Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Journal of visualized experiments : JoVE publish this investigation article
May 2nd, 2018
Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This "evolutionary rescue" approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains
Escudero JA., MacLean RC. and San Millan A.
 | Departamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM). |
| Servicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM). |
 | Department of Zoology. University of Oxford. |
 | Servicio de Microbiología. Hospital Universitario Ramón y Cajal. Salud Madrid. Comunidad de Madrid. |
 | Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS). Universidad Complutense (UCM). Universidad Autónoma de Madrid (UAM). Universidad de Alcalá (UAH). Salud Madrid. Comunidad de Madrid. |
 | Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP). Instituto de Salud Carlos III (ISCIII). |
