Dissolved Fe(III) defines a stabilizing window for tetracycline resistance evolution under environmentally relevant...

Dissolved iron and sub-inhibitory tetracycline frequently coexist in natural waters and biological treatment units of wastewater treatment plants, but how iron availability regulates tetracycline resistance evolution under environmentally relevant conditions remains poorly understood. Here, we investigated resistance evolution in Escherichia coli MG1655 under co-exposure to dissolved Fe(III) and tetracycline using long-term serial passaging combined with short-term physiological assays. Resistance evolution showed a clear non-monotonic response to iron concentration, decreasing at low Fe levels and increasing again under iron overload. A micromolar Fe interval (1-4 μM) was identified as a stabilizing window in which resistance evolution was minimized. To explain this pattern, we established an exposure-damage-homeostatic cost (E-D-C) framework integrating antibiotic exposure chemistry, cellular stress responses, and metabolic burden. A descriptive E-D-C attribution analysis suggested a damage-dominant pattern, with cellular damage showing the largest positive contribution to resistance evolution (β_D = 0.9276, P = 0.0015), followed by effective antibiotic exposure (β_E = 0.4609, P = 0.0288), whereas homeostatic cost exerted a weaker constraining effect (β_C = -0.3052, P = 0.0628). Under iron limitation, resistance evolution was primarily driven by elevated intracellular tetracycline exposure and iron acquisition burden. Under iron overload, it was dominated by oxidative and membrane damage despite reduced intracellular antibiotic levels. Molecular analyses revealed regime-specific resistance strategies, with efflux associated genes enriched under iron limitation and ribosomal protection or antibiotic inactivation mechanisms prevailing under iron overload. Coagulation simulations further showed that residual dissolved Fe under conventional treatment conditions overlapped with the stabilizing window, highlighting the environmental relevance of dissolved iron in shaping tetracycline resistance selection in aquatic treatment systems.