Cross-linked agarose-gelatine beads as a substrate for investigating biofilms of bacterial pathogens

Treating chronic bacterial infections is challenging, primarily due to the formation of biofilms, which render bacteria less susceptible and more recalcitrant to antimicrobials. Despite the significance of biofilms in natural and medical settings, in vitro models are very limited in replicating bacterial biofilm physiology during infection. To address this challenge, we have developed a new approach using a surface hydrogel to bring the in vitro studies of biofilms closer to in vivo situations. Our hydrogel substrate combines cross-linked agarose and gelatine. These biogel beads have several attractive properties, such as stability and autoclaving resistance, and can be rapidly colonised by bacterial pathogens when submerged in liquid culture. We tested this new substrate using three model bacterial pathogens: E. coli, P. aeruginosa, and S. aureus . The biofilms formed on the agarose-gelatine beads exhibited more robust growth than those on glass beads for all three species. Moreover, biofilms formed on the agarose-gelatine beads exhibited increased virulence factor production, as indicated by their distinct proteomic signatures. In addition, these beads also acted as a reservoir for biofilm quorum sensing molecules, such as acyl-homoserine lactone. These unique features closely resemble clinical situations, suggesting that our new substrate provides a more accurate representation of biofilm-associated infections than current approaches, such as glass beads. The advantages of our new substrate over current methods are clear, making it an up-and-coming model for studying biofilms of bacterial pathogens and offering a practical and convenient solution to the research community.