Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool

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 cas9 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 Escherichia coli . 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 Pseudomonas . 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.

Interspecific competition can drive plasmid loss from a focal species in a microbial community

“…Using pKJK5 as a template, conjugative plasmid pKJK5:: gfp PL was constructed by ƛ-red mediated recombineering as described in [ 19 ], with the exception that the gene cassette constitutively expressing GFP, Streptococcus pyogenes Cas9, and sgRNA was inserted into pKJK5’s intI1 gene (Supplementary Fig. S1b ).…”

One Earth: The Equilibrium between the Human and the Bacterial Worlds

“…Alternatives to tackle bacterial infections include, at least, the following approaches: (i) employment of inhibitors of protein-protein interactions; (ii) use of antisense RNAs; (iii) adoption of phage therapies; (iv) usage of nanoparticles as delivery agents; (v) employment of biofilm inhibitors; (vi) engineering Toxin-Antitoxin systems to use the toxins as alternative drugs; (vii) engineering broad-host-range plasmids equipped with CRISPR-cas9 genes to block entry of plasmid DNA; and (viii) targeting ubiquitous genes by the use of the interference system CRISPRi. All these ways could be considered valuable tools to co-participate in controlling the spread of AMR [6,88,[91][92][93][94]. Furthermore, monoclonal antibody-based strategies are being explored to treat bacterial sepsis and septic shock [95].…”

Microbial Musings: Spring 2023

“…Is it possible for bio-engineers to use microbe–microbe interactions to modify the functional traits of microbiomes in situ ? This was the question asked by Walker-Sünderhauf and colleagues [9], who sought to artificially combine naturally occurring microbiological machineries to address the problem of antimicrobial resistance (AMR). Genes encoding resistance to antimicrobials can be found all over the world, and the problem cannot be simply reversed by refraining from use of antimicrobials, even if this were possible.…”