Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae

<i>Haemophilus influenzae</i> is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in <i>H. influenzae</i> necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for <i>H. influenzae</i> using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (<i>N</i> = 14) and high-quality draft <i>H. influenzae</i> genomes (<i>N</i> = 2297). The draft genome dataset was divided into a development dataset (<i>N</i> = 921) and a validation dataset (<i>N</i> = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (<i>R</i> ² = 0.945). We used this cgMLST scheme to define a high-resolution population structure for <i>H. influenzae</i>, which enhances the genomic analysis of this clinically relevant human pathogen.