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

Environmental conditions strongly influence interactions between bacteria and bacteriophages (phages). Here, we examined how osmolality (solute concentration) shapes the in vitro co-evolution of T4 phage and its host Escherichia coli 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.