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

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.

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

“…The greenhouse scale of the present study afforded examination of a wider array of factors, while comparing the portion of the experiment that was analogous to the field study afforded some degree of validation. Overall, the similarity of trends relative to those observed in the Wind et al (2021) study was striking. Notably, both studies reported relative abundance of total ARGs increasing from amendment to soil to both lettuce and radish when manure-derived amendments were applied.…”

“…Overall, the similarity of trends relative to those observed in the Wind et al . (2021) study was striking. Notably, both studies reported relative abundance of total ARGs increasing from amendment to soil to both lettuce and radish when manure‐derived amendments were applied.…”

Potential of Meta-Omics to Provide Modern Microbial Indicators for Monitoring Soil Quality and Securing Food Production

“…Metagenomics outcompetes metabarcoding in that it provides insights into the metabolic, virulence or resistance potential of microbial communities. A good example is the study of Wind and colleagues ( Wind et al, 2021 ) focused on antibiotic resistance genes (ARG) across a vegetable production system. The study was interested on three pre-harvest “control-points,” i.e., manure and compost amendments, soils, and lettuce samples, to track antibiotic resistance and the associated microbiomes in order to identify the potential impacts of key agricultural management strategies.…”

Pseudomonas aeruginosa Detection Using Conventional PCR and Quantitative Real-Time PCR Based on Species-Specific Novel Gene Targets Identified by Pangenome Analysis

“…DNA fingerprinting and 16S DNA-based analyses were used to identify the harm of plant derived P. aeruginosa to humans and animals, which is complex and requires very professional inspectors ( Ambreetha et al, 2021 ). Biosensor method and 16r RNA gene amplicon sequencing, which had high detection efficiency, were used to analyze P. aeruginosa of food microorganisms, but these methods need complex pretreatment ( Zhong et al, 2020 ; Wind et al, 2021 ). Illumina whole gene sequencing has great advantages in accuracy, was used to analyze the distribution of P. aeruginosa after pasteurized milk, but it takes a lot of testing costs ( Maske et al, 2021 ).…”