Prophage ϕSA169 Enhances Vancomycin Persistence in Methicillin-Resistant Staphylococcus aureus (MRSA)

<b>Background:</b> Persistent methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) endovascular infections present a significant clinical therapeutic challenge. Prophages are increasingly recognized as important genetic factors influencing the pathogenicity of <i>S. aureus</i>, yet their role in antibiotic persistence in MRSA remains underexplored. Our previous work demonstrated that prophage ϕSA169 promotes vancomycin (VAN) persistence in an experimental model of endocarditis caused by MRSA strains with a clonal complex (CC) 45 genetic background. However, it is unknown whether this persistence-promoting effect of ϕSA169 extends to other clinically relevant MRSA lineages. This study aims to elucidate the role of ϕSA169 in influencing VAN persistence across diverse MRSA genetic backgrounds. <b>Methods:</b> A pilot analysis of clinical data suggested that patients infected by MRSA containing ϕSA169-like prophage appear to have worse clinical outcomes. Thus, we lysogenized representative clinical resolving bacteremia (RB) MRSA strains with ϕSA169 and evaluated phenotypes closely associated with VAN persistence, including VAN susceptibility, biofilm formation, and the efficacy of VAN treatment in an experimental infective endocarditis (IE) model. Each ϕSA169 lysogenic strain was compared to its isogenic MRSA parental counterpart. <b>Results:</b> ϕSA169 lysogeny significantly promotes biofilm formation and enhances survival to VAN exposure under human-mimicking conditions for RB strains from CC5 and CC30. ϕSA169 lysogeny significantly reduces VAN effectiveness in the IE model due to RB lysogen from CC5 despite no detectable impact on VAN MICs. <b>Conclusions:</b> These results indicate that ϕSA169 promotes VAN persistence across clonal backgrounds, likely through biofilm formation and VAN tolerance. Targeting prophage could provide new strategies to combat persistent MRSA infections.