Groundwater as an overlooked hotspot of antibiotic resistance in hydrologically connected agricultural catchments

The intensifying use of antibiotics in agriculture is accelerating the dissemination of antibiotic resistance genes (ARGs) in aquatic environments, posing an increasing threat to global water security. Despite their intrinsic link, surface water and groundwater are frequently studied as isolated compartments, overlooking the role of hydrological connectivity and the combined influence of agricultural pressures on ARG transport and risks. 

This study investigated the antibiotic resistome within a hydrologically connected surface-groundwater system in an agricultural catchment characterized by intensive crop cultivation, livestock farming, and aquaculture. Using metagenomic sequencing and resistance risk assessment, we reveal that groundwater, generally assumed to be protected by natural filtration, is actually a critical yet overlooked hotspot of antibiotic resistance.

Groundwater exhibited approximately twofold higher ARG abundance, diversity, and resistance risk than surface water, dominated by multidrug resistance genes. While surface water resistome was more homogenized by hydrodynamic mixing, groundwater ARG profiles were strongly shaped by hydrogeological conditions and agricultural activity intensity. Livestock-impacted units showed the highest ARG loads and resistance risks in groundwater, reflecting intensive antibiotic usage and manure-derived inputs. Notably, aquaculture impacts were strongly influenced by hydrogeological conditions, with significantly higher ARG abundance and risks observed in high-permeability sandy aquifers compared to clay-dominated settings, likely reflecting rapid vertical infiltration and limited adsorption during subsurface transport.

Our findings identify groundwater as a major reservoir of antibiotic resistance in agricultural regions, emphasizing the urgent need to integrate groundwater into resistance monitoring frameworks and strengthen manure and aquaculture waste management to protect water security.