Plasmid biology underpinning development of a novel plasmid displacement technology to eliminate antibiotic resistance genