Antibiotic Resistance from Wastewater to Agricultural Soils and Food Crops

The overall objective of this application is to identify the abundance and diversity of antibiotic resistance in wastewater and biosolids across different seasonal and regional variations, and further determine the impact of the application of wastewater and biosolids on growth and dissemination of antibiotic-resistant bacteria and genes in soils and on vegetable crops. The central hypothesis underlying this research effort is that the abundance and diversity of antibiotic-resistant genes and the mobile genetic elements in wastewater and biosolids, when used in agricultural practices, impact their persistence in soils and on food crops. This hypothesis was formulated based on our preliminary findings on the impact of soil type and inoculation with antibiotic-resistant bacteria on the growth and persistence of antibiotic resistance in soil. Moreover, our unpublished data using minimum inhibitory concentration assays - a metric for antibiotic resistance informing on the thresholds for resistance and the human health exposures - suggests the development of antibiotic resistance through chronic exposure to antibiotics in soil microbiota.We plan to test our central hypothesis objectively by pursuing the following two specific aims:Research objective 1: Identify the variability of antibiotics and antibiotic resistance in wastewater treatment facilities in Oregon.Our working hypothesis is that seasonal (winter or summer) and geographical (coastal, valley, or high desert, and urban or rural) variations impact the abundance and diversity of antibiotics and antibiotic-resistant genes in wastewater influent, as well as the treatment efficiency, hence the variability of antibiotics and antibiotic-resistant genes in wastewater effluent and biosolids.Irrigation with wastewater effluent and fertilization with biosolids is commonly practiced across the US. The knowledge on the variability of antibiotics and antibiotic resistance in municipal wastewater, the associated treatment efficiencies at wastewater treatment facilities, and their variability in the wastewater effluent and biosolids is limited. The goal of the research proposed under research objective 1 is to identify and characterize the abundance and diversity of antibiotics and antibiotic-resistant genes in wastewater treatment facilities in Oregon. To achieve this goal, it is necessary to determine the impact of seasonal and geographical differences on the variability of antibiotics and antibiotic resistance in wastewater influent, the treated wastewater effluent, and biosolids. Our working hypothesis is that seasonal (winter or summer) and geographical (coastal, valley, or high desert, and urban or rural) variations impacts the abundance and diversity of antibiotics and antibiotic-resistant genes in wastewater influent, as well as the treatment efficiency, hence the variability of antibiotics and antibiotic-resistant genes in wastewater effluent and biosolids. We will test our working hypothesis by using the experimental approach of characterizing the variability of antibiotics and antibiotic-resistant genes in wastewater treatment facilities across Oregon in winter and summer (seasonal impact), in urban and rural areas (population density impact), and in the coastal, valley, and high desert regions (geographical impact). The rationale that underlies research objective 1 is that knowledge on the abundance and diversity of antibiotics and antibiotic-resistant genes in municipal wastewater, the treated effluent, and biosolids in different seasons and regions will elucidate the range of contaminants received by the agricultural fields and food crops. At the completion of the proposed study for research objective 1, we expect as the overall outcome to have determined how seasonal and geographical differences in Oregon impact the variability of antibiotics and antibiotic resistance in municipal wastewater and the treated effluent and biosolids.Research objective 2: Determine the impact of irrigation with wastewater effluent and fertilization with biosolids on the prevalence and dissemination of antibiotic resistance in soil.Our working hypothesis is that irrigating with wastewater effluent or fertilizing with biosolids promotes the persistence of antibiotic-resistant bacteria and determinant genes in soils and food crops.The knowledge on the impact of wastewater irrigation and biosolids amendment on the prevalence and growth of antibiotic-resistant bacteria, and the dissemination of mobile genetic elements is limited. The goal of the proposed study under research objective 2 is to determine how the wastewater and biosolids application to agricultural soil affect the prevalence and growth of enteric antibiotic-resistant bacteria and determinant genes, and the spread of mobile genetic elements. In order to understand this, it will be necessary to monitor the microbiological content of soil and harvested crops after the application of wastewater or biosolids. Our working hypothesis is that irrigating with wastewater effluent or fertilizing with biosolids promotes the persistence of antibiotic-resistant bacteria, determinant genes, and mobile genetic elements in soils and food crops. To test our working hypothesis, our approach will be to apply different combinations of water, wastewater effluent, class A or class B biosolids, and autoclaved wastewater and biosolids to different soil types (i.e., sand and silt) during the growth season of carrots (i.e., 70-80 days) in a greenhouse. We will monitor E. coli - as fecal indicator bacteria, resistant-E. coli to different antibiotic families (i.e., aminoglycosides, beta lactams, macrolides, quinolones, sulfonamides, tetracyclines), antibiotics, antibiotic-resistant genes, and genes associated with mobile genetic elements (i.e., integrons, plasmids, transposons, and insertion sequences). The rationale underlying this objective is that determining the impact of wastewater and biosolids application to soils on the prevalence, growth, persistence, and dissemination of antibiotic resistance in agricultural soils and food crops will improve our understanding of the emergence and transmission modes of antimicrobial resistance in agriculture-impacted environments. Without such new knowledge, it is highly unlikely to prevent and control the spread of antibiotic resistance associated with wastewater and biosolids application in agricultural practices that impact food safety and human health. When the proposed studies for research objective 2 have been completed, we expect as the overall outcome to have identified how wastewater irrigation and biosolids application promote the growth and persistence of antibiotic resistance in agricultural soil and food crops. Findings are expected to have significant impact on the field as a result of our improved understanding of human health risks and food safety associated with enteric antibiotic-resistant infections due to wastewater and biosolids application in agricultural practices.