Knowledge Resource Center for Ecological Environment in Arid Area
| 项目编号 | 1015099 |
| Antibiotic Resistance from Wastewater to Agricultural Soils and Food Crops | |
| Navab-Daneshmand, Tala | |
| 主持机构 | OREGON STATE UNIVERSITY |
| 开始日期 | 2018 |
| 结束日期 | 2021 |
| 资助机构 | US-NIFA（美国食品与农业研究所） |
| 语种 | 英语 |
| 国家 | 美国 |
| 中文简介 | 2020 - Engineering |
| 英文简介 | Goals / Objectives 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 Moreover, our unpublished data using minimum inhibitory concentration assays - a metric for antibiotic resistance informing on the thresholds for resistance and the human health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.Project Methods Methods for research objective 1:Wastewater influent, wastewater effluent, and biosolids will be collected from 18 wastewater treatment facilities across Oregon from urban and rural areas in the coastal, valley, and high desert regions. Samples will be collected from all the 18 wastewater treatment facilities in both summer and winter over project years 1 and 2 to include the seasonal variations in the influent. In addition to wastewater and biosolids samples, we will collect data from the wastewater treatment facilities on their serving population, the presence of hospitals in their service area, and their treatments processes.Prior to DNA extraction, samples will be treated with propidium monoazide (PMA) to inhibit the amplification of dead or membrane compromised DNA. PMA-treated DNA of wastewater influent, wastewater effluent, and biosolids samples will be extracted, and target antibiotic-resistant genes and mobile genetic elements (16S-rRNA, aac(3)-IV, blaSHV, blaCMY, blaKPC, qnrA, sul1, tet(A), tet(B), intI1, oriT-incN, IS6, orf37-IS26) will be quantified. Findings will identify the impact of seasonal, regional, and population density variations on the abundance of target antibiotic-resistant genes in wastewater influent, treated effluent, and biosolids in Oregon. In addition, results on the abundance of mobile genetic elements (integrons, plasmids, transposons, and insertion sequences) will determine the potential for dissemination of antibiotic-resistant genes in the wastewater system and the receiving environments.PMA-treated DNA samples will be submitted for next generation Illumina high-throughput DNA sequencing for further analyses of the microbial community. Antibiotic-resistant genes and other mobile genetic elements can be detected and quantified by searching against online databases using publically available Comprehensive Antibiotic Resistance Database project. The obtained results will identify the key antibiotic-resistant players (i.e., genes), and the impact of seasonal, regional, and population density variations on microbial community profiles in the wastewater system across Oregon.Aliquots of wastewater and biosolids will be filtered and prepared by solid phase extraction. Samples will be analyzed for 21 antibiotics by liquid chromatographyGoals /mass spectrometry, and by high resolutionGoals /mass spectrometry. Results will describe the variability of antibiotics in wastewater treatment systems.Correlation analyses, factorial analysis of variance (factorial ANOVA), and multivariate analyses of variance will determine the associations between seasonal, regional, and population density variations, as well as different treatment processes in the wastewater treatment facilities on the abundance and diversity of antibiotics, antibiotic-resistant genes, and mobile genetic elements in wastewater and biosolids. The strength of correlations will be determined by Spearman ρ coefficients.Methods for research objective 2:A set of experiments will be performed at pilot-scale in a greenhouse at Oregon State University. 4' × 4' growth trays will be placed on mesh grid tables to collect and contain contaminated runoff. Polyvinyl chloride pipe cages with mosquito netting will be constructed around each tray to prevent flying insects from accessing contaminated plants and soil. We will obtain sandy and silty soil from commercial agricultural fields with no history of wastewater irrigation or biosolidsGoals /manure fertilization from the Willamette Valley area in Oregon. Soil will be hand ground and distributed in growth pots. Wastewater effluent and biosolids samples (both class A and class B microbiological quality) will be collected from treatment facilities that had the highest antibiotic-resistant content as evaluated in research objective 1. For sample combinations with biosolids amendment, biosolids will be added and mixed with soil. In order to evaluate the impact of biosolids nutrient and texture without the microbial community, we will add autoclaved biosolids to some pots as control. Carrot seeds will be planted in growth pots, and irrigated every other day or as needed with autoclaved nanopure water or wastewater effluent following the experimental setup. To assess the impact of nutrients in wastewater effluent without the microbial community, we will irrigate selected pots with autoclaved wastewater effluent as control. We will have triplicates of each of the 16 sample types in three separate growth pots. For additional control, we will have growth pots filled with soil without any planted carrot seeds or wastewater and biosolids addition. Soil cores will be collected using a T-sampler from each growth pot every 3 days until harvest. Carrots will be harvested when ready.MacConkey agar will be used to enumerate E. coli. To quantify antibiotic-resistant E. coli, MacConkey agar plates will be supplemented with fixed concentrations of antibiotics from different families and chemical structure, specifically gentamicin, ampicillin, meropenem, ciprofloxacin, sulfamethoxazole, and tetracycline. The E. coli and antibiotic-resistant E. coli colony forming units will then be quantified. At least three isolated colonies will be selected from each plate with antibiotic supplements and stored for further processing. Findings on the concentration of E. coli and antibiotic-resistant E. coli is expected to determine the impact of irrigation with wastewater effluent or fertilization with biosolids on the presence, abundance, and persistence of antibiotic-resistant E. coli in soils and on harvested root crops.Phenotypic characterization of susceptibility or resistance to antibiotics of collected isolates will be analyzed using the broth macrodilution assay to determine the minimum inhibitory concentrations. Results are expected to determine the impact of wastewater and biosolids application to soil, and the persistence of antibiotic-resistant bacteria on the minimum antibiotic concentration that completely inhibits growth of E. coli in soil and on harvested carrots.A subset of isolated antibiotic-resistant colonies will be tested against a library of antibiotic-resistant genes and genes associated with mobile genetic elements. Findings will identify how wastewater irrigation and biosolids land application promotes the persistence and dissemination of antibiotic-resistant genes and mobile genetic elements in agricultural soils and food crops.Following the methods in research objective 1, DNA of soil and carrot rinsate pellets will be extracted, sequenced, and target genes will be quantified. Quantified target genes will determine the impact of wastewater and biosolids application to soils on the growth and survival of antibiotic-resistant genes and other mobile genetic elements in soils and crops. Microbial community analyses during growth season will describe the impact of wastewater and biosolids use on the abundance and diversity of antibiotic resistance in soils and food crops.Statistical analyses will be performed on the obtained data as descirbed under research objective 1. Correlation analyses, factorial ANOVA, and MANOVA will be performed to determine the associations between soil types and soil amendments with the prevalence, growth, persistence and dissemination of antibiotics, antibiotic-resistant E. coli, and target genes. |
| 英文关键词 | agriculture antibiotic resistance biosolids land application crops soil wastewater irrigation |
| 来源学科分类 | 2020 - Engineering |
| 资源类型 | 项目 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/356107 |
| 推荐引用方式 GB/T 7714 | Navab-Daneshmand, Tala.Antibiotic Resistance from Wastewater to Agricultural Soils and Food Crops.2018. |
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