Transient restriction promotes intracellular bacterial community formation.

86-028NP that was restricted (A, B) for heme-iron was individually inoculated into the chinchilla middle ears. Four days post inoculation, infected middle ear bullae was fixed and processed for histological examination. Scale bar = 10 µm. 86-028NP/pGM1.1 was cultured for 24 hours in the presence or absence of heme-iron to generate “replete” and “restricted” populations (CE or TR). Nutritionally conditioned NTHI were co-cultured with chinchilla middle ear epithelial cells for 48 hours. Epithelial cell surface was labeled with wheat germ agglutinin conjugated to Alexa-Fluor 594 (red). Biofilms were visualized by GFP fluorescence (green), imaged using optical sections and reconstructed to generate a 3D view from the top (large pane) including orthogonal views of the side (top and left side) (C, D). Optical sections that depict additional representative images of the localization of NTHI based upon nutritional conditioning (E, F). Scale bar = 10 µm.