Integrated Metagenomic Assessment of Multiple Pre-harvest Control Points on Lettuce Resistomes at Field-Scale.

An integrated understanding of factors influencing the occurrence, distribution, and fate of antibiotic resistance genes (ARGs) in vegetable production systems is needed to inform the design and development of strategies for mitigating the potential for antibiotic resistance propagation in the food chain. The goal of the present study was to holistically track antibiotic resistance and associated microbiomes at three distinct pre-harvest control points in an agroecosystem in order to identify the potential impacts of key agricultural management strategies. Samples were collected over the course of a single growing season (67 days) from field-scale plots amended with various organic and inorganic amendments at agronomic rates. Dairy-derived manure and compost amendment samples (n = 14), soil samples (n = 27), and lettuce samples (n = 12) were analyzed via shotgun metagenomics to assess multiple pre-harvest factors as hypothetical control points that shape lettuce resistomes. Pre-harvest factors of interest included manure collection during/post antibiotic use, manure composting, and soil amended with organic (stockpiled manure/compost) versus chemical fertilizer. Microbial community resistome and taxonomic compositions were unique from amendment to soil to lettuce surface according to dissimilarity analysis. The highest resistome alpha diversity (i.e., unique ARGs, n = 642) was detected in amendment samples prior to soil application, while the composted manure had the lowest total ARG relative abundance (i.e., 16S rRNA gene-normalized). Regardless of amendment type, soils acted as an apparent ecological buffer, i.e., soil resistome and taxonomic profiles returned to background conditions 67 d-post amendment application. Effects of amendment conditions surprisingly re-emerged in lettuce phyllosphere resistomes, with the highest total ARG relative abundances recovered on the surface of lettuce plants grown in organically-fertilized soils (i.e., compost- and manure-amended soils). Co-occurrence analysis identified 55 unique ARGs found both in the soil amendments and on lettuce surfaces. Among these, arnA and pmrF were the most abundant ARGs co-occurring with mobile genetic elements (MGE). Other prominent ARG-MGE co-occurrences throughout this pre-harvest lettuce production chain included: TetM to transposon (Clostridiodies difficile) in the manure amendment and TriC to plasmid (Ralstonia solanacearum) on the lettuce surfaces. This suggests that, even with imposing manure management and post-amendment wait periods in agricultural systems, ARGs originating from manure can still be found on crop surfaces. This study demonstrates a comprehensive approach to identifying key control points for the propagation of ARGs in vegetable production systems, identifying potential ARG-MGE combinations that could inform future surveillance. The findings suggest that additional pre-harvest and potentially post-harvest interventions may be warranted to minimize risk of propagating antibiotic resistance in the food chain.

Cross-comparison of methods for quantifying antibiotic resistance in agricultural soils amended with dairy manure and compost.

Agricultural soils are often amended with livestock manure, making them a key reservoir of antibiotic resistance genes (ARGs). Given that soils are among the most microbially-diverse environments on the planet; effective characterization and quantification of the effects of manure-derived amendments on soil resistomes is a major challenge. This study examined the effects of dairy manure-derived amendments on agricultural soils via two strategies: quantification of anthropogenic ARG markers via qPCR and shotgun metagenomic resistome profiling; and these strategies were compared to a previously published antibiotic resistant fecal coliform dataset. Soil samples were collected throughout a 120 day complete block field experiment to compare the effects of amendment type on antibiotic resistance. Results of all three measurements were consistent with the hypothesis that the application of composted manure reduced antibiotic resistance in soil relative to the application of raw manure, although some differences were noted in comparing the patterns of the three measurements with time. Raw dairy manure-amended soils yielded high sul1 and tet(W) relative abundances on Day 0 (following amendment application), but significantly decreased to background levels by Day 67 (harvest) and Day 120 (study completion). Shotgun metagenomics similarly detected a decrease in the relative abundances of sulfonamide and tetracycline-associated ARGs over time in the raw manure- and compost-amended soils; however, these levels were significantly lower than those estimated by qPCR. Interestingly, although patterns of sulfonamide and tetracycline resistance among culturable fecal coliforms echoed those observed via qPCR and metagenomics; erythromycin resistant coliforms were directly recovered by culture in amended soils, but corresponding ARGs were not detected by qPCR or metagenomics. This study supports both composting and time restrictions as means of reducing the potential for antibiotic resistance in manure to spread via soil application. Results suggest some differences in finer conclusions drawn depending on which antibiotic resistance monitoring target is selected.

Fecal Indicator Bacteria and Antibiotic Resistance Genes in Storm Runoff from Dairy Manure and Compost-Amended Vegetable Plots.

