A suite of modular, all-synthetic suicide vectors for allelic exchange mutagenesis in multidrug resistant Acinetobacter strains

Alaska Pokhrel, Liping Li, Francesca L. Short*, Ian T. Paulsen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Background: Acinetobacter baumannii is an opportunistic human pathogen that causes a variety of infections in immunosuppressed individuals and patients in intensive care units. The success of this pathogen in nosocomial settings can be directly attributed to its persistent nature and its ability to rapidly acquire multidrug resistance. It is now considered to be one of the top priority pathogens for development of novel therapeutic approaches. Several high-throughput techniques have been utilised to identify the genetic determinants contributing to the success of A. baumannii as a global pathogen. However, targeted gene-function studies remain challenging due to the lack of appropriate genetic tools.

Results: Here, we have constructed a series of all-synthetic allelic exchange vectors – pALFI1, pALFI2 and pALFI3 – with suitable selection markers for targeted genetic studies in highly drug resistant A. baumannii isolates. The vectors follow the Standard European Vector Architecture (SEVA) framework for easy replacement of components. This method allows for rapid plasmid construction with the mutant allele, efficient conjugational transfer using a diaminopimelic acid-dependent Escherichia coli donor strain, efficient positive selection using the suitable selection markers and finally, sucrose-dependent counter-selection to obtain double-crossovers. 

Conclusions: We have used this method to create scar-less deletion mutants in three different strains of A. baumannii, which resulted in up to 75% deletion frequency of the targeted gene. We believe this method can be effectively used to perform genetic manipulation studies in multidrug resistant Gram-negative bacterial strains.

Original languageEnglish
Article number137
Pages (from-to)1-7
Number of pages7
JournalBMC Microbiology
Volume23
Issue number1
DOIs
Publication statusPublished - 18 May 2023

Bibliographical note

Copyright the Author(s) 2023. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • Acinetobacter baumannii
  • Allelic-exchange mutagenesis
  • Conjugation
  • Genetic manipulation
  • Homologous recombination
  • MDR
  • Scar-less deletion
  • SEVA

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