Mechanistic Origins and Evolutionary Erosion of Collateral Sensitivity in a β-lactamase.

As antibiotic discovery stalls, exploiting collateral sensitivity, where resistance to one drug increases sensitivity to another, offers a promising route to extend the lifespan of existing drugs. However, the molecular origins and robustness of such trade-offs at the level of single resistance determinants remain poorly understood. Here, we examined a previously evolved trajectory of the β-lactamase OXA-48 to Q4 (A33V/F72L/T212A/S213A) in Escherichia coli. Compared to OXA-48, Q4 conferred 40-fold increase in ceftazidime resistance, but a 27-fold lower piperacillin resistance. This trade-off was caused by the introduction of F72L. We challenged the stability of this collateral sensitivity network by subjecting Q4 to directed evolution followed by co-selection from both ceftazidime and piperacillin. The emerging substitution V120G alleviated the piperacillin trade-off while maintaining elevated resistance to ceftazidime in genetic backgrounds harboring F72L. Structural and computational analyses revealed that evolution introduced substantial conformational changes in the Ω-loop, likely leading to less productive piperacillin binding poses. V120G counteracted the effect of F72L by decreasing the Ω-loop's conformational freedom, thereby partially restoring piperacillin resistance. Finally, we show that other substitutions at position 120 can exert similar mitigating effects. Taken together, our results provide a mechanistic understanding of how adaptive solutions both generate and erode collateral sensitivity, knowledge crucial for predicting the long-term stability of these networks.