Otitis media is the most common disease for which children visit a healthcare provider, undergo surgery with anesthesia and receive antibiotics, Acute OM (AOM) is characterized by constitutional symptoms including purulence, otalgia, and fever, and has been etiologically associated with Haemophilus influenzae (Hi), Streptococcus pneumoniae and Moraxella catarrhalis. Yet these species are also constituents of the normal pharyngeal microbiota. We focus on the nontypeable Hi (NTHi) as they are increasingly associated with virulence. In cross-sectional studies we have documented enormous differences among NTHi clinical isolates with regard to the severity of both otologic and systemic disease. These phenotypic differences mirror the enormous genomic plasticity we have also documented whereby every strain pair, on average, differs by the possession of ~400 genes, and at the species level more than 60% of the supragenome is composed of distributed genes. In spite of these observations, the means by which bacterial virulence arises from within a largely commensal species is poorly understood. We do know that horizontal gene transfer (HGT) is principally responsible for strain evolution in this naturally competent species, but how or why this leads to the development of pathogenesis is unknown. Thus we will perform a natural history study wherein we follow NTHi population dynamics via whole genome sequencing of multiple strains from each of multiple time-points from two cohorts of children: those that are AOM-prone, and those that are healthy. This is designed to test the hypotheses that virulence increases over time among strains that are already pathogenic, but that nonpathogenic carriage strains do not tend to acquire virulence traits. The rationale is to determine if once strains start acquiring virulence traits via HGT(albeit initially by chance) does this essentially place them in a separate ecological niche from carriage strains, and thus provide an environmental driver that selects for increasing pathogenicity. We are cognizant that there are limitations on virulence progression, and therefore our emphasis will be on identifying moderately virulent strains early in infection and tracking their evolution and pathogenicity over the course of the natural pediatric infectious process. This study will be performed in parallel with a first-ever bacterial supragenome wide association study (SGWAS) wherein we will use our validated NTHi supragenome comparative genome hybridization (CGH) chip to determine the gene possession makeup of a library of greater than 700 clinically phenotyped (as to disease type or carriage) NTHi strains. Using the gene possession data from this study we will perform a statistical genetic analysis to identify NTHi virulence gene candidates associated with various pathogenic traits and look for the appearance of any of these genes in the cohorts' strains as they evolve in situ. Finally, we will compare the predominate predecessor strains to the predominate evolved strains from each child using the OMID model to determine actual virulence levels. We will also test for the expression of the candidate virulence genes both in the host and in the OMID model using NanoString technology.