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.
In support of Objective 2, batch experiments were conducted to examine the dynamics of bacterial conjugation with a multi-antibiotic and metal resistant plasmid in the presence and absence of antibiotics (Cefotaxime and Ampicillin) in growing (lysogeny broth at 37°celsius (C)) and non-growing (9.1 millimolar sodium chloride at 37°C) cultures of Escherichia coli (E. coli). Conjugation data exhibited a lag phase, rapid conjugation, a plateau phase in growing and non-growing cultures, and then a decay phase at later times under non-growing conditions due to bacterial die-off. Results indicated that there was a two to four hour window for conjugation after donor and recipient cells were mixed. Published models did not provide accurate descriptions of conjugation under non-growing conditions. A modified modeling approach was developed that accurately described the observed conjugation behavior. Ongoing experiments are examining the influence of other environmental stressors on survival and conjugation behavior. Under Objectives 1 and 2, monthly water samples were collected from Mill Creek wetland in the middle Santa Ana River watershed for 15 months to quantify antibiotic resistant Enterococcus species. Wastewater treatment plant effluent serves as influent for this wetland. Results revealed high resistances to multiple antimicrobials associated with wastewater treatment plant effluent. Additional analysis revealed that the wetland could only remove less than 75 percent of Enterococcus species entering the wetland. Additional sequencing studies will be done on these samples to understand the mechanism of horizontal gene transfer within the wetland. A parallel study is being conducted with E. coli, but the removal rate of E. coli by this wetland is more than 95 percent. Under Objective 3, a greenhouse study was conducted using multi-soil-layering (MSL) technology for removal of antibiotic, antibiotic resistant bacteria, and antibiotic resistance genes from soil columns. Spinach was used as a model plant to quantify the removal efficiency of antibiotics, since it is consumed fresh by humans. Quantification of antibiotics from soil, roots, and leaf as well as antibiotic resistance genes in bacteria is in progress.
1. Assessment of E. coli species carrying multiple antibiotic resistance genes from different animal sources. An ARS researcher from Riverside, California, and collaborators from seven additional ARS locations, examined different animal sources under different management systems to determine the severity of occurrence of multi-drug resistant Escherichia coli (E. coli). Whole genome sequencing was used to examine the presence of multi-drug resistant E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the U.S. Results revealed that one of the E. coli isolates from swine was resistant to nine antibiotics and carries more than 28 virulent factors, and this isolate has been shown to belong to an international high-risk clone. The data suggest that multi-drug resistant E. coli are widely distributed in different animal sources, but swine and dairy may be their main reservoir. This assessment will lead to the development of management practices that will reduce the transfer of antibiotic resistance genes and antibiotic resistant bacteria from swine or cattle manure to the environment.
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