Topography and Expansion Patterns at the Biofilm-Agar Interface in Bacillus subtilis Biofilms

Abstract

Bacterial biofilms are complex microbial communities which are formed on various natural and synthetic surfaces. In contrast to bacteria in their planktonic form, biofilms are characterized by their relatively low susceptibility to anti-microbial treatments, in part due to limited diffusion throughout the biofilm and the complex distribution of bacterial cells within. The virulence of biofilms is therefore a combination of structural properties and patterns of adhesion that anchor them to their host surface. In this paper, we analyze the topographical properties of Bacillus subtilis’ biofilm-agar interface across different growth conditions. B. subtilis colonies were grown to maturity on biofilm-promoting agar-based media (LBGM), under standard and stress-inducing growth conditions. The biofilm-agar interface of the colony type biofilms was modeled using confocal microscopy and computational analysis. Profilometry data was obtained from the macrocolonies and used for the analysis of surface topography as it relates to the adhesion modes present at the biofilm-agar interface. Fluorescent microspheres were utilized to monitor the expansion patterns present at the interface between the macrocolonies and the solid growth medium. Contact surface analysis reveals topographical changes that could have a direct effect on the adhesion strength of the biofilm to its host surface, thus affecting its potential susceptibility to anti-microbial agents. The topographical characteristics of the biofilm-agar interface partially define the macrocolony structure and may have significant effects on bacterial survival and virulence.

Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness

“…Finally, the variation in channel width inside mature biofilms was investigated: channel width increased non-linearly along the radial direction from the centre of the biofilm under all nutrient conditions. We hypothesise that channel width is larger at the edge of the colony due to rapid cell growth in nutrient sufficient conditions, resulting in non-uniform radial expansion, as previously observed for B. subtilis [40] and V. cholerae [4]. Remarkably, channels were on average 50% wider at the centre of carbon-limited media than inside nitrogen-limited media.…”

“…We speculate that the increase in channel width at the edge of the colony is due to rapid cell growth in nutrient-rich conditions [36]. This would result in, non-uniform radial expansion, as previously observed for B. subtilis [31] and V. cholerae [6], suggesting that variations in channel width are an emergent property of E. coli biofilm growth.…”

“…Channels formed on substrates with limiting carbon or nitrogen concentrations expanded radially outwards, in approximately straight lines. This could be because bacteria growing on nutrient-depleted substrates adhere less strongly on the surface of attachment, and expand more rapidly [40], or may be indicative of nutrient foraging behaviour. On the other hand, channels formed on nutrient-rich substrates formed complex fractal patterns.…”

An Open-Source Computational Tool for Measuring Bacterial Biofilm Morphology and Growth Kinetics upon One-Sided Exposure to an Anti-Microbial Source

“…Mature B. subtilis macrocolonies are characterized by a well-defined central core that differs morphologically from colony periphery. Furthermore, B. subtilis biofilms have been found to be highly adaptive in terms of their morphology to changing environmental conditions 5,6 .…”

The role of surface adhesion on the macroscopic wrinkling of biofilms

“…Recent studies found that the adhesive strength and friction between biofilm and substrate might play a role in virulence as well as the structural evolution of the biofilm ( Fei et al, 2020 ; Cont et al, 2020 ). However, many experimental studies use agar as a substrate where adhesion appears to be spatially and temporally heterogeneous ( Gingichashvili et al, 2021 ). Therefore, systematic investigations of the interplay between adhesive strength and the formation of folds are needed to understand better the mechanical instabilities that govern biofilm morphology.…”