Alterations in the Ser/Thr phosphatase StpA confer van-independent reduced susceptibility to dalbavancin in Enterococcus faecium

Dalbavancin is a long-acting lipoglycopeptide increasingly used against difficult-to-treat Gram-positive infections, including enterococcal infections. However, mechanisms conferring reduced susceptibility in Enterococcus faecium remain uncharacterized. We selected in vitro spontaneous mutants with decreased susceptibility to dalbavancin in two distinct vancomycin-susceptible E. faecium backgrounds and identified independent missense mutations in the flap domain of the Ser/Thr phosphatase StpA. Deletion of stpA increased dalbavancin MICs, and complementation with the wild-type allele restored susceptibility, linking StpA to the reduced-susceptibility phenotype. Mutants displayed reduced cell size, thicker cell wall enriched in 3-3 cross-links, concentration-dependent reduction of dalbavancin binding, elevated levels of MurA and other envelope-biosynthesis proteins, and global hyperphosphorylation of several cell-division regulators. Despite only a modest increase in MIC, dalbavancin’s in vivo efficacy was abolished in a Galleria mellonella model. These findings suggest that StpA-dependent envelope remodeling represents an original pathway for reducing susceptibility to dalbavancin in E. faecium independent of canonical van -mediated resistance.