Non-typeable Haemophilus influenzae biofilm formation on primary human airway epithelia cultured at air-liquid interface

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Introduction Primary ciliary dyskinesia (PCD) is an inherited heterogeneous disorder associated with defective motile cilia on airway epithelia, resulting in impaired mucociliary function and ultimately, bronchiectasis. Non-typeable Haemophilus influenzae (NTHi) is frequently isolated from PCD airways where it resides in biofilms. It is unclear if biological pathways in response to NTHi infection differ in PCD, contributing to increased susceptibility to biofilm-associated infection. Aim The development of a biologically representative co-culture model to investigate the interactions between PCD airway epithelia and NTHi biofilms. Methods Air-liquid interface (ALI)-cultured non-PCD primary nasal cells were grown in transwells. Barrier properties were assessed by trans-epithelial electrical resistance (TEER), FITC-dextran passage and tight junction staining. Ciliary function was assessed by high-speed video microscopy at 37°C. ALI cultures were infected with a PCD NTHi isolate at multiplicity of infections (MOIs) 10, 50 or 100 and co-cultured to form biofilms over 3 days. TEER was measured pre- and post-infection, NTHi recoverability assessed by conventional culture, and biofilm formation confirmed by scanning electron microscopy (SEM). Results Epithelial culture at ALI was successful, with tight barriers being formed and normal ciliary beat pattern and frequency recorded (mean 14Hz). Following co-culture, TEER increased relative to pre-infection at all MOIs but maximum NTHi recoverability was at MOI 50 (median colony forming units, 6.8x106per cm2). SEM confirmed NTHi biofilm formation at MOI 50. Conclusions A successful NTHi biofilm co-culture model on human primary epithelia was established. This will be used with PCD epithelia to investigate host-pathogen interactions during NTHi biofilm colonisation.