Department of Microbiology, Instituto de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, Montevideo, Uruguay.
J Microbiol Methods. 2011 Nov;87(2):234-40. doi: 10.1016/j.mimet.2011.07.021. Epub 2011 Aug 12.
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.
本研究基于共聚焦激光扫描显微镜 (CLSM) 堆栈和图像处理程序计算的形态拓扑描述符,研究了批培养奇异变形菌生物膜的 3D 结构的发展。在形态拓扑层面上精确理解生物膜的组织架构对于理解与环境的新兴相互作用以及功能不同的游动细胞后代的出现是必要的。奇异变形菌生物膜在 LB 肉汤中培养 7 天,在玻璃盖玻片上生长,并进行原位免疫荧光。在对识别单个细菌和细胞外物质的感兴趣区域 (ROI) 进行分割之前,对共聚焦图像堆栈进行反卷积,然后进行 3D 重建和不同形态拓扑关键描述符的计算。结果表明,奇异变形菌生物膜的形成经历了五个阶段:(i)可逆地附着在表面,其特征为生长缓慢、存在伸长的细菌和不存在细胞外物质,(ii)不可逆的细菌附着伴随着伸长的减少和细胞外聚合物的产生,(iii)细菌生长的加速伴随着不断减少的伸长和细胞外聚合物的生产停止,(iv)生物膜的成熟定义为最大细菌密度、体积、最小伸长、最大细胞外物质和最高的压实度,以及(v)通过脱落和分散导致细菌密度和细胞外物质减少。在应用条件下,游动细胞在奇异变形菌生物膜形成中不起作用。我们的方法为使用动态体内模型和不同环境条件研究 3D 生物膜结构奠定了基础,这些条件评估了奇异变形菌生物膜的临床影响。