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Purpose: To describe the epidemiologic features, risk factors, and antibiotic susceptibilities for gram-negative conjunctivitis among neonatal intensive care unit (NICU) patients., Design: Retrospective, observational study., Methods: In a University Tertiary Care Hospital with a level III-IV NICU, 65 NICU infants (< eight weeks of age) with positive culture results for conjunctivitis between January 1, 2001 and June 1, 2007, were included. Patient demographics, clinical examination results, device usage, and antibiotic susceptibility data were compared between infants with gram-negative conjunctivitis and infants without gram-negative conjunctivitis., Results: One or more episodes of gram-negative conjunctivitis occurred in 38% (n = 25/65) of NICU infants. Predominant pathogens included Klebsiella species (23%), Escherichia coli (17%), Serratia marcescens (17%), Pseudomonas aeruginosa (3%), and Enterobacter species (2%). Birth weight and gestational age were significantly less in infants with gram-negative conjunctivitis than in infants without gram-negative conjunctivitis (P = .008 and P = .008, respectively). With multivariate analysis, birth weight less than 1500 g (odds ratio [OR], 4.35; 95% confidence interval [CI], 1.42 to 13.36), and gestational age of 29 weeks or fewer (OR, 5.60; 95% CI, 1.88 to 16.66) were identified as risk factors for the development of gram-negative conjunctivitis. Antibiotic resistance of gram-negative organisms to ampicillin, cefazolin, gentamicin, and levofloxacin were 96%, 52%, 13%, and 7% respectively. Of the Klebsiella isolates, two (25%) of eight were resistant to gentamicin., Conclusions: Low birth weight and low gestational age in NICU infants with clinical signs of conjunctivitis should raise the suspicion for a gram-negative cause. Given the observed resistance patterns of Klebsiella isolates, gentamicin resistance should be considered when designing empiric treatment.