<p>Strains, plasmids, and oligonucleotides used.</p>

Using a laboratory strain, knockouts of candidate “anti-OM” operon <i>potE-speF</i> were tested. <i>In vitro</i> growth assays varying pH and supplementation with polyamines showed that the presence of <i>potE-speF</i> conferred alkaline sensitivity to cells in the presence of ornithine. Measurements of human middle ear fluid pH found higher alkalinity in specimens where <i>H. influenzae</i> was collected over those with other OM pathogens, supporting the hypothesis that the alkaline middle ear microenvironment in OM arises from or selects for <i>H. influenzae</i> infection.

Experiments using a mouse OM model found that absence of <i>potE-speF</i> 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 <i>H. influenzae</i> lacking <i>potE-speF</i> 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.</p></div>