Unde venis? Bacterial resistance from environmental reservoirs to lettuce: tracking microbiome and resistome over a growth period

Fresh produce is suggested to contribute highly to shaping the gut resistome. We investigated the impact of pig manure and irrigation water quality on microbiome and resistome of field-grown lettuce over an entire growth period. Lettuce was grown under four regimes, combining soil amendment with manure (with/without) with sprinkler irrigation using river water with an upstream wastewater input, disinfected by UV (with/without). Lettuce leaves, soil, and water samples were collected weekly and analysed by bacterial cultivation, 16S rRNA gene amplicon sequencing, and shotgun metagenomics from total community DNA. Cultivation yielded only few clinically relevant antibiotic-resistant bacteria (ARB), but numbers of ARB on lettuce increased over time, while no treatment-dependent changes were observed. Microbiome analysis confirmed a temporal trend. Antibiotic resistance genes (ARGs) unique to lettuce and water included multidrug and β-lactam ARGs, whereas lettuce and soil uniquely shared mainly glycopeptide and tetracycline ARGs. Surface water carried clinically relevant ARB (e.g. ESBL-producing Escherichia coli or Serratia fonticola) without affecting the overall lettuce resistome significantly. Resistance markers including biocide and metal resistance were increased in lettuce grown with manure, especially young lettuce (increased soil contact). Overall, while all investigated environments had their share as sources of the lettuce resistome, manure was the main source especially on young plants. We therefore suggest minimizing soil-vegetable contact to minimize resistance markers on fresh produce.