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.

Public Health Relevance

Middle-ear disease in children is the number one reason they see a healthcare provider, receive antibiotics and undergo surgery. Most middle-ear disease is caused by bacteria from the back of the throat moving into the middle ear and causing an infection. However, these bacteria are often members of the normal human bacterial population (the microbiota) that have acquired extra genes that make them cause disease. This grant application aims to determine how and why some normally good bacteria evolve to become disease causing bacteria.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Clinical Research and Field Studies of Infectious Diseases Study Section (CRFS)
Program Officer
Watson, Bracie
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Drexel University
Schools of Medicine
United States
Zip Code
Janto, Benjamin A; Hiller, N Luisa; Eutsey, Rory A et al. (2014) Development and validation of an Haemophilus influenzae supragenome hybridization (SGH) array for transcriptomic analyses. PLoS One 9:e105493
Eutsey, Rory A; Hiller, N Luisa; Earl, Joshua P et al. (2013) Design and validation of a supragenome array for determination of the genomic content of Haemophilus influenzae isolates. BMC Genomics 14:484
Frazao, Nelson; Hiller, N Luisa; Powell, Evan et al. (2013) Virulence potential and genome-wide characterization of drug resistant Streptococcus pneumoniae clones selected in vivo by the 7-valent pneumococcal conjugate vaccine. PLoS One 8:e74867
Nistico, L; Kreft, R; Gieseke, A et al. (2011) Adenoid reservoir for pathogenic biofilm bacteria. J Clin Microbiol 49:1411-20
Davie, Jeremiah J; Earl, Josh; de Vries, Stefan P W et al. (2011) Comparative analysis and supragenome modeling of twelve Moraxella catarrhalis clinical isolates. BMC Genomics 12:70
Boissy, Robert; Ahmed, Azad; Janto, Benjamin et al. (2011) Comparative supragenomic analyses among the pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae using a modification of the finite supragenome model. BMC Genomics 12:187
Tuttle, Marie S; Mostow, Eliot; Mukherjee, Pranab et al. (2011) Characterization of bacterial communities in venous insufficiency wounds by use of conventional culture and molecular diagnostic methods. J Clin Microbiol 49:3812-9
Hiller, N Luisa; Eutsey, Rory A; Powell, Evan et al. (2011) Differences in genotype and virulence among four multidrug-resistant Streptococcus pneumoniae isolates belonging to the PMEN1 clone. PLoS One 6:e28850
Janto, Benjamin; Ahmed, Azad; Ito, Masahiro et al. (2011) Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4. Environ Microbiol 13:3289-309
Kerschner, Joseph E; Erdos, Geza; Hu, Fen Ze et al. (2010) Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa. Ann Otol Rhinol Laryngol 119:270-8

Showing the most recent 10 out of 57 publications