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We sought new insights into mechanisms of pathogenesis by identifying bacterial genes associated with OM disease. We whole-genome sequenced &gt;200 clinical isolates collected over time from 9 healthy and 12 OM-prone children. Genome analysis first annotated and clustered homologous genes from across assemblies, inferred the phylogeny, and identified closely related set of strains, or clonal lineages, finding both transient and persistent colonization by distinct lineages. Phylogeny-informed association tests then identified 7 strong associations of genes\/operons with health or disease. Remarkably, 5 associations were with gene absences and OM, and all 5 had predicted functions consistent with roles in pH homeostasis and nitrogen metabolism, specifically in transport and modification of polyamines. Using a laboratory strain, knockouts of candidate \u201canti-OM\u201d operon\n                    <jats:italic>potE-speF<\/jats:italic>\n                    were tested.\n                    <jats:italic>In vitro<\/jats:italic>\n                    growth assays varying pH and supplementation with polyamines showed that the presence of\n                    <jats:italic>potE-speF<\/jats:italic>\n                    conferred alkaline sensitivity to cells in the presence of ornithine. Measurements of human middle ear fluid pH found higher alkalinity in specimens where\n                    <jats:italic>H. influenzae<\/jats:italic>\n                    was collected over those with other OM pathogens, supporting the hypothesis that the alkaline middle ear microenvironment in OM arises from or selects for\n                    <jats:italic>H. influenzae<\/jats:italic>\n                    infection. Experiments using a mouse OM model found that absence of\n                    <jats:italic>potE-speF<\/jats:italic>\n                    caused a\u2009~\u200963-fold increase in bacterial disease burden in middle ear washes. By contrast, a mouse lung infection model showed no significant difference. We conclude that\n                    <jats:italic>H. influenzae<\/jats:italic>\n                    lacking\n                    <jats:italic>potE-speF<\/jats:italic>\n                    have a selective advantage in OM disease pathogenesis, because these strains thrive in the otherwise hostile alkaline environment that occurs in the infected middle ears of children.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1014649","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T17:59:24Z","timestamp":1790359164000},"page":"e1014649","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A bacterial pangenome association study identifies a role for polyamine metabolism and alkaline tolerance in middle ear infections"],"prefix":"10.1371","volume":"22","author":[{"given":"Jocelyn A.","family":"Hammond","sequence":"first","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel L.","family":"Ehrlich","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ariel","family":"Gonzalez","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bego\u00f1a","family":"Euba","sequence":"additional","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda, Consejo Superior de Investigaciones Cient\u00edficas-Gobierno de Navarra","place":["Mutilva, Spain"]},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red Enfermedades Respiratorias (CIBERES)","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin M.","family":"Raible","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jill M.","family":"Lawrence","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1124-9210","authenticated-orcid":true,"given":"Ari B.","family":"Gordon","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evangeline","family":"Williams","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mary C.","family":"Marino","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Limbo","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven","family":"Lang","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bhaswati","family":"Sen","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergey V.","family":"Balashov","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Azad","family":"Ahmed","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junkal","family":"Garmendia","sequence":"additional","affiliation":[{"name":"Instituto de Agrobiotecnolog\u00eda, Consejo Superior de Investigaciones Cient\u00edficas-Gobierno de Navarra","place":["Mutilva, Spain"]},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica en Red Enfermedades Respiratorias (CIBERES)","place":["Madrid, Spain"]},{"name":"Conexi\u00f3n Antimicrobial Resistance-AMR, CSIC","place":["Madrid, Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joris","family":"Beld","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Garth D.","family":"Ehrlich","sequence":"additional","affiliation":[{"name":"Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Microbiology & Immunology, Drexel University College of Medicine"]},{"name":"Drexel University College of Medicine","place":["Philadelphia, Pennsylvania, United States of America"],"department":["Department of Otolaryngology \u2013 Head and Neck Surgery"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael E.","family":"Pichichero","sequence":"additional","affiliation":[{"name":"Center for Infectious Diseases and Immunology, Rochester General Hospital Research Institute, Rochester General Hospital","place":["Rochester, NewYork, United States of America"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravinder","family":"Kaur","sequence":"additional","affiliation":[{"name":"Center for Infectious Diseases and Immunology, Rochester General Hospital Research Institute, Rochester General Hospital","place":["Rochester, NewYork, United States of America"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0222-5636","authenticated-orcid":true,"given":"Joshua 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Receiving Long-Term Azithromycin Treatment","volume":"11","author":"A Carrera-Salinas","year":"2023","journal-title":"Microbiol Spectr"},{"issue":"4","key":"ppat.1014649.ref039","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0001969","article-title":"Strain-specific virulence phenotypes of Streptococcus pneumoniae assessed using the Chinchilla laniger model of otitis media","volume":"3","author":"ML Forbes","year":"2008","journal-title":"PLoS One"},{"key":"ppat.1014649.ref040","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1186\/1471-2180-8-173","article-title":"Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates","volume":"8","author":"L Hall-Stoodley","year":"2008","journal-title":"BMC Microbiol"},{"key":"ppat.1014649.ref041","first-page":"91","article-title":"Molecular and translational research approaches for the study of bacterial pathogenesis in otitis 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