Osmotic conditions shape fitness gains and resistance mechanisms during <i>E. coli</i> and T4 phage co-evolution

during serial passage. When evolved independently, we observed substantial fitness gains in both bacteria and phages, particularly in high-osmotic conditions. During co-evolution, however, fitness gains were limited, bacterial populations consistently evolved phage resistance, and several phage populations went extinct. Furthermore, the resistance mechanisms varied by osmolality. In lower osmolalities, mutations disrupted phage-binding sites, conferring strong resistance. In higher osmolalities, mutations led to increased colonic acid production, producing a mucoid phenotype with weaker resistance. Because mucoidy has been associated with increased bacterial virulence, these findings suggest that gut-relevant osmotic conditions may constrain evolutionary trajectories, favoring resistance strategies that are less effective against phage but potentially more virulent, with important implications for phage therapy design.Phages offer a promising alternative to antibiotics, but their safety and efficacy strongly depend on the environmental conditions where the bacteria and phages interact. In the human gut, for instance, solute concentrations can vary widely due to factors such as food intolerances or laxative use. In this study, we show that such variations significantly impact how bacteria and phages co-evolve. In particular, we find that in higher osmolalities, bacteria evolve phage resistance through mucoidy-a phenotype linked with increased bacterial virulence-rather than receptor loss. This highlights the need to consider environmental factors when developing phage therapies.