Given the presence of antibiotics and resistant bacteria in livestock manures, it is important to identify the key pathways by which land-applied manure-derived soil amendments potentially spread resistance. The goal of this field-scale study was to identify the effects of different types of soil amendments (raw manure from cows treated with cephapirin and pirlimycin, compost from antibiotic-treated or antibiotic-free cows, or chemical fertilizer only) and crop type (lettuce [ L.] or radish [ L.]) on the transport of two antibiotic resistance genes (ARGs; 1 and ) via storm runoff from six naturally occurring storms. Concurrent quantification of sediment and fecal indicator bacteria (FIB; and enterococci) in runoff permitted comparison to traditional agricultural water quality targets that may be driving factors of ARG presence. Storm characteristics (total rainfall volume, storm duration, etc.) significantly influenced FIB concentration (two-way ANOVA, < 0.05), although both effects from individual storm events (Kruskal-Wallis, < 0.05) and vegetative cover influenced sediment levels. Composted and raw manure-amended plots both yielded significantly higher 1 and B levels in runoff for early storms, at least 8 wk following initial planting, relative to fertilizer-only or unamended barren plots. There was no significant difference between 1 or B levels in runoff from plots treated with compost derived from antibiotic-treated versus antibiotic-free dairy cattle. Our findings indicate that agricultural fields receiving manure-derived amendments release higher quantities of these two "indicator" ARGs in runoff, particularly during the early stages of the growing season, and that composting did not reduce effects of ARG loading in runoff.

Plants mediate precipitation-driven transport of a neonicotinoid pesticide.

Neonicotinoid insecticides provide crop protection via water solubility and systemicity, yet these chemical characteristics, combined with high toxicity to non-target invertebrates (e.g., honeybees), elicit concern of environmental transport. Neonicotinoids have been detected in soil and surface water throughout North America; however, no investigation has defined a direct connection to planted seed dressings. We quantified the physical transport of thiamethoxam (TMX), a neonicotinoid, under field conditions. We planted TMX-coated corn seeds and maintained plots with and without viable crops (n = 3 plots per treatment) to determine plant influence on pesticide transport. TMX concentrations were measured in soil and drainage throughout the growing season. Storm-generated runoff was the dominant transport mechanism (maximum TMX concentration 1.72 ±â€¯0.605 µg L-1; no viable plants), followed by shallow (<72 cm) lateral drainage (0.570 ±â€¯0.170 µg L-1; no viable plants), and deep (110 cm) drainage (0.170 ±â€¯0.265 µg L-1; viable plants). Soil samples confirmed vertical and lateral movement within 23 and 36 days of planting, respectively. Plants facilitated downward migration of TMX in soil but restricted TMX drainage. Altogether, these study results revealed that neonicotinoids can be transported from seed coatings both above and through the soil profile, which may enable migration into surrounding ecosystems.

Macroinvertebrate sensitivity thresholds for sediment in Virginia streams.

Sediment is the most commonly identified pollutant associated with macroinvertebrate community impairments in freshwater streams nationwide. Management of this physical stressor is complicated by the multiple measures of sediment available (e.g., suspended, dissolved, bedded) and the variability in natural "healthy" sediment loadings across ecoregions. Here we examine the relative importance of 9 sediment parameters on macroinvertebrate community health as measured by the Virginia Stream Condition Index (VSCI) across 5 ecoregions. In combination, sediment parameters explained 27.4% of variance in the VSCI in a multiregion data set and from 20.2% to 76.4% of variance for individual ecoregions. Bedded sediment parameters had a stronger influence on VSCI than did dissolved or suspended parameters in the multiregion assessment. However, assessments of individual ecoregions revealed conductivity had a key influence on VSCI in the Central Appalachian, Northern Piedmont and Piedmont ecoregions. In no case was a single sediment parameter sufficient to predict VSCI scores or individual biological metrics. Given the identification of embeddedness and conductivity as key parameters for predicting biological condition, we developed family-level sensitivity thresholds for these parameters, based on extirpation. Resulting thresholds for embeddedness were 68% for combined ecoregions, 65% for the Mountain bioregion (composed of Central Appalachian, Ridge and Valley, and Blue Ridge ecoregions), and 88% for the Piedmont bioregion (composed of Northern Piedmont and Piedmont ecoregions). Thresholds for conductivity were 366 µS/cm for combined ecoregions, 391 µS/cm for the Mountain bioregion, and 136 µS/cm for the Piedmont bioregion. These thresholds may help water quality professionals identify impaired and at-risk waters designated to support aquatic life and develop regional strategies to manage sediment-impaired streams. Inclusion of embeddedness as a restoration endpoint may be warranted; this could be facilitated by application of more quantitative, less time-intensive measurement approaches. We encourage refinement of thresholds as additional data and genus-based metrics become available. Integr Environ Assess Manag 2019;15:77-92. Published 2018. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

Fate of Pirlimycin and Antibiotic-Resistant Fecal Coliforms in Field Plots Amended with Dairy Manure or Compost during Vegetable Cultivation.

