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Community-wide use of conjugated heptavalent pneumococcal vaccine (PCV7) in children <2 years of age could affect the microbiology of acute otitis media (AOM) in vaccinees, particularly for penicillin-nonsusceptible Streptococcus pneumoniae (PNSP).Since Summer 2000, 94% of young children cared for by this 7-clinician, pediatric practice in rural central Kentucky received 3 or 4 doses of PCV7 in the first 18 months of life.To determine changes in microbiology of AOM before and after community-wide routine implementation of PCV7.Among children 7–24 months old with severe or refractory AOM, we compared 336 AOM isolates from 1992–1998 with 83 AOM isolates from 2000–2003 in children who had received 3 or 4 doses of PCV7.Comparing each cohort (1992–1998 versus 2000–2003), the proportion of S. pneumoniae decreased from 48% to 31% (P = 0.009; relative risk, 0.754; 95% confidence interval, 0.628–0.906), and nontypable Haemophilus influenzae increased from 41% to 56% (P = 0.01; relative risk, 1.87; 95% confidence interval, 1.15–3.04; β-lactamase-positive, 56% versus 64%, not significant). The proportions of intermediate PNSP and resistant PNSP, respectively, were 16% and 9% versus 13% and 6% pre- and post-PCV7, respectively. Vaccine and vaccine-related serotypes, respectively, comprised 70% and 8% versus 36% and 32% of S. pneumoniae strains (P = 0.003). Post-PCV7, Gram-negative bacteria and β-lactamase-producing organisms accounted for two-thirds and one-half of all AOM isolates, respectively.The overall proportion of S. pneumoniae isolates and vaccine serotypes in AOM were significantly reduced by community-wide use of PCV7 vaccine in our practice. The proportion of Gram-negative bacteria became 2-fold more frequent than S. pneumoniae in AOM in PCV7-vaccinated young children where PCV7 uptake was community-wide and supply was adequate.