Biofilm morphogenesis on soft hydrogels

Bacteria often colonize their environment in the form of surface attached multicellular communities called biofilms. Biofilms grow from surface-attached cells that undergo division while self-embedding in a viscoelastic matrix. Biofilms grow at the surface of biotic and abiotic materials with a wide range of mechanical properties. In particular, during infections and in microbiota, the association of the bacterial cells with the surface of soft tissues is of fundamental importance for successful colonization. For a long time, the field of microbiology has focused on biochemical aspects of infection and biofilm formation. There is now evidence that mechanical forces play critical roles in bacterial physiology and impact biofilm formation and stability. For example, evidence is emerging that fluid flow and physicochemical substrate material properties impact biofilm formation. We are however still missing a rigorous investigation of how the mechanical properties of a substrate material impacts biofilm morphogenesis and its relevance in the context of infection. In the laboratory, bacteria are traditionally grown in liquid cultures, on agar plates or in flow cells with glass or hard plastic as a surface. However, these systems do not recapitulate the mechanical complexity of a real infection environment, where bacteria most often colonize soft tissues while experiencing flow. To solve these technical limitations, I combined synthetic PEGDA hydrogels with microfluidics which enabled high-resolution live imaging of single bacteria and biofilm formation while interacting with soft substrates in flow.In chapter 2, I show that the pathogens Vibrio cholerae and Pseudomonas aeruginosa deform the synthetic soft gels. This behavior is the result of a buckling instability generated by the buildup of compressive mechanical stress inside the growing biofilm and its adhesion to the substrate. By using mutants in matrix components and comparing overproducer strains with wild type ones we showed that cell-cell cohesion and cell-substrate adhesion simultaneously drive deformation. In addition, we found that buckling biofilms can exert forces that compromise the integrity of soft epithelial cells monolayers, thus suggesting that biofilm can mechanically compromise host integrity.In chapter 3, I investigated the effect of substrate mechanical properties on P. aeruginosa biofilm morphology. We showed that biofilms take different shapes as a function of substrate mesh size due to differences in exploratory twitching motility. The mechanical control of single-cell twitching speed, gives rise to a range of architectures that vary from compact, dome shaped biofilms to flat and dispersed ones, ultimately influencing both their tolerance to antibiotics and the spatial structure of different lineages. Overall, our results show that the mechanical coupling with soft substrates impacts bacterial phenotypes such as surface motility and biofilm architecture and can play a role in the outcome of an infection both by modulating the biofilm susceptibility to antibiotics or by actively becoming a source of virulence.