Extreme low-flow conditions in a dual-chamber denitrification bioreactor contribute to pollution swapping with low landscape-scale impact.

Denitrification bioreactors are an effective edge-of-field conservation practice for nitrate (NO3) reduction from subsurface drainage. However, these systems may produce other pollutants and greenhouse gases during NO3 removal. Here a dual-chamber woodchip bioreactor system experiencing extreme low-flow conditions was monitored for its spatiotemporal NO3 and total organic carbon dynamics in the drainage water. Near complete removal of NO3 was observed in both bioreactor chambers in the first two years of monitoring (2019-2020) and in the third year of monitoring in chamber A, with significant (p < 0.01) reduction of the NO3-N each year in both chambers with 8.6-11.4 mg NO3-N L-1 removed on average. Based on the NO3 removal observed, spatial monitoring of sulfate (SO4), dissolved methane (CH4), and dissolved nitrous oxide (N2O) gases was added in the third year of monitoring (2021). In 2021, chambers A and B had median hydraulic residence times (HRTs) of 64 h and 39 h, respectively, due to varying elevations of the chambers, with drought conditions making the differences more pronounced. In 2021, significant production of dissolved CH4 was observed at rates of 0.54 g CH4-C m-3 d-1 and 0.07 g CH4-C m-3 d-1 in chambers A and B, respectively. In chamber A, significant removal (p < 0.01) of SO4 (0.23 g SO4 m-3 d-1) and dissolved N2O (0.21 mg N2O-N m-2 d-1) were observed, whereas chamber B produced N2O (0.36 mg N2O-N m-2 d-1). Considering the carbon dioxide equivalents (CO2e) on an annual basis, chamber A had loads (~12,000 kg CO2e ha-1 y-1) greater than comparable poorly drained agricultural soils; however, the landscape-scale impact was small (<1 % change in CO2e) when expressed over the drainage area treated by the bioreactor. Under low-flow conditions, pollution swapping in woodchip bioreactors can be reduced at HRTs <50 h and NO3 concentrations >2 mg N L-1.

Allelic variation of Escherichia coli outer membrane protein A: Impact on cell surface properties, stress tolerance and allele distribution.

Outer membrane protein A (OmpA) is one of the most abundant outer membrane proteins of Gram-negative bacteria and is known to have patterns of sequence variations at certain amino acids-allelic variation-in Escherichia coli. Here we subjected seven exemplar OmpA alleles expressed in a K-12 (MG1655) ΔompA background to further characterization. These alleles were observed to significantly impact cell surface charge (zeta potential), cell surface hydrophobicity, biofilm formation, sensitivity to killing by neutrophil elastase, and specific growth rate at 42°C and in the presence of acetate, demonstrating that OmpA is an attractive target for engineering cell surface properties and industrial phenotypes. It was also observed that cell surface charge and biofilm formation both significantly correlate with cell surface hydrophobicity, a cell property that is increasingly intriguing for bioproduction. While there was poor alignment between the observed experimental values relative to the known sequence variation, differences in hydrophobicity and biofilm formation did correspond to the identity of residue 203 (N vs T), located within the proposed dimerization domain. The relative abundance of the (I, δ) allele was increased in extraintestinal pathogenic E. coli (ExPEC) isolates relative to environmental isolates, with a corresponding decrease in (I, α) alleles in ExPEC relative to environmental isolates. The (I, α) and (I, δ) alleles differ at positions 203 and 251. Variations in distribution were also observed among ExPEC types and phylotypes. Thus, OmpA allelic variation and its influence on OmpA function warrant further investigation.

Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples.

Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a method implementing TruSeq high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant gene targets representing a full-spectrum of antibiotic resistance classes common to environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within complex environmental samples originated from manure, soil, and livestock feces, in addition to a mock-community reference to assess sensitivity and specificity. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate some of the obstacles that many researchers in this area currently face.

Comparison of microbial communities in replicated woodchip bioreactors.