Identification of agricultural practices that mitigate the environmental dissemination of antibiotics is a key need in reducing the prevalence of antibiotic-resistant bacteria of human health concern. Here, we aimed to compare the effects of crop (lettuce [ L.] or radish [ L.]), soil amendment type (inorganic fertilizer, raw dairy manure, composted dairy manure, or no amendment), and prior antibiotic use history (no antibiotics during previous lactation cycles vs. manure mixed from cows administered pirlimycin or cephapirin) of manure-derived amendments on the incidence of culturable antibiotic-resistant fecal coliforms in agricultural soils through a controlled field-plot experiment. Antibiotic-resistant culturable fecal coliforms were recoverable from soils across all treatments immediately after application, although persistence throughout the experiment varied by antibiotic class and time. The magnitude of observed coliform counts differed by soil amendment type. Compost-amended soils had the highest levels of cephalosporin-resistant fecal coliforms, regardless of whether the cows from which the manure was derived were administered antibiotics. Samples from control plots or those treated with inorganic fertilizer trended toward lower counts of resistant coliforms, although these differences were not statistically significant. No statistical differences were observed between soils that grew leafy (lettuce) versus rooted (radish) crops. Only pirlimycin was detectable past amendment application in raw manure-amended soils, dissipating 12 to 25% by Day 28. Consequently, no quantifiable correlations between coliform count and antibiotic magnitude could be identified. This study demonstrates that antibiotic-resistant fecal coliforms can become elevated in soils receiving manure-derived amendments, but that a variety of factors likely contribute to their long-term persistence under typical field conditions.

Invertebrate-Based Water Quality Impairments and Associated Stressors Identified through the US Clean Water Act.

Macroinvertebrate community assessment is used in most US states to evaluate stream health under the Clean Water Act. While water quality assessment and impairment determinations are reported to the US Environmental Protection Agency, there is no national summary of biological assessment findings. The objective of this work was to determine the national extent of invertebrate-based impairments and to identify pollutants primarily responsible for those impairments. Evaluation of state data in the US Environmental Protection Agency's Assessment and Total Maximum Daily Load Tracking and Implementation System database revealed considerable differences in reporting approaches and terminologies including differences in if and how states report specific biological assessment findings. Only 15% of waters impaired for aquatic life could be identified as having impairments determined by biological assessments (e.g., invertebrates, fish, periphyton); approximately one-third of these were associated with macroinvertebrate bioassessment. Nearly 650 invertebrate-impaired waters were identified nationwide, and sediment was the most common pollutant in bedded (63%) and suspended (9%) forms. This finding is not unexpected, given previous work on the negative impacts of sediment on aquatic life, and highlights the need to more specifically identify the mechanisms driving sediment impairments in order to design effective remediation plans. It also reinforces the importance of efforts to derive sediment-specific biological indices and numerical sediment quality guidelines. Standardization of state reporting approaches and terminology would significantly increase the potential application of water quality assessment data, reveal national trends, and encourage sharing of best practices to facilitate the attainment of water quality goals.

Stormwater loadings of antibiotic resistance genes in an urban stream.

Antibiotic resistance presents a critical public health challenge and the transmission of antibiotic resistance via environmental pathways continues to gain attention. Factors driving the spread of antibiotic resistance genes (ARGs) in surface water and sources of ARGs in urban stormwater have not been well-characterized. In this study, five ARGs (sul1, sul2, tet(O), tet(W), and erm(F)) were quantified throughout the duration of three storm runoff events in an urban inland stream. Storm loads of all five ARGs were significantly greater than during equivalent background periods. Neither fecal indicator bacteria measured (E. coli or enterococci) was significantly correlated with sul1, sul2, or erm(F), regardless of whether ARG concentration was absolute or normalized to 16S rRNA levels. Both E. coli and enterococci were correlated with the tetracycline resistance genes, tet(O) and tet(W). Next-generation shotgun metagenomic sequencing was conducted to more thoroughly characterize the resistome (i.e., full complement of ARGs) and profile the occurrence of all ARGs described in current databases in storm runoff in order to inform future watershed monitoring and management. Between 37 and 121 different ARGs were detected in each stream sample, though the ARG profiles differed among storms. This study establishes that storm-driven transport of ARGs comprises a considerable fraction of overall downstream loadings and broadly characterizes the urban stormwater resistome to identify potential marker ARGs indicative of impact.

Seasonal Variation in Floodplain Biogeochemical Processing in a Restored Headwater Stream.

