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

Staphylococcus capitis 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 Staphylococcus aureus, 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, S. capitis acquired DAP-R more rapidly and robustly than S. aureus or S. epidermidis under subinhibitory concentrations of either daptomycin or vancomycin. Resequencing after in vitro DAP-R evolution in S. capitis identified newly acquired mutations in cell envelope-associated genes, including walK and mprF. These results indicate that S. capitis NRCS-A rapidly evolves resistance via polygenic, cell-envelope-driven mechanisms distinct from those in S. aureus. The high prevalence and adaptive capacity of DAP-R in neonatal isolates raise concern for therapeutic failure in neonatal intensive care.