Gingichashvili Sarah, Duanis-Assaf Danielle, Shemesh Moshe, Featherstone John D B, Feuerstein Osnat, Steinberg Doron
Biofilm Research Laboratory, Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University-Hadassah, Jerusalem, Israel.
Department of Prosthodontics, Faculty of Dental Medicine, Hebrew University-Hadassah, Jerusalem, Israel.
Front Microbiol. 2017 Nov 7;8:2072. doi: 10.3389/fmicb.2017.02072. eCollection 2017.
Biofilm is commonly defined as accumulation of microbes, embedded in a self-secreted extra-cellular matrix, on solid surfaces or liquid interfaces. In this study, we analyze several aspects of biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing biofilm complexity across different growth conditions. GFP-labeled cells were cultured on agar surfaces over a 4-day period during which microscopic images of developing colonies were taken at equal time intervals. The images were used to perform a computerized analysis of few aspects of biofilm development, based on features that characterize the different phenotypes of colonies. Specifically, the analysis focused on the segmented structure of the colonies, consisting of two different regions of sub-populations that comprise the biofilm - a central "core" region and an "expanding" region surrounding it. Our results demonstrate that complex biofilm of grown on biofilm-promoting medium [standard lysogeny broth (LB) supplemented with manganese and glycerol] is characterized by rapidly developing three-dimensional complex structure observed at its core compared to biofilm grown on standard LB. As the biofilm develops, the core size remains largely unchanged during development and colony expansion is mostly attributed to the expansion in area of outer cell sub-populations. Moreover, when comparing the bacterial growth on biofilm-promoting agar to that of colonies grown on LB, we found a significant decrease in the GFP production of colonies that formed a more complex biofilm. This suggests that complex biofilm formation has a diminishing effect on cell populations at the biofilm core, likely due to a combination of reduced metabolic rate and increased levels of cell death within this region.
生物膜通常被定义为微生物在固体表面或液体界面上的积聚，这些微生物嵌入自我分泌的细胞外基质中。在本研究中，我们使用图像处理领域的工具分析生物膜形成的几个方面。具体而言，我们表征了菌落型生物膜形成的生长动力学和形态特征，并比较了在两种不同类型固体培养基上生长的菌落中的这些特征。此外，我们提出了一个模型，用于评估不同生长条件下生物膜的复杂性。将绿色荧光蛋白（GFP）标记的细胞在琼脂表面培养4天，在此期间，以相等的时间间隔拍摄发育中菌落的显微图像。这些图像用于基于表征菌落不同表型的特征，对生物膜发育的几个方面进行计算机分析。具体而言，分析集中在菌落的分段结构上，该结构由构成生物膜的两个不同亚群区域组成——一个中央“核心”区域和围绕它的“扩展”区域。我们的结果表明，与在标准LB培养基上生长的生物膜相比，在促进生物膜形成的培养基[添加了锰和甘油的标准溶原肉汤（LB）]上生长的复杂生物膜的特征是在其核心观察到快速发展的三维复杂结构。随着生物膜的发育，核心大小在发育过程中基本保持不变，菌落扩展主要归因于外部细胞亚群面积的扩大。此外，当比较促进生物膜形成的琼脂上的细菌生长与LB上生长的菌落时，我们发现形成更复杂生物膜的菌落的GFP产量显著降低。这表明复杂生物膜的形成对生物膜核心处的细胞群体有递减效应，这可能是由于该区域代谢率降低和细胞死亡水平增加共同作用的结果。
