Bacterial filamentation mediates cross-protection against β-lactam antibiotics in droplet microreactors

Bacterial strains that are resistant to antibiotics may protect not only themselves, but also sensitive bacteria nearby if resistance involves antibiotic degradation. Such cross-protection poses a challenge to effective antibiotic therapy by enhancing the long-term survival of bacterial infections. In this study, we utilize an automated nanoliter droplet analyzer to study the interactions between Escherichia coli strains expressing a β-lactamase (resistant) and those not expressing it (sensitive) when exposed to the β-lactam antibiotic cefotaxime (CTX), with the aim to define criteria contributing to cross-protection. We observed a cross-protection window of CTX concentrations for the sensitive strain, extending up to approximately 100 times its minimal inhibitory concentration (MIC). Through both microscopy and enzyme activity analyses, we demonstrate that filamentation of bacterial cells, triggered by antibiotic stress contributes to cross-protection through increased extracellular β-lactamase activity, resulting from cell lysis and/or β-lactamase leakage due to greater cell wall permeability. The antibiotic concentration window for cross-protection depends on the difference in β-lactamase activity between co-cultured strains: larger differences shift the ‘cross-protection window’ towards higher CTX concentrations. Our findings highlight the crucial role of bacterial filamentation in community-wide antibiotic resistance and emphasize the need for intervention therapies that consider -and potentially suppress- filamentation