Population dynamics of 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, however, the current understanding is limited. In this study, we utilize an automated nanoliter droplet analyzer to study the interactions between 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 bacterial filaments, triggered by antibiotic stress, contribute to cross-protection. 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' toward higher CTX concentrations. Our findings highlight the dependence of opportunities for cross-protection on the relative resistance levels of the strains involved and suggest a possible specific role for filamentation.