Phage Intolerance Impacts Antibiotic Susceptibility and Virulence in Staphylococcus aureus

Phage therapy is a promising alternative to the growing problem of antibiotic-resistance. However, bacterial phage-resistance may develop, compromising therapy. Phage-resistance has primarily been associated with changes in phage receptors that in the human pathogen, Staphylococcus aureus, are the cell-wall linked wall teichoic acids (WTA) which can be modified by glycosylation. With the aim of identifying factors contributing to phage-resistance, we exposed S. aureus to lytic K-type myo-viruses, namely phage K, ϕIPLA-RODI and Stab21 to obtain resistant clones. Out of 9 phage-resistant mutants, a third harbored mutations in cell-wall genes previously linked to phage resistance, namely in femA, involved in peptidoglycan crossbridge formation, and tagO, encoding the initiator of the WTA biosynthesis. The remaining mutants had mutations in pathways not previously associated with phage-resistance, with three in deoC1 involved in nucleoside catabolism, two in potA and potB, respectively, involved in polyamine import and one in the RNA helicase, cshA. When assessing virulence in Galleria mellonella and antibiotic susceptibility as well as WTA glycosylation, our results showed diverse effects. As expected, mutations in the wall teichoic acid synthesis pathway increased β-lactam sensitivity and attenuated virulence in a G. mellonella model. In contrast, the cshA mutation increased both virulence and susceptibility to β-lactams. Increased virulence was also seen for a mutant with several mutations including femA. Further phage susceptibility appeared not to be strictly correlated with WTA glycosylation patterns. Our findings show that in S. aureus reduced phage susceptibility can be caused by mutations affecting central metabolic processes and can have unpredictable consequences for antibiotic susceptibility and virulence. Our results emphasize the need for evaluating evolutionary trade-offs before clinical phage therapy deployment.