Molecular Analysis of Pathogens in Otitis Media by PCR

Drexel University, Philadelphia PA

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Abstract

Otitis media (OM) is the most common reason that an ill child visits a health care provider or undergoes a general anesthetic. OM is also the most common reason that a child receives an oral antibiotic and the over-treatment of patients with OM has been suspected of contributing to the development of antimicrobial- resistant organisms. OM disproportionately affects socio-economically disadvantaged children, Native American 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 chronic OM with the ultimate goal of developing more effective treatments. During this time, we have shown that chronic OM is not a purely inflammatory process, but rather is a bacterial biofilm illness. The recognition that OM is a biofilm disease then led to a novel hypothesis: The Distributed Genome Hypothesis. This hypothesis states that there is a supra-genome for pathogenic bacteria and that each individual bacterium possesses only a subset of genes from the supra-genome. The supra-genome is a reservoir for a panoply of contingency genes that collectively provides a significant survival benefit for the population-at-large. In this continuing application we specifically test the Distributed Genome Hypothesis in Haemophilus invluenzae (HI) with four Specific Aims: 1) Perform comparative genomic studies among clinical isolates of H! to characterize the extent of genomic plasticity; 2) Determine the extent of HI inter-isolate recombination during the infectious process; 3) Prepare transformation knockouts of HI and compare their survival time in vivo with wild-type congenic strains; 4) Phenotypic characterization of HI biofilms. These Specific Aims will be 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 a microarray library composed of 10 clinical isolates of H. influenzae demonstrated that H. influenzae does indeed have a supra-genome that is twice the Size of a single bacterium. These findings shed light on many aspects of OM, including disease persistence in the fact of antibiotic treatment, and provide an explanation for 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, PA Center for Biofilm Engineering, Montana State University (MSU), Bozeman, MT KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Ehdich, Garth D. ASRI Post, J. Christopher ASRI Costerton, J. William MSU Erdos, Geza ASRI Kathju, Sandeep ASRI Hu, Fen Ze ASRI Stewart, Philip S. MSU in the format shown below. Role on Project Principal Investigator Co-Principal Investigator Subcontract Principal Investigator Co-Principal Investigator Co-Investigator Co-Investigator and Operations Director Subcontract Co-Investigator Disclosure Permission StatemenL Applicable to SBIR/STTR Only. See instructions. _ Yes r-] No PHS 398 (Rev. 05/01) Page 2 Form Page 2 Principal 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 GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description, Performance Sites, and Personnel ................................................................................... 2- Table of Contents ..................................................................................................................................... 3 Detailed Budget for Initial Budget Period (or Modular Budget) ........................................................... 4 Budget for Entire Proposed Period of Support (not applicable with Modular Budget) ........................... 6 1/ Budgets Pertaining to ConsortiumlContractual Arrangements (not applicable with Modular Budget) 20 Biographical SketchMPrincipal Investigator/Program Director (Not to exceed four pages) .................. Other Biographical Sketches (Not to exceed four pages for each - See instructions)) ........................ 24 Resources ................................................................................................................................................. 54 Research Plan Introduction to Revised Application (Not to exceed 3 pages) ............................................................................................................... 61 Introduction 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 if Appendix (Five collated sets. No page numbefing necessary for Appendix.) Appendix is Included Appendices 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