S pneumoniae

Isolates used in this study were obtained from the American Type Culture Collection (ATCC): H. influenzae (ATCC 49766), S. aureus, (ATCC 29213), S. pneumoniae (ATCC 49619) and P. aeruginosa (ATCC 27853). All isolates were stored at -80 °C prior to inoculation onto chocolate blood agar (H. influenzae: Oxoid, Basingstoke, UK) or blood agar (S. aureus, S. pneumoniae, P. aeruginosa: Oxoid, Basingstoke, UK) and incubated at 37 °C in 5% CO₂ (H. influenzae, S. pneumoniae), or in air (S. aureus, P. aeruginosa).

Sputum plugs collected from five donors with no significant growth of potentially pathogenic respiratory organisms on culture (PID: 1052, 5, 1055, 315, 187) were homogenized with DTT as above and pooled together to generate a baseline sample for all spiking conditions. The pooled sample was then split for spiking simultaneously with Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis and Pseudomonas aeruginosa. Bacteria were cultured overnight (37°C with 5% CO₂, shaking at 180 rpm) in 10 mL culture broth as indicated by species (H. influenzae in BHI with 10 μg/mL NAD and hemin [Sigma-Aldrich, Dorset, UK], S. pneumoniae in BHI and S. aureus/M. catarrhalis/P. aeruginosa in nutrient broth [Oxoid]). Once optical density at 600 nm reached 0.5 (exponential phase) bacteria were pelleted and resuspended in BHI or nutrient broth containing 10% glycerol and stored at −80°C until use. Serial dilutions (in phosphate-buffered saline) and confirmatory CFU counts were performed to determine bacterial stock concentrations. The five cultured bacterial species were spiked simultaneously from stock solutions into the pooled homogenized sputum samples, producing three spiked samples containing 10⁴, 10⁶ or 10⁸ CFU/mL of each bacterial species. Microbial DNA was extracted, as described previously, from the unspiked and spiked pooled samples.