Denitrification in woodchip bioreactors is a microbial process, but the effects of variations in bioreactors operation on microbial community structure are not well understood. Here, our goals were to understand hydraulic retention time (HRT) as a factor that influences woodchip bioreactor microbial community variation and structure in replicated field bioreactors and to evaluate relationships between microbial community membership and marker genes for denitrification. We used a combination of quantitative polymerase chain reaction of nirS, nirK, nosZI, and nosZII and 16S rRNA amplicon sequencing to characterize the microbial communities of nine pilot-scale woodchip bioreactors located at Iowa State University. Our results showed dynamic microbial communities but with persistent taxa between two sampling years and three HRTs. Similarities between functional gene copy numbers across sampling year and HRT indicate that the potential for denitrification is conserved despite differences in the microbial communities. These results are evidence that there are specific and persistent taxa within replicated bioreactors. Woodchip bioreactor microbial community membership is recommended to be the focus of future studies to better understand the relationship between microbial community functions and bioreactor management.

Denitrifying bioreactor microbiome: Understanding pollution swapping and potential for improved performance.

Denitrifying woodchip bioreactors are a best management practice to reduce nitrate-nitrogen (NO3 -N) loading to surface waters from agricultural subsurface drainage. Their effectiveness has been proven in many studies, although variable results with respect to performance indicators have been observed. This paper serves the purpose of synthesizing the current state of the science in terms of the microbial community, its impact on the consistency of bioreactor performance, and its role in the production of potential harmful by-products including greenhouse gases, sulfate reduction, and methylmercury. Microbial processes other than denitrification have been observed in these bioreactor systems, including dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic ammonium oxidation (anammox). Specific gene targets for denitrification, DNRA, anammox, and the production of harmful by-products are identified from bioreactor studies and other environmentally relevant systems for application in bioreactor studies. Lastly, cellulose depletion has been observed over time via increasing ligno-cellulose indices, therefore, the microbial metabolism of cellulose is an important function for bioreactor performance and management. Future work should draw from the knowledge of soil and wetland ecology to inform the study of bioreactor microbiomes.

Impact of Swine and Cattle Manure Treatment on the Microbial Composition and Resistome of Soil and Drainage Water.

Evaluating potential environmental and clinical impacts of industrial antibiotic use is critical in mitigating the spread of antimicrobial resistance. Using soil columns to simulate field application of swine or cattle manure and subsequent rain events, and a targeted qPCR-based approach, we tracked resistance genes from source manures and identified important differences in antimicrobial resistance gene transport and enrichment over time in the soil and water of artificially drained cropland. The source manures had distinct microbial community and resistance gene profiles, and these differences were also reflected in the soil columns after manure application. Antibiotic resistance genes (ARGs) were only significantly enriched in effluent samples following the first rain event (day 11) for both soil types compared to the control columns, illustrating the high background level of resistance present in the control soils chosen. For swine, the genes tetQ, tet(36), tet44, tetM, sul2 and ant(6)-ib persisted in the soil columns, whereas tetO, strB and sul1 persisted in effluent samples. Conversely, for cattle manure sul2 and strB persisted in both soil and effluent. The distinct temporal dynamics of ARG distribution between soil and effluent water for each manure type can be used to inform potential mitigation strategies in the future.

Antimicrobial resistance in integrated agroecosystems: State of the science and future opportunities.

As the Journal of Environmental Quality (JEQ) celebrates 50 years of publication, the division of environmental microbiology is one of the newest additions to the journal. During this time, significant advances in understanding of the interconnected microbial community and impact of the microbiome on natural and designed environmental systems have occurred. In this review, we highlight the intractable challenge of antimicrobial resistance (AMR) on humans, animals, and the environment, with particular emphasis on the role of integrated agroecosystems and by highlighting contributions published in JEQ. From early studies of phenotypic resistance of indicator organisms in waters systems to current calls for integrating AMR assessment across "One Health," publications in JEQ have advanced our understanding of AMR. As we reflect on the state of the science, we emphasize future opportunities. First, integration of phenotypic and molecular tools for assessing environmental spread of AMR and human health risk continues to be an urgent research need for a one health approach to AMR. Second, monitoring AMR levels in manure is recommended to understand inputs and potential spread through agroecosystems. Third, baseline knowledge of AMR levels is important to realize the impact of manure inputs on water quality and public health risk; this can be achieved through background monitoring or identifying the source-related genes or organisms. And finally, conservation practices designed to meet nutrient reduction goals should be explored for AMR reduction potential.

Antibiotic resistance gene dissipation in soil microcosms amended with antibiotics and swine manure.

