Evolutionary paths to antibiotic resistance under dynamically sustained drug selection

Abstract

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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Figure 1: The morbidostat is a continuous-culture device that automatically tunes drug concentration to maintain constant growth inhibition.
Figure 2: Parallel populations attain high levels of drug resistance in similar adaptive trajectories.
Figure 3: Unique and common genetic changes identified by whole-genome sequencing.
Figure 4: Semi-ordered acquisition of trimethoprim resistance mutations.

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Acknowledgements

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.

Author information

E.T., A.V., R.C., D.L.H. and R.K. designed the project. E.T. and A.V. performed the experiments and E.T., A.V., J.-B.M. and R.K. analyzed the data. All authors contributed to preparing the manuscript.

Correspondence to Roy Kishony.

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Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Note. (PDF 2730 kb)

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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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