Emergence of 3D Biofilms at Solid/Air Interfaces - What is the role of Water and Surface Geometry?

LARGE, B.; EHRIG, S.; SERRA, DIEGO O.; BLANK, K.G.; BERTINETTI, L.; HENGGE, REGINE; FRATZL, PETER; BIDAN, CECILE M.

Simposio; International Symposium on Biophysics of Microbial Adhesion (BioPhysAdh); 2018

Comité organizador del Simposio: Biophysics of Microbial Adhesion (BioPhysAdh)

Biofilms are complex 3D structures forming as bacteria get embedded in a matrix of self-produced amyloid and cellulose fibers. These living 3D structures impair human health and industrial processes in numerous ways and understanding their formation and architecture is crucial for biofilms prevention and destruction. Whereas many studies focus on the emergence of these complex structures at the protein and cell levels, quantitative methods designed to characterize the formation of entire biofilms and the underlying biophysical cues are largely lacking. Moreover, nano- and micro-topographies are known to affect bacteria adhesion and biofilm formation but the role of larger geometrical constrains is still unclear.Here we culture E. coli bacteria from the strain AR3110 on salt-free LB agar plates for 5 to 7 days to obtain three-dimensional biofilms containing both amyloid and cellulose fibres. Controlling the concentration of agar in the medium allows for tuning the initial moisture content in the substrate, whereas using different semi-spherical templates allows for shaping the agar surfaces with different curvatures. Biofilm spreading is monitored macroscopically using a flatbed scanner. Agar plates are also supplemented with the fluorescent marker ThioflavinS of extracellular polymeric substance (EPS) to visualize biofilm spreading and wrinkle emergence using a fluorescence stereomicroscope. After 7 days of growth, the biofilms are fixed in 4% paraformaldehyde and scanned by micro-computer tomography for further characterization of their complex 3D geometry. Our preliminary results confirm that the presence of water is a key parameter for biofilm spreading, and suggest that it greatly determines the wrinkling pattern of E. coli biofilms. Indeed, E. coli (AR3110) grow into large biofilms with a few but large radial wrinkles on soft agar with high water content, whereas they expand much less and rather form many small and less oriented wrinkles when grown on stiffer agar with lower water content. These distinct phenotypes are associated with different spreading kinetics, as calculated from the projected area on the experimental images. The present methods established to quantify the potential influence of surface curvature on biofilm spreading kinetics tend to show a faster spreading of the few biofilms grown on concave surfaces compared to biofilms grown on flat surfaces.Comparing our macroscopic data with simple growth models of bidimensional systems and of cellular tissues will be of high interest for identifying common principles as well as fundamental differences between matrix formation and shaping in bacterial and mammalian systems.