Self-Adaptation of Pseudomonas fluorescens Biofilms to Hydrodynamic Stress

Abstract

In some conditions, bacteria self-organize into biofilms, supracellular structures made of a self-produced embedding matrix, mainly composed of polysaccharides, DNA, proteins, and lipids. It is known that bacteria change their colony/matrix ratio in the presence of external stimuli such as hydrodynamic stress. However, little is still known about the molecular mechanisms driving this self-adaptation. In this work, we monitor structural features of Pseudomonas fluorescens biofilms grown with and without hydrodynamic stress. Our measurements show that the hydrodynamic stress concomitantly increases the cell density population and the matrix production. At short growth timescales, the matrix mediates a weak cell-cell attractive interaction due to the depletion forces originated by the polymer constituents. Using a population dynamics model, we conclude that hydrodynamic stress causes a faster diffusion of nutrients and a higher incorporation of planktonic bacteria to the already formed microcolonies. This results in the formation of more mechanically stable biofilms due to an increase of the number of crosslinks, as shown by computer simulations. The mechanical stability also relies on a change in the chemical compositions of the matrix, which becomes enriched in carbohydrates, known to display adhering properties. Overall, we demonstrate that bacteria are capable of self-adapting to hostile hydrodynamic stress by tailoring the biofilm chemical composition, thus affecting both the mesoscale structure of the matrix and its viscoelastic properties that ultimately regulate the bacteria-polymer interactions.

Keywords: NMR; Pseudomonas fluorescens; active matter; biofilms; computer simulations; extracellular matrix; mechanical properties.

Figure 1

Evolution of Pseudomonas fluorescens B52 biofilms parameters under shaking (blue) and static conditions (red). (A) Attached cell population. (B) % of surface coverage. (C) Images of Coomassie blue stained coupons over time. (D) Optical density proportional to the biomass production (OD; cells + matrix). Asterisks indicate statistically significant differences between shaking and static biofilms (n = 10; p < 0.05).

Figure 2

CLSM images of different sections of 48 h P. fluorescens biofilms developed at 20°C under shaking (top panel) and static conditions (down panel). The red channel corresponds to the zenital 3D view of P. fluorescens biofilm matrix stained with Sypro. The green channel corresponds to the zenital 3D view of P. fluorescens biofilm cells stained with Syto. Matrix appears in red and cells in green in merged images (scale bars are 20 μm). The z cross-sections show the width of a representative biofilm of those obtained, being the average of maximum height (28 ± 4) μm and (17 ± 3) μm in shaking and static biofilms, respectively (n = 3; scale bars are 20 μm). Pie charts represent the percentage of the volume occupied by cells (in green) and by matrix (in red) for each type of biofilm. Concretely, (53 ± 4) and (47 ± 4) for shaking biofilms and (72 ± 7) and (28 ± 7) for static ones (n = 3).

Figure 3

Model for biofilm formation. Population density in the biofilm (green thick line) and in the plankton (gray thin line), nutrient concentration in the biofilm (green dashed line) and in the plankton (gray dashed line). Experimental values (blue and red symbols, shaking and static, respectively) normalized to the cell count at 24 h. (A) Shaking: to mimic an efficient diffusion of nutrients and bacteria and strong adhesion respectively, we consider a high transition rate from plankton to biofilm (attachment) and diffusion (k⁺ = 1 h⁻¹, D = 1 h⁻¹) and low transition rate from biofilm to plankton (detachment; k⁻ = 1 Å~10⁻³ h⁻¹). (B) Static: to mimic a weak diffusion of nutrients/bacteria and poor adhesion, we consider a low attachment rate (k⁺ = 25 Å~10⁻³ h⁻¹) and diffusion (D = 0.05 h⁻¹) and high detachment rate (k⁻ = 1 h⁻¹; low diffusion and low adhesion). (C) Number of bacteria (log scale) versus time as calculated from full atomistic simulations (black line), reported together with the short time data in panels (A,B). (D) Onset of the biofilm formation, as in atomistic simulations. The elongated red particles mimic the bacteria, while polymers are simulated as an implicit effective attraction between bacteria since the first stages of bacterial growth happen on a surface, bacteria have been simulated in two dimensions. Data correspond to the average ± SD (n = 10).

Figure 4

Mechanical properties of P. fluorescens biofilms as measured by rheology and computer simulations. (A) Stress-strain plot P. fluorescens at different incubation times under oscillatory shear (f = 1 Hz and T = 25°C). Biofilms were grown under shaking (left panel) and static (right panel) conditions. The mechanical response is found linear (dashed line) up to shear deformations of nearly ~20 and ~100% respectively, where a yield point is clearly visible in the σ-γ plots. Beyond this limit, the responses are nonlinear, and the two systems behave as a plastic body that yields under further stress. (B) G', G'', and η values plotted as a function of the incubation time for P. fluorescens biofilms grown under shaking (blue) and static (red) conditions. Stars indicate that similar values were obtained for pure water. (C) Snapshots of the simulated biofilm grown under static (up) and shaking (down) conditions. Green beads are bacteria, and the yellow beads are polymers. The beads size has been chosen for visualization. (D) Ratio between the stress response of the biofilm grown under shaking and the one of the statically-grown biofilm, as a function of the ratio between the number of crosslinks in the two systems. Data computed with numerical simulations (Supplementary Material), and referring to different shear amplitudes within the linear regime response. The quadratic fitting function is 0.75x² + 4.4x − 1.14 (black dashed curve).

Figure 5

¹³C CPMAS spectra of biofilm samples. (A) Spectrum of a biofilm grown under shaking conditions. (B) Spectrum of a biofilm grown under static conditions.