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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Journal of visualized experiments : JoVE publica este artículo de investigación

2 de mayo de 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. y San Millan A.




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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance


Participantes:

Universidad ComplutenseDepartamento de Sanidad Animal. Facultad de Veterinaria. Universidad Complutense (UCM).

Universidad ComplutenseServicio de Zoonosis de Transmisión Alimentaria y Resistencia a Antimicrobianos (ZTA). Centro de Vigilancia Sanitaria Veterinaria (VISAVET). Universidad Complutense (UCM).

University of OxfordDepartment of Zoology. University of Oxford.

Comunidad de MadridServicio de Microbiología. Hospital Universitario Ramón y Cajal. Salud Madrid. Comunidad de Madrid.

Comunidad de MadridInstituto Ramón y Cajal de Investigación Sanitaria (IRYCIS). Universidad Complutense (UCM). Universidad Autónoma de Madrid (UAM). Universidad de Alcalá (UAH). Salud Madrid. Comunidad de Madrid.

Instituto de Salud Carlos IIICentro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP). Instituto de Salud Carlos III (ISCIII).







FACTOR YEAR Q
1.108 2018

NLMID: 101313252

PMID: 29781985

ISSN: 1940-087X



TÍTULO: Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance


REVISTA: J Vis Exp


NUMERACIÓN: 135:1


AÑO: 2018


EDITORIAL: Boston, Mass. : MYJoVE Corporation, 2006]


AUTORES: Escudero JA., MacLean RC. and San Millan A.


PARTICIPANTES VISAVET


José Antonio Escudero García-Calderón

DOI: https://doi.org/10.3791/57386


CITA ESTA PUBLICACIÓN:

Escudero JA., MacLean RC. y San Millan A. Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance. Journal of visualized experiments : JoVE. 135:1. 2018. (A). ISSN: 1940-087X. DOI: 10.3791/57386


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