A bacterial pangenome association study identifies a role for polyamine metabolism and alkaline tolerance in middle ear infections
6 affiliations
- 1Department of Microbiology & Immunology, Drexel University College of Medicine;Center for Genomic Sciences, Institute of Molecular Medicine and Infectious Disease; Drexel University, Philadelphia, Pennsylvania, United States of America
- 2Center for Infectious Diseases and Immunology, Rochester General Hospital Research Institute, Rochester General Hospital, Rochester, NewYork, United States of America
- 3Instituto de Agrobiotecnología, Consejo Superior de Investigaciones Científicas-Gobierno de Navarra, Mutilva, Spain
- 4Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), Madrid, Spain
- 5Conexión Antimicrobial Resistance-AMR, CSIC, Madrid, Spain
- 6Department of Otolaryngology – Head and Neck Surgery;Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America
Abstract
Infection of the middle ear, or otitis media (OM), is a disease that commonly afflicts children, and Haemophilus influenzae is the most prevalent OM-associated bacterium. We sought new insights into mechanisms of pathogenesis by identifying bacterial genes associated with OM disease. We whole-genome sequenced >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 “anti-OM” operon potE-speF were tested. In vitro growth assays varying pH and supplementation with polyamines showed that the presence of potE-speF conferred alkaline sensitivity to cells in the presence of ornithine. Measurements of human middle ear fluid pH found higher alkalinity in specimens where H. influenzae was collected over those with other OM pathogens, supporting the hypothesis that the alkaline middle ear microenvironment in OM arises from or selects for H. influenzae infection. Experiments using a mouse OM model found that absence of potE-speF caused a ~ 63-fold increase in bacterial disease burden in middle ear washes. By contrast, a mouse lung infection model showed no significant difference. We conclude that H. influenzae lacking potE-speF 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. Author summary: Many bacteria that inhabit the healthy human microbiome also cause disease. How bacteria transition into pathogens remains poorly understood and results from a complex interplay among microbial, host, and environmental factors. Among such bacteria are species that are found in the healthy human nasopharynx but also cause middle-ear infections (otitis media, or OM), as well as other diseases. Most people experience at least one OM episode in early childhood, and many have persistent disease with potentially lifelong consequences from delayed language acquisition or hearing loss. Here we present evidence that the absence of certain genes of the bacterium Haemophilus influenzae is associated with OM disease. Prior work showed that children with recurrent OM who later receive tympanostomy tube surgery often have a middle ear microenvironment characterized by alkaline pH. Here we show that the absence of a two-gene unit involved in polyamine transport and conversion, potE-speF, results in an alkaline-tolerant phenotype. We show that strains without these genes increase the bacterial disease burden 63-fold in a mouse model of OM but not in a lung infection model. The results point to unexplored host-mediated mechanisms for limiting pathogen burden by creating an alkaline environment in the middle ears of children.
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BibTeX
@article{hammond2026bacterial,
title = {{A bacterial pangenome association study identifies a role for polyamine metabolism and alkaline tolerance in middle ear infections}},
author = {Jocelyn A. Hammond and Rachel L. Ehrlich and Ariel Gonzalez and Begoña Euba and Kevin M. Raible and Jill M. Lawrence and Ari B. Gordon and Evangeline Williams and Mary C. Marino and Jason Limbo and Steven Lang and Bhaswati Sen and Sergey V. Balashov and Azad Ahmed and Junkal Garmendia and Joris Beld and Garth D. Ehrlich and Michael E. Pichichero and Ravinder Kaur and Joshua Chang Mell},
journal = {PLOS Pathogens},
year = {2026},
month = sep,
doi = {10.1371/journal.ppat.1014649},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014649},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A bacterial pangenome association study identifies a role for polyamine metabolism and alkaline tolerance in middle ear infections
AU - Jocelyn A. Hammond
AU - Rachel L. Ehrlich
AU - Ariel Gonzalez
AU - Begoña Euba
AU - Kevin M. Raible
AU - Jill M. Lawrence
AU - Ari B. Gordon
AU - Evangeline Williams
AU - Mary C. Marino
AU - Jason Limbo
AU - Steven Lang
AU - Bhaswati Sen
AU - Sergey V. Balashov
AU - Azad Ahmed
AU - Junkal Garmendia
AU - Joris Beld
AU - Garth D. Ehrlich
AU - Michael E. Pichichero
AU - Ravinder Kaur
AU - Joshua Chang Mell
PY - 2026
DA - 2026/09/25
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014649
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014649
AB - Infection of the middle ear, or otitis media (OM), is a disease that commonly afflicts children, and Haemophilus influenzae is the most prevalent OM-associated bacterium. We sought new insights into mechanisms of pathogenesis by identifying bacterial genes associated with OM disease. We whole-genome sequenced >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 “anti-OM” operon potE-speF were tested. In vitro growth assays varying pH and supplementation with polyamines showed that the presence of potE-speF conferred alkaline sensitivity to cells in the presence of ornithine. Measurements of human middle ear fluid pH found higher alkalinity in specimens where H. influenzae was collected over those with other OM pathogens, supporting the hypothesis that the alkaline middle ear microenvironment in OM arises from or selects for H. influenzae infection. Experiments using a mouse OM model found that absence of potE-speF caused a ~ 63-fold increase in bacterial disease burden in middle ear washes. By contrast, a mouse lung infection model showed no significant difference. We conclude that H. influenzae lacking potE-speF 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. Author summary: Many bacteria that inhabit the healthy human microbiome also cause disease. How bacteria transition into pathogens remains poorly understood and results from a complex interplay among microbial, host, and environmental factors. Among such bacteria are species that are found in the healthy human nasopharynx but also cause middle-ear infections (otitis media, or OM), as well as other diseases. Most people experience at least one OM episode in early childhood, and many have persistent disease with potentially lifelong consequences from delayed language acquisition or hearing loss. Here we present evidence that the absence of certain genes of the bacterium Haemophilus influenzae is associated with OM disease. Prior work showed that children with recurrent OM who later receive tympanostomy tube surgery often have a middle ear microenvironment characterized by alkaline pH. Here we show that the absence of a two-gene unit involved in polyamine transport and conversion, potE-speF, results in an alkaline-tolerant phenotype. We show that strains without these genes increase the bacterial disease burden 63-fold in a mouse model of OM but not in a lung infection model. The results point to unexplored host-mediated mechanisms for limiting pathogen burden by creating an alkaline environment in the middle ears of children.
SN - 1553-7374
ER -