Biofilm streamers cause catastrophic disruption of flow with consequences for environmental and medical systems

Abstract

Biofilms are antibiotic-resistant, sessile bacterial communities that occupy most moist surfaces on Earth and cause chronic and medical device-associated infections. Despite their importance, basic information about biofilm dynamics in common ecological environments is lacking. Here, we demonstrate that flow through soil-like porous materials, industrial filters, and medical stents dramatically modifies the morphology of Pseudomonas aeruginosa biofilms to form 3D streamers, which, over time, bridge the spaces between obstacles and corners in nonuniform environments. We discovered that accumulation of surface-attached biofilm has little effect on flow through such environments, whereas biofilm streamers cause sudden and rapid clogging. We demonstrate that flow-induced shedding of extracellular matrix from surface-attached biofilms generates a sieve-like network that captures cells and other biomass, which add to the existing network, causing exponentially fast clogging independent of growth. These results suggest that biofilm streamers are ubiquitous in nature and strongly affect flow through porous materials in environmental, industrial, and medical systems.

Fig. 1.

Biofilm streamers cause rapid and sudden clogging. (A) A constant pressure difference Δp drives a suspension of P. aeruginosa cells through the model microfluidic channel, which is 200 μm wide and 90 μm high. (B) Measurement of flow rate versus time. The flow rate through this channel only changes slowly during biofilm buildup on the walls of the channel for the time period T. Channel walls are indicated by dashed white lines, and cells constitutively express gfp. Biofilm streamers expand rapidly and cause clogging over a short time τ.

Fig. 2.

Cell growth sets T, while τ is due to a transport process. (A) Semilogarithmic plot of the accumulation of cells on the walls, measured via GFP fluorescence. Different colors represent data from n = 10 independent experiments. (B) T depends on flow rate, and can be prolonged by slowing growth with a low concentration of the growth-inhibitor tetracycline (tet). (C) Tetracycline has no effect on τ. (D) For the first 43 h, cells expressing gfp are flowed through the channel at a rate 18.1 ± 0.05 μL/min. Subsequently, the in-flowing culture is exchanged to contain only cells producing the red fluorescent protein mCherry. Biofilm streamers are exclusively composed of red cells, whereas very few red cells attach to the resident green biofilm on the wall, indicating that streamers consist of cells that were transported to the eventual clogging site by flow (Movie S1).

Fig. 3.

Biofilm filaments form a sieve-like network that captures cells flowing through. (A) A model based on a nonporous biofilm streamer oriented transverse to the flow direction predicts slow growth rates for the streamer radius R. (B) A model based on a porous streamer, which grows by capturing cells that flow through it, predicts exponential growth for R (SI Text). (C) Image of the biofilm during the clogging transition for an initial flow rate 1.5 ± 0.05 μL/min. P. aeruginosa cells are shown in red, EPS is visualized with green fluorescent dyes conjugated to polysaccharide-binding lectins and a green fluorescent DNA stain. Yellow regions result from the superposition of green and red channels. White arrows point to smaller biofilm streamers that form a network. The thick streamer structures are interspersed with dark regions, indicating that these structures are porous. The porosity is further illustrated by Fig. S3 and Movie S2. (D) Clogging duration τ for different flow rates (which are proportional to U, the average flow speed before streamers emerge) at a fixed concentration of ≈ 2 × 10⁸ cfu/mL, corresponding to midlogarithmic-phase growth. (E) τ for different cell concentrations at a fixed flow rate of 4.8 ± 0.8 μL/min. Error bars: SD of n = 8 independent measurements.

Fig. 4.

Mutants that affect biofilm formation in nonuniform environments. (A, B) Comparison of the time until clogging T and the duration of the clogging transition τ for different mutants: ΔpelA lacks the major component of the EPS, ΔflgK is nonmotile due to an incomplete flagellum (21), ΔpilC has no type IV pili, and ΔlasR lacks the quorum sensing master regulator. (C) ΔpelA produces no significant biofilm during 210 h of observation. (D) ΔflgK produces biofilm streamers similar to the wild type. (E) ΔpilC forms no streamers, but does form thick biofilms on the walls of the channel. (F) ΔlasR forms biofilms on the walls of the channel, which detach, slowly deform, and reattach to clog the channel. The image lookup table is the same for C–F. Scale bars: 200 μm.

Fig. 5.

Biofilm streamers form in diverse environments. (A) Time series of biofilm buildup in a 3D soil-like porous material made from transparent Nafion granules (outlined by red dashed lines). Green indicates P. aeruginosa cells constitutively expressing gfp. Arrows point toward streamers, which are heterogeneous in thickness at this magnification. (B) Networks of biofilm streamers form in a feed spacer mesh, which is a component of spiral-wound reverse osmosis water filters. The image is a maximum-intensity projection of a confocal z-stack, which visualizes biofilms on the surface of the mesh, located outside the white dashed lines. (C) Biofilm streamers form in bare-metal stents. White arrows point to streamers; green arrows point to wire mesh of the stent. The image is stitched together from the maximum intensity projections of 83 z-stacks. A false-color scheme is used to illustrate that different color intensity scales were used for visualizing the stent surface and the streamers because the fluorescence from the stent surface was significantly brighter due to the large amount of biomass on the surface. The resulting two images of the stent surface and the streamers were overlaid, giving the displayed image. Green indicates P. aeruginosa cells constitutively expressing gfp. Arrows point toward streamers, which are heterogeneous in thickness and biomass at this magnification.