Intra-host genomic variation of serologically nontypeable <i>Haemophilus influenzae</i> isolates from otitis media

Serologically nontypeable <i>Haemophilus influenzae</i> is a human pathogen that causes infections ranging in severity from mild otitis media and sinusitis to life-threatening pneumonia, bacteremia, and meningitis. Although intra-host genomic variation in infected humans has been studied, many important questions remain unanswered. To address this knowledge deficit, we sequenced the genomes of 500 isolates recovered from ear drainage fluid collected from 11 Icelandic children with otorrhea. We discovered substantial genomic diversity among the <i>H. influenzae</i> strains infecting each patient. In total, we identified 88 genes that acquired nonsynonymous (amino acid-changing) or nonsense (protein-truncating) single-nucleotide polymorphisms, insertions, or deletions in at least one isolate. Of these, 13 genes were recurrently polymorphic. The polymorphic genes encoded proteins with varied inferred functions, including carbohydrate metabolism, cell wall biosynthesis, environmental stress response, glycolipid metabolism, iron metabolism, recombination, small molecule transport, and transcription and translation. No amino acid substitutions or protein truncations were identified in proven <i>H. influenzae</i> virulence factors or major transcription regulators. However, many of the polymorphic genes likely contribute to fitness, virulence, or host-pathogen molecular interactions. Our study of intra-host variation in otitis media provides a framework for understanding the genomic adaptability of <i>H. influenzae</i> during human infections.IMPORTANCESerologically nontypeable <i>H. influenzae</i> is a human pathogen responsible for a range of diseases, including mild otitis media (middle ear infection) and sinusitis, and severe pneumonia, bacteremia, and meningitis. While research has begun advancing our understanding of the population genomic structure of <i>H. influenza</i> strains infecting humans, little is known about intra-host genomic variation. To address this knowledge gap, we sequenced the genomes of 500 <i>H</i>. <i>influenzae</i> isolates recovered from ear drainage fluid of Icelandic children diagnosed with otitis media. Our findings revealed that intra-host genomic variation involves many different genes encoding proteins with diverse functions. The data provide novel information bearing on the complexity of intra-host diversity and improve our understanding of <i>H. influenzae</i> strain fitness and molecular pathogenesis. This information could generate new hypotheses bearing on host-pathogen interactions and identify new therapeutic and vaccine targets.