Title: Pneumococcal phenotype and interaction with nontypeable Haemophilus influenzae as determinants of otitis media progression Running title: Otitis media progression by S. pneumoniae and NTHi

Background: Mixed-species otitis media caused by Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi) is associated with complex manifestations that are less common in pneumococcal infections without NTHi. We analyzed the microbiological composition of middle ear fluid (MEF) cultures and nasopharyngeal samples obtained before PCV7/13 rollout to identify bacterial factors influencing singleand mixed-species disease progression. Methods: Data included OM episodes submitted for MEF cultures during a 10-year prospective study in southern Israel and nasopharyngeal samples from unvaccinated asymptomatic children in the same population. We compared pneumococcal serotype diversity across carriage and disease isolates with and without NTHi co-isolation. We also measured associations between pneumococcal phenotype and rate of progression from colonization to OM in the presence and absence of NTHi. Results: Whereas pneumococcal serotype diversity in single-species OM is lower than in singlespecies colonization, serotype diversity does not differ significantly between colonization and OM in mixed-species episodes. Moreover, the pneumococcal serotype distribution of mixedspecies OM corresponds more closely to that of mixed-species carriage than to the distribution of pneumococcal serotypes colonizing without NTHi. Pneumococcal phenotypes predicting prolonged carriage duration—such as efficient metabolic properties and strong negative surface charge—are associated with higher rate of progression to single-species OM. These factors are weaker predictors of the progression of mixed-species episodes. Conclusions: Immune-evasive pneumococcal serotypes able to persist in the nasopharynx have elevated rates of progression to OM. However, these serotype differences in progression rate are attenuated in the presence of NTHi. peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/200733 doi: bioRxiv preprint first posted online Oct. 11, 2017;