Stenosis triggers spread of helical Pseudomonas biofilms in cylindrical flow systems

Abstract

Biofilms are multicellular bacterial structures that adhere to surfaces and often endow the bacterial population with tolerance to antibiotics and other environmental insults. Biofilms frequently colonize the tubing of medical devices through mechanisms that are poorly understood. Here we studied the helicoidal spread of Pseudomonas putida biofilms through cylindrical conduits of varied diameters in slow laminar flow regimes. Numerical simulations of such flows reveal vortical motion at stenoses and junctions, which enhances bacterial adhesion and fosters formation of filamentous structures. Formation of long, downstream-flowing bacterial threads that stem from narrowings and connections was detected experimentally, as predicted by our model. Accumulation of bacterial biomass makes the resulting filaments undergo a helical instability. These incipient helices then coarsened until constrained by the tubing walls, and spread along the whole tube length without obstructing the flow. A three-dimensional discrete filament model supports this coarsening mechanism and yields simulations of helix dynamics in accordance with our experimental observations. These findings describe an unanticipated mechanism for bacterial spreading in tubing networks which might be involved in some hospital-acquired infections and bacterial contamination of catheters.

Figure 1. Experimental setup description.

(a) Sketch of an experimental setup. Diameter variations in the tubes: (b) adaptor, (c) T-junction, (d) vertically oriented clamp. (e) Detail of the roller pump ISMATEC IP-C 8 used in the experiments. (f) Scheme of the device used for the drip flow experiment.

Figure 2. Wild-type P. putida KT2440 helical biofilms wrapped inside a 2 mm inner diameter silicone tube.

Differences in local geometry or curvature prompt the formation of (a) Single helix or (b) overlapping helices. Photographs were taken after 4 days of continuous pulsatile pumping generated by a roller multiport pump at a flow rate of 0.45 ml/min. Helices are visible to the naked eye. Brightness and contrast were adjusted to enhance the images.

Figure 3. An advancing air bubble destroyed a helical biofilm, and debris flowing downstream showed that helices are located near the tube wall.

Fully developed helices reside near the tube walls. Brightness and contrast were adjusted to enhance the image.

Figure 4. Image showing a broken helical thread split into sequences of rings.

Brightness and contrast were adjusted to enhance the image.

Figure 5. Scheme showing stages of helix formation over time (illustrations supported by experimental images selected from different experiments because of their clarity).

(a–b) Nucleated straight biofilm thread (green arrows) flows downstream. (c–d) Helical instability bends the biofilm thread to shape an initial wavy pattern. (e–f) Biomass accumulation and subsequent deformation foster radial expansion of the wavy pattern resulting in a sequence of large bent loops. (g–h) Tube radius constrains helix enlargement and molds the thread into the final helical shape. Brightness and contrast were adjusted to enhance the images.

Figure 6. Numerical solution and description of the three-dimensional discrete filament model.

Helix evolution showing: (a) Emergence of the helical instability and (b) coarsening due to the increase in length.

Figure 7. Effect of the stenoses in helix structure and evolution.

Replacing (a) circular connectors by (b) vertically oriented clamps (see Fig. 1d) deforms the symmetry in the tube cross-section, forcing the nucleated thread to expand along the bottom of the tubes and to adopt an asymmetric and irregular shape. Brightness and contrast were adjusted to enhance the images.

Figure 8. Numerical plot of fluid streamlines at different stenosis points.

(a) Steady drip flow and (b) pulsatile flow at a fixed time through a tube adaptor. The size of the vortical region in (b) expands and shrinks periodically over time. Colored slices indicate the direction of the x component of velocity. (c) Streamline and (d) y component of fluid velocity chart inside a T-Junction. Note in addition to the vortices after the stenoses, the secondary vortices formed at the corners by the streamlines in (c).

Figure 9. Centerline and material frame representing filament deformation.

(a) Continuous description and (b) discrete description. The Bishop frame {t, u, v} defines the “rest orientation” at any point of the centerline. The material frame characterizing the local orientation {t(s), m₁(s), m₂(s)} is obtained rotating {u(s), v(s)} an angle θ around t(s). The centerline is discretized into a set of points {x₀, x₁, …, x_n+1} and segments eⁱ = x_i+1 − x_i. A local orthonormal material frame {tⁱ, m₁ⁱ, m₂ⁱ} is assigned to each point setting tⁱ = eⁱ/||eⁱ||, the unit tangent vector per edge.