Evolutionary interplay between Staphylococcus aureus and phages: strategies to target biofilm and overcome resistance

Multidrug resistance made conventional antibiotic treatments ineffective against critical pathogens such as Staphylococcus aureus, and therefore, alternative approaches are needed. Among them, phage therapy represents a promising one. The present thesis examines the therapeutic applicability of bacteriophages, focusing on important challenges: characterisation of novel phages, evading bacterial resistance, interactions with biofilms, and the role of human serum regarding phage effectiveness. The novel lytic phage Zeno infects 95% of the tested S. aureus strains, including multidrug-resistant isolates, and has been shown to be stable over a wide range of pH and temperature (pH 4-11; 37-60°C). Directed evolution resulted in increased lytic activity of Zeno but at the expense of narrower host range specificity. The current work also demonstrated how human serum in vitro inhibited phage activity due to lipid components interfering with the bacterial cell wall. This preliminary study highlights how serum can affect phage therapy efficacy, suggesting the need for further investigation through in vivo studies. Moreover, a directed evolution protocol to improve phage antibiofilm activity was designed, and an enhanced activity of mutated phages was achieved, demonstrating how baseplate proteins were responsible for the improved efficacy. Finally, the novel temperate phage Hesat was isolated, and pathogenicity islands and virulence factors were identified. The role of temperate phages is of great importance because they may interfere with therapeutic phages due to mechanisms such as superinfection exclusion, where the lysogenised bacteria become resistant to other phage infections.