Cooperative resistance varies among β-lactamases in E. coli, with some enabling cross-protection and sustained extracellular activity.

β-lactamases confer bacteria resistance to β-lactam antibiotics, and interestingly, this protective effect can extend to neighboring susceptible cells. However, knowledge of this cooperative resistance remains limited. Here, we investigated the underlying factors of cooperative resistance to assess commonalities and differences among the highly diverse group of β-lactamases. We first analyzed β-lactamase genes from 2637 Escherichia coli genomes, followed by experimental characterization of seven prevalent β-lactamase genes. Larger plasmids, particularly conjugative ones, commonly encoded β-lactamases. All seven genes had strong wildtype promoters, and plasmid-based expression rescued more susceptible bacteria than chromosomal expression. Cooperative resistance positively correlated with β-lactamase activity and minimal inhibitory concentrations. Cross-protection could be established between different β-lactamase producers, challenging the effectiveness of therapies combining β-lactams. Extracellular activity varied among β-lactamases and, when high, resulted in a legacy resistance effect in the environment. These findings advance our understanding of β-lactam resistance and highlight important implications for antibiotic treatment strategies.