Microscopic bacterial microcolonies are responsible for the formation of granulomas.

<p>A) BALB/c mice (n = 3 per group) received 10⁹ (red) or 10^{9 Highly purified single-cell} (purple) NbGFP CFUs and were weighted daily. B) Lungs from mice in A were analysed at day 4 post infection by whole lung imaging at λ 488 nm. Representative images of two lungs per group are shown. C) BALB/c mice (n = 5 per group) received 10⁹ (red) CFUs or ≈100 (teal) NbGFP microcolonies and were weighted daily. D) Lungs from mice in D were analysed at day 4 post infection by whole lung imaging at λ 488 nm. Representative images of two lungs per group are shown. White arrows point to GFP⁺ lesions. E) Representative histology of lungs in mice receiving 10^{9 Highly purified single-cell} CFUs at day 4 post infection. Kinyoun's stain. F) Representative images of lesions present in mice receiving microcolonies. Kinyoun's stain. Red stained NbGFP is evident at the core of the lesion. G) Representative H&E image of lesions present in mice 21 days after infection with purified microcolonies. White arrows: NbGFP, black arrows: activated macrophages, black arrowheads: lymphocytes.</p

Neither alveolar macrophages nor eosinophils nor neutrophils are targets for NbGFP infection in the lung.

<p>A) Bone marrow derived macrophages (BMDM) or alveolar macrophages (AM) were incubated <i>in vitro</i> with NbGFP (1:10) and imaged at 2 hours and at 48 hrs post infection. Cells from BALF of infected mice were imaged 48 hrs after infection to look for infected alveolar macrophages. Representative images are shown. B) BALF analysis of eosinophils by flow cytometry. Left panel shows non-specific events detected within the eosinophil gate in the absence of CCR3 staining. Right panel shows no increase in GFP+ events amongst eosinophils stained with an anti-CCR3 antibody. C) BALF analysis of granulocytes by flow cytometry at 24 (left panel) and 48 (right panel) hours post infection.</p

NbGFP microcolonies are present in the lungs of infected mice as early as 6 hours post infection.

<p>A) Lungs from mice receiving 10⁹ CFUs were analysed at 6 hours, 1 day and 4 days post infection by whole lung imaging at λ 488 nm. Representative images of two lungs per time point are shown. B) Confocal microscopy images of lung sections at 6 and 24 hours post infection. C) Growth curve of NbGFP under constant stirring to prevent biofilm formation.</p