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

Bacteriophages, or phages, are viruses that infect bacteria and offer a promising approach to combating antimicrobial resistance in the gut by selectively targeting harmful bacteria while preserving the broader microbiota. This study examines how variations in osmolality (solute concentration), which can occur in the gut due to factors such as food intolerances or laxative use, affect the in vitro co-evolution of T4 phage and its host Escherichia coli. When evolved independently, we observed substantial fitness gains in both bacteria (growth rate) and phages (productivity), especially at higher osmolalities. During co-evolution, phage resistance emerged in all co-evolved bacterial populations. However, the resistance mechanism varied by osmolality: in lower osmolalities, mutations disrupted phage binding sites, conferring strong resistance to phages. Conversely, in higher osmolalities mutations led to increased colonic acid production, resulting in a mucoid phenotype associated with weaker phage resistance. This bacterial phenotype has been linked with increased bacterial virulence, underscoring the need to consider environmental factors when designing phage therapies. SignificancePhage therapy, which uses viruses to target and kill harmful bacteria, is a promising alternative to antibiotics, particularly in the fight against antibiotic-resistant infections. However, the success of phage therapy depends on how well phages can adapt to different environments, such as those found in the human gut, where factors such as solute concentration (osmolality) fluctuate rapidly. In this study, we demonstrate that shifts in osmolality can dramatically affect the evolution of phage resistance and phage counter-adaptation. In particular, elevated osmolality induced the emergence of mucoid bacterial phenotypes that increased resistance to phages and may be associated with increased bacterial virulence. Our findings highlight the need to consider environmental factors when designing phage therapies to ensure they remain effective in diverse conditions, including within the human body.