Confined MechanicalMicroenvironment Regulated AntibioticResistance in 3D Biofilm Aggregates Probed by Scanning ElectrochemicalMicroscopy

Antibiotic resistance is a significant global concern. Clinical trials have highlighted discrepancies in antibiotic doses between <i>in vivo</i> three-dimensional (3D) biofilms and <i>in vitro</i> two-dimensional biofilm models. A critical factor often overlooked is the confined mechanical microenvironment (e.g., host extracellular matrix (ECM) stiffness) surrounding the <i>in vivo</i> biofilms, leading to inaccurate diagnosis and increased antibiotic resistance. Herein, we designed a 3D agarose-gel-based <i>in vitro</i> biofilm model and applied scanning electrochemical microscopy (SECM) to monitor the metabolic dynamics <i>in situ</i>, including cellular respiration and reactive oxygen species of an embedded single biofilm aggregate. We discovered distinct respiration patterns for biofilm aggregates embedded in stiff and soft gels at the single aggregate level, which was corroborated by transcriptional analysis. Our findings indicate that mechanical cues mediate antibiotic tolerance by reducing metabolic activity and increasing the production of extracellular polymeric substances (EPS). Additionally, we identified that metabolite glycine enhances the tricarboxylic acid cycle, suggesting its potential as an adjuvant to improve antibiotic efficacy. Knocking out the upregulated EPS-related gene (Δ<i>yjbE</i>) results in significantly reduced survival rates of Δ<i>yjbE</i> mutants in stiff agarose gels compared to the wild type, thereby enhancing antibiotic efficacy. Overall, our study demonstrates the versatility of the SECM-based strategy for investigating both metabolic dynamics and antibiotic resistance in biofilms and uncovers the role of ECM stiffness in mediating antibiotic resistance in 3D biofilms, paving the way for improved clinical strategies in antibiotic treatment.