Emergence of three-dimensional order and structure in growing biofilms

Abstract

Surface-attached bacterial biofilms are self-replicating active liquid crystals and the dominant form of bacterial life on earth ( 1 – 4 ). In conventional liquid crystals and solid-state materials, the interaction potentials between the molecules that comprise the system determine the material properties. However, for growth-active biofilms it is unclear whether potential-based descriptions can account for the experimentally observed morphologies, and which potentials would be relevant. Here, we overcame previous limitations of single-cell imaging techniques ( 5 , 6 ) to reconstruct and track all individual cells inside growing three-dimensional (3D) biofilms with up to 10,000 individuals. Based on these data, we identify, constrain, and provide a microscopic basis for an effective cell-cell interaction potential, which captures and predicts the growth dynamics, emergent architecture, and local liquid crystalline order of Vibrio cholerae biofilms. Furthermore, we show how external fluid flows control the microscopic structure and 3D morphology of biofilms. Our analysis implies that local cellular order and global biofilm architecture in these active bacterial communities can arise from mechanical cell-cell interactions, which cells can modulate by regulating the production of particular matrix components. These results establish an experimentally validated foundation for improved continuum theories of active matter and thereby contribute to solving the important problem of controlling biofilm growth.

Topological and geometrical quantities in active cellular structures

“…However, other forms are possible as well, such as short range adhesive interactions or even more realistic effective interaction potentials, as e.g. experimentally determined in 29 and shown to be essential for local cellular order. Here our approach differs from other phase field studies, e.g.…”

BiofilmQ, a software tool for quantitative image analysis of microbial biofilm communities

“…The strains and plasmids used in this study are listed in Supplementary Tables 2 and 3. Flow chamber biofilm experiments were performed in M9 minimal medium with 0.5% (w/v) glucose for V. cholerae, and tryptone broth (10 g L -1 ) for E. coli. To grow flow chamber biofilms of V. cholerae and E. coli, microfluidic chambers of 7 mm length and 500 µm x 100 µm crosssection were used 18,21 , and a flow rate of 0.1 µL min -1 was set using a syringe pump (Pico Plus, Harvard Apparatus). To inoculate flow chambers, overnight cultures were back-diluted 1:200 in LB medium for V. cholerae, and 1:80 in 0.9% (w/v) NaCl for E. coli, and grown to an optical density at 600 nm of OD600 = 0.5.…”

“…2d). Biofilm segmentation algorithms have received significant attention in the literature [15][16][17][18][19][20][21] . BiofilmQ therefore offers different segmentation workflows: either semi-manual segmentation supported by immediate visual feedback, or via automatic segmentation algorithms, such as Otsu, Ridler-Calvard, robust background, or maximum correlation thresholding.…”

Geometric control of bacterial surface accumulation

“…Such accumulation increases the possibility cell-surface attachment, facilitating undesirable secondary effects like biofouling and biofilm formation [11,12]. Surface-attached microbial communities [13,14] cause widespread problems to a broad range of industrial equipment and infrastructure, such as food processing facilities [15,16], ships and pipes [17], and surgical equipment and medical implants [18][19][20]. In the medical context, these surface-attached microbial colonies are especially harmful because they can lead to persistent infection [21].…”