Self-induced mechanical stress can trigger biofilm formation in uropathogenic Escherichia coli

Abstract

Bacterial biofilms represent an important medical problem; however, the mechanisms of the onset of biofilm formation are poorly understood. Here, using new controlled methods allowing high-throughput and reproducible biofilm growth, we show that biofilm formation is linked to self-imposed mechanical stress. In growing uropathogenic Escherichia coli colonies, we report that mechanical stress can initially emerge from the physical stress accompanying colony confinement within micro-cavities or hydrogel environments reminiscent of the cytosol of host cells. Biofilm formation can then be enhanced by a nutrient access-modulated feedback loop, in which biofilm matrix deposition can be particularly high in areas of increased mechanical and biological stress, with the deposited matrix further enhancing the stress levels. This feedback regulation can lead to adaptive and diverse biofilm formation guided by the environmental stresses. Our results suggest previously unappreciated mechanisms of the onset and progression of biofilm growth.

Fig. 1

Pressure buildup as a result of confined growth leads to biochemical stress response. a Schematic cross-section of the microfluidic device, illustrating the deformation of the flexible roof due to bacterial colony growth. b The deformation of the roof [black area between blue (Alexa Fluor 647) and green (GFP-expressing E. coli) layers] is visualized with CLSM. Scale bar for the vertical dimension, 20 μm. c Maximum pressures produced by different bacteria; E. coli JM105 (n = 9); E. coli CFT073 (n = 7); P. aeruguinosa (n = 4); S. cohnii (n = 5). d Distributions of rpoH expression-reporting GFP fluorescence intensity per pixel in a WFM image of a chamber (in arbitrary units ranged 25 to 225) at various time points, starting 3.5 h after seeding cells into the chamber. The arrow (t = 4.5 h) indicates when the chamber is completely filled with cells. The color of the plot shows the mean intensity at the corresponding time point. e The integral fluorescence of bacterial colony in the chamber divided by the chamber volume measured at the same time point. The data is normalized to the time point just before the roof deformation becomes detectable (t = 4.5 h in Fig. 1d) (n = 3). f GFP expression due to rpoH upregulation measured from xy-sections of confocal imaging. The data was normalized to the time point when roof deformation started to become measurable (t = 6.5 h in Fig. 1b, indicated by the blue arrow) (n = 3). Error bars are SD

Fig. 2

Spatial correlation between expression of biofilm-associated cell surface structures and biochemical stress response. a Expression of biofilm-associated cell-surface structures, EPS d-(+)-glucose and mannose groups (red) and curli (orange), as detected using rhodamine-labeled concanavalin A (10 µg/mL) and Congo Red dye (10 µg/mL), respectively, before (top row) and after (bottom row) roof deformation in uropathogenic E. coli populations. Scale bar, 10 µm. Bar graphs show major increases in expression of biofilm-related factors after roof deformation (n = 5). Error bars are SD. b Spatial distribution of GFP, as a reporter of rpoH expression, and of rhodamine-labeled concanavalin A (EPS stain) in E. coli JM105 after roof deformation. Dashed lines show boundaries of the deformable roof. Scale bars, 50 µm. c Agent-based (top) and mean field (bottom) simulations of the stress response distribution reproduce the characteristic patterns observed in GFP and EPS expression. The intensity of green color encodes the stress response. The spatial correlation between the experimentally measured expressions of GFP and EPS and simulated stress responses (agent-based: black dotted curve; mean field: black dashed curve) further supports the connection between mechanical stress and biofilm formation

Fig. 3

Confinement-dependent formation of a selective penetration barrier confers increased antibiotic tolerance. SYTO 9 (green, left) dye, mimicking positively-charged antibiotics was used to assess the penetrability of bacterial colonies at low cell density (top), high cell density before roof deformation (middle), and high density after deformation (bottom). Separately, antibiotic tolerance was assessed with 10x MIC gentamicin (15 µg/mL) treatment for 3 h followed by PI (red, right, to stain dead cells with compromised membranes) staining under identical density conditions. Dotted line boxes indicate regions proximal to the flow-through channels. Bar graphs indicate extent of SYTO 9 penetration after roof deformation and the percentage of PI-stained areas in regions proximal to the flow-through channels after antibiotic treatment in 100 and 150 µm wide chambers. Error bars are SD (n = 3); **P < 0.01; two-tailed Student’s t-test. Scale bar, 50 µm

Fig. 4

Biofilm growth in environment with mechanical resistance. a Filmstrip of a simulated colony growing in hydrogel. The green color scale encodes the level of stress response. b Spatial distributions of stress-reporting GFP and biofilm-like cell-surface structures in E. coli (JM105) colonies grown in 1% PuraMatrix hydrogel. CLSM was used to visualize both the rhodamine-labeled concanavalin A-stained EPS d-(+)-glucose and mannose groups and the Congo Red-stained curli after ~6 h of growth. Scale bar, 20 µm. c Viability comparison between planktonic and 6 h hydrogel cultures after exposure to 20 μg/mL of kanamycin (JM105) (n = 2 for control, n = 3 for gel), 20 μg/mL of gentamicin (CFT073) (n = 3), and 10 μg/mL of ampicillin (CFT073) (n = 3 for control, n = 4 for gel). Error bars are SD