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Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains

An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several gene...

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Autores principales: Bhattacharjee, Disha, Sorg, Joseph A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056809/
https://www.ncbi.nlm.nih.gov/pubmed/32132157
http://dx.doi.org/10.1128/mSphere.00941-19
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author Bhattacharjee, Disha
Sorg, Joseph A.
author_facet Bhattacharjee, Disha
Sorg, Joseph A.
author_sort Bhattacharjee, Disha
collection PubMed
description An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study C. difficile in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into C. difficile can only be performed by conjugation using Escherichia coli or Bacillus subtilis as conjugal donors. Here we report a method to introduce plasmid DNA into C. difficile using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the C. difficile strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab. IMPORTANCE Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to Clostridioides difficile researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into C. difficile. Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different C. difficile strains.
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spelling pubmed-70568092020-03-06 Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains Bhattacharjee, Disha Sorg, Joseph A. mSphere Research Article An important risk factor for acquiring Clostridioides difficile infection is antibiotic use. Therefore, a detailed knowledge of the physiology and the virulence factors can help drive the development of new diagnostic tools and nonantibiotic therapeutic agents to combat these organisms. Several genetic systems are available to study C. difficile in the laboratory environment, and all rely on stably replicating or segregationally unstable plasmids. Currently, the transfer of plasmids into C. difficile can only be performed by conjugation using Escherichia coli or Bacillus subtilis as conjugal donors. Here we report a method to introduce plasmid DNA into C. difficile using electroporation and test factors that might contribute to higher transformation efficiencies: osmolyte used to stabilize weakened cells, DNA concentration, and recovery time postelectroporation. Depending on the C. difficile strain and plasmid used, this transformation protocol achieves between 20 and 200 colonies per microgram of DNA and is mostly influenced by the recovery time postelectroporation. Based on our findings, we recommend that each strain be tested for the optimum recovery time in each lab. IMPORTANCE Understanding the underlying biology of pathogens is essential to develop novel treatment options. To drive this understanding, genetic tools are essential. In recent years, the genetic toolbox available to Clostridioides difficile researchers has expanded significantly but still requires the conjugal transfer of DNA from a donor strain into C. difficile. Here we describe an electroporation-based transformation protocol that was effective at introducing existing genetic tools into different C. difficile strains. American Society for Microbiology 2020-03-04 /pmc/articles/PMC7056809/ /pubmed/32132157 http://dx.doi.org/10.1128/mSphere.00941-19 Text en Copyright © 2020 Bhattacharjee and Sorg. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Bhattacharjee, Disha
Sorg, Joseph A.
Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains
title Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains
title_full Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains
title_fullStr Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains
title_full_unstemmed Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains
title_short Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains
title_sort factors and conditions that impact electroporation of clostridioides difficile strains
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056809/
https://www.ncbi.nlm.nih.gov/pubmed/32132157
http://dx.doi.org/10.1128/mSphere.00941-19
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