High density waves of the bacterium Pseudomonas aeruginosa in propagating swarms result in efficient colonization of surfaces

Abstract

This work describes a new, to our knowledge, strategy of efficient colonization and community development where bacteria substantially alter their physical environment. Many bacteria move in groups, in a mode described as swarming, to colonize surfaces and form biofilms to survive external stresses, including exposure to antibiotics. One such bacterium is Pseudomonas aeruginosa, which is an opportunistic pathogen responsible for both acute and persistent infections in susceptible individuals, as exampled by those for burn victims and people with cystic fibrosis. Pseudomonas aeruginosa often, but not always, forms branched tendril patterns during swarming; this phenomena occurs only when bacteria produce rhamnolipid, which is regulated by population-dependent signaling called quorum sensing. The experimental results of this work show that P. aeruginosa cells propagate as high density waves that move symmetrically as rings within swarms toward the extending tendrils. Biologically justified cell-based multiscale model simulations suggest a mechanism of wave propagation as well as a branched tendril formation at the edge of the population that depends upon competition between the changing viscosity of the bacterial liquid suspension and the liquid film boundary expansion caused by Marangoni forces. Therefore, P. aeruginosa efficiently colonizes surfaces by controlling the physical forces responsible for expansion of thin liquid film and by propagating toward the tendril tips. The model predictions of wave speed and swarm expansion rate as well as cell alignment in tendrils were confirmed experimentally. The study results suggest that P. aeruginosa responds to environmental cues on a very short timescale by actively exploiting local physical phenomena to develop communities and efficiently colonize new surfaces.

Figure 1

(A) Schematic diagram of a cell representation with three nodes connected together characterized by basic parameters. (B) Cell search box of a cell to make cell-cell alignment.

Figure 2

Pseudomonas aeruginosa swarming and RL concentration over time. Swarms on soft agar form tendrils when high concentrations of RL are present (snapshots taken from Movie S1). Both P. aeruginosa cell density (blue-green) and RL (red-fire) concentrations distribute as a wave (white) through the developing swarm. Images at 6-h intervals (from 12 to 30 h) are presented (Scale bar = 15 mm).

Figure 3

(A) Area of bacterial swarms as a function of time. Green and dark red triangles represent swarming on hard agar plates, and the red line represents swarming on soft agar plates (Movie S1). (B) Image intensity profiles of the swarming P. aeruginosa colony on soft agar as functions of the radial distance at time intervals of 111.25 min. Image intensity values were normalized with respect to the value at the center of the bacterial colony. Assuming that intensity is proportional to the bacterial density, the shown profiles demonstrate the dynamics of the cell density along the radial direction of the colony.

Figure 4

(A) Positions of the internal wave (IW) and of the swarm edge (SE) on soft agar as a function of time. Speeds of SE expansion are 0.05 and 0.18 cm/h in experiments (black line) and simulation (light blue dashed line), respectively. Speeds of IW propagation are 0.08 and 0.24 cm/h in experiments (purple line) and simulation (dark blue line), respectively. (B) Nondimensional bacterial density as a function of time. IW corresponds to the internal wave, and SE corresponds to the density near the swarm edge.

Figure 5

Calculation of orientation correlation of P. aeruginosa cells at the swarm edge and in the internal wave. Experimental images show one representative horizontal plane acquired using confocal microscopy to obtain multiple planes (in the z-direction) of green fluorescent protein-labeled cells. (A) Cells are closely packed and form rafts in a tendril at the swarm edge. The tendril is divided into four regions (as marked in the figure). Orientation correlation for each region was computed. (B) Patterning of cells within one plane within the cell wave. Cells also form rafts. Although cells in the wave of high cell density show certain alignment with neighboring cells, cells at some distance from each other display random orientation. (C) Orientation correlations for each region indicated by green-dotted, cyan-dotted, purple-dotted, and blue-dotted lines, respectively, and red-dotted line for the region of the wave of high cell density. Calculation of orientation correlations reveals that cells in the tendril show strong alignment with each other, whereas cells in the wave of high cell density display random orientation with respect to each other.

Figure 6

Simulation of primary and secondary tendril development. (A) Water height profile in a simulation at t = 7 h. (B) Cell density distribution in a simulation at t = 7 h. (C) Experimental image of a swarm on soft agar for comparison. Primary and secondary tendrils are seen to develop at the swarm edge.

Figure 7

Comparison of swarm area dynamics in experiments and simulations. Black and blue lines indicate model predictions for soft and hard agar cases, respectively. Triangles represent data for swarming on hard agar plates. Experimental data for swarming on soft agar plates, represented by diamonds, agree well with the simulated data represented by a black line. Experimental data (triangles) and simulated data (blue line) also agree well with each other.