Directed evolution of a staphylophage under biofilm and planktonic conditions.

The rise of multidrug-resistant bacteria, particularly biofilm-forming pathogens such as Staphylococcus epidermidis, highlights the urgent need for alternative antimicrobial strategies. Phage therapy, which uses phages to selectively infect and lyse bacterial cells, offers a promising solution. In this study, we evolved the lytic phage vB_Sep_Steph1 under both biofilm and planktonic conditions, using varying initial phage inoculum titers. Whole-genome sequencing of evolved populations revealed recurrent condition-dependent mutations in holins and structural genes with putative depolymerase activity-critical for host recognition and biofilm degradation. Phenotypic improvements in traits such as antibacterial efficacy and replicative fitness were observed to be highly dependent on both the presence of biofilm and the initial phage titer during evolution. Furthermore, some evolved phage lineages could delay bacterial resistance better than the ancestral strain. These findings support the utility of directed phage evolution to improve therapeutic efficacy and robustness, particularly against biofilm-associated infections.