Crosslinked agarose-gelatine beads as a substrate for investigating biofilms of bacterial pathogens.

Treating chronic bacterial infections remains challenging because biofilm formation reduces bacterial susceptibility to antimicrobial agents. However, many in vitro biofilm models rely on abiotic surfaces that poorly reflect host-associated environments. Here, we developed a crosslinked agarose-gelatine hydrogel substrate in the form of autoclavable beads for biofilm cultivation. Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus rapidly colonised the beads and formed significantly greater biofilm biomass than on glass beads. Quantitative proteomic analyses revealed substrate-dependent physiological differences, including increased abundance of several virulence-associated proteins in biofilms grown on agarose-gelatine beads. In addition, the hydrogel matrix functioned as a reservoir for quorum-sensing molecules and diffusible pigments. Together, these findings indicate that agarose-gelatine beads provide a practical and physiologically relevant platform for investigating biofilm-associated bacterial phenotypes beyond conventional abiotic surfaces.