- Background: The pneumococcal conjugated vaccines (PCVs) impact on complex otitis media (including recurrent, non-responsive, spontaneously draining and chronic OM with effusion) was greater than that against simple, acute-OM in previous studies. Since complex OM is often a polymicrobial disease, we speculated that reduction of complex OM by PCVs will be associated in addition with reduction of non-pneumococcal OM. Methods: In a prospective, population-based, active surveillance, all OM episodes submitted for middle ear fluid (MEF) culture in children <3 years, during 2004-2015 were included. Three sub-periods were defined: pre-PCV, PCV7, and PCV13 periods. Incidence rate ratios (IRRs) comparing the three periods were calculated for pneumococcal, non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis, Streptococcus pyogenes and culture-negative OM. Results: Overall, 7,475 episodes were included. Of all NTHi cases in the pre-PCV period, 34% were mixed with Streptococcus pneumoniae IRRs (95% confidence interval) comparing the pre-PCV to the PCV13 period were 0.02 (0.01-0.04), 0.12 (0.08-0.20) and 0.18 (0.15-0.21) for PCV7+6A serotypes, 5 additional PCV13 serotypes and overall pneumococcal OM, respectively; non-PCV13 serotype episodes were not significantly reduced; The respective IRRs for single NTHi, mixed NTHi+S. pneumoniae and all-NTHi OM were 0.30 (0.25-0.35), 0.18 (0.13-0.24) and 0.25 (0.22-0.29). M. catarrhalis, S. pyogenes and culture-negative episodes were also significantly reduced. Conclusions: Both pneumococcal and non-pneumococcal OM episodes, enriched with complex cases, declined substantially in children <3 years old, following sequential PCV7/PCV13 introduction. The reduction in non-pneumococcal episodes may be attributed to prevention of early-OM episodes, resulting in a lower rate of complex, often non-pneumococcal OM.