance genes, antibiotic resistant bacteria, and mobile genetic elements) from both animal manure and treated municipal wastewater into soil, plants (spinach and radish crops), and earthworms. The main antibiotic compounds of interest were oxytetracycline (prevalent in the cattle manure used) and sulfamethoxazole (prevalent in the treated municipal wastewater used). In addition to using manure and treated wastewater as-collected, additional treatments were conducted in which the antibiotic concentrations were increased via spiking. The biological transfers of the compounds are being quantified (e.g., through bioaccumulation factors) and will be considered in relation to the development and spread of antibiotic resistance in the various system compartments. In addition, leaching of the antibiotic compounds from the base of the pots is being quantified to assess the potential for system looses via this pathway. The data will indicate the potential for antibiotic resistance transfers through and beyond agricultural systems under realistic (outdoor) conditions typical of vegetable growing regions. Under Objective 2, a growth chamber pot experiment was conducted to quantify the effect of biochar addition to soil on the uptake of antibiotic compounds by spinach plants. Two differing biochars (one from a pine wood feedstock and the other from a biosolids feedstock) were soil applied at a 1% application rate (by mass). The pots were bottom-irrigated with treated municipal wastewater (used as-collected and also spiked with the antibiotics sulfamethoxazole, trimethoprim, and cephalexin). The biological transfers of the compounds are being quantified (e.g., through bioaccumulation factors) and will be considered in relation to the development and spread of antibiotic resistance in the various system compartments. The potential of biochar addition to soil as a low-cost strategy for mitigating these transfers and thus lowering the risk of antibiotic resistance dissemination through the food production environment will be assessed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Bhattacharjee A. S., Phan D., Zheng, C., Ashworth, D., Schmidt M., Men, Y., Ferreira, J. F. S., Muir, G., Hasan, N. A., Ibekwe, A. M., Dissemination of antibiotic resistance genes through soilplantearthworm continuum in the food production environment. Environment International, 183, 108374. https://doi.org/10.1016/j.envint.2023.108374
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Phan, D, Bhattacharjee, A. S., Hanan, D., Ibekwe, A. M., Zheng, C., Ashworth, D., Schmidt, M., Men, Y., Ferreira, J. F. S. Spread of Antimicrobial Resistance in the Agri-Food Production Environment. (poster presentation) ASM 2023, June 15-19, Houston, TX.
|
Progress 11/01/21 to 10/31/22
Outputs
Target Audience:During the reporting period, we continue reaching out to local wastewater treatmetn plants, which applied the treated wastewater for agricultural irrigation.We will be taking water samples from those facilities and conducted the research objectives.We also reached out to local farms to get manure for small and large pot experimetns. Findings will be disseminated to managers of the facilities, as well as farmers who are using the treated wastewater or manure in agricultural practices. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One PhD student (Chujing Zheng) started in Fall 2021, who has bee trained with the detection of antibiotic resistance genes and the cultivation of antibiotic resistant bacteria and will be involved in future pot experiments to understand the fate and transport of antibiotic resistance determinants during water reuse in agriculture irrigation. One undergraduate researcher (Daisy Herrera) recruited during the past year continued as a PhD student and just started in October2022. The PhD student will be trained with field and lab experiment skills by postdocs and involve in future pot experiments in this project. One student scientist (Yue Xing) working onthis project last year was recruited as a research scientist to continue the work on lab exposure experimentsin October 2022 after PhD graduation. The person leftfor a new job position in a biotech company in March 2022. Two postdocs (Ananda Bhattacharjee and Duc Phan) were involved in this project, who have been trained with research, presentation, writing, and mentoring skills. They have presented and will be presenting research outcomes at conferences, universities, and ARS seminars. They are training graduate students and undergraduate researchers. One has led another NIFA proposal, which has been selected for funding. How have the results been disseminated to communities of interest?Two research articles have been publishedin high impact journals. One manuscript "Dissemination risk of anthropogenically induced antimicrobial resistance in the food production environment" based on omic analysis of food production environmental samplesin prepration.We have also disseminated our findings to communities of interest at conferences, including American Society for Microbiology in June 2022 and American Chemical Society Meeting in August 2022. What do you plan to do during the next reporting period to accomplish the goals? Under Task 1.2, a large scale, outdoor pot experiment will be conducted in Spring 2023 to compare treated wastewater and animal manure as sources of antibiotics to cropped soils. Antibiotic uptake by (i) plants (spinach and radish) and (ii) earthworms from the soil will be assessed. The development and dissemination of ARGs and ARB within these systems will be quantified. Under Task 2, a greenhouse pot experiment will be conducted to assess the potential of biochar amendment of soil in limiting antibiotic uptake by spinach. Again, the development and dissemination of ARGs and ARB within these systems will be quantified.
