Flagellum-driven motility enhances Pseudomonas aeruginosa biofilm formation by altering cell orientation

Abstract

Bacterial motility plays a crucial role in biofilm development, yet the underlying mechanism remains not fully understood. Here, we demonstrate that the flagellum-driven motility of Pseudomonas aeruginosa enhances biofilm formation by altering the orientation of bacterial cells, an effect controlled by shear stress rather than shear rate. By tracking wild-type P. aeruginosa and its non-motile mutants in a microfluidic channel, we demonstrate that while non-motile cells align with the flow, many motile cells can orient toward the channel sidewalls, enhancing cell surface attachment and increasing biofilm cell density by up to 10-fold. Experiments with varying fluid viscosities further demonstrate that bacterial swimming speed decreases with increasing fluid viscosity, and the cell orientation scales with the shear stress rather than shear rate. Our results provide a quantitative framework to predict the role of motility in the orientation and biofilm development under different flow conditions and viscosities.IMPORTANCEBiofilms are ubiquitous in rivers, water pipes, and medical devices, impacting the environment and human health. While bacterial motility plays a crucial role in biofilm development, a mechanistic understanding remains limited, hindering our ability to predict and control biofilms. Here, we reveal how the motility of Pseudomonas aeruginosa, a common pathogen, influences biofilm formation through systematically controlled microfluidic experiments with confocal and high-speed microscopy. We demonstrate that the orientation of bacterial cells is controlled by shear stress. While non-motile cells primarily align with the flow, many motile cells overcome the fluid shear forces and reorient toward the channel sidewalls, increasing biofilm cell density by up to 10-fold. Our findings provide insights into how bacterial transition from free-swimming to surface-attached states under varying flow conditions, emphasizing the role of cell orientation in biofilm establishment. These results enhance our understanding of bacterial behavior in flow environments, informing strategies for biofilm management and control.

Keywords: Pseudomonas aeruginosa; biofilms; cell motility; fluid flow; shear.

Fig 1

Biofilm formation by motile and non-motile bacterial cells under varying flow conditions. (a) Schematic of the experimental setup. (b) Fluorescence microscopy image showing biofilms formed by motile Pseudomonas aeruginosa (wild-type) cells, labeled with GFP. (c) Fluorescence microscopy image showing scattered distribution of non-motile GFP-labeled P. aeruginosa (ΔfliC) cells. (d) Fluorescence microscopy image showing biofilms formed by motile but pilus-deficient (ΔpilA) cells. Biofilms were stained with dyes specific to exopolymeric substances. (e) Fluorescence microscopy image showing scattered distribution of non-motile (ΔmotAB motCD) cells stained with SYTO-9. Scale bars represent 10 µm for panels b–e. The shear stress is 12 mPa for panels b–e. (f) Comparison of cell density within biofilms (cells/mm²) among motile (WT and ΔpilA) and non-motile (ΔfliC, ΔmotAB motCD) cells under varying shear stress over a 15 hour injection period. Error bars indicate the standard error of the mean from three replicate experiments.

Fig 2

Representative trajectories of motile and non-motile Pseudomonas aeruginosa cells near the sidewall. (a) Sequential fluorescent microscopy images capture the attachment of motile GFP-labeled cells to the biofilm. The white triangles point to one representative cell that moved toward and got attached to the biofilm (the green blur), with its trajectory shown by a solid line. The circles and crosses denote the start and end points of the trajectory, respectively. The green blur represents the biofilm structure slightly out of the focal plane. Scale bars represent 10 µm. (b) Representative sequential fluorescent images showing the trajectory (solid line) of a non-motile (ΔfliC) cell flowing along a streamline and not attaching to the sidewall. The circles and crosses denote the start and end points, respectively. Scale bars represent 5 µm. The circles and crosses represent the start and end points of the trajectories, respectively. y = 0 represents the PDMS sidewall surface.

Fig 3

Cell wall-normal angle in response to fluid flow. (a) Time-lapsed fluorescence microscopy images demonstrate typical behavior of motile cells moving toward and attaching to the PDMS sidewall under shear stress of 12 mPa during a 0.2 s period. Scale bar represents 5 µm. Panels b and c illustrate the probability distribution of the wall-normal angle for wild-type and non-motile (ΔfliC) cells under the same shear stress of 12 mPa, respectively. Insets show the definition of cell wall-normal angle: the angle between the cell’s axis and the y-axis. Panels d and e show the probability distribution of the wall-normal angle for wild-type and non-motile (ΔfliC) cells under the same shear stress of 120 mPa, respectively. Each statistical analysis used around 300 trajectories from three biological replicates. (f) The cumulative success rate to reach the biofilm-forming region (defined as regions where the local velocity u is less than the cell swimming speed v_s) as a function of the average wall-normal angle θ_avg (averaged for each single trajectory) under shear stress of 12 mPa. The inset depicts the definition of the biofilm-forming region. (g) The percentage of cells in the biofilm-forming region as a function of shear stress.

Fig 4

Shear stress-induced cell orientation and enhanced biofilm formation. (a) Fluorescence microscopy images of biofilms formed by wild-type Pseudomonas aeruginosa at increasing Ficoll concentrations (5%, 10%, 15%, and 20%), which increases viscosity while maintaining constant shear rate. Scale bar: 10 µm. (b) Cell density within biofilms (cells/mm²) as a function of wall shear stress for wild-type P. aeruginosa under Ficoll-supplemented conditions (open diamonds). (c) Relative viscosity as a function of the corresponding bacterial swimming speed measured across Ficoll concentrations. The fitted trend line follows: y = − 44log₁₀x + 50. (d) Average wall-normal angle of bacterial cells as a function of wall shear rate. (e) Average wall-normal angle of bacterial cells as a function of wall shear stress. (f) Wall-normal angle as a function of the non-dimensional relative velocity u^*. The fitted trend line follows: y = 18log₁₀x + 50.

Fig 5

The competitive advantage of motility on biofilm formation. (a) Overlap of GFP and mCherry images showing the dominance of motile cells in the biofilm. Yellow color indicates the co-existence of the GFP- and mCherry-labeled cells. The experiment involved injecting GFP-labeled motile and mCherry-labeled non-motile Pseudomonas aeruginosa cells into the microfluidic chamber at the same cell density under a shear stress of 12 mPa for a 15 hour injection period. (b) Formation of a dense biofilm by GFP-labeled motile cells after a 15 hour experiment. (c) Distribution of scattered aggregates of mCherry-labeled non-motile cells (ΔfliC) within the microfluidic chamber. Scale bars represent 20 µm.