Transient restriction promotes intracellular bacterial community formation.

<p>86-028NP that was restricted (<b>A, B</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) (<b>C, D</b>). Optical sections that depict additional representative images of the localization of NTHI based upon nutritional conditioning (<b>E, F</b>). Scale bar = 10 µm.