This work studies the development of the 3D architecture of batch culture P. mirabilis biofilms on the basis of morpho-topological descriptors calculated from confocal laser scanning microscopy (CLSM) stacks with image processing routines. A precise architectonical understanding of biofilm organization on a morpho-topological level is necessary to understand emergent interactions with the environment and the appearance of functionally different progeny swarmer cells. P. mirabilis biofilms were grown on glass coverslips for seven days on LB broth and subjected to in situ immunofluorescence. Confocal image stacks were deconvolved prior to segmentation of regions of interest (ROI) that identify individual bacteria and extracellular material, followed by 3D reconstruction and calculation of different morpho-topological key descriptors. Results showed that P. mirabilis biofilm formation followed a five stage process: (i) reversible adhesion to the surface characterized by slow growth, presence of elongated bacteria, and absence of extracellular material, (ii) irreversible bacterial adhesion concomitant to decreasing elongation, and the beginning of extracellular polymer production, (iii) accelerated bacterial growth concomitant to continuously decreasing elongation and halting of extracellular polymer production, (iv) maturation of biofilm defined by maximum bacterial density, volume, minimum elongation, maximum extracellular material, and highest compaction, and (v) decreased bacterial density and extracellular material through detachment and dispersion. Swarmer cells do not play a role in P. mirabilis biofilm formation under the applied conditions. Our approach sets the basis for future studies of 3D biofilm architecture using dynamic in vivo models and different environmental conditions that assess clinical impacts of P. mirabilis biofilm.
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