The use of antibiotics in animal agriculture has exacerbated the presence of both antibiotic resistance genes (ARGs) and residual antibiotics excreted in animal manure. Field application of this manure is a common practice because its nutrient rich material can benefit crop growth. However, this practice can also introduce antibiotics and ARGs into nonagricultural settings. The integration of prairie buffer strips within and at the edge of crop fields is a potential management solution to reduce concentrations of ARGs commonly transported via water runoff and infiltration. An incubation experiment was conducted to investigate the fate of ARGs in directly manured crop field soils and the surrounding affected prairie strip soils. Row crop and prairie strip soils sampled from three sites received either an antibiotic spike and swine manure addition or a control water addition. The concentrations of select ARGs were then monitored over a 72-d period. Although soil vegetation and site location were not observed to influence ARG dissipation, the select genes did display different half-lives from one another. For example, tetM demonstrated the fastest dissipation of the genes quantified (average half-life, 5.18 d). Conversely, sul1 did not conform to the first-order linear regression kinetics used to describe the other investigated genes and was highly abundant in control prairie strip soils. The quantified half-lives of these select ARGs are comparable to previous studies and can inform monitoring and mitigative efforts aimed at reducing the spread of ARGs in the environment.

Midwestern cropping system effects on drainage water quality and crop yields.

Grain producers are challenged to maximize crop production while utilizing nutrients efficiently and minimizing negative impacts on water quality. There is a particular concern about nutrient export to the Gulf of Mexico via loss from subsurface drainage systems. The objective of this study was to investigate the effects of crop rotation, tillage, crop residue removal, swine manure applications, and cereal rye (Secale cereale L.) cover crops on nitrate-N (NO3 -N) and total reactive phosphorus (TRP) loss via subsurface drainage. The study was evaluated from 2008 through 2015 using 36 0.4-ha plots outfitted with a subsurface drainage water quality monitoring system. Results showed that when swine manure was applied before both corn (Zea mays L.) and soybean [Glycine max (L.) Merr.], drainage water had significantly higher 8-yr-average flow-weighted NO3 -N concentrations compared with swine manure applied before corn only in a corn-soybean rotation. The lowest NO3 -N loss was 15.2 kg N ha-1  yr-1 from a no-till corn-soybean treatment with rye cover crop and spring application of urea-ammonium nitrate (UAN) to corn. The highest NO3 -N loss was 29.5 kg N ha-1  yr-1 from swine manure applied to both corn and soybean. A rye cover crop reduced NO3 -N loss, whereas tillage and residue management had little impact on NO3 -N loss. Losses of TRP averaged <32 g P ha-1  yr-1 from all treatments. Corn yield was negatively affected by both no-till management and cereal rye cover crops. Results showed that cropping management affected N leaching but impacts on P leaching were minimal.

Seasonal variations in export of antibiotic resistance genes and bacteria in runoff from an agricultural watershed in Iowa.

Seasonal variations of antimicrobial resistance (AMR) indicators in runoff water can help improve our understanding of AMR sources and transport within an agricultural watershed. This study aimed to monitor multiple areas throughout the Black Hawk Lake (BHL) watershed (5324 ha) in central Iowa during 2017 and 2018 that consists of both swine and cattle feeding operations as well as known areas with manure application. The measured indicators included plate counts for fecal indicator bacteria (FIB) E. coli, Enterococcus, antibiotic resistant fecal indicator bacteria (ARBs) tylosin resistant Enterococcus, tetracycline resistant Enterococcus, and antibiotic resistance genes (ARGs): ermB, ermF (macrolide), tetA, tetM, tetO, tetW (tetracycline), sul1, sul2 (sulfonamide), aadA2 (aminoglycoside), vgaA, and vgaB (pleuromutilin). Both the plate count and the ARG analyses showed seasonal trends. Plate counts were significantly greater during the growing season, while the ARGs were greater in the pre-planting and post-harvest seasons (Wilcoxon Rank-Sum Test p < 0.05). The ermB gene concentration was significantly correlated (p < 0.05) with E. coli and Enterococcus concentrations in 2017, suggesting a potential use of this ARG as an indicator of environmental AMR and human health risk. Flow rate was not a significant contributor to annual variations in bacteria and AMR indicators. Based on observed seasonal patterns, we concluded that manure application was the likely contributor to elevated ARG indicators observed in the BHL watershed, while the driver of elevated ARB indictors in the growing season can only be speculated. Understanding AMR export patterns in agricultural watersheds provides public health officials knowledge of seasonal periods of higher AMR load to recreational waters.

