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The purpose of the present paper was to prospectively determine the rate of β-lactam antibiotic resistance in commensal fecal flora of newborns and the risk factors leading to this colonization.One hundred and eighteen newborns in the neonatal intensive care unit (NICU) group (n = 38), the neonatal ward group (n = 36), and the control group (n = 44) were enrolled. Three or four stool samples were obtained from each infant, 15 days apart. Bacterial growth in Eozin Methylene Blue (EMB) agar + 10 μg ampicillin/mL was considered to be ampicillin-resistant bacteria, and antibiotic susceptibility and extended spectrum β-lactamases (ESBL) production was investigated in those bacteria.Colonization with ampicillin-resistant commensal fecal flora microorganisms was determined in 75.2% of 367 stool samples. Klebsiella spp. and Escherichia coli were identified in 59% and 41% of the samples, respectively. The lowest rate of ampicillin-resistant bacterial colonization was determined in the NICU group. Microorganisms producing ESBL were identified in 33.7% of 367 stool samples. Fifty-one and 73 of ampicillin-resistant E. coli and Klebsiella spp. isolates were determined to produce ESBL, respectively. There was no difference with respect to colonization with ESBL-producing microorganisms between the three groups. When risk factors related to colonization with ESBL-producing microorganisms in stool samples were evaluated through the whole study period, very low birthweight, vaginal delivery, infant antibiotic usage, maternal antibiotic usage, male sex and premature rupture of membranes were determined as risk factors, while feeding with nasogastric tube was identified as a protective factor. When the risk factors related to colonization with ESBL-producing bacteria in stool samples after discharge from the hospital were evaluated, failure to feed breast milk was determined as the only risk factor.To decrease mortality and morbidity due to infection caused by resistant microorganisms colonized in the intestine flora of the infant, protection of normal non-pathogenic bacterial colonization is important. This can be provided by feeding neonates with breast milk, application of infection control measures efficiently, and limitation of antibiotic usage only to strict clinical indications.