Polymer-induced microcolony compaction in early biofilms: A computer simulation study

Microscopic organisms, such as bacteria, have the ability of colonizing surfaces and developing biofilms that can determine diseases and infections. Most bacteria secrete a significant amount of extracellular polymer substances that are relevant for biofilm stabilization and growth. In this work, we apply computer simulation and perform experiments to investigate the impact of polymer size and concentration on early biofilm formation and growth. We observe as bacterial cells formed loose, disorganized clusters whenever the effect of diffusion exceeded that of cell growth and division. Addition of model polymeric molecules induced particle self-assembly and aggregation to form compact clusters in a polymer size- and concentration-dependent fashion. We also find that large polymer size or concentration lead to the development of intriguing stripe-like and dendritic colonies. The results obtained by Brownian dynamic simulation closely resemble the morphologies that we experimentally observe in biofilms of a Pseudomonas Putida strain with added polymers. The analysis of the Brownian dynamic simulation results suggests the existence of a threshold polymer concentration that distinguishes between two growth regimes. Below this threshold, the main force driving polymer-induced compaction is the hindrance of bacterial cell diffusion, while collective effects play a minor role. Above this threshold, especially for large polymers, polymer-induced compaction is a collective phenomenon driven by depletion forces. Well above this concentration threshold, severely limited diffusion drives the formation of filaments and dendritic colonies.

Figure 1

Top row, from left to right: snapshots of configurations in absence of polymers at $\mathrm{\Gamma }=1.67\times {10}^{-2}$, containing 8, 16, 32, and 64 bacteria halfway through the lengthening cycle ($L^{*}=3.9$). The color gradient indicates different bacterial orientations. Bottom row, left frame: coverage profile, $g\left(r\right)$, of colonies containing 8, 16, 32, and 64 bacteria at $L^{*}=3.9$; middle and right frames: mean-square displacement, ${\langle \mathrm{\Delta }{r}^2\rangle }_{N_B}$, and orientational autocorrelation function, ${\langle E_2\left(t\right)\rangle }_{{\mathrm{N}}_{\mathrm{B}}}$, as a function of the bacterium aspect ratio, $L^{*}$, in colonies containing 1 ($•$), 2 ($\begin{array}{c}◯\end{array}$), 4 ($\square$), 8 ($◇$), 16 ($▵$), 32 ($◃$), and 64 ($▿$) bacteria. Error bars are standard deviations.

Figure 2

Clusters of 8, 16, 32, and 64 bacteria halfway through the lengthening cycle ($L^{*}=3.9$) containing polymer particles of diameter ${\sigma }_p=0.05\sigma$. From top to bottom: $N_p={10}^3,{10}^4,5\times {10}^4$, and ${10}^5$. The color gradient indicates different bacterial orientations. Polymer particles are not shown.

Figure 3

Surface coverage profiles $g\left(r\right)$ for colonies of 8 (circles), 16 (square), 32 (diamonds), and 64 (triangles) bacteria with $L^{*}=3.9$. The number of polymers is $N_p={10}^4$ (blue lines and symbols), $5\times {10}^4$ (red lines and symbols) and ${10}^5$ (green line and symbols). The diameter of the polymer particles is ${\sigma }_p=0.05\sigma$ (left frame), ${\sigma }_p=0.1\sigma$ (middle frame), and ${\sigma }_p=0.5\sigma$ right frame.

Figure 4

Colonies of 8, 16, 32, and 64 bacteria halfway through the lengthening cycle ($L^{*}=3.9$) containing polymer particles of diameter ${\sigma }_p=0.5\sigma$. From top to bottom: $N_p={10}^3,{10}^4,5\times {10}^4$, and ${10}^5$. The color gradient indicates different bacterial orientations. Polymer particles are not shown.

Figure 5

Micrographs of microcolonies containing $40$ cells of $\mathrm{\Delta }$fleQ P. putida strain MRB52 at different dextran sulfate concentration.

Figure 6

MSDs as a function of bacteria aspect ratio $L^{*}$ for colonies containing 1 to 64 bacteria and $N_p={10}^4$ (left), $N_p=5\times {10}^4$ (middle), and $N_p={10}^5$ (right) polymer particles with diameter ${\sigma }_p=0.1\sigma$. Symbols refer to colonies containing 1 ($•$), 2 ($\begin{array}{c}◯\end{array}$), 4 ($\square$), 8 ($◇$), 16 ($▵$), 32 ($◃$), and 64 ($▿$) bacteria. The inset in the right frame magnifies the panel where it is included. Solid lines are guides for the eye and error bars represent the standard deviation of the mean.

Figure 7

MSDs as a function of bacterium aspect ratio $L^{*}$ for colonies containing 1 to 64 bacteria and $N_p={10}^4$ (left), $N_p=5\times {10}^4$ (middle) or $N_p={10}^5$ (right) polymer particles with diameter ${\sigma }_p=0.5\sigma$. Symbols refer to colonies containing 1 ($•$), 2 ($\begin{array}{c}◯\end{array}$), 4 ($\square$), 8 ($◇$), 16 ($▵$), 32 ($◃$), and 64 ($▿$) bacteria. The insets in each frame magnify the panel where they are included. Solid lines are guides for the eye and error bars represent the standard deviation of the mean.

Figure 8

Orientational correlation function, ${\langle E_2\left(t\right)\rangle }_{{\mathrm{N}}_{\mathrm{B}}}$, as a function of bacterium aspect ratio $L^{*}$ for colonies containing 1 to 64 bacteria and $N_p={10}^4$ (left), $N_p=5\times {10}^4$ (middle), or $N_p={10}^5$ polymer particles (diameter ${\sigma }_p=0.5\sigma$). Symbols refer to colonies containing 2 ($\begin{array}{c}◯\end{array}$), 4 ($\square$), 8 ($◇$), 16 ($▵$), 32 ($◃$), and 64 ($▿$) bacteria. Solid lines are guides for the eye and error bars represent the standard deviation of the mean.