Spatial and temporal distribution of E. coli contamination on three inland lake and recreational beach systems in the upper Midwestern United States.

Swimming advisories are commonly posted at public beaches across the United States every year. In Iowa, weekly monitoring of public swimming areas at state and county beaches have resulted in the impairment of numerous lakes for fecal indicator bacteria (FIB) contamination, as detected by E. coli. An extensive study was established to assess the relationships between E. coli contamination of nearshore beach water environments, open lake conditions and beach sands in three recreational beach/lake systems currently impaired for FIB contamination across Iowa. A transect/grab sample based sampling design was implemented across the systems with collections spanning from April through October of 2015 and 2016. Collections of E. coli along water transects identified strong near to far shore gradients of decreasing concentrations in all systems. Results indicate that concentrations of E. coli observed in swimming waters consistently disassociate with concentrations in the broader lake environment. Swimming water E. coli concentrations correlated with elevated beach sand E. coli, samples collected from beach sands uncovered concentrations up to 86,500 times higher than adjacent swimming waters. Results from this study indicate that foreshore beach sands and other beach proximate FIB sources serve as the major contributing source for swimming zone advisories. The current methodology used by state and federal officials includes impairing entire lake waterbodies for FIB contamination of the swimming area. These impairment listings do not accurately reflect the condition(s) of the larger lake environment outside the swimming area and fail to account for beach proximate conditions in the assessment process. Further, this approach provides potentially misleading information to the public and may undermine implementation strategies deployed by resource managers aimed at addressing FIB contamination at recreational swimming areas. Views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the Iowa Department of Natural Resources.

Catchment-scale export of antibiotic resistance genes and bacteria from an agricultural watershed in central Iowa.

Antibiotics are administered to livestock in animal feeding operations (AFOs) for the control, prevention, and treatment of disease. Manure from antibiotic treated livestock contains unmetabolized antibiotics that provide selective pressure on bacteria, facilitating the expression of anti-microbial resistance (AMR). Manure application on row crops is an agronomic practice used by growers to meet crop nutrient needs; however, it can be a source of AMR to the soil and water environment. This study in central Iowa aims to directly compare AMR indicators in outlet runoff from two adjacent (221 to 229 ha) manured and non-manured catchments (manure comparison), and among three catchments (600 to 804 ha) with manure influence, no known manure application (control), and urban influences (mixed land use comparison). Monitored AMR indicators included antibiotic resistance genes (ARGs) ermB, ermF (macrolide), tetA, tetM, tetO, tetW (tetracycline), sul1, sul2 (sulfonamide), aadA2 (aminoglycoside), vgaA, and vgaB (pleuromutilin), and tylosin and tetracycline resistant enterococci bacteria. Results of the manure comparison showed significantly higher (p<0.05) tetracycline and tylosin resistant bacteria from the catchment with manure application in 2017, but no differences in 2018, possibly due to changes in antibiotic use resulting from the Veterinary Feed Directive. Moreover, the ARG analysis indicated a larger diversity of ARGs at the manure amended catchment. The mixed land use comparison showed the manure amended catchment had significantly higher (p<0.05) tetracycline resistant bacteria in 2017 and significantly higher tylosin resistant bacteria in 2017 and 2018 than the urban influenced catchment. The urban influenced catchment had significantly higher ermB concentrations in both sampling years, however the manure applied catchment runoff consisted of higher relative abundance of total ARGs. Additionally, both catchments showed higher AMR indicators compared to the control catchment. This study identifies four ARGs that might be specific to AMR as a result of agricultural sources (tetM, tetW, sul1, sul2) and optimal for use in watershed scale monitoring studies for tracking resistance in the environment.

Long-term impact of poultry manure on crop yield, soil and water quality, and crop revenue.

