Bacillus subtilis Biofilm Development – A Computerized Study of Morphology and Kinetics

Abstract

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 Bacillus subtilis biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of B. subtilis colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing B. subtilis biofilm complexity across different growth conditions. GFP-labeled B. subtilis 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 B. subtilis 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 B. subtillis 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.

The Adaptive Morphology of Bacillus subtilis Biofilms: A Defense Mechanism against Bacterial Starvation

“…Similar to colonies grown on agar that lacks glycerol and manganese (non-biofilm-promoting, [21]), the nutrient-deprived colonies also demonstrated a more stable bacterial GFP signal production at their core with a smaller characteristic decrease of GFP signal over time ( Figure 3B). In both cases, the loss of complex wrinkled formations at the colony core was accompanied by a smaller decrease in the GFP signal produced by the bacterial cells at the colony core, supporting a previous suggestion that there is an inherent "cost" to the formation of the network of wrinkles in the long term [21].…”

“…B. subtilis biofilms, for example, when grown under biofilm-promoting conditions, develop a complex network of such wrinkles but only at the colony core, not the periphery. Under standard biofilm-promoting conditions, the wrinkled structure is a reflection of the biofilm's elasticity [21]. Conversely, the channels that develop under nutrient-limiting conditions appear straight and span the entire colony from the center to the very edge of the biofilm.…”

Biological Activity and Antibiofilm Molecular Profile of Citrus aurantium Essential Oil and Its Application in a Food Model

“…Bacilus subtilis growth kinetics and morphological features were characterized for colony type biofilm formation previously [ 64 ]. The complexity of B. subtillis biofilm is characterized by a rapidly developing three-dimensional complex structure with the core size remains largely unchanged while the colony expansion is mostly attributed to the growth in the area of outer cell subpopulations [ 65 ]. Oliveira et al [ 66 ] indicated that CAEOs were effective in controlling multi-species biofilms.…”

Antioxidant, Antimicrobial and Antibiofilm Activity of Coriander (Coriandrum sativum L.) Essential Oil for Its Application in Foods

“…subtilis is a well-known model microorganism to study the molecular principles of biofilm development. B. subtilis growth kinetics and morphological features were characterized with a colony type biofilm formation [37]. Biofilm of B. subtillis is quickly developing into a three-dimensional complex structure with an unchanged core and expansion, which is mostly related to the outer cell subpopulations enlargement.…”