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Background   Haemophilus influenzae  is an opportunistic bacterial pathogen that exclusively colonises humans and is associated with both acute and chronic disease. Despite its clinical significance, accurate identification of  H. influenzae  is a non-trivial endeavour.  H. haemolyticus  can be misidentified as  H. influenzae  from clinical specimens using selective culturing methods, reflecting both the shared environmental niche and phenotypic similarities of these species. On the molecular level, frequent genetic exchange amongst  Haemophilus  spp. has confounded accurate identification of  H. influenzae , leading to both false-positive and false-negative results with existing speciation assays .Results  Whole-genome single-nucleotide polymorphism data from 246 closely related global  Haemophilus  isolates, including 107 Australian isolate genomes generated in this study, were used to construct a whole-genome phylogeny. Based on this phylogeny,  H. influenzae  could be differentiated from closely related species. Next, a  H. influenzae -specific locus,  fucP , was identified, and a novel TaqMan real-time PCR assay targeting  fucP  was designed. PCR specificity screening across a panel of clinically relevant species, coupled with  in silico  analysis of all species within the order Pasteurellales, demonstrated that the  fucP  assay was 100 % specific for  H. influenzae ; all other examined species failed to amplify.    Conclusions  This study is the first of its kind to use large-scale comparative genomic analysis of  Haemophilus  spp. to accurately delineate  H. influenzae  and to identify a species-specific molecular signature for this species. The  fucP  assay outperforms existing  H. influenzae  targets, most of which were identified prior to the next-generation genomics era and thus lack validation across a large number of  Haemophilus  spp. We recommend use of the  fucP  assay in clinical and research laboratories for the most accurate detection and diagnosis of  H. influenzae  infection and colonisation.    Electronic supplementary material  The online version of this article (doi:10.1186/s12864-015-1857-x) contains supplementary material, which is available to authorized users.

Haemophilus influenzae: using comparative genomics to accurately identify a highly recombinogenic human pathogen