Trained phages constrain evolutionary routes to antibiotic resistance in Escherichia coli

Bacteriophages are major drivers of bacterial evolution and can impose strong selection on traits that mediate resistance and fitness. This creates the possibility of using phage to steer microbial adaptation toward predictable phenotypic outcomes. Yet bacterial populations often evolve phage resistance through diverse genetic routes, not all of which impose meaningful costs or trade-offs. Such evolutionary flexibility can allow bacteria to escape phage selection without acquiring the desired phenotype, limiting the reliability of phage-based evolutionary steering. Here, we develop a directed phage evolution strategy that eliminates cost-free resistance pathways, forcing bacteria into evolutionary trajectories that restore antibiotic susceptibility. We isolated two novel Microviridae phages (BLS2 and BLS5) that infect E. coli BL21 and found that bacteria evolved resistance through two distinct mechanisms: mutations in lipopolysaccharide (LPS) biosynthesis genes, which carried substantial fitness costs and conferred sensitivity to erythromycin, and loss-of-function mutations in yajC , which imposed no fitness penalty and maintained full antibiotic resistance. We show that YajC is a previously uncharacterised accessory receptor required for phage genome translocation across the inner membrane. We then used this mechanistic understanding to design a phage training regime that evolved phages capable of infecting yajC mutants, thereby closing this cost-free escape route. Bacteria challenged with trained phages were constrained to evolve LPS-based resistance, and were consequently resensitised to erythromycin. Our results demonstrate that directed phage evolution can reshape the fitness landscape of bacterial resistance, channelling adaptation along trajectories that carry predictable phenotypic costs – with broad implications for phage-mediated manipulation of microbial populations.