Microbial adaptation and environmental co-selection drive antimicrobial resistance risk in agricultural ditches, evidence from the multilevel ditch-river continuum.

Channel connectivity reshapes chemical and microbial compositions. About 94% of ditches discharged to natural streams flowing to the ocean, facilitating the downstream transport of antibiotics and antimicrobial resistance (AMR), and thereby, posing potential public health risks. Interconnected collector ditch, branch ditch, and main ditch link to the connect river, forming the multilevel ditch-river continuum (MDRC) system, which intensifies the hydrologic connectivity and further promotes the accumulation of antibiotics and AMR in sediments. However, the composition, distribution, and risk of AMR in the sediments of such an aquatic continuum remain unclear. Here, a total of 48 sediment samples were collected from 13 different MDRC systems in the North China Plain (NCP), a representative agricultural plain with widely constructed ditch systems. By quantifying 50 typical antibiotics and profiling the resistomes, their risk quotient was integrated with antibiotic resistance genes (ARGs) pathogenicity and mobility into a multi-indicator risk framework using random-forest modeling. The result revealed that MDRC sediments serve as an emerging source of AMR, with the collector ditch occupying the highest risk. Importantly, it is the microbial adaption to environmental stress and environmental co-selection drive the AMR risk rather than the antibiotics. Furthermore, bacA, ceoB, and MexE were identified as key ARGs indicators for monitoring the MDRC system. This work conducted the very first multiple evaluation of AMR risk in the various MDRC systems, providing a semi-quantitative and integrated AMR risk index for improving the environmental management practices in the ditch-river aquatic continuum.