Dynamic bacterial growth modulation in structurally distinct and functionally tuneable agarose hydrogels

Bacterial adaptability to diverse environments drives infection, persistence, and antibiotic resistance. Although hydrogels are increasingly used to model such conditions, the factors governing hydrogel-dependent bacterial growth is complex. Here, we focus on agarose hydrogels and investigate how their material properties influence bacterial proliferation. Using two agarose types – hydroxyethyl substituted and unsubstituted – at varying concentrations, we tested four bacterial species (E. coli, P. fluorescens, S. aureus, B. subtilis) across five nutrient media yielding 120 conditions. Growth consistently decreased with increasing hydrogel stiffness and water loss in unsubstituted and substituted agarose hydrogels, regardless of species. Media effects were largely due to their impact on hydrogel properties rather than nutrient content. Furthermore, electrostatic repulsion between Gram positive bacteria and anionic unsubstituted agarose suppressed growth in high concentration gels. These findings demonstrate that bacterial growth in agarose systems is primarily shaped by gel mechanics and surface interactions, informing the design of infection models and antibacterial materials. The factors that govern hydrogel-dependent bacterial growth are complex. Here, bacteria proliferation in two agarose hydrogels is investigated for 120 different growth conditions, finding that growth decreased with increasing hydrogel stiffness and with water loss.