Heterogeneity Governs 3D-Cultures of Clinically Relevant Microbial Communities

The intrinsic heterogeneity of bacterial niches should be retained in in vitrocultures to represent the complex microbial ecology. As a case study,mucin-containing hydrogels -CF-Mu3Gel - are generated by diffusion-inducedgelation, bioinspired on cystic fibrosis (CF) mucus, and a microbial nichechallenging current therapeutic strategies. At breathing frequency, CF-Mu3Gelexhibits aG′andG′′equal to 24 and 3.2 Pa, respectively. Notably, CF-Mu3Gelexhibits structural gradients with a gradual reduction of oxygen tensionacross its thickness (280–194μmol L−1). Over the culture period, a steepdecline in oxygen concentration occurs just a few millimeters below theair–mucus interface in CF-Mu3Gel, similar to those of CF airway mucus.Importantly, the distinctive features of CF-Mu3Gel significantly influencebacterial organization and antimicrobial tolerance in mono- and co-cultures ofStaphylococcus aureusandPseudomonas aeruginosathat standard culturesare unable to emulate. The antimicrobial susceptibility determined inCF-Mu3Gel corroborates the mismatch on the efficacy of antimicrobialtreatment between planktonically cultured bacteria and those in patients.With this example-based research, new light is shed on the understanding ofhow the substrate influences microbial behavior, paving the way for improvedfundamental microbiology studies and more effective drug testing anddevelopment.