Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence

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The evolution of antibiotic resistance is a major threat to society and has been predicted to lead to 10 million casualties annually by 20501. Further aggravating the problem, multidrug tolerance in bacteria not only relies on the build-up of resistance mutations, but also on some cells epigenetically switching to a non–growing antibiotic-tolerant ‘persister’ state26. Yet, despite its importance, we know little of how persistence evolves in the face of antibiotic treatment7. Our evolution experiments in Escherichia coli demonstrate that extremely high levels of multidrug tolerance (20–100%) are achieved by single point mutations in one of several genes and readily emerge under conditions approximating clinical, once-daily dosing schemes. In contrast, reversion to low persistence in the absence of antibiotic treatment is relatively slow and only partially effective. Moreover, and in support of previous mathematical models810, we show that bacterial persistence quickly adapts to drug treatment frequency and that the observed rates of switching to the persister state can be understood in the context of ‘bet-hedging’ theory. We conclude that persistence is a major component of the evolutionary response to antibiotics that urgently needs to be considered in both diagnostic testing and treatment design in the battle against multidrug tolerance.

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Figure 1: Frequent antibiotic exposure leads to the fast emergence of high survival levels.
Figure 2: Single mutations in evolved clones cause multidrug tolerance, not by increased antibiotic resistance but by increased persistence.
Figure 3: Costs and benefits of persistence under contrasting levels of antibiotic exposure.
Figure 4: Persister levels quickly adapt in response to the frequency of antibiotic treatment.


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B.V.D.B. is a Research Foundation - Flanders (FWO)-fellow and J.E.M. is a fellow of the Agency for Innovation by Science and Technology (IWT). The research was supported by the KU Leuven Research Council (PF/10/010; PF/10/07; IDO/09/010; IDO/13/008; CREA/13/019; DBOF/12/035; DBOF/14/049), Interuniversity Attraction Poles–Belgian Science Policy Office (IAP-BELSPO) (IAP P7/28), ERC (241426), Human Frontier Science Program (HFSP) (RGP0050/2013), FWO (G047112N, KAN2014, Flanders Institute for Biotechnology(VIB) and the European Molecular Biology organisation (EMBO). We thank S. Xie for providing the ancestor strains and E. Toprak and S. Diggle for their comments on this manuscript.

Author information

B.V.D.B. designed and performed the experiments, analysed the data and wrote the manuscript. J.E.M. helped in performing the experiments and analysing the data and edited the manuscript. T.W. made the evolutionary model, helped in analysing the data and edited the manuscript. E.M.W. helped in analysing the evolutionary model. P.V.B. and D.K. helped in performing the experiments. L.D.M. and K.J.V. edited the manuscript. N.V., M.F. and J.M. designed the experiments and edited the manuscript. J.E.M. and T.W. contributed equally. M.F. and J.M. contributed equally as senior authors.

Correspondence to Jan Michiels.

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Supplementary Figures 1-8, Tables 1 and 2, Methods and References. (PDF 1311 kb)

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Van den Bergh, B., Michiels, J., Wenseleers, T. et al. Frequency of antibiotic application drives rapid evolutionary adaptation of Escherichia coli persistence. Nat Microbiol 1, 16020 (2016) doi:10.1038/nmicrobiol.2016.20

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