A long-term poultry manure fertilizer study was initiated in 1998 and continued until 2009 under corn-soybean (CS) rotation. To match changing landscape trends, the plots were switched to continuous corn (CC) from 2010 to 2017. In both CS and CC phases, poultry manure (PM) was applied at the crop rotation recommended agronomic N rate and either half (CC phase) or double (CS phase) the recommended rate. Urea-ammonium nitrate (UAN) was applied to comparison plots at the crop recommended application rate (168 kg N ha-1 and 224 kg N ha-1 for the CS and CC phases, respectively) throughout the study. The objectives of this study include evaluation of the economic benefits of long-term PM application at various rates (PM2, PM, and PM0.5), and the impact of poultry manure application on soil health and nutrient levels, crop yield, and drainage water quality. Lower NO3-N concentrations were reported in drainage from PM treated plots when compared to UAN fertilizer applied at the same agronomic rate. Of the parameters tested for soil health analysis after twenty years of repeat application, particulate organic matter (POM) present was significantly greater in the PM treated soils (6.1-6.7 g kg soil-1) when compared to UAN plots (4.6 g kg soil-1), showing potential for stabilized soil particles, increased infiltration and water-holding capacity. The results show a consistent positive impact of manure application on corn and soybean yields when compared to yields observed in UAN treated plots. During the CS phase, we estimated the same average revenue per dollar spent for PM and UAN treatments, while the average return rate for PM2 was 1% lower; during CC phase,15% increased return rates were observed when PM0.5 and PM were compared against the UAN treatment. When managed properly, PM application to cropland is a sustainable option for diversifying agroecosystems, improving soil health and improving farm economics.

Investigating the dispersal of antibiotic resistance associated genes from manure application to soil and drainage waters in simulated agricultural farmland systems.

Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment.

Nitrous oxide and methane production from denitrifying woodchip bioreactors at three hydraulic residence times.

Denitrifying bioreactors remove nitrate (NO3-) from agricultural drainage and are slated to be an integral part of nitrogen reduction strategies in the Mississippi River Basin. However, incomplete denitrification can result in nitrous oxide (N2O) production and anaerobic conditions within bioreactors may be conducive to methane (CH4) production via methanogenesis. Greenhouse gas production has the potential to trade excess NO3- in surface water with excess greenhouses gases in the atmosphere. Our study examined N2O and CH4 production from pilot scale (6.38 m3) bioreactors across three hydraulic residence times (HRTs), 2, 8, and 16 h. Production was measured from both the surface of the bioreactors and dissolved in the bioreactor effluent. Nitrous oxide and CH4 was produced across all HRTs, with the majority dissolved in the effluent. Nitrous oxide production was significantly greater (P < 0.05) from 2 h HRTs (478.43 mg N2O m-3 day-1) than from 8 (29.95 mg N2O m-3 day-1) and 16 (36.61 mg N2O m-3 day-1) hour HRTs. Methane production was significantly less (P < 0.05) from 2 h HRTs (0.51 g C m3 day-1) compared to 8 (1.50 g C m3 day-1) and 16 (1.69 g C m3 day-1) hour HRTs. The 2 h HRTs had significantly greater (P = 0.05) impacts to climate change compared to 8 and 16 h HRTs. Results from this study suggest managing HRTs between 6 and 8 h in field bioreactors could minimize total greenhouse gas production and maximize NO3- removal.

Catchment-scale Phosphorus Export through Surface and Drainage Pathways.

The site-specific nature of P fate and transport in drained areas exemplifies the need for additional data to guide implementation of conservation practices at the catchment scale. Total P (TP), dissolved reactive P (DRP), and total suspended solids (TSS) were monitored at five sites-two streams, two tile outlets, and a grassed waterway-in three agricultural subwatersheds (221.2-822.5 ha) draining to Black Hawk Lake in western Iowa. Median TP concentrations ranged from 0.034 to 1.490 and 0.008 to 0.055 mg P L for event and baseflow samples, respectively. The majority of P and TSS export occurred during precipitation events and high-flow conditions with greater than 75% of DRP, 66% of TP, and 59% of TSS export occurring during the top 25% of flows from all sites. In one subwatershed, a single event (annual recurrence interval < 1 yr) was responsible for 46.6, 84.0, and 81.0% of the annual export of TP, DRP, and TSS, respectively, indicating that frequent, small storms have the potential to result in extreme losses. Isolated monitoring of surface and drainage transport pathways indicated significant P and TSS losses occurring through drainage; over the 2-yr study period, the drainage pathway was responsible for 69.8, 59.2, and 82.6% of the cumulative TP, DRP, and TSS export, respectively. Finally, the results provided evidence that particulate P losses in drainage were greater than dissolved P losses. Understanding relationships between flow, precipitation, transport pathway, and P fraction at the catchment scale is needed for effective conservation practice implementation.

Water and Sediment Microbial Quality of Mountain and Agricultural Streams.

Increased public health risk caused by pathogen contamination in streams is a serious issue, and mitigating the risk requires improvement in existing microbial monitoring of streams. To improve understanding of microbial contamination in streams, we monitored in stream water columns and streambed sediment. Two distinct streams and their subwatersheds were studied: (i) a mountain stream (Merced River, California), which represents pristine and wild conditions, and (ii) an agricultural stream (Squaw Creek, Iowa), which represents an agricultural setting (i.e., crop, manure application, cattle access). Stream water column and sediment samples were collected in multiple locations in the Merced River and Squaw Creek watersheds. Compared with the mountain stream, water column concentrations in the agricultural stream were considerably higher. In both mountain and agricultural streams, concentrations in bed sediment were higher than the water column, and principal component analysis indicates that land use affected water column levels significantly ( < 0.05). The cluster analysis showed grouping of subwatersheds for each basin, indicating unique land use features of each watershed. In general, water column levels in the mountain stream were lower than the USEPA's existing water quality criteria for bacteria. However, the levels in the agricultural stream exceeded the USEPA's microbial water quality criteria by several fold, which substantiated that increased agricultural activities, use of animal waste as fertilizers, and combined effect of rainfall and temperature may act as potential determining factors behind the elevated levels in agriculture streams.

Correction to: Escherichia coli persistence kinetics in dairy manure at moderate, mesophilic, and thermophilic temperatures under aerobic and anaerobic environments.

Escherichia coli persistence kinetics in dairy manure at moderate, mesophilic, and thermophilic temperatures.

Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance.

Use of antibiotics in human and animal medicine has applied selective pressure for the global dissemination of antibiotic-resistant bacteria. Therefore, it is of interest to develop strategies to mitigate the continued amplification and transmission of resistance genes in environmental reservoirs such as farms, hospitals and watersheds. However, the efficacy of mitigation strategies is difficult to evaluate because it is unclear which resistance genes are important to monitor, and which primers to use to detect those genes. Here, we evaluated the diversity of one type of macrolide antibiotic resistance gene (erm) in one type of environment (manure) to determine which primers would be most informative to use in a mitigation study of that environment. We analyzed all known erm genes and assessed the ability of previously published erm primers to detect the diversity. The results showed that all known erm resistance genes group into 66 clusters, and 25 of these clusters (40%) can be targeted with primers found in the literature. These primers can target 74%-85% of the erm gene diversity in the manures analyzed.

Seasonal variation of macrolide resistance gene abundances in the South Fork Iowa River Watershed.

The Midwestern United States is dominated by agricultural production with high concentrations of swine, leading to application of swine manure onto lands with artificial subsurface drainage. Previous reports have indicated elevated levels of antibiotic resistance genes (ARGs) in surface water and groundwater around confined animal feeding operations which administer antimicrobials. While previous studies have examined the occurrence of ARGs around confined swine feeding operations, little information is known how their transport from tile-drained fields receiving swine manure application impacts downstream environments. To further our knowledge in this area, water samples were collected from five locations in the agriculturally dominated South Fork Iowa River Watershed with approximately 840,000 swine present in the 76,000ha basin. Samples were collected monthly from three stream sites and two main artificial subsurface drainage outlets. Samples were analyzed for macrolide resistance genes ermB, ermF and 16S rRNAgene abundance using qPCR. Abundance of erm genes ranged from below limits of quantification to >107 copies 100mL-1 water. Eighty-nine percent of stream water samples contained one of these two ARGs. Results indicate significantly more ermB and ermF in main drainage outlets than stream samples when normalized by 16S rRNA abundance (p<0.0001). Both artificial drainage locations revealed temporal trends for ermB and ermF abundance when normalized to 16S rRNA abundance. The higher resistance gene concentrations identified in artificial drainage samples occurring mid-Spring and late-Fall are likely due to manure application.