Impacts
What was accomplished under these goals?
Under Task 1 (Processes of Dissemination), we focused on Task 1.1 in the past year. A greenhouse pot experiment was conducted to assess the role by soil-plant, and plant-animal (earthworm) in the dissemination of antimicrobial resistance in the agriculture. Spinach and Radish plants (representing vegetables with above and below ground edible portions, that are typically eaten raw) were grown and irrigated with deionized water (with and without antibiotics) and treated municipal wastewater (with and without antibiotics) in the greenhouse. The harvested plant material was then fed to earthworms (Eisenia fetida). DNA was extracted from water, bulk soil, plant phyllosphere, plant endosphere, and earthworm feces to elucidate the microbial composition, antibiotic resistance determinants (ARDs, such as antibiotic-resistance genes (ARGs), virulence factor (VF) genes antibiotic-resistant bacteria (ARBs), and mobile genetic elements (MGEs)). The good-quality DNA samples were aliquoted in three subsamples for a) 16SrRNA amplicon sequencing, b) shotgun sequencing, and c) high-throughput-qPCR (HT-qPCR) for 16S RNA genes, ARGs, and MGEs. The 16S RNA amplicon raw sequences were processed with the DADA2 pipeline. The taxonomy was assigned to amplicon sequence variants with the 16SrRNA gene SILVA database. Similarly, the raw shotgun sequences were quality trimmed and mapped to an antibiotic resistance database. The mapping identified and quantified the ARDs in the samples. The taxonomic affiliations of the sequences were performed by mapping to a curated microbial database. The ARDs for each sample were statistically compared to delineate the development of antibiotic resistance. The antibiotic-resistant bacterial genomes were extracted and identified within the samples. A metabolic reconstruction of the ARBs was performed to annotate gene function and antibiotic resistance mechanisms. In addition to sequencing, 295 ARGs (including MGEs) and one 16SrRNA gene were qualified for the samples with HT-qPCR assay. This work has allowed us to identify the development of antibiotic resistance within the soil, together with the transfer of ARDs to plants and earthworms. The findings on the dissemination of ARDs in soil-plant-animal will assist us with the large scale, outdoor pot experiment (under Task 1.2). In addition, we also conducted lab experiments to further examine the effect of exposure to both antibiotics and pesticides on the development of stronger antibiotic resistance in different bacterial genera. We observed the same synergistic effect of the co-exposure to pathogenic Escherichia coli species, but not another two genera, i.e., Pseudomonas and Staphylococcus. This finding suggests that the induction of stronger antibiotic resistance by the co-exposure to antibiotics and pesticides was specific to E. coli species, including pathogenic ones. Further studies are going on to tackle the individual pesticides causing the synergistic effect, the mechanisms. Moreover, pot experiments will be used to investigate whether the same stimulation effect could be observed in agricultural soils irrigated with treated wastewater, which may contain a mixture of antibiotics and pesticides. Under Task 2 (Mitigation Using Biochar), several biochar materials have been assessed to determine their antibiotics adsorption potential (Kd and Qmax values). The physical and chemical characteristics (e.g., pH, specific surface area, cation exchange capacity, elemental composition, surface charge, functional group content, volatile and ash contents) of the biochars have also been quantified and related to the adsorption properties. Biochars from differing feedstocks (rice husk, dairy manure, pistachio shells, and date palm leaves) have been studied, and the effects of pyrolysis temperature and post-production modification with acid and base have also been considered. This work has allowed us to identify the biochar characteristics most suitable for our planned experiments looking at the mitigating impact of biochar on antibiotic uptake by plants under greenhouse conditions (also under Task 2). As such two biochars will be selected for use in these pot experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Ashworth, D. J., Ibekwe, A.M., Men, Y, Ferreira, J. F. S. 2023. Dissemination of antibiotics through the wastewatersoilplantearthworm continuum. Science of the Total Environment, 858 (3) 159841.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Xing, Y., Herrera D., Zhang, S., Kang X., Men, Y.* (2022). Site-specific target-modification mutations exclusively induced by the coexposure to low levels of pesticides and streptomycin caused strong streptomycin resistance in clinically relevant Escherichia coli. Journal of Hazardous Materials Advances 7: 100141.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Ananda S. Bhattacharjee, Duc Phan, Chujing Zheng, Daniel J. Ashworth, Michael Schmidt, Yujie Men , Jorge F. S. Ferreira, Gabrielle Muir, Nur. A. Hasan, and Abasiofiok M. Ibekwe (2022).Dissemination and Risk of Anthropogenically Induced Antimicrobial Resistance in the Food Production Environment. American Society Microbiology (ASM) Microbe 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Daniel Ashworth, Michael Schmidt, Abasiofiok Ibekwe, Yujie Men, Ananda Bhattacharjee, Duc Phan (2022). Biochar-based polishing technology for the removal of antibiotics and other emerging contaminants from wastewater. American Chemical Society (ACS) Fall 2022.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Michael Schmidt, Daniel Ashworth, Nydia Celis, Abasiofiok Ibekwe, Yujie Men, Ananda Bhattacharjee, Duc Phan (2022). Biochar feedstock and modification influences on physicochemical properties: Implications for antibiotic adsorption from treated wastewater streams. American Chemical Society (ACS) Fall 2022.
