Heterogeneity Governs 3D‐Cultures of Clinically Relevant Microbial Communities

The intrinsic heterogeneity of bacterial niches should be retained in in vitro cultures to represent the complex microbial ecology. As a case study, mucin-containing hydrogels -CF-Mu3Gel - are generated by diffusion-induced gelation, bioinspired on cystic fibrosis (CF) mucus, and a microbial niche challenging current therapeutic strategies. At breathing frequency, CF-Mu3Gel exhibits a G′ and G″ equal to 24 and 3.2 Pa, respectively. Notably, CF-Mu3Gel exhibits structural gradients with a gradual reduction of oxygen tension across its thickness (280–194 µmol L−1). Over the culture period, a steep decline in oxygen concentration occurs just a few millimeters below the air–mucus interface in CF-Mu3Gel, similar to those of CF airway mucus. Importantly, the distinctive features of CF-Mu3Gel significantly influence bacterial organization and antimicrobial tolerance in mono- and co-cultures of Staphylococcus aureus and Pseudomonas aeruginosa that standard cultures are unable to emulate. The antimicrobial susceptibility determined in CF-Mu3Gel corroborates the mismatch on the efficacy of antimicrobial treatment between planktonically cultured bacteria and those in patients. With this example-based research, new light is shed on the understanding of how the substrate influences microbial behavior, paving the way for improved fundamental microbiology studies and more effective drug testing and development.