Bacterial and resistome dynamics in reclaimed water and lysimeter-irrigated soil under real-field conditions

Reclaimed water (RW) offers a sustainable solution for agricultural irrigation and freshwater conservation, but its microbial and chemical composition, shaped by treatment and storage processes, requires careful consideration for environmental and public health impacts. This study compared two RW types-conventional activated sludge with sand filtration and chlorination (CAS+SFC-RW) and membrane bioreactor (MBR-RW)-to a tube well water control. The goal was to assess how storage conditions influence the microbial composition, resistome, and mobilome, and their subsequent impact on microbial communities and antibiotic resistance profiles in lysimeter soils irrigated with RW during lettuce cultivation. Total bacterial communities were profiled using 16S rRNA gene sequencing and ddPCR, while antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) were quantified by ddPCR and metagenomics. Initial RW samples had 1-1.5 orders of magnitude more 16S rRNA copies compared to the control, with significant differences in bacterial community structures and ARG/MGE profiles. Storage reduced these differences, leading to convergence toward the tube well water profile. Daily RW irrigation altered soil bacterial composition, with MBR-RW having a greater impact. These results highlight the need for careful management of RW treatment and storage to ensure safe, resilient agricultural practices.