Haemophilus test media

The bacterial reference strains used in this study included type strain H. influenzae DSM 4690, a range of H. influenzae capsular subtypes (a–f), and closely related Haemophilus strains, with N. meningitidis NCTC 10025 and S. pneumoniae DSM 20566 type strains (Table S1). Haemophilus test media (Oxoid, Hampshire, UK) was used to culture all Haemophilus strains, and brain heart infusion media (Oxoid) was used to culture the N. meningitidis and S. pneumoniae type strains. Bacterial incubation was carried out at 37 °C for 18 h under microaerophilic conditions. The DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) and Qubit dsDNA broad range kit (Life Technologies, Warrington, UK) were used as per manufacture instructions to extract and quantify DNA from all cultured strains. All bacterial strains and extracted DNA were stored at −80 °C prior to use. Genome copy number values for DNA extracted from each strain were generated using resulting DNA concentrations with approximate genome size standards of 1.83 Mb, 2.2 Mb and 2.1 Mb for Haemophilus, N. meningitidis, and S. pneumoniae strains, respectively.

A total of 168 bacterial reference strains and clinical isolates were evaluated in this study (Supplemental Table S1). Clinical isolates were collected from culture-confirmed cases of bloodstream infection as part of routine diagnostic service at Galway University Hospital (GUH). All strains, stored at −80 °C, were cultured in brain heart infusion (BHI) media (Oxoid, Hampshire, UK) and incubated at 37 °C for 18 h, under microaerophilic conditions, excluding Haemophilus strains which were cultured using Haemophilus test media (Oxoid). Genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) and quantified using the Qubit dsDNA broad range/high sensitivity assay kits and Qubit 2.0 fluorometer (Life Technologies, Warrington, UK), according to manufacturer’s instructions. Resulting DNA concentrations were converted to genome copy values using genome size standards of 2.1 Mb, 2.2 Mb and 1.83 Mb for S. pneumoniae, N. meningitidis and H. influenzae, respectively.