Lace-like architecture and towers arise from SulA-mediated filamentous morphotypes.

Restricted or replete 86-028NP were cultured in medium containing 2 µg heme mL−1 for 48 hours on glass chamber slides and visualized by fluorescent microscopy using a live/dead stain (A, B). Restricted 86-028NP/pGM1.1 was grown in the presence 2 µg heme mL−1 for 6 hours and subjected to centrifugation through a 5 µm pore filter to enrich for bacillary and filamentous subpopulations. Enriched bacillary (C) and filamentous (D) populations were used to initiate biofilm formation on glass chamber slides in the presence of 2 µg heme mL−1 for 48 hours. Scale bar = 10 µm. Restricted 86-028NP/pGM1.1) (E) and restricted sulA (86-028NP ΔsulA/pGM1.1) (F) were used to initiate biofilm formation for 48 hours. The height of biofilm base or tower formed by CE or TR NTHI was measured in 20 random fields of view from a representative 48-hour biofilm (G). Statistical analysis was performed using a two-tailed t-test. 86-028NP/pGZRS39A and 86-028NP ΔsulA/pSPEC1 were restricted of heme-iron for 24 hours. Both strains were normalized for viability and equivalent numbers of each strain were co-inoculated into the middle ears of chinchillas. Effusions were collected on day 4, 7 and 10 days following inoculation and plated on selective media (H). Dashed line indicated limit of detection. Statistical analysis was performed using a Mann-Whitney U-test.