Virulence factors in nontypeable <italic>Haemophilus influenzae</italic> contributing to otitis media.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of acute otitis media (OM), infection of the middle ear. Although the pathogenic mechanisms are not well understood, NTHi may contain special virulence features that facilitate development of otitis media. To identify unique genetic regions specific for the genome of an OM pathogenic nontypeable isolate, G622, compared to the genome of a nonpathogenic nontypeable throat strain (TH), 23221, genomic subtraction was used in this study. Subsequently, the resultant middle ear specific gene regions were used to determine their relative prevalences among a large panel of OM and TH strains using dot blot hybridization. With this approach, nine new genetic regions were significantly more prevalent in OM strains compared to TH strains (sJPX124, 84, 163, 161, 147, 173, 101, 120, 132). Classification tree analysis of lic2b, hmwA, and the nine regions reveals two H. influenzae pathotypes associated with OM. To determine length, location and possible allelic variation of each of these genetic regions, we performed PCR based genomic walking on OM strain G622 and TH strain 23221. Follow up experiments in region sJPX132, associated with histidine biosynthesis, revealed a 7.5 kb deletion associated with the complete his operon. Isolates lacking this operon were unable to grow properly in histidine restricted liquid culture. This observation suggests that histidine biosynthesis may be important in bacterial survival in the middle ear where nutrients, such as histidine, are found in limited amounts. Further characterization of the remaining eight genetic regions suggest that many of these genetic regions identified through genomic subtraction may be markers for larger more complex regions that may be associated with OM pathogenesis. Most of these regions contain gene(s) involved with more conserved functions such as amino acid biosynthesis, nutrient uptake and energy metabolism rather than the traditionally defined virulence factors in NTHi such as adhesins, IgA protease, and lipooligosaccharides. These findings suggest that these previously thought conserved genes may be an added benefit to survival in certain niches, such as the middle ear, rather than strictly essential in H. influenzae.Ph.D.Biological SciencesEpidemiologyHealth and Environmental SciencesMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126068/2/3224917.pd