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We observed an increase in methicillin-susceptible Staphylococcus aureus (MSSA) infections at a Dutch neonatal intensive care unit. Weekly neonatal MSSA carriage surveillance and cross-sectional screenings of health care workers (HCWs) were available for outbreak tracing. Traditional clustering of MSSA isolates by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA) suggested that nosocomial transmission had contributed to the infections. We investigated whether whole-genome sequencing (WGS) of MSSA surveillance w