Metagenomic tracking of antibiotic resistance genes through a pre-harvest vegetable production system: an integrated lab-, microcosm- and greenhouse-scale analysis.

Prior research demonstrated the potential for agricultural production systems to contribute to the environmental spread of antibiotic resistance genes (ARGs). However, there is a need for integrated assessment of critical management points for minimizing this potential. Shotgun metagenomic sequencing data were analysed to comprehensively compare total ARG profiles characteristic of amendments (manure or compost) derived from either beef or dairy cattle (with and without dosing antibiotics according to conventional practice), soil (loamy sand or silty clay loam) and vegetable (lettuce or radish) samples collected across studies carried out at laboratory-, microcosm- and greenhouse-scale. Vegetables carried the greatest diversity of ARGs (n = 838) as well as the most ARG-mobile genetic element co-occurrences (n = 945). Radishes grown in manure- or compost-amended soils harboured a higher relative abundance of total (0.91 and 0.91 ARGs/16S rRNA gene) and clinically relevant ARGs than vegetables from other experimental conditions (average: 0.36 ARGs/16S rRNA gene). Lettuce carried the highest relative abundance of pathogen gene markers among the metagenomes examined. Total ARG relative abundances were highest on vegetables grown in loamy sand receiving antibiotic-treated beef amendments. The findings emphasize that additional barriers, such as post-harvest processes, merit further study to minimize potential exposure to consumers.

AgroSeek: a system for computational analysis of environmental metagenomic data and associated metadata.

BACKGROUND: Metagenomics is gaining attention as a powerful tool for identifying how agricultural management practices influence human and animal health, especially in terms of potential to contribute to the spread of antibiotic resistance. However, the ability to compare the distribution and prevalence of antibiotic resistance genes (ARGs) across multiple studies and environments is currently impossible without a complete re-analysis of published datasets. This challenge must be addressed for metagenomics to realize its potential for helping guide effective policy and practice measures relevant to agricultural ecosystems, for example, identifying critical control points for mitigating the spread of antibiotic resistance. RESULTS: Here we introduce AgroSeek, a centralized web-based system that provides computational tools for analysis and comparison of metagenomic data sets tailored specifically to researchers and other users in the agricultural sector interested in tracking and mitigating the spread of ARGs. AgroSeek draws from rich, user-provided metagenomic data and metadata to facilitate analysis, comparison, and prediction in a user-friendly fashion. Further, AgroSeek draws from publicly-contributed data sets to provide a point of comparison and context for data analysis. To incorporate metadata into our analysis and comparison procedures, we provide flexible metadata templates, including user-customized metadata attributes to facilitate data sharing, while maintaining the metadata in a comparable fashion for the broader user community and to support large-scale comparative and predictive analysis. CONCLUSION: AgroSeek provides an easy-to-use tool for environmental metagenomic analysis and comparison, based on both gene annotations and associated metadata, with this initial demonstration focusing on control of antibiotic resistance in agricultural ecosystems. Agroseek creates a space for metagenomic data sharing and collaboration to assist policy makers, stakeholders, and the public in decision-making. AgroSeek is publicly-available at https://agroseek.cs.vt.edu/ .

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.

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.

Finding What Is Inaccessible: Antimicrobial Resistance Language Use among the One Health Domains.

The success of a One Health approach to combating antimicrobial resistance (AMR) requires effective data sharing across the three One Health domains (human, animal, and environment). To investigate if there are differences in language use across the One Health domains, we examined the peer-reviewed literature using a combination of text data mining and natural language processing techniques on 20,000 open-access articles related to AMR and One Health. Evaluating AMR key term frequency from the European PubMed Collection published between 1990 and 2019 showed distinct AMR language usage within each domain and incongruent language usage across domains, with significant differences in key term usage frequencies when articles were grouped by the One Health sub-specialties (2-way ANOVA; p < 0.001). Over the 29-year period, "antibiotic resistance" and "AR" were used 18 times more than "antimicrobial resistance" and "AMR". The discord of language use across One Health potentially weakens the effectiveness of interdisciplinary research by creating accessibility issues for researchers using search engines. This research was the first to quantify this disparate language use within One Health, which inhibits collaboration and crosstalk between domains. We suggest the following for authors publishing AMR-related research within the One Health context: (1) increase title/abstract searchability by including both antimicrobial and antibiotic resistance related search terms; (2) include "One Health" in the title/abstract; and (3) prioritize open-access publication.

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.

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.