Stream and river restoration activities have recently begun to emphasize the enhancement of biogeochemical processing within river networks through the restoration of river-floodplain connectivity. It is generally accepted that this practice removes pollutants such as nitrogen and phosphorus because the increased contact time of nutrient-rich floodwaters with reactive floodplain sediments. Our study examines this assumption in the floodplain of a recently restored, low-order stream through five seasonal experiments. During each experiment, a floodplain slough was artificially inundated for 3 h. Both the net flux of dissolved nutrients and nitrogen uptake rate were measured during each experiment. The slough was typically a source of dissolved phosphorus and dissolved organic matter, a sink of NO3(-), and variable source/sink of ammonium. NO3(-) uptake rates were relatively high when compared to riverine uptake, especially during the spring and summer experiments. However, when scaled up to the entire 1 km restoration reach with a simple inundation model, less than 0.5-1.5% of the annual NO3(-) load would be removed because of the short duration of river-floodplain connectivity. These results suggest that restoring river-floodplain connectivity is not necessarily an appropriate best management practice for nutrient removal in low-order streams with legacy soil nutrients from past agricultural landuse.

Effect of manure application on abundance of antibiotic resistance genes and their attenuation rates in soil: field-scale mass balance approach.

The development of models for understanding antibiotic resistance gene (ARG) persistence and transport is a critical next step toward informing mitigation strategies to prevent the spread of antibiotic resistance in the environment. A field study was performed that used a mass balance approach to gain insight into the transport and dissipation of ARGs following land application of manure. Soil from a small drainage plot including a manure application site, an unmanured control site, and an adjacent stream and buffer zone were sampled for ARGs and metals before and after application of dairy manure slurry and a dry stack mixture of equine, bovine, and ovine manure. Results of mass balance suggest growth of bacterial hosts containing ARGs and/or horizontal gene transfer immediately following slurry application with respect to ermF, sul1, and sul2 and following a lag (13 days) for dry-stack-amended soils. Generally no effects on tet(G), tet(O), or tet(W) soil concentrations were observed despite the presence of these genes in applied manure. Dissipation rates were fastest for ermF in slurry-treated soils (logarithmic decay coefficient of -3.5) and for sul1 and sul2 in dry-stack-amended soils (logarithmic decay coefficients of -0.54 and -0.48, respectively), and evidence for surface and subsurface transport was not observed. Results provide a mass balance approach for tracking ARG fate and insights to inform modeling and limiting the transport of manure-borne ARGs to neighboring surface water.

Riparian deforestation, stream narrowing, and loss of stream ecosystem services.

A study of 16 streams in eastern North America shows that riparian deforestation causes channel narrowing, which reduces the total amount of stream habitat and ecosystem per unit channel length and compromises in-stream processing of pollutants. Wide forest reaches had more macroinvertebrates, total ecosystem processing of organic matter, and nitrogen uptake per unit channel length than contiguous narrow deforested reaches. Stream narrowing nullified any potential advantages of deforestation regarding abundance of fish, quality of dissolved organic matter, and pesticide degradation. These findings show that forested stream channels have a wider and more natural configuration, which significantly affects the total in-stream amount and activity of the ecosystem, including the processing of pollutants. The results reinforce both current policy of the United States that endorses riparian forest buffers as best management practice and federal and state programs that subsidize riparian reforestation for stream restoration and water quality. Not only do forest buffers prevent nonpoint source pollutants from entering small streams, they also enhance the in-stream processing of both nonpoint and point source pollutants, thereby reducing their impact on downstream rivers and estuaries.

Understanding stream geomorphic state in relation to ecological integrity: evidence using habitat assessments and macroinvertebrates.

Scientists have long assumed that the physical structure and condition of stream and river channels have pervasive effects on biological communities and processes, but specific tests are few. To investigate the influence of the stream-reach geomorphic state on in-stream habitat and aquatic macroinvertebrate communities, we compared measures of habitat conditions and macroinvertebrate community composition between stable and unstable stream reaches in a paired-study design. We also explored potential associations between these ecological measures and individual geomorphic characteristics and channel adjustment processes (degradation, aggradation, overwidening, and change in planform). We found that habitat quality and heterogeneity were closely tied to stream stability, with geomorphically stable reaches supporting better habitat than unstable reaches. Geomorphic and habitat assessment scores were highly correlated (r = 0.624, P < 0.006, n = 18). Stable reaches did not support significantly greater macroinvertebrate densities than unstable reaches (t = -0.415, P > 0.689, df = 8). However, the percent of the macroinvertebrate community in the Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa was significantly correlated with the overall habitat assessment scores as well as with individual measures of geomorphic condition and habitat quality. While there is a clear need for more work in classifying and quantifying the responses of aquatic and aquatic-dependent biota to various geomorphic states and processes, this study provides solid preliminary evidence that macroinvertebrate communities are affected by the geomorphic condition of the stream reaches they inhabit and that geomorphic assessment approaches can be used as a tool for evaluating ecological integrity.