The sodA gene as a target for phylogenetic dissection of the genus Haemophilus and accurate identification of human clinical isolates

The genus Haemophilus constitutes a heterogeneous group of Pasteurellaceae species, and conventional identification of isolates other than Haemophilus influenzae and Haemophilus parainfluenzae is often challenging. Here, simple colony-PCR and sequencing assays with the same pair of degenerate primers were used to characterize a 449- to 458-bp fragment (sodA_int) internal to the sodA gene encoding the manganese-dependent superoxide dismutase in type strains of all 15 Haemophilus species and Actinobacillus actinomycetemcomitans. The topology of a sodA_int-based phylogenetic tree was in general agreement with that inferred from the analysis of 16S rRNA and other housekeeping gene sequences, but allowed more confident delineation of the main clusters of species. The sodA_int sequences showed a markedly higher divergence than those of the corresponding 16S rRNA genes, and 38 independent human clinical isolates were identified by comparing their sodA_int sequence to those of the type species. Except for one Haemophilus aphrophilus strain, all isolates were unambiguously characterized in spite of a high intraspecific sodA_int sequence diversity. This study provides a comprehensive sequence-based phylogenetic analysis of the entire genus Haemophilus, and confirms that sodA is a potent target for the identification of clinical isolates of Pasteurellaceae. This approach might contribute to the taxonomic reappraisal of this family, and to the development of diagnostic tools.