Abstract
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.
Highlights
Antibiotic resistance is recognized as a critical threat to human health, food security, and global development (O’Neill, 2016)
The use of antibiotics as growth promoters on EU farms was banned in 2006 (European Commission, 2005) and more recently (2017) on United States farms as part of the Veterinary Feed Directive, which requires veterinary authorization and oversight, rather than simple over-the-counter distribution, for the use of antimicrobials in livestock (FDA, 2013, 2017, 2018). Despite this progress in antibiotic stewardship, it is worth noting that antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are still regularly recovered from EU and United States livestock herds and the United States and some EU countries remain the top users of antibiotics for livestock (Aarestrup et al, 2001; Van Boeckel et al, 2015; Elliott et al, 2017)
Of the 854 unique ARGs annotated across all metagenomes, 302 were detected in all three sample types (Figure 2A) and a total of 121, 55, and 114 ARGs were unique to the amendment, soil, and lettuce samples, respectively (Shannon diversity, p < 0.0001)
Summary
Antibiotic resistance is recognized as a critical threat to human health, food security, and global development (O’Neill, 2016). The use of antibiotics as growth promoters on EU farms was banned in 2006 (European Commission, 2005) and more recently (2017) on United States farms as part of the Veterinary Feed Directive (final rule #213), which requires veterinary authorization and oversight, rather than simple over-the-counter distribution, for the use of antimicrobials in livestock (FDA, 2013, 2017, 2018). Despite this progress in antibiotic stewardship, it is worth noting that antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are still regularly recovered from EU and United States livestock herds and the United States and some EU countries remain the top users of antibiotics for livestock (Aarestrup et al, 2001; Van Boeckel et al, 2015; Elliott et al, 2017). Knowledge is lacking with respect to how use of livestock manure-derived soil amendments in vegetable production systems could influence carriage of antibiotic resistance in the food-chain
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