Evolutionary paths to antibiotic resistance under dynamically sustained drug selection

Antibiotic resistance can evolve through the sequential accumulation of multiple mutations1. To study such gradual evolution, we developed a selection device, the 'morbidostat', that continuously monitors bacterial growth and dynamically regulates drug concentrations, such that the evolving population is constantly challenged ^2-5. We analyzed the evolution of resistance in Escherichia coli under selection with single drugs, including chloramphenicol, doxycycline and 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 resistance to a particular drug and shared in resistance to multiple drugs. Chloramphenicol and doxycycline resistance evolved smoothly through diverse combinations of mutations in genes involved in translation, transcription and transport ³. In contrast, trimethoprim resistance evolved in a stepwise manner ^1,6, through mutations restricted to the gene encoding the enzyme dihydrofolate reductase (DHFR) ^7,8. Sequencing of DHFR over the time course of the experiment showed that parallel populations evolved similar mutations and acquired them in a similar order ⁹.