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Empiric parenteral ampicillin has traditionally been used to treat listeria and enterococcal serious bacterial infections (SBI) in neonates 28 days of age or younger. Anecdotal experience suggests that these infections are rare. Existing data suggest an increasing resistance to ampicillin. Guidelines advocating the routine use of empiric ampicillin may need to be revisited.This study aimed to describe the epidemiology and ampicillin sensitivity of listeria and enterococcal infections in neonates 28 days of age and younger who presented to 2 pediatric emergency departments (ED) in Michigan.We conducted a 2-center, retrospective chart review (2006–2010) of neonates 28 days of age or younger who were evaluated for SBI in the ED. We abstracted and compared relevant demographic, historical and physical details, laboratory test results, and antibiotic sensitivity patterns to ampicillin from the eligible patient records.We identified SBI in 6% (72/1192) of neonates 28 days of age or younger who were evaluated for SBI, of which 0.08% (1/1192) neonates had enterococcal bacteremia and 0.08% (1/1192) neonates had listeria bacteremia. A total of 1.4% (15/1192) of patients had enterococcal urinary tract infection (UTI). Urinalysis is less helpful as a screening tool for enterococcal UTI when compared with Escherichia coli UTI (P < 0.001). Seventy-three percent (11/15) of urine isolates had an increase of minimal inhibitory concentrations, which indicate gradual development of resistance to ampicillin.Listeria is an uncommon cause of neonatal SBI in febrile neonates who presented to the ED. Empiric use of ampicillin may need to be reconsidered if national data confirm very low listeria and enterococcal prevalence and high ampicillin resistance patterns.