Multicopy plasmids potentiate the evolution of antibiotic resistance in bacteria


Plasmids are thought to play a key role in bacterial evolution by acting as vehicles for horizontal gene transfer, but the role of plasmids as catalysts of gene evolution remains unexplored. We challenged populations of Escherichia coli carrying the blaTEM-1 β-lactamase gene on either the chromosome or a multicopy plasmid (19 copies per cell) with increasing concentrations of ceftazidime. The plasmid accelerated resistance evolution by increasing the rate of appearance of novel TEM-1 mutations, thereby conferring resistance to ceftazidime, and then by amplifying the effect of TEM-1 mutations due to the increased gene dosage. Crucially, this dual effect was necessary and sufficient for the evolution of clinically relevant levels of resistance. Subsequent evolution occurred by mutations in a regulatory RNA that increased the plasmid copy number, resulting in marginal gains in ceftazidime resistance. These results uncover a role for multicopy plasmids as catalysts for the evolution of antibiotic resistance in bacteria.

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Figure 1: Effects of plasmid pBGT on bacterial fitness.
Figure 2: Survival curves under increasing concentrations of ceftazidime.
Figure 3: pBGT mutations in the ceftazidime resistant populations.
Figure 4: Characterization of the mutations in blaTEM-1 and RNAI.


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A.S.M. thanks E. Frago for his help with the statistical analyses. This work was supported by funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant (StG-2011-281591). A.S.M. is supported by a Miguel Servet fellowship from the Instituto de Salud Carlos III (MS15/00012) co-financed by the European Social Fund and The European Development Regional Fund ‘A way to achieve Europe’ (ERDF). J.A.E. is supported by a Marie Curie Intra-European Fellowship for Career Development (FP-7-PEOPLE-2011-IEF, ICADIGE). Work in the Mazel lab was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR3525) and the European Union Seventh Framework Programme (FP7-HEALTH- 2011-single-stage), the ‘Evolution and Transfer of Antibiotic Resistance’ (EvoTAR, FP7- HEALTH-282004).

We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics funded by Wellcome Trust grant reference 090532/Z/09/Z and Medical Research Council Hub grant G0900747 91070 for generation of the high-throughput sequencing data.

Author information

A.S.M. and R.C.M. were responsible for the conceptualization of this study; A.S.M., J.A.E., and D.R.G. designed the methodology; formal analysis was by D.R.G.; A.S.M. and J.A.E. carried out the investigations; A.S.M. and R.C.M. prepared the original draft of the manuscript and also undertook the reviewing and editing; R.C.M. and D.M. were responsible for funding acquisition and for supervision; D.M. oversaw the resources; project administration was performed by A.S.M.

Correspondence to Alvaro San Millan.

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Supplementary information

Supplementary Information

Supplementary Figures 1,2, Supplementary Tables 1–3 and Supplementary References. (PDF 214 kb)

Supplementary Data 1

Description of the mutations affecting the chromosome and pBGT in the different populations and clones of MG, MG::blaTEM-1 and MG/pBGT analysed in this work. The position and nature of the mutations are indicated, as well as the genes affected and their activity. The frequencies of the mutations in the different populations and clones are also indicated. (XLSX 70 kb)

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San Millan, A., Escudero, J., Gifford, D. et al. Multicopy plasmids potentiate the evolution of antibiotic resistance in bacteria. Nat Ecol Evol 1, 0010 (2017).

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