Biofilm Formation of Non-Typeable Haemophilus influenzae

50 µl from frozen stock cultures of non-typeable Haemophilus influenzae strains PittAA, PittEE, PittGG, PittII [42] (link), [43] (link), 2019 [44] (link) and 9274 [45] (link) were spread onto chocolate agar plates (Hardy Diagnostics, Santa Maria, CA, USA) and incubated at 37°C in an atmosphere of 5% CO₂ with 95% relative humidity. After 24-hour incubation, multiple colonies were used to inoculate BHI broth (Bacto Brain Heart Infusion; BD Diagnostics, Sparks, MD, USA) supplemented with hemin (Sigma-Aldrich Inc., St. Louis, MO, USA) at 10 µg/ml and nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich Inc., St. Louis, MO, USA) at 2 µg/ml (sBHI) [46] . The bacterial suspension was grown to stationary phase (1×10⁹ CFU/ml) at 37°C, 5% CO₂, with 95% relative humidity. Biofilms in this study were prepared by diluting 1∶200 bacterial suspensions at stationary phase with fresh sBHI, aliquoting into culture vessels appropriate for subsequent applications, and incubating at 37°C with 5% CO₂ and 95% relative humidity for 24 hours. All NTHi strains were isolated from chronic otitis media patients, with the exception of 2019, which was isolated from a patient with chronic bronchitis.