Extracellular Matrix Rigidities Regulate the Tricarboxylic Acid Cycle and Antibiotic Resistance of Three‐Dimensionally Confined Bacterial Microcolonies

As a major cause of clinical chronic infection, microbial biofilms/microcolonies in host tissues essentially live in 3D-constrained microenvironments, which potentially modulate their spatial self-organization and morphodynamics. However, it still remains unclear whether and how mechanical cues of 3D confined microenvironments, for example, extracellular matrix (ECM) stiffness, exert an impact on antibiotic resistance of bacterial biofilms/microcolonies. With a high-throughput antibiotic sensitivity testing (AST) platform, it is revealed that 3D ECM rigidities greatly modulate their resistance to diverse antibiotics. The microcolonies in 3D ECM with human tissue-specific rigidities varying from 0.5 to 20 kPa show a ≈2-10 000-fold increase in minimum inhibitory concentration, depending on the types of antibiotics. The authors subsequently identified that the increase in 3D ECM rigidities leads to the downregulation of the tricarboxylic acid (TCA) cycle, which is responsible for enhanced antibiotic resistance. Further, it is shown that fumarate, as a potentiator of TCA cycle activity, can alleviate the elevated antibiotic resistance and thus remarkably improve the efficacy of antibiotics against bacterial microcolonies in 3D confined ECM, as confirmed in the chronic infection mice model. These findings suggest fumarate can be employed as an antibiotic adjuvant to effectively treat infections induced by bacterial biofilms/microcolonies in a 3D-confined environment.