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Purpose: Little is known about the molecular epidemiology of Staphylococcus aureus in Chinese neonatal intensive care units (NICUs). We describe the molecular epidemiology of S. aureus isolated from neonates on admission to Beijing Children's Hospital. Methods: From May 2015-March 2016, nasal swabs were obtained on admission from 536 neonates. Cultures were also obtained from body sites with suspected infections. S. aureus isolates were characterized by staphylococcal chromosomal cassette (SCCmec) type, staphylococcal protein A (spa) type, multilocus sequence type (MLST), sasX gene, antimicrobial susceptibility and cytotoxicity. Logistic regression assessed risk factors for colonization. Results: Overall, 92 (17%) infants were colonized with S. aureus and 20 (3.7%) were diagnosed with culture-positive S. aureus infection. Of the colonized infants, 70% (64/92) harbored methicillin-susceptible S. aureus (MSSA), 30% (28/92) harbored methicillin-resistant S. aureus (MRSA) while 70% (14/20) of infected infants were culture-positive for MRSA, 30% (6/20) were culture-positive for MSSA. Risk factors for colonization included female sex, age 7–28 days, higher birthweight (3270 IQR [2020–3655] grams) and vaginal delivery (p<0.05). The most common MRSA and MSSA clones were community-associated ST59-SCCmecIVa-t437 (60%) and ST188-t189 (15%), respectively. The sasX gene was not detected. Some MSSA isolates (16%) were penicillin-susceptible and some MRSA isolates (18%) were oxacillin-susceptible. MRSA and MSSA had similar cytotoxicity, but colonizing strains were less cytotoxic than strains associated with infections. Conclusions: S. aureus colonization was common in infants admitted to our NICU and two community-associated clones predominated. Several non-modifiable risk factors for S. aureus colonization were identified. These results suggest that screening infants for S. aureus upon admission and targeting decolonization of high-risk infants and/or those colonized with high-risk clones could be useful to prevent transmission. [ABSTRACT FROM AUTHOR]