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Most very low birth weight (VLBW, birth weight <1500 g) infants receive empiric antibiotics for risk of early-onset sepsis (EOS). The objective of this study was to determine the characteristics of VLBW infants with culture-confirmed EOS at a single center during 25 years and to identify opportunities for antibiotic stewardship.Retrospective cohort study includes VLBW infants admitted from 1990 to 2015. EOS was defined as isolation of a pathogen in blood or cerebrospinal fluid culture obtained at <72 hours of age. Clinical and microbiologic characteristics of EOS case infants were obtained by review of medical, laboratory and administrative records. Blood culture, antibiotic initiation and maternal discharge code data were available for all VLBW infants born between 1999 and 2013.One-hundred nine EOS cases (20.5/1000 VLBW births) occurred during the study period. Preterm labor, preterm rupture of membranes and/or the obstetrical diagnosis of chorioamnionitis were present in 106/109 cases (97%). Obligate anaerobic organisms accounted for 16% of cases. Time to culture positivity was 36 hours for 88% and 48 hours for 98% of cases. From 1999 to 2013, 97% of VLBW infants were evaluated for EOS and 90% administered empiric antibiotics; 22% of these infants were born by cesarean section to mothers with preeclampsia and without preterm labor or chorioamnionitis and had a 12-fold lower incidence of EOS compared with the remaining infants.Decisions to initiate and discontinue empiric antibiotics among VLBW infants can be informed by the delivery characteristics of infected infants and by local microbiologic data.