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 two-fold 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.
Highlights
Antibiotic resistance in bacteria is an increasingly urgent problem that is one of the key global challenges to public health [1]
With pCURE-F-RK2 none of the initial transconjugant colonies were completely free of F’prolac (Fig 3B) but after growing cultures from single colonies and a second round of screening, displacement of F’prolac increased to >85% while RK2 without the anti-IncF cassette as well as RK2Δaph did not affect F’prolac stability at all (Fig 3 and Table 1). This indicates that the anti-IncF cassette, when inserted into the IncP-1 plasmid to give pCURE-F-RK2, is able to sustain the unidirectional displacement of F-like plasmids but works less efficiently than when carried by the high copy number (>40 copies per chromosome) pCURE2 plasmid [12] which is based on the pMB1 replicon
The results showed that pCURE plasmids spread rapidly and that pCURE-F-307 reduced the target F plasmid in a reciprocal way, with F’-positive bacteria falling to less than 0.1% of the target population (Fig 5A)
Summary
Antibiotic resistance in bacteria is an increasingly urgent problem that is one of the key global challenges to public health [1]. The rise of resistance is due to the selective pressure from. A broad-host-range conjugative vector for plasmid curing. The funders provided support in the form of salaries for authors [Wellcome Trust, CEM; BBSRC, GSL, ERS; NHMRC, MK], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions
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