The Efflux–Two-Component System (TCS)–Virulence Axis Drives Resistance–Virulence Convergence in Aquatic “Superhost Precursors” under Pollution Stress

The coexpression of antibiotic resistance and virulence traits in aquatic bacteria represents an emerging ecological and public health threat, yet the mechanisms underlying their coordinated regulation under complex environmental pressures remain unclear. In this work, we integrated metagenomic, proteomic, and metabolomic data sets from surface water samples across the Yangtze River Basin in Jiangsu Province to elucidate the drivers of resistance-virulence convergence under multipollutant stress. Among 392 multidrug-resistant (MDR; resistant to ≥3 antibiotic classes) isolates, approximately 5% were identified as "culturable superhost precursors" exhibiting pandrug-resistant (PDR; resistant to ≥10 antibiotic classes) phenotypes. Multiomics analyses indicated frequent colocalization and synchronous activation of antibiotic resistance genes (ARGs) and virulence factors (VFs) in these environmental reservoirs. Functional assays under subinhibitory antibiotic exposure demonstrated enhanced cytotoxicity and efflux activity, accompanied by the upregulation of <i>tolC</i> and two-component regulators <i>evgA/evgS</i>. Together, these results characterize a putative redox-coupled efflux-two-component system (TCS)-virulence functional axis that synchronizes adaptive gene expression under pollution stress. Crucially, our findings challenge traditional antimicrobial resistance (AMR) surveillance approaches, which rely primarily on static gene abundance metrics, by demonstrating that the dynamic regulatory activation of this axis provides a more sensitive indicator of environmental health risks. Furthermore, <i>tolC</i> and <i>evgA</i> were identified as potential transcript-level biomarkers, providing a proof of concept for environmental antimicrobial resistance early warning tools within the One Health framework.