Otitis media (OM) is the most common reason that an ill child visits a health care provider or undergoes ageneral anesthetic. OM is also the most common reason that a child receives an oral antibiotic and theover-treatment of patients with OM has been suspected of contributing to the development of antimicrobial-resistant organisms. OM disproportionately affects socio-economically disadvantaged children, NativeAmerican children and is a factor that inhibits women from full participation in the workforce.The major focus of our laboratory for the past decade has been elucidating the pathophysiology of chronicOM with the ultimate goal of developing more effective treatments. During this time, we have shown thatchronic OM is not a purely inflammatory process, but rather is a bacterial biofilm illness. The recognition thatOM is a biofilm disease then led to a novel hypothesis: The Distributed Genome Hypothesis. Thishypothesis states that there is a supra-genome for pathogenic bacteria and that each individual bacteriumpossesses only a subset of genes from the supra-genome. The supra-genome is a reservoir for a panoplyof contingency genes that collectively provides a significant survival benefit for the population-at-large. Inthis continuing application we specifically test the Distributed Genome Hypothesis in Haemophilusinvluenzae (HI) with four Specific Aims: 1) Perform comparative genomic studies among clinical isolates ofH! to characterize the extent of genomic plasticity; 2) Determine the extent of HI inter-isolate recombinationduring the infectious process; 3) Prepare transformation knockouts of HI and compare their survival time invivo with wild-type congenic strains; 4) Phenotypic characterization of HI biofilms.
These Specific Aims willbe accomplished by experiments using state-of-the-art high throughput genomics, molecular biology,imaging and modeling techniques, as well as investigations in children. Preliminary data generated from amicroarray library composed of 10 clinical isolates of H. influenzae demonstrated that H. influenzae doesindeed have a supra-genome that is twice the Size of a single bacterium. These findings shed light on manyaspects of OM, including disease persistence in the fact of antibiotic treatment, and provide an explanationfor the success of adenoidectomy in the management of OM.PERFORMANCE SiTE(S) (organization, city, state)Center for Genomic Sciences, Allegheny-Singer Research Institute (ASRI), Pittsburgh, PACenter for Biofilm Engineering, Montana State University (MSU), Bozeman, MTKEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required informationStart with Principal Investigator. List all other key personnel in alphabetical order, last name first.Name OrganizationEhdich, Garth D. ASRIPost, J. Christopher ASRICosterton, J. William MSUErdos, Geza ASRIKathju, Sandeep ASRIHu, Fen Ze ASRIStewart, Philip S. MSUin the format shown below.Role on ProjectPrincipal InvestigatorCo-Principal InvestigatorSubcontract PrincipalInvestigatorCo-Principal InvestigatorCo-InvestigatorCo-Investigator andOperations DirectorSubcontract Co-InvestigatorDisclosure Permission StatemenL Applicable to SBIR/STTR Only. See instructions. _ Yes r-] NoPHS 398 (Rev. 05/01) Page 2 Form Page 2Principal Investigator/Program Director (Last, first, middle): EHRLICH, G&rth D.The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page.RESEARCH GRANTTABLE OF CONTENTS Page NumbersFace Page .................................................................................................................................................. 1Description, Performance Sites, and Personnel ................................................................................... 2-Table of Contents ..................................................................................................................................... 3Detailed Budget for Initial Budget Period (or Modular Budget) ........................................................... 4 Budget for Entire Proposed Period of Support (not applicable with Modular Budget) ........................... 61'/Budgets Pertaining to ConsortiumlContractual Arrangements (not applicable with Modular Budget)20Biographical SketchMPrincipal Investigator/Program Director (Not to exceed four pages) ..................Other Biographical Sketches (Not to exceed four pages for each - See instructions)) ........................ 24Resources ................................................................................................................................................. 54Research PlanIntroduction to Revised Application (Not to exceed 3 pages) ............................................................................................................... 61Introduction to Supplemental Application (Not to exceed one page) ...................................................................................................64 A.
Specific Aims .. ....................................................................... _. ...................................................................................._5 B. Background and Significance ................................................ '''t .................................................................................... _'68 C. Preliminary Studies/Progress Report/ _ (Items A-D: not to exceed 25 pages*) ._ Phase I Progress Report (SBIR/STTR Phase II ONLY) I * SBIR/STTR Phase I: Items A-D limited to 15 pages. I I I 82 D, Research Design and Methods ............................................. _ ..................................................................................... E. Human Subjects ................................................................................................................................................................. 90 Protection of Human Subjects (Required if item 4 on the Face Page is marked 'yes') Inclusion of Women (Required if item 4 on the Face Page is marked 'Yes') ................................................................. Inclusion of Minorities (Required if Item 4 on the Face Page is marked 'Yes') ............................................................... Inclusion of Children (Required if Item 4 on the Face Page is marked 'Yes') ................................................................. Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked 'Yes' anda Phase I, II, or III clinical trial is proposed ...................................................................................................................................................... F. Vertebrate Animals ............................................................................................................................................................. 91 G. Literature Cited ................................................................................................................................................................... 92 H. Consortium/Contractual Arrangements ............................................................................................................................... 97 I. Letters of Support (e.g., Consultants) ........................................................................................................................................ 98 J. Product Development Plan (SBIPJSTTR Phase II and Fast-Track ONLY) ..........................................................................Checklist .................................................................................................................................................... 101 Check ifAppendix (Five collated sets. No page numbefing necessary for Appendix.) Appendix isIncludedAppendices NOT PERMITTED for Phase I SBIR/STTR unless specifically solicited.Number of publications and manuscripts accepted for publication (not to exceed 10)Other items (list):PHS 398 (Rev. 05101) Page ...... 3 Form Page 3

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
7R01DC002148-18
Application #
8791355
Study Section
Clinical Research and Field Studies of Infectious Diseases Study Section (CRFS)
Program Officer
Watson, Bracie
Project Start
1993-12-01
Project End
2018-03-31
Budget Start
2013-12-01
Budget End
2014-03-31
Support Year
18
Fiscal Year
2013
Total Cost
$548,173
Indirect Cost
$139,761
Name
Drexel University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
002604817
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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

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