Abstract
BackgroundHaemophilus 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.ResultsWhole-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.ConclusionsThis 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 materialThe online version of this article (doi:10.1186/s12864-015-1857-x) contains supplementary material, which is available to authorized users.
Highlights
Haemophilus influenzae is an opportunistic bacterial pathogen that exclusively colonises humans and is associated with both acute and chronic disease
nontypeable” H. influenzae (NTHi) is a common coloniser of the upper respiratory tract in healthy individuals but can cause otitis media, conjunctivitis, sinusitis, and lower respiratory infections in children, exacerbations of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) in adults, and sepsis in neonates and immunocompromised adults [4]
Assay design has conventionally been thwarted by high levels of recombination between H. influenzae and other Haemophilus species, and has even been documented between Haemophilus and Neisseria meningitidis [39]
Summary
Haemophilus influenzae is an opportunistic bacterial pathogen that exclusively colonises humans and is associated with both acute and chronic disease. The Gram-negative Haemophilus spp. bacteria comprise a diverse group containing at least 12 currently recognised species, all of which are commensal or pathogenic to humans or animals. Other H. influenzae serotypes (a; c-f ), and nonencapsulated, “nontypeable” H. influenzae (NTHi), which are not targeted by the Hib vaccine, are recognised as important causes of primarily mucosal acute and chronic infections [3]. NTHi is a common coloniser of the upper respiratory tract in healthy individuals but can cause otitis media, conjunctivitis, sinusitis, and lower respiratory infections in children, exacerbations of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) in adults, and sepsis in neonates and immunocompromised adults [4]. Far less common than H. influenzae, other Haemophilus species have the potential to cause human disease including H. haemolyticus, H. parainfluenzae, H. aegyptius (a biogroup of H. influenzae), H. pittmaniae, H. parahaemolyticus and H. paraphrohaemolyticus [5,6,7,8,9,10,11,12,13,14]
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