title: Haemophilus influenzae: using comparative genomics to accurately identify a highly recombinogenic human pathogen.
abstract: 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.
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. erin.price@menzies.edu.au.|Menzies School of Health Research, PO Box 41096, Casuarina, NT, 0811, Australia. erin.price@menzies.edu.au.
authors: Erin P
authors: EP
authors: Price
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. derek.sarovich@menzies.edu.au.
authors: Derek S
authors: DS
authors: Sarovich
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. elizabeth.nosworthy@menzies.edu.au.
authors: Elizabeth
authors: E
authors: Nosworthy
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. jemima.beissbarth@menzies.edu.au.
authors: Jemima
authors: J
authors: Beissbarth
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. robyn.marsh@menzies.edu.au.
authors: Robyn L
authors: RL
authors: Marsh
authors: University of Western Australia, Perth, WA, Australia. janessa.pickering@uwa.edu.au.
authors: Janessa
authors: J
authors: Pickering
authors: University of Western Australia, Perth, WA, Australia. lea-ann.kirkham@uwa.edu.au.
authors: Lea-Ann S
authors: LA
authors: Kirkham
authors: Department of Microbiology, PathWest Laboratory Medicine WA, Princess Margaret Hospital for Children and King Edward Memorial Hospital for Women, Perth, WA, Australia. anthony.keil@health.wa.gov.au.
authors: Anthony D
authors: AD
authors: Keil
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. anne.chang@menzies.edu.au.
authors: Anne B
authors: AB
authors: Chang
authors: Child Health Division, Menzies School of Health Research, Darwin, NT, Australia. heidi.smith-vaughan@menzies.edu.au.
authors: Heidi C
authors: HC
authors: Smith-Vaughan
mesh_terms: D016000:Cluster Analysis; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D006193:Haemophilus influenzae / Q000145:classification / Q000235:genetics*; D006801:Humans; D010802:Phylogeny; D020641:Polymorphism, Single Nucleotide; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA