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.
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.
This is the first report for this new project which began in October of 2016. Please see the report for the previous project entitled “Protection of Food and Water Supplies from Pathogen Contamination” (2036-32000-004-00D) for additional information. Considerable research has been conducted to establish methods necessary to quantify and mathematically model the development of antibiotic resistance bacteria (e.g., horizontal gene transfer via conjugation) in laboratory studies under various environmental stressors (antibiotics, heavy metal, and biocidal organics) and physicochemical (salinity, environmental stressors, temperature, and nutrient conditions, water velocity, etc.) conditions. In particular, we have genetically modified an Escherichia coli strain (e.g., genes for yellow or blue fluorescence and counter selection, and inserted a plasmid which confers resistance to several antibiotics and environmental stressors) so that the numbers of antibiotic resistant and nonresistant bacteria can be quantitatively determined. We have purchased a flow cytometer for the detection and quantification of the blue and yellow fluorescing E. coli strains and are currently in the process of optimizing our experimental protocols with the flow cytometer. Preliminary results from batch experiments suggest that population sizes, physicochemical conditions, and survival behavior govern the development of antibiotic resistance, not the presence of antibiotic stressors. Transfer of bacteria with resistance to multiple antibiotics from the intestinal track of different animals to the environment has led to a critical discussion about worldwide antibiotic resistance patterns. E. coli isolates collected from fresh fecal samples from dairy and beef cattle, swine, and poultry were screened for markers known to carry antibiotic resistance genes from bacteria to bacteria. Precise measurements were made on a collection of well-characterized and genetically diverse isolates of E. coli to determine the animal source(s) with the highest potential for gene transfer to the environment. We are collaborating with ARS scientists in Athens, Georgia; Lincoln, Nebraska; Ames, Iowa; Kimberly, Idaho; and Florence, South Carolina, to determine if these markers are universal or specific to certain regions and to develop mitigation measures to minimize their transfer to the environment.
1. Quantification of Escherichia coli O157 in environmental samples. An improved approach for quantifying low concentrations of the pathogen E. coli O157 in environmental samples was evaluated by an ARS scientist in Riverside, California. The specificity and detection limit of E. coli O157 in inoculated samples of fresh produce, soil, and water was determined. The assay was further applied to swine, dairy, beef, and poultry manure, and wastewater effluent collected from a dairy wetland over a twelve-month period. The assay quantified E. coli O157 with concentrations below 100 cells per gram of soil or manure. The accuracy of this assay will enable the quantification of low cell numbers of E. coli O157 in environmental samples, providing a valuable tool to growers and researchers to protect humans from E. coli O157 contamination.
2. Velocity dependency of bacteria transport and retention. An understanding of factors that influence the fate of bacteria in soils and groundwater are needed to design more efficient remediation strategies and to mitigate the risks of disease causing bacteria on human health. An ARS researcher in Riverside, California, and collaborators, investigated the causes and complexities associated with the velocity dependency of bacteria retention and release parameters under different solution chemistry conditions. Results indicated that bacteria retention parameters were strong functions of water velocity due to changes in the adhesive interaction with time and differences in forces and torques that act on bacteria near solid surfaces. This information indicates that the mobility of bacteria in soils and groundwater will decrease under low velocity conditions, and that subsequent increases in velocity will not remobilize retained bacteria.
3. Differences in virus transport with temperature. Variations in the temperature of groundwater can occur as a result of seasonal changes, and mixing with surface water supplies (recharge and injection wells). An ARS researcher from Riverside, California, and collaborators, performed experimental studies and theoretical calculations to investigate the influence of temperature on virus and nanoparticle (NP) transport and retention. Results showed that an increase in temperature from 4 degrees Celsius (C) to 20 degrees C increased the retention of viruses and NPs in porous media under intermediate ionic strength (IS) conditions, but not when the IS was too low or too high. These findings were explained by differences in the energy barrier height with IS on heterogeneous surfaces, and a small reduction in the energy barrier with increasing temperature. This information indicates that low temperature conditions will increase the mobility of viruses and NPs under some environmental conditions.
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Sasidharan, S., Torkzaban, S., Bradford, S.A., Cook, P., Vadakattu, G.V. 2016. Temperature dependency of virus and nanoparticle transport and retention in saturated porous media. Journal of Contaminant Hydrology. 196:10-20. doi: 10.1016/j.jconhyd.2016.11.004.
Sasidharan, S., Bradford, S.A., Torkzaban, S., Ye, X., Vanderzalm, J., Du, X., Page, D. 2017. Unraveling complexities of velocity dependent retention and release parameters for E. coli in saturated porous media. Science of the Total Environment. 603:406-415. doi: 10.1016/j.scitotenv.2017.06.091.
Ibekwe, A.M., Murinda, S.E., Murry, M.A., Shwartz, G., Lundquist, T. 2016. Microbial community structures in algae cultivation ponds for bioconversion of agricultural wastes from livestock industry for feed production. Science of the Total Environment. 580(2017):1185-1196. doi: 10.1016/j.scitotenv.2016.12.076.
Ibekwe, A.M., Ma, J., Murinda, S., Reddy, G.B. 2017. Microbial diversity of bacteria, archaea, and fungi communities in a continuous flow constructed wetland for the treatment of swine waste. Hydrology: Current Research. 8(2):1-8. doi: 10.4172/2157-7587.1000277.
Ibekwe, A.M., Ors, S., Ferreira, J.F., Liu, X., Suarez, D.L. 2016. Seasonal induced changes in spinach rhizosphere microbial community structure with varying salinity and drought. Science of the Total Environment. 579:1485-1495. doi: 10.1016/j.scitotenv.2016.11.151.