Assessing variations and spatial patterns of antibiotic resistance genes and water quality in irrigation pond water

Irrigation waters can act as reservoirs and transmission pathways for antimicrobial resistant (AMR) bacteria, contributing to the broader global AMR crisis. The current extent of AMR in irrigation waters is poorly understood. This is especially true for small waters such as farm ponds. The objectives of this work were to determine if stable spatial patterns in the concentration data exist that can be used to inform monitoring design. Water sampling was conducted on 9 dates from June to September 2023 at 20 locations within an irrigation pond in Maryland, USA. The antibiotic resistant genes (ARGs) tetA and bla_CTX-M were enumerated using digital PCR (dPCR) in all collected samples. Spatial variation of tetA concentrations was time-dependent with coefficients of variation ranging from 97 to 377%. Average concentrations steadily declined throughout the observation period. Rainfall events did not result in higher concentrations of tetA. Stable spatial patterns in the concentration data were detected using the mean relative difference analysis. Samples collected at the banks contained higher tetA concentrations than those collected in the interior. Elevated ARG concentrations at bank sites were attributed to on-land activities as well as hydrological conditions within the water body. Sampling sites were identified which best represented the spatiotemporal average of the concentrations. Spatial patterns of tetA were negatively correlated with those of water temperature, electrical conductance, and pH, while correlations for all other measurements were positive. This work is the first to evaluate fine-scale spatial variation of ARGs in lentic waters used for irrigation. The results show that the choice of where to sample for AMR enumeration in ponds or lakes should not be made arbitrarily.