Directed evolution of phage Romulus in biofilm-embedded Staphylococcus aureus: mutations in baseplate proteins enhanced its antibiofilm activity

The ability of <i>Staphylococcus aureus</i> to form biofilm and the emergence of multidrug-resistant strains make staphylococcal infections often chronic and difficult to treat. To face these challenges, alternative or adjunct strategies to antibiotics are urgently required. In this context, phage therapy gained renewed interest as promising approach to target multidrug-resistant bacteria. To enhance their efficacy as natural phages, they can undergo directed evolution via serial host passages. To date, most protocols focus on planktonic cultures, while the effects towards biofilm-targeted evolution remain poorly explored. Our study aims at investigating the potential of a new directed evolution protocol designed to specifically enhance the efficacy of phage Romulus to target staphylococcal sessile communities and to identify whether specific phage proteins are involved in this process. The method involved 31 serial passages with a two-step incubation: 1 h for phage adsorption and infection, followed by 8 h for its amplification. Mutant phages were isolated, sequenced, and phenotypically characterised. Mutations emerged in two baseplate proteins (gp54 and gp58), involved in host adsorption. Three mutants (R31, R31p2, R31p5) showed enhanced bactericidal activity against planktonic cells and improved efficacy against biofilm, achieving up to a 4-log₁₀ reduction. While their host range remained consistent with the wildtype, phage Romulus mutants exhibited higher efficiency of plating against the nine out of 21 sensitive <i>S. aureus</i> strains. Overall, our results underscore the potential of biofilm-adapted phages to improve phage efficacy towards both planktonic and sessile cells, without impacting on the phage host range. The analysis of mutations suggested that the baseplate plays a crucial role in targeting biofilm-embedded cells, even if further investigation is necessary to explain the molecular basis responsible for the enhanced lytic efficacy.