Location: Agricultural Water Efficiency and Salinity Research Unit
2021 Annual Report
Objectives
The overall goal of the project is to develop improved understandings and new tools for the protection of food and water supplies from contamination by ARBs and ARGs associated with fecal indicator bacteria (FIB), and ARGs from CAFOs, WWTPs effluent, and urban runoff.
Research Tasks – Three tasks crosscut the research objectives creating a subtask matrix. The subtasks are listed under each corresponding objective.
Task I: Mechanistic studies of conjugation - Mechanistically study and model the transport, retention, and release of NRB, ARB containing ARGs in the presence of various environmental stressors under different physicochemical conditions at the laboratory scale.
Task II: Runoff Studies with sediment from the SAR Watershed - Investigate factors that influence the development, spread, and mitigation of ARB, ARGs, and pathogenic E. coli and Salmonella in sediment/runoff water from the SAR watershed.
Task III: Root zone transport and uptake studies - Investigate the influence of environmental stressors on the development, spread, and mitigation of ARB and ARGs in the root zone and in food crops.
Objective 1: Identify and quantify microbial contaminants, antibiotics and antibiotic resistance genes, and develop methods and tools for tracking their transport and fate.
Subtask Ia. Identification of environmental conditions and stressors concentrations that promote HGT and the transport of ARB in idealized systems.
Subtask Ib. Create models to simulate the transport and fate of ARB and HGT.
Subtask IIa. Identification of environmental conditions and stressors concentrations that promote HGT and the transport of ARB in runoff water.
Subtask IIb. Apply models to simulate the transport and fate of ARB and HGT.
Subtask IIIa. Identification of environmental conditions and stressors concentrations that promote HGT and the transport of ARB in the root zone and in food crops.
Subtask IIIb. Apply models to simulate the transport and fate of aRB and HGT.
Objective 2: Evaluation of metagenomics and culture methods to identify specific pathogens, antibiotics, ARGs and their mechanisms of transfer (e.g., horizontal gene transfer (HGT)) in the environment from contamination sources to water, food, and humans.
Subtask Ic. Development of procedures to quantitatively study HGR under idealized systems.
Subtask IIc. Isolation, identification, and quantification of ARGs in indicator microbes, pathogens, and the microbial community in runoff water and natural sediment.
Subtask IIIc. Isolation, identification, and quantification of aRGs in the root zone and food crops.
Objective 3: Evaluation of effective methods and practices to protect crops often eaten raw from antibiotics, antibiotic resistance genes, and pathogen contamination.
Subtask Id. Models developed in Task I will be used in Task II and II to simulate HGT and ARB in runoff water and the root zone.
Subtask IId. Develop strategies to manage ARB and HGT in runoff water and sediment that is used to irrigate crops.
Subtask IIId. Develop strategies to manage ARB and HGT in the root zone.
Approach
Mechanistical studies (batch and column, runoff chamber, and lysimeter scales) will be conducted to investigate the influence of environmental factors and stressors (heavy metals and biocidal organics) on the development and migration of ARB, ARGs, and gene transfer between indicator microorganisms and pathogenic bacteria in soils, recharge water, sediments, runoff water, the root zone, and food crops. New mathematical modeling tools to better understand and simulate the transport, fate, and transfer of ARBs/ARGs will be developed. Furthermore, state-of-the-art detection protocols will be implemented to quantify the types, amounts and distribution of ARB and ARGs.
Progress Report
This is the final report for the project 2036-32000-005-000D which will terminate in October 2021. The new project, 2036-12320-001-000D, titled “Protection of Food and Water Supplies from Pathogens and Human Induced Chemicals of Emerging Concern” just completed NP212 OSQR review. A summary of results for all the expiring project Objectives are described below.
Under Objectives 1 and 2, monthly water samples were collected from Mill Creek wetland in the middle Santa Ana River watershed in California for 15 months to quantify antibiotic resistant Enterococcus and Escherichia coli (E. coli). Genomic DNA was also extracted from surface water to determine total bacterial composition, their resistance genes, and other contaminants. Quantification of antibiotic resistance genes in bacterial community and in bacteria is in progress. Also, susceptibility test of Enterococcus and E. coli to a panel of 18 antibiotics is in progress.
In support of Objective 2, batch experiments examining conjugation dynamics in growing and non-growing E. coli cultures were completed. Experiments examining conjugation dynamics in the rhizosphere and the physiochemical conditions that allow for conjugation to occur on biological surfaces such as roots, which have been shown to concentrate bacteria in agricultural soils relative to bulk sediment started in early 2020. However, due to impact of the maximized telework requirement, this portion of the study was not completed, and thereafter, the scientist relocated to another ARS location.
