Evolutionary paths to strong antibiotic resistance under dynamically sustained drug stress

Antibiotic resistance can evolve through sequential accumulation of multiple mutations1. To study such gradual evolution, we developed a selection device, the morbidostat, which continuously monitors bacterial growth and dynamically regulates drug concentrations such that the evolving population is constantly challenged 2-5. We analyzed evolutionary trajectories of Escherichia coli populations towards resistance to chloramphenicol, doxycycline, or trimethoprim. Over a period of ~20 days, resistance levels increased dramatically, with parallel populations showing similar phenotypic trajectories. Whole-genome sequencing of the evolved strains identified mutations both specific to and shared between drugs. Chloramphenicol and doxycycline resistance evolved through diverse combinations of mutations in genes involved in translation, transcription, and transport3. In contrast, trimethoprim resistance evolved in a stepwise manner1,6, through mutations restricted to the target enzyme dihydrofolate reductase (DHFR)7,8. Sequencing DHFR over time showed that parallel populations evolved similar mutations and acquired them in similar order9.