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Progress 11/01/20 to 10/31/21
Outputs
Target Audience:During the reporting period, we have reached out to local wastewater treatmetn plants, which applied the treated wastewater for agricultural irrigation. We will be taking water samples from those facilities and conducted the research objectives. Findings will be disseminated to managers of the facilities, as well as farmers who are using the treated wastewater for irrigation. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two UCR undergraduate students have been employed at the USDA US Salinity Lab under co-PIs Ashworth and Ibekwe to train in the experimental and analytical techniques used in the project. A PhD student funded by the project enrolled at UC Riverside in August 2021 and has begun training in experimental/analytical techniques relevant to the project. One visiting PhD student at UC Riversidewas partially supported by the project, who has been working on the effect of lab exposure to antibiotics and non-antibiotic micropollutants (e.g., pesticides) on the development and transmission of antibiotic resistance. How have the results been disseminated to communities of interest?Owing to COVID-19, the normal route for disseminating the results (i.e., conference presentations) has not been possible due to travel restrictions. One manuscript on the effect of lab exposure to antibiotics and non-anbitoitic micropollutants at environmental levels on the development of antimicrobial resistance has been published.Another manuscript on the data from the pot experiments is currently being prepared to disseminate the results to a scientific audience. National Program Leaders within USDA-ARS have expressed an interest in the work and we have liaised with them in writing a "highlight" piece for an upcoming Antimicrobial Resistance document being developed. What do you plan to do during the next reporting period to accomplish the goals?Experimental work will be continued in support of the main objective, i.e., to understand processes and mechanisms controlling the transfers of antibiotics and ARDs through food chains. Experimental work will also be undertaken to assess the effectiveness of biochar in mitigating these transfers.
Impacts
What was accomplished under these goals?
We first demonstrated that lab exposure to pesticide and antibiotics (i.e., streptomycin) may stimulate the genetic evolution of Escherichacoli populations towards stronger resistance compared to the exposure to antibiotics only. The distinct genetic mutations conferring the stronger antibiotic resistance have been identified. Moreover, the stronger resistant mutants selected by the co-exposure might have higher growth fitness than mild resistant mutants selected by the antibiotic-only exposure. These results have been published in ISME communications. In practice, the application of waste materials such as animal manure and municipal wastewater to agricultural soils can lead to the introduction of antibiotic compounds and antibiotic resistance determinants (ARDs) into farming systems. The potential for the subsequent development and spread of antibiotic resistance through food chains is of primal importance to human health. This project aims to better understand the mechanisms and processes by which the spread of antibiotic resistance may occur through a model food chain, i.e., the soil-plant-earthworm food chain. It then aims to assess whether this spread can be mitigated by biochar application to soils. This data will be important to regulators and growers when evaluating irrigation waters and manures and to ensure crops are not inadvertently contaminated with antibiotics and ARDs. Government researchers, university scientists, cooperative extension personnel, and public agencies such as the Center for Disease Control and Environmental Protection Agency will also benefit from an improved understanding of the processes underlying the spread of antibiotic resistance in the environment. Under the main objective of the project, a pot experiment was conducted in a greenhouse during the reporting period, followed by an earthworm bioavailability study. In the pot experiment, radish and spinach plants were grown in soils irrigated using treated municipal wastewater (TMW) from a municipal wastewater treatment plant in Riverside, CA. The antibiotics sulfamethoxazole, sulfapyridine, and trimethoprim were identified in the TMW - but at different and low (e.g., less than 0.3 ng mL−1) concentrations. To explore the fates of these antibiotics, experiments were conducted using the as-collected TMW as well as TMW spiked with trace levels (10 and 100 ng mL−1) of each antibiotic. These treated wastewaters were used to irrigate the plants. The fate and concentration of each antibiotic were determined in roots and shoots of the crop plants studied, and the soil used. The antibiotics were extracted from the experimental materials (e.g., soils and plant organs) using traditional solvent