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Background Otitis media (OM), a prevalent pediatric infectious disease, is mainly caused by Streptococcus pneumoniae (S.pn). Neutrophil extracellular traps (NETs), a novel antimicrobial strategy, were reported in 2004. We found that NETs formed in the middle ear with acute otitis media (AOM) induced by S.pn. However, the mechanisms of NETs formation are not entirely clear. Methods We stimulated neutrophils isolated from mouse bone marrow with S.pn clinical stain 19F in vitro, and established mouse model of AOM via transbullar injection