Dissemination of antibiotic resistance genes through soil-plant-earthworm continuum in the food production environment

Treated municipal wastewater (TMW) can provide a reliable source of irrigation water for crops, which is especially important in arid areas where water resources are limited or prone to drought. Nonetheless, TMW may contain residual antibiotics, potentially exposing the crops to these substances. The goal of this study was to investigate the dissemination of antibiotics resistance genes (ARGs) in the soil-plant-earthworm continuum after irrigation of spinach and radish plants with TMW containing trimethoprim, sulfamethoxazole, and sulfapyridine in a greenhouse experiment, followed by feeding of earthworms with harvested plant materials. Our results showed that antibiotic resistance genes (ARGs) were enriched in the soil-plant-earthworm microbiomes irrigated with TMW and TMW spiked with higher concentrations of antibiotics. The number of ARGs and antibiotic-resistant bacteria (ARB) enrichment varied with plant type, with spinach harboring a significantly higher amount of ARGs and ARB compared to radish. Our data showed that bulk and rhizosphere soils of spinach and radish plants irrigated with MilliQ water, TMW, TMW10, or TMW100 had significant differences in bacterial community (p < 0.001), ARG (p < 0.001), and virulence factor gene (VFG) (p < 0.001) diversities. The abundance of ARGs significantly decreased from bulk soil to rhizosphere to phyllosphere and endosphere. Using metagenome assembled genomes (MAGs), we recovered many bacterial MAGs and a near complete genome (>90 %) of bacterial MAG of genus Leclercia adecarboxylata B from the fecal microbiome of earthworm that was fed harvested radish tubers and spinach leaves grown on TMW10 irrigated waters, and this bacterium has been shown to be an emerging pathogen causing infection in immunocompromised patients that may lead to health complications and death. Therefore, crops irrigated with TMW containing residual antibiotics and ARGs may lead to increased incidences of enrichment of ARB in the soil-plant-earthworm continuum.