Chiral antibiotics amplify the phage-associated antibiotic resistome in salinized soil

Anthropogenic global change is intensifying antibiotic pollution and soil salinization, yet how salinity shapes bacteria-phage interactions to drive the dissemination of antibiotic resistance genes (ARGs) under chiral antibiotic pressure remains poorly understood. Using viral metagenomic analyses and laboratory validation, we show that salinity amplifies the enrichment of total and high-risk ARGs along gradients of racemic ofloxacin and its S-enantiomer levofloxacin, concomitant with the parallel accumulation of mobile genetic elements and virulence factors. High-concentration antibiotic treatment in saline soils selected for stress-tolerant, fast-growing antibiotic-resistant bacteria (ARBs) carrying abundant prophages and diverse defense systems, thereby promoting tight ARB-phage interactions. Under these dual extreme stresses, lysogenic phages were enriched, enhancing their carriage of ARGs, auxiliary metabolic genes (AMGs) that bolster host survival, and antidefense genes via a "piggyback-the-winner" strategy, thereby increasing ARG dissemination potential. Notably, high-concentration levofloxacin exerted stronger selective pressure than ofloxacin, increasing virus-to-host ratios and phage-associated ARG accumulation, as supported by incubation assays. Collectively, our findings support a mechanistic framework in which soil salinization interacts with chiral antibiotic exposure, particularly levofloxacin, to increase phage-associated ARG dissemination potential. This highlights a long-underappreciated, Anthropocene-driven pathway intensifying environmental resistance risks and posing new challenges for the One Health framework.