Polygenic, cell-envelope adaptations drive high-frequency daptomycin resistance in <i>Staphylococcus capitis</i> NRCS-A from neonatal sepsis and NEC

<i>Staphylococcus capitis</i> NRCS-A is a major cause of neonatal sepsis worldwide and exhibits resistance to multiple antibiotics. We assessed the prevalence and mechanisms of daptomycin resistance (DAP-R) in bloodstream isolates from a German neonatal intensive care unit. Ten of 11 NRCS-A isolates (91%) were resistant to daptomycin, and 18% displayed decreased susceptibility to vancomycin. Genomic analysis revealed diverse polymorphisms in genes associated with DAP-R in <i>Staphylococcus aureus,</i> but no single amino acid variant explained resistance. Phospholipid composition remained unchanged, whereas isolates displayed increased cell surface charge and cell wall thickening. Consistently, BODIPY-labeled daptomycin binding was reduced and more diffusely distributed in DAP-R NRCS-A with weaker septal enrichment. In serial passaging experiments, <i>S. capitis</i> acquired DAP-R more rapidly and robustly than <i>S. aureus</i> or <i>S. epidermidis</i> under subinhibitory concentrations of either daptomycin or vancomycin. Resequencing after <i>in vitro</i> DAP-R evolution in <i>S. capitis</i> identified newly acquired mutations in cell envelope-associated genes, including <i>walK</i> and <i>mprF</i>. These results indicate that <i>S. capitis</i> NRCS-A rapidly evolves resistance via polygenic, cell-envelope-driven mechanisms distinct from those in <i>S. aureus</i>. The high prevalence and adaptive capacity of DAP-R in neonatal isolates raise concern for therapeutic failure in neonatal intensive care.