Substrate stiffness-dependent bacterial growth dynamics in 3D*

Complex microenvironments inside our body—such as the gut mucosal layer—host diverse microbial communities that symbiotically regulate our metabolism and health. Our daily diet, immune response, and physical activities dynamically alter the mechanical properties of the gut mucosal layer. Interestingly, in 2D culture, the variation in mechanical stiffness of soft agar substrates significantly affects bacterial growth, morphology, and pathogenicity. However, despite extensive metagenomic profiling of gut microbiota, bacterial growth dynamics in 3D and its dependence on the stiffness of their natural habitat remain poorly understood. Here, we employ a porous growth medium made from jammed polyelectrolyte microgels, which replicates the viscoelastic properties of mucus. We use different microbial strains isolated from red flour beetles and directly quantify their population scale growth dynamics within 3D medium of varying stiffness. The optically translucent 3D growth medium supports direct visualization of bacterial proliferation through live-cell imaging and absorbance-based growth curves. By combining our experimental results with an agent-based simulation, we will present how increase in stiffness differentially affect the growth of various bacterial strains.