1887

Abstract

Antimicrobial resistance (AMR) is a growing threat to healthcare. Many AMR-encoding genes are carried on accessory genetic elements like plasmids, which spread between phylogenetically distant members of bacterial communities. CRISPR-Cas based technologies may help combat the spread of AMR genes by removal of such plasmids. To optimise this technology, implementation of a delivery method which can reach a diverse range of bacteria is needed. Therefore, we engineered broad host-range conjugative plasmid pKJK5 to express Cas9. pKJK5::Cas[GmR] encodes a guide RNA which targets Gentamicin resistance gene aacC1, while pKJK5::Cas[nt] encodes a non-targeting guide RNA. After confirming its Ca9 activity by electroporation of targeted and untargeted plasmids, we set up a proof-of-concept experiment to conjugatively deliver pKJK5::Cas from a donor Escherichia coli strain DH5α to unrelated recipient E. coli K12, and to remove Gentamicin-resistance encoding target plasmid pHERD30T. For these means, we mixed donors and recipients in liquid media and incubated them overnight; proportions of the total population were enumerated by differential plating. We found that Gentamicin-resistant recipients were reduced by 33 % when treated with pKJK5::Cas[GmR] compared to treatment with pKJK5::Cas[nt], indicating that pKJK5::Cas[GmR] has the ability to remove AMR plasmids from recipient cells. This proof-of-concept experiment shows how an engineered broad host-range conjugative plasmid is an effective means of removing AMR-encoding plasmids and may be a viable approach to remove resistance genes from complex bacterial communities. To make this method more effective, community experiments as well as optimisation of Cas9 activity are needed.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Loading

Article metrics loading...

/content/journal/acmi/10.1099/acmi.ac2019.po0082
2019-04-08
2024-12-14
Loading full text...

Full text loading...

/content/journal/acmi/10.1099/acmi.ac2019.po0082
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error