Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR

Abstract

Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about twofold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure.

A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection

“…For example, the presence of malfunctioning CRISPR plasmid in recipients could prevent the entry of functioning CRISPR plasmids. Future study should explore the approaches for vector self-clearance (Lazdins et al, 2020). Third, the use of multi-plasmid systems could lead to unexpected plasmid recombination.…”

“…Here, we demonstrated highly efficient CRISPR antimicrobial delivery systems based on conjugation and mobilization of multi-plasmids. The systems consisted of an RK2 (Kolatka et al, 2010) based self-transmissible helper plasmid and an R1162 (Meyer et al, 1985) based mobilizable plasmid encoding CRISPR/Cas cassettes. These two plasmids can be co-transferred to the majority of recipient cells in the absence of antibiotic selection and even at a low (1:180) donor-to-recipient ratio.…”

Biological Functions of Type II Toxin-Antitoxin Systems in Bacteria

“…Antibiotic resistance or virulence plasmids can be cured of bacteria by using plasmid incompatibility, but plasmid-free cells can be killed off by stable toxins from plasmid-mediated TA systems. To circumvent this problem, curing plasmids have been constructed to provide excess antitoxin in trans by engineering TA modules (deletion of toxin gene) with plasmid incompatibility to cure target antibiotic resistance plasmids in vitro and in vivo successfully [172,173]. This opens a new area for selective neutralisation of TA modules to cure antibiotic resistance and virulence plasmids without killing bacterial populations.…”

In situ, in vivo, and in vitro approaches for studying AMR plasmid conjugation in the gut microbiome

“…As an example, Palencia-Gándara et al( 2021 ) demonstrated that synthetic fatty acid 2-hexadecynoic acid reduced conjugation of an IncF and IncW plasmid between laboratory E. coli strains 50-fold in the intestines of C57BL/6 mice. Other examples include elucidating the role of conjugation in bacteriocin-producing Enterococci (Kommineni et al 2016 ), and determining the in vivo antibiotic selection dependence of a plasmid displacement vector for reducing the prevalence of an IncK plasmid in E. coli within BALB/c mice (Lazdins et al 2020 ).…”