Nontypeable (NT) Haemophilus influenzae strains cause significant respiratory illness and are isolated from up to half of middle ear aspirates from children with acute otitis media. Previous studies have identified two genes, lic2B and hmwA, that are associated with NT H. influenzae strains isolated from the middle ears of children with otitis media but that are not associated with NT H. influenzae strains isolated from the throats of healthy children, suggesting that they may play a role in virulence in otitis media. In this study, genomic subtraction was used to identify additional genetic regions unique to middle ear strains. The genome of NT H. influenzae middle ear strain G622 was subtracted from that of NT H. influenzae throat strain 23221, and the resultant gene regions unique to the middle ear strain were identified. Subsequently, the relative prevalence of the middle ear-specific gene regions among a large panel of otitis media and throat strains was determined by dot blot hybridization. By this approach, nine genetic regions were found to be significantly more prevalent in otitis media strains. Classification tree analysis of lic2B, hmwA, and the nine new potential otitis media virulence genes revealed two H. influenzae pathotypes associated with otitis media.