Under Objective 3, a growth chamber study was conducted to determine the transfer of antibiotics (trimethoprim, sulfamethoxazole, and sulfapyridine) and antibiotics resistance genes from the spinach and radish materials harvested from the pot experiment to earthworms, i.e., to study transfers between trophic levels. Microcosms containing moist (12% gravimetric moisture content), non-contaminated soil were established in glass beakers and a single earthworm added to each. The soil was augmented with the finely chopped plant material (spinach leaves or radish tubers) from each of the pot experiment treatments on a weekly basis as a food source for the earthworms (0.8 g per week). After three weeks, the worms were removed from the soil and placed in petri dishes to purge their guts. The purged soil was retained for antibiotics resistance genes analysis while the worms were then weighed and frozen for the analysis of antibiotics and their metabolites.
Additionally in support of Objective 3, laboratory and growth chamber studies were conducted to track antibiotic transfer from plant to earthworm. Given the low concentrations present in the as-collected wastewater in this experiment, these transfers were trackable only when antibiotic spiking was performed (i.e., spiking of the wastewater or spiking of the vegetable material). Nevertheless, the spikes are considered to represent environmentally significant concentrations and so offer a very useful insight into the transfer potential. The transfer of antibiotic resistance determinants will be considered through the same continuum and to use more heavily contaminated wastewaters in which contaminants already present in the water. Moreover, the potential for biomagnification through this continuum will also be investigated.
Accomplishments
1. Uptake of antibiotics by plants. The prevalence and transmission of antibiotics and antibiotic resistance genes in the environment can pose a threat to public health. Methods to limit their spread from the environment to humans is limited. ARS scientists in Riverside, California, have identified mechanisms whereby antibiotics are uptaken by plants from soil irrigated with treated municipal wastewater. Antibiotics sulfamethoxazole, sulfapyridine, and trimethoprim were identified in the wastewater at different concentrations, and low degradation rate was the main mechanism for the translocation of antibiotics from soil to the edible portion of plants. In the edible portions, uptake was greatest for trimethoprim followed by sulfamethoxazole and sulfapyridine in both spinach and radish, with translocation in spinach being consistently greater than radish.
Review Publications
Liao, J., Li, J., Han, Z., Lyu, G., Ibekwe, A.M., Ma, J. 2021. Persistence of Salmonella Typhimurium in apple-pear (Pyrus bretschneideri Rehd.) orchard soils influenced by bacterial communities and soil properties. Science of the Total Environment. 768. Article 144458. https://doi.org/10.1016/j.scitotenv.2020.144458.
Ibekwe, A.M., Murinda, S.E., Park, S., Obayiuwana, A., Murry, M.A., Shwartz, G., Lundquist, T. 2020. Comparative use of quantitative PCR (qPCR), droplet digital PCR (ddPCR) and Recombinase Polymerase Amplification (RPA) in detection of Shiga Toxin-producing E. coli (STEC) in environmental samples. Water. 12(2). Article 3507. https://doi.org/10.3390/w12123507.
Obayiuwana, A., Ibekwe, A.M. 2020. Antibiotic resistance genes occurrence in wastewaters from selected pharmaceutical facilities in Nigeria. Water. 12(7). Article 1897. https://doi.org/10.3390/w12071897.
Murry, M.A., Murinda, S.E., Huang, S., Ibekwe, A.M., Schwartz, G., Lundquist, T. 2019. Bioconversion of agricultural wastes from the livestock industry for biofuel and feed production. In: Hosseini, M., editor. Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts. United Kingdom:Woodhead Publishing. p. 225-247. https://doi.org/10.1016/B978-0-12-817941-3.00012-7.
Ziming, H., Ma, J., Yang, C., Ibekwe, A.M. 2020. Soil salinity, pH, and indigenous bacterial community interactively influence the survival of E. coli O157:H7 revealed by multivariate statistics. Environmental Science and Pollution Research. 28:5575-5586. https://doi.org/10.1007/s11356-020-10942-6.
Bradford, S.A., Sasidharan, S., Kim, H., Gomez-Flores, A., Li, T., Shen, C. 2021. Colloid interaction energies for surfaces with steric effects and incompressible and/or compressible roughness. Langmuir. 37(4):1501-1510. https://doi.org/10.1021/acs.langmuir.0c03029.
Headd, B.J., Bradford, S.A. 2020. The conjugation window in an Escherichia coli K-12 strain with an IncFII plasmid. Applied and Environmental Microbiology. 86(17). Article e00948-20. https://doi.org/10.1128/AEM.00948-20.
Ibekwe, A.M., Durso, L.M., Ducey, T.F., Oladeinde, A., Jackson, C.R., Frye, J.G., Dungan, R.S., Moorman, T.B., Brooks, J.P., Obayiuwana, A., Karathia, H., Fanelli, B., Hasan, N.A. 2021. Diversity of plasmids and genes encoding resistance to extended-spectrum ß-Lactamase in Escherichia coli from different animal sources. Microorganisms. 9(5). Article 1057. https://doi.org/10.3390/microorganisms9051057.
Ibekwe, A.M., Ors, S., Ferreira, J.F., Liu, X., Suarez, D.L. 2021. Influence of seasonal changes and salinity on spinach phyllosphere bacterial functional assemblage. PLoS ONE. 16(6). Article e0252242. https://doi.org/10.1371/journal.pone.0252242.
