Dual Sub‑MIC Copper-Gentamicin Stress Drives Strain‑Specific, Non‑Additive Phenotypic Shifts in Pseudomonas...

Exposure to redox‑active metals and sub‑inhibitory antibiotics represents a significant selective pressure shaping bacterial physiology and antimicrobial susceptibility. Here, we investigated how four genetically and phenotypically distinct Pseudomonas aeruginosa strains respond to sub‑MIC copper (Cu) and gentamicin (GE) stress, individually and in combination (Cu + GE). Across all strains, Cu acted as the dominant envelope‑active stressor, increasing biofilm biomass and extracellular DNA (eDNA) release, suppressing twitching and swarming motility, reducing quorum‑sensing‑linked protease activity, and enhancing pyomelanin production. GE alone produced limited physiological changes but modulated Cu‑driven outputs during co‑exposure, including attenuation of Cu‑induced eDNA release and strain‑specific shifts in motility and pigmentation. Early transcriptional profiling revealed consistent Cu‑dependent repression of lasI and mvfR, induction of the metal‑responsive regulator czcR, and downregulation of oprD under Cu or Cu + GE, corresponding to reduced imipenem inhibition zones when Cu was present during susceptibility testing. Combined Cu + GE exposure produced non‑additive, emergent effects that diverged from single‑stressor responses and varied across strains. These findings demonstrate that sub‑inhibitory Cu creates an envelope‑centered regulatory landscape into which gentamicin‑derived signals are integrated, generating heterogeneous and context‑dependent phenotypes. This work underscores the importance of metal-antibiotic interactions in shaping bacterial adaptation and highlights limitations of single‑stressor models for predicting antimicrobial behavior in combination.