Abstract
Understanding all sources of selective pressure that contribute to the emergence of antibiotic resistance is essential for developing sustainable antimicrobial strategies. Here, we investigated the interaction between phage T4 and Escherichia coli MG1655 to determine whether mutations conferring phage resistance also shape the genetic background for β-lactam resistance. Using experimental evolution, whole-genome sequencing, and targeted genetic reconstructions, we identified mutations in porins and lipopolysaccharide (LPS) biosynthesis as the predominant routes to phage T4 resistance. Precise allelic replacements and isogenic strain comparisons demonstrated that these mutations not only protect against phage predation but also create a genetic context that facilitates the emergence of β-lactam resistance, including resistance to carbapenems. Together, these findings provide compelling evidence that phage-driven selection can establish bacterial genetic backgrounds predisposed to antibiotic resistance. This work highlights the evolutionary risks associated with phage therapy and underscores the need to account for genetic trade-offs when developing alternative antimicrobial strategies.