Context-dependent evolutionary dynamics toward bacterial survival during sequential antibiotic treatment

The antibiotic resistance crisis is global and growing. Stagnant development pipelines for new antibiotics have motivated increased efforts toward optimizing the use of our current antibiotic arsenal for treatment efficacy and resistance risk mitigation through investigations of evolutionary dynamics during treatments using laboratory evolutions. Valuable insights into collateral sensitivity and cross resistance interactions have been observed and characterized. These however are limited as the represent only the phenotypic consequences of susceptibility following preceding antibiotic treatments. During sequential treatments, antibiotics with remaining susceptibility can also drive resistance evolution, and it remains unclear the contributions of the earlier evolutionary dynamics on those afforded to bacteria during sequential treatments. With numerous clinical indications requiring the sequential use of antibiotics, and the rates of resistant and multi-drug resistant infections rising, it is crucial to evaluate these effects as well. To do this, we developed a novel laboratory evolution protocol that uses clinically relevant doses and limits population bottlenecks, a limitation of conventional approaches. We find that this generates diverse survival phenotypes of resistance, tolerance, and persistence. We observe context-dependent collateral effects. When comparing mutational routes toward survival between single and sequential uses of antibiotics, we find context-dependent evolutionary dynamics, allele-specific susceptibility, and shifts from divergent to convergent evolution. These dynamics have the potential to improve antibiotic choice and require further investigation.