In Vivo Emergence of Podovirus Resistance via tarS Mutation During Phage-Antibiotic Treatment of Experimental MSSA Endocarditis

Phage therapy shows promise as an adjunct to antibiotics for treating Staphylococcus aureus infections. We previously reported a combined flucloxacillin/two-phage cocktail treatment selected for resistance to podovirus phage 66 in a rodent model of methicillin-susceptible <i>S. aureus</i> (MSSA) endocarditis. Here we show that resistant clones harbor mutations in <i>tarS</i>, which encodes a glycosyltransferase essential for β-GlcNAcylation of wall teichoic acid (WTA). This WTA modification has been described in vitro as critical for <i>podoviruses</i> adsorption. Transcriptomics confirmed continued <i>tarS</i> expression in resistant clones, supporting a loss-of-function mechanism. Accordingly, phage 66 binding and killing were restored by WT <i>tarS</i> complementation. In addition, we investigated the counterintuitive innate susceptibility to phage 66 of the <i>tarM</i> + Laus102 strain used in the endocarditis model. We show that it likely results from a significant lower <i>tarM</i> expression, in contrast to the innate resistant strain RN4220. Our findings demonstrate that <i>tarS</i>-mediated WTA β-GlcNAcylation is critical for podovirus infection also in vivo and identify <i>tarM</i> transcriptional defect as a new mechanism of podoviruses susceptibility in <i>S. aureus</i>. Moreover, and since <i>tarS</i> disruption has been previously shown to enhance β-lactam susceptibility, our results support the development of combined podovirus/antibiotic strategies for the management of MRSA infections.