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This study was undertaken to determine whether a change in the frequency or distribution of the causative pathogens in persistent acute otitis media (AOM) and AOM treatment failure (AOMTF) occurred after publication of the Centers for Disease Control and Prevention AOM treatment guidelines advocating high dose amoxicillin in 1998 and the universal use of the pneumococcal conjugate vaccine in 2000.This was a 9-year prospective study in a suburban, community-based private practice. To identify bacterial isolate(s), 551 children with AOM who had not responded after 1 or 2 empiric antimicrobial treatment courses (termed persistent AOM) and those who were failures after 48 h on treatment (AOMTF) underwent tympanocentesis. Three periods were compared: (1) 1995–1997 when all enrolled received standard dose amoxicillin (40–50 mg/kg/day divided 3 times daily) as the initial empiric treatment; (2) 1998–2000 when all received high dose amoxicillin (80–100 mg/kg/day divided twice daily); and (3) 2001–2003 when high dose amoxicillin and pneumococcal conjugate vaccinations were used.Persistent AOM or AOMTF for which tympanocentesis was performed occurred in 195 (16.2%) of 1207, 204 (16.1%) of 1278 and 152 (12.3%) of 1232 AOM visits for 1995–1997, 1998–2000 and 2001–2003, respectively; the 24% decline in 2001–2003 in persistent AOM and AOMTF was significant (P = 0.007). Middle ear aspirates grew Streptococcus pneumoniae (48, 44 and 31%) and Haemophilus influenzae (38, 43 and 57%) for time periods 1, 2 and 3, respectively. There was a significant decline in S. pneumoniae (P = 0.017) and increase in H. influenzae (P = 0.012) isolations and of H. influenzae that were β-lactamase-producing (P = 0.04) among middle ear fluid isolates. Also there was a trend for an increased proportion of S. pneumoniae in 2001–2003 that were penicillin-susceptible (P = 0.17).In our experience, persistent AOM and AOMTF has decreased in frequency since the introduction of high dose amoxicillin therapy and pneumococcal conjugate vaccination. It appears that H. influenzae has become the predominant pathogen of persistent AOM and AOMTF since universal immunization with the pneumococcal conjugate vaccine. Fewer S. pneumoniae AOM isolates are penicillin-resistant and more H. influenzae are β-lactamase-producing.