A bacterial pangenome association study identifies a role for polyamine metabolism and alkaline tolerance in middle ear infections

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 middle ears of children.