extraction, concentration, and cleanup methods, with analysis by state-of-the-art high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results obtained with this high-sensitivity detection system showed that for the spiked TMW (10 and 100 ng mL−1), the antibiotics persisted (owing to their relatively slow degradation rates and low degree of adsorption to the sandy soil medium) and were available for uptake from soil water into the edible portions of the spinach and radish crops. In the edible portions, the highest levels detected in both crops were for trimethoprim, followed by sulfamethoxazole and sulfapyridine (with the levels in spinach being consistently greater than those in radish). It should be noted that while the antibiotics in the original as-collected TMW were detected at low levels, they were not found at detectable concentrations in the tissues of plants irrigated with this water. In subsequent experiments, the vegetable materials from the pot experiment were fed toEisenia fetidaearthworms (e.g., as might happen during crop plant residue senescence following harvest). In a 3-week experiment with weekly feeding, the use of spinach and radish materials from the spiked TMW (100 ng mL−1) treatment, as well as materials surface-spiked post-harvest to concentrations of 1 and 10 µg g−1, led to antibiotics being detected in earthworm tissues (no uptake was detected in earthworms fed plant materials irrigated with the as-collected TMW or the spiked TMW (10 ng mL−1) treatment). These findings indicate the potential for antibiotic transfers across trophic levels, i.e., through a food chain, at the higher concentrations used. However, the data did not suggest a "biomagnification" effect, i.e., the antibiotic concentrations observed in the earthworms were lower than those in the plant materials used as food. These experimental findings demonstrate that if higher levels of these antibiotics persist in wastewaters and these waters are used for irrigation, the antibiotics may be translocated to edible plant tissues, as seen in the spiked TMW (10 and 100 ng mL−1) treatments, and then potentially to animals feeding on those plants, as seen in the spiked TMW (100 ng mL−1) treatment and the post-harvest treatments spiked with 1 and 10 µg g−1of each antibiotic. Data on the dissemination of antibiotic resistance genes (ARGs), antibiotic resistant bacteria (ARB), and mobile genetic elements (MGEs) were also collected to support data on the dissemination of antibiotic residues mentioned above.Microbial communities and ARGs in the soil-plant- earthworm continuum were analyzed using Illumina shotgun sequencing and the dissemination of ARGs and ARB through these matrices were characterized. The focus of this section of the study was to understand the dissemination of antibiotic resistance via micro-organisms that colonize the rhizosphere and non-rhizosphere soils, phyllosphere, endophyte tissues of vegetable plants, and gut of earthworm irrigated with treated wastewaters. Genomic DNA (gDNA) was extracted from each sample in triplicates and sequenced on an Illumina HiseqX10 sequencer for metagenomics shotgun sequencing. For each sample, functional annotations were obtained by matching each read, using DIAMOND, against the KEGG database. The abundance of antibiotic resistance genes was higher in vegetable crops irrigated with treated wastewater and spiked with antibiotics than the non-irrigated samples. This suggests that irrigation of agricultural soil and crops with treated wastewater can select for resistant bacteria and become a reservoir of ARB and ARGs. Overall, the work conducted during this reporting period has shown potential for antibiotic transfers through a food chain if the initial concentrations in the wastewater are relatively high. It has also demonstrated that the use of TMW can lead to the development of antibiotic resistance within agricultural systems. These findings directly address the main objective of the project. The tertiary-treated municipal wastewater used in this initial set of experiments was relatively clean (obtained from a state-of-the-art wastewater treatment facility); in future experiments, less-processed wastewaters, as well as the continued use of spiked wastewaters, will be used to simulate worst-case scenarios.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Dissemination of antibiotic compounds and antibiotic resistance in the agricultural environment. Abstract accepted for Soil Science Society of America Meeting, Nov 2021, Salt Lake City (Presenter: Co-PI Daniel Ashworth) (later withdrawn owing to USDA Covid policy preventing travel).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Xing, Y., Kang, X., Zhang, S., Men, Y.* (2021) Pesticides drive evolutionary trajectories toward strong and irreversible antibiotic resistance in Escherichia coli populations under low-level streptomycin selection. ISME Commun. 1: Article number 39. DOI: 10.1038/s43705-021-00041-z
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