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Colonization of infants with methicillin-resistant Staphylococcus aureus (MRSA) carries specific toxin genes. In particular, Panton–Valentine leukocidin (PVL) are a risk factor for subsequent infection during hospitalization. This prospective study investigated important epidemiological characteristics of Staphylococcus aureus colonizing the nares and intestines of Jordanian infants.A total of 860 nasal and stool specimens were obtained from each of the 430 infants admitted to the neonatal intensive care unit or referred to outpatient clinics of Jordan University Hospital. All specimens were cultured to recover S. aureus, all isolates were tested for antimicrobial susceptibility and the MRSA strains for presence of specific toxin genes and SCCmec using polymerase chain reaction.Eighty of the 430 (18.6%) infants were colonized with S. aureus, of these, 27 (6.3%) harbored the organism in both the nose and intestine. The frequency of S. aureus nasal and intestinal carriage in outpatient infants compared to inpatients admitted to the neonatal intensive care unit was significantly higher (27.3% vs 2.8%) and (17.1% vs 2.3%), respectively. MRSA accounted for 57/107 (53.3%) of all isolates, and of these 16/57 (28%) were PVL-positive and carried SCCmec type IV, except one, which was type III. All nasal and intestinal MRSA carried at least one toxin gene (tst, eta, seb), but few carried two toxin genes.This study demonstrates that S. aureus strains are more frequently colonizing Jordanian outpatient infants than inpatients and all MRSA strains carried 1–3 clinically important staphylococcal toxin genes. Further studies are needed to investigate the role of these toxins in hospitalized infants.