Evolved extracellular polymeric substances act as a frontline shield against antibiotic tolerance and resistance in Escherichia coli

The evolution of antibiotic resistance is a severe global threat. Investigating this issue, instead of merely confining our research to bacterial resistance based on membrane-associated and intracellular mechanisms (for example, reduced permeability and antibiotic efflux), we have shed light on the role of novel extracellular mechanisms, namely, the extracellular polymeric substance (EPS) shield. We used experimental evolution to identify significant modifications in EPS compositions and properties of Escherichia coli, even under low-level antibiotic exposure. Heterogeneous EPS–antibiotic interactions universally increased the minimum inhibitory concentration by 1.4- to 1.7-fold and tolerance capabilities by 1.2–3.2 log10 in resistant evolved strains. Conversely, the resistance of susceptible isolates was retained irrespective of the presence or absence of EPS. The EPS–antibiotic interaction was unequivocally characterized by integrated microscopy–spectroscopy, while multi-omics analysis further emphasized the contribution of EPS to resistance. Collectively, the results of this study highlight that evolved EPS, as a frontline shield against antibiotic treatment, is currently a neglected key determinant of antibiotic tolerance and resistance. Antibiotic resistance is a growing global threat, yet the role of extracellular defence mechanisms remains underexplored. This study reveals that evolved extracellular polymeric substances act as a frontline shield, substantially boosting Escherichia coli antibiotic resistance, demonstrating their key but overlooked role in antibiotic resistance.