Research Project: Identifying, Quantifying and Tracking Microbial Contaminants, Antibiotics and Antibiotic Resistance Genes in Order to Protect Food and Water Supplies

Location: Agricultural Water Efficiency and Salinity Research Unit

2020 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
In support of Objective 2, batch experiments examining conjugation dynamics in growing and non-growing E. coli cultures were completed. These experiments found that E. coli only conjugate after they have finished growing and before they transition to a non-growing phase. Batch studies in sediment microcosms found that conjugation is severely restricted in this environment because the larger surface area of sand particles relative to the size of bacteria results in donor and recipient bacteria colonizing the particle at distances that are too great for conjugation to occur. A computer model was developed to model conjugation in E. coli under growing and non-growing conditions taking into account non clonal growth and accurately describing the observed behavior. Experiments are now 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. Under Objective 3, a greenhouse study was conducted to understand the risk of dissemination of antibiotic compounds and antibiotic resistance determinants from treated wastewater in the soil–plant–earthworm gut continuum. Radish and spinach (vegetables typically eaten raw and representing those with below- and above-ground edible portions, respectively) were used for the study. Four antibiotics and other compounds of emerging concern were identified from the treated wastewater. Quantification of these compounds and antibiotic resistance genes from soil, roots, leaf, and earthworm is in progress.

Accomplishments
1. Assessment of strategies to remove antibiotics from wastewater. Understanding the removal mechanism of antibiotic compounds and antibiotic resistance determinants in agricultural systems is a global challenge that is important in the protection of human health. An ARS researcher from Riverside, California, and collaborator from University of California, Riverside, designed and tested a system of layered environmental media (consisting of gravel, sand, soil, and soil+biochar) through which antibiotic-laden water was pumped. Overall removal efficiencies of the antibiotics amoxicillin, cefalexin, sulfadiazine, and tetracycline were 81, 91, 51, and 98%, respectively. Amoxicillin and cefalexin removal were largely controlled by chemical degradation within the gravel layer, whereas sulfadiazine was largely removed via both chemical and microbial degradation in the soil+biochar layer and tetracycline was lost via hydrolysis reactions in the gravel layer. Increasing the hydraulic retention time of the system improved removal efficiency, especially for amoxicillin and cefalexin, which had half-lives that were much shorter than the hydraulic retention time. Overall, the results from this lab-scale proof of concept system indicate the potential of the system for antibiotic removal from wastewater and highlight ways in which improvements to removal efficacy may be scalable for broad applicability. The results of this study will be used by wastewater treatment facilities, World Health Organization, researchers, and other local municipalities in many developing countries.

Review Publications
Shen, C., Bradford, S.A., Flury, M., Huang, Y., Wang, Z., Li, B. 2018. DLVO Interaction energies for hollow particles: The filling matters. Langmuir. 34(43):12764-12775. https://doi.org/10.1021/acs.langmuir.8b02547.
Adrian, Y.F., Schneidewind, U., Bradford, S.A., Simunek, J., Klumpp, E., Azzam, R. 2019. Transport and retention of engineered silver nanoparticles in carbonate-rich sediments in the presence and absence of soil organic matter. Environmental Pollution. 255. https://doi.org/10.1016/j.envpol.2019.113124.
Gomez-Flores, A., Bradford, S.A., Hwang, G., Choi, S., Tong, M., Kim, H. 2020. Shape and orientation of bare silica particles influence their deposition under intermediate ionic strength: A study with QCM–D and DLVO theory. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 599. https://doi.org/10.1016/j.colsurfa.2020.124921.
Gomez-Flores, A., Bradford, S.A., Hwang, G., Heyes, G.W., Kim, H. 2020. Particle-bubble interaction energies for particles with physical and chemical heterogeneities. Minerals Engineering. 155. https://doi.org/10.1016/j.mineng.2020.106472.
Murinda, S.E., Ibekwe, A.M., Rodriguez, N.G., Quiroz, K.L., Mujica, A.P., Osmon, K. 2019. Shiga Toxin-producing Escherichia coli in Mastitis: An international perspective. Foodborne Pathogens and Disease. 16(4):229-243. https://doi.org/10.1089/fpd.2018.2491.
Ducey, T.F., Durso, L.M., Ibekwe, A.M., Dungan, R.S., Jackson, C.R., Frye, J.G., Castleberry, B., Rashash, D.M., Rothrock Jr, M.J., Boykin, D.L., Whitehead, T.R., Ramos, Z.D., McManus, M.N., Cook, K.L. 2020. A newly developed Escherichia coli isolate panel from a cross section of U.S. animal production systems reveals geographic and commodity-based differences in antibiotic resistance gene carriage. Journal of Hazardous Materials. 382. https://doi.org/10.1016/j.jhazmat.2019.120991.
Liang, Y., Zhou, J., Dong, Y., Klumpp, E., Simunek, J., Bradford, S.A. 2020. Evidence for the critical role of nanoscale surface roughness on the retention and release of silver nanoparticles in porous media. Environmental Pollution. 258. https://doi.org/10.1016/j.envpol.2019.113803.
Yuan, X., Zeng, Q., Xu, J., Severin, G.B., Zhou, X., Waters, C.M., Ibekwe, A.M., Liu, F., Yang, C. 2020. Systematic study of the TCA cycle in virulence regulation links cyclic di-GMP signaling in Dickeya dadantii. Molecular Plant-Microbe Interactions. 33(2):296-307. https://doi.org/10.1094/MPMI-07-19-0203-R.
Ibekwe, A.M., Ors, S., Ferreira, J.F., Liu, X., Suarez, D.L., Ma, J., Ghasemimianaei, A., Yang, C. 2020. Functional relationships between aboveground and belowground spinach (Spinacia oleracea L., cv. Racoon) microbiomes impacted by salinity and drought. Science of the Total Environment. 717. https://doi.org/10.1016/j.scitotenv.2020.137207.
Ibekwe, A.M., Murinda, S.E. 2019. Linking microbial community composition in treated wastewater with water quality in distribution systems and subsequent health effects. Microorganisms. 7(12). https://doi.org/10.3390/microorganisms7120660.
Li, T., Shen, C., Wu, S., Jin, C., Bradford, S.A. 2020. Synergies of surface roughness and hydration on colloid detachment in saturated porous media: Column and atomic force microscopy studies. Water Research. 183. https://doi.org/10.1016/j.watres.2020.116068.
Gomez-Flores, A., Bradford, S.A., Wu, L., Kim, H. 2019. Interaction energies for hollow and solid cylinders: Role of aspect ratio and particle orientation. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 580. https://doi.org/10.1016/j.colsurfa.2019.123781.
Ashworth, D.J., Ibekwe, A.M. 2020. System of multi-layered environmental media for the removal of antibiotics from wastewater. Journal of Environmental Chemical Engineering. 8(5). https://doi.org/10.1016/j.jece.2020.104206.