Flow-Induced Symmetry Breaking in Growing Bacterial Biofilms

Philip Pearce, Boya Song, Dominic J. Skinner, Rachel Mok, Raimo Hartmann, Praveen K. Singh, Hannah Jeckel, Jeffrey S. Oishi, Knut Drescher, and Jörn Dunkel

Phys. Rev. Lett. 123, 258101 – Published 20 December 2019

Abstract

Bacterial biofilms represent a major form of microbial life on Earth and serve as a model active nematic system, in which activity results from growth of the rod-shaped bacterial cells. In their natural environments, ranging from human organs to industrial pipelines, biofilms have evolved to grow robustly under significant fluid shear. Despite intense practical and theoretical interest, it is unclear how strong fluid flow alters the local and global architectures of biofilms. Here, we combine highly time-resolved single-cell live imaging with 3D multiscale modeling to investigate the mechanisms by which flow affects the dynamics of all individual cells in growing biofilms. Our experiments and cell-based simulations reveal three quantitatively different growth phases in strong external flow and the transitions between them. In the initial stages of biofilm development, flow induces a downstream gradient in cell orientation, causing asymmetrical dropletlike biofilm shapes. In the later developmental stages, when the majority of cells are sheltered from the flow by the surrounding extracellular matrix, buckling-induced cell verticalization in the biofilm core restores radially symmetric biofilm growth, in agreement with predictions of a 3D continuum model.

Received 2 May 2019

DOI:https://doi.org/10.1103/PhysRevLett.123.258101

Physics Subject Headings (PhySH)

Bacteria Biofilms

Physics of Living Systems

Authors & Affiliations

Philip Pearce ¹, Boya Song¹, Dominic J. Skinner¹, Rachel Mok^1,2, Raimo Hartmann³, Praveen K. Singh³, Hannah Jeckel^3,5, Jeffrey S. Oishi ^1,4, Knut Drescher ^3,5,*, and Jörn Dunkel ^1,†

¹Department of Mathematics, Massachusetts Institute of Technology, Cambridge Massachusetts 02139-4307, USA
²Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA
³Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany
⁴Department of Physics, Bates College, Lewiston, Maine 04240, USA
⁵Department of Physics, Philipps-Universität Marburg, 35043 Marburg, Germany

^*k.drescher@mpi-marburg.mpg.de
^†dunkel@mit.edu

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Vol. 123, Iss. 25 — 20 December 2019

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Images

Figure 1
The three phases of V. cholerae biofilm growth and hydrodynamic cell alignment mechanisms in strong flow. (a) The transition from the 2D surface-dominated growth phase 1 (light green) to the 3D bulk-dominated phase 2 (green) occurs when the volume of the biofilm shell (light blue circles) equals the volume of the biofilm core (dark blue circles); see Supplemental Material [27] for details of the volume measurements. The transition to the verticalization phase 3 (dark green) occurs when the average cell-orientation angles with $z$ (light red triangles) and the flow direction (dark red triangles) cross. The diagram shows the combined data from $n = 3$ independent biofilm experiments, with typical snapshots of biofilms in each phase above (Video S1). (b),(c) Flow-induced cell reorientation dynamics in the initial phase of biofilm growth. (b) Cells in direct contact with the surface align with the flow as a result of a torque generated by a combination of the fluid drag and asymmetrical attachment to their parent cell at the pole; see also Fig. S10 for additional intermediate snapshots [27]. Fluorescence images show projection of a confocal $z$ stack. (c) Cells at the front of the biofilm (red) align vertically as a result of the torques $τ_{drag}$ and $τ_{shear}$ (see also Fig. S11). When a vertically oriented cell (red) divides, the daughter cell (blue), if exposed to shear, aligns with the flow.
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Figure 2
A wave of cell verticalization travels downstream through the biofilm in the second phase of biofilm growth. (a) At the beginning of this phase, a small group of cells is verticalized at the front of the biofilm by a combination of cell-cell interactions and fluid shear. (b) The fraction of vertical cells increases as the biofilm grows. The downstream region of flow-aligned cells leads to distinctive dropletlike shapes that are captured in (a) cell-based (Video S2) and (b) continuum simulations (Video S3) [27]. For the experiments ( $n = 3)$ and cell-based simulations ( $n = 10$ ), the gray area denotes the region inside the convex hull around grid points with a cell number density higher than $0.1 μ m^{- 3}$ per biofilm. (c) 3D renderings of shapes generated by the continuum simulations. The isosurface $ϕ = 0$ of the phase-field variable $ϕ$ is shown (Supplemental Material [27]). (d) In low-flow environments, a central core of the biofilm is verticalized owing to a buckling instability induced by growth and surface attachment. (e) Strong flow causes symmetry breaking and a growing group of vertically aligned cells at the front of the biofilm. Error bars show the standard error for grid points spaced $2 μ m$ throughout $n = 3$ biofilms in each case.
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Figure 3
Biofilms transition from asymmetric to symmetric growth in the final phase of their development in flow. (a) In the final phase of growth, most of the cells in the biofilm are vertical, with only a small region of flow-aligned cells on the downstream side. The gray area denotes the region inside the convex hull around grid points with a cell number density higher than $0.1 μ m^{- 3}$ per biofilm ( $n = 3$ ). (b) Growth-induced cumulative biomass flux through the $y z$ plane at each downstream distance; positive and negative values correspond to flux in the downstream and upstream direction, respectively. In low flow, growth is always symmetric (left). In high flow (right), growth symmetry is broken in the early phases, but as more cells verticalize, the biofilm transitions to symmetric growth, which is also captured by continuum simulations (bottom). In experiments (top, $n = 3$ ), biomass flux measurements were obtained using optical flow [17]. In continuum simulations (bottom), the solution for the flow field inside the biofilm was used to calculate cumulative biomass flux.
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