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 challenged2,3,4,5. 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 transport3. In contrast, trimethoprim resistance evolved in a stepwise manner1,6, 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 order9.
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The authors thank M. Baym, S. Bershtein, T. Bollenbach, M. Ernebjerg, Y. Gerardin, J. Horn, A. Kocabas, C. Kocabas, D. Landgraf, R. Milo, B. Okumus, A. Palmer, J.M. Pedraza, M. Shuman, I. Wapinski, R. Ward, P. Yeh and all members of the Kishony laboratory for technical help and discussions. This work was supported in part by grants from the US National Institutes of Health (GM081617 to R.K. and GM079536 to D.L.H.) and The New England Regional Center of Excellence for Biodefense and Emerging Infectious Diseases (AI057159 to R.K.). J.-B.M. is supported by a Foundational Questions in Evolutionary Biology Fellowship.
The authors declare no competing financial interests.
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Toprak, E., Veres, A., Michel, J. et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. Nat Genet 44, 101–105 (2012). https://doi.org/10.1038/ng.1034
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