Abstract
BackgroundBacteria belonging to the genus Haemophilus cause a wide range of diseases in humans. Recently, H. influenzae was classified by the WHO as priority pathogen due to the wide spread of ampicillin resistant strains. However, other Haemophilus spp. are often misclassified as H. influenzae. Therefore, we established an accurate and rapid whole genome sequencing (WGS) based classification and serotyping algorithm and combined it with the detection of resistance genes.MethodsA gene presence/absence-based classification algorithm was developed, which employs the open-source gene-detection tool SRST2 and a new classification database comprising 36 genes, including capsule loci for serotyping. These genes were identified using a comparative genome analysis of 215 strains belonging to ten human-related Haemophilus (sub)species (training dataset). The algorithm was evaluated on 1329 public short read datasets (evaluation dataset) and used to reclassify 262 clinical Haemophilus spp. isolates from 250 patients (German cohort). In addition, the presence of antibiotic resistance genes within the German dataset was evaluated with SRST2 and correlated with results of traditional phenotyping assays.ResultsThe newly developed algorithm can differentiate between clinically relevant Haemophilus species including, but not limited to, H. influenzae, H. haemolyticus, and H. parainfluenzae. It can also identify putative haemin-independent H. haemolyticus strains and determine the serotype of typeable Haemophilus strains. The algorithm performed excellently in the evaluation dataset (99.6% concordance with reported species classification and 99.5% with reported serotype) and revealed several misclassifications. Additionally, 83 out of 262 (31.7%) suspected H. influenzae strains from the German cohort were in fact H. haemolyticus strains, some of which associated with mouth abscesses and lower respiratory tract infections.Resistance genes were detected in 16 out of 262 datasets from the German cohort. Prediction of ampicillin resistance, associated with blaTEM-1D, and tetracycline resistance, associated with tetB, correlated well with available phenotypic data.ConclusionsOur new classification database and algorithm have the potential to improve diagnosis and surveillance of Haemophilus spp. and can easily be coupled with other public genotyping and antimicrobial resistance databases. Our data also point towards a possible pathogenic role of H. haemolyticus strains, which needs to be further investigated.
Highlights
Bacteria belonging to the genus Haemophilus cause a wide range of diseases in humans
Differentiation between H. influenzae and H. haemolyticus strains Misclassifications of H. haemolyticus strains as H. influ‐ enzae are clinically the most relevant [3, 21,22,23,24]
We first employed a pangenome-wide association study using all (n=61) publicly available H. haemolyticus genome assemblies and all (n=68) publicly available fully closed H. influenzae genomes (Additional file 1: Table S1 and Additional file 2: Table S2), to identify new marker genes that discriminate between these two species
Summary
Bacteria belonging to the genus Haemophilus cause a wide range of diseases in humans. The global expansion of ampicillin resistant H. influenzae strains led to the WHO classification of a priority pathogen for research and development of new antibiotics [10]. Other species, such as H. haemo‐ lyticus and H. parainfluenzae have been considered as harmless respiratory tract commensals; evidence is accumulating that strains of these species should be considered as opportunistic pathogens with the potential of causing a wide range of infections [8, 11, 12]. The classification of H. paraphrohaemolyticus, H. aegyptius, and H. quentini, which are closely related to H. parahaemo‐ lyticus, H. influenzae, and H. haemolyticus, respectively, as separate species is still under debate [3, 15, 19, 20]
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