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Abstract

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from . Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of . This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

Funding
This study was supported by the:
  • H2020 European Research Council (Award ERC-STG-2016-714478)
    • Principle Award Recipient: EdzeR Westra
  • Bundesministerium für Bildung und Forschung (Award 01LC1904A)
    • Principle Award Recipient: UliKlümper
  • JPI-AMR HARISSA (Award MISTAR)
    • Principle Award Recipient: Stinekevan Houte
  • Lister Institute of Preventive Medicine
    • Principle Award Recipient: Stinekevan Houte
  • Biotechnology and Biological Sciences Research Council (Award BB/S017674/1)
    • Principle Award Recipient: Stinekevan Houte
  • Biotechnology and Biological Sciences Research Council (Award BB/R010781/1)
    • Principle Award Recipient: Stinekevan Houte
  • Medical Research Council (Award MR/N0137941/1)
    • Principle Award Recipient: DavidWalker-Sünderhauf
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-05-25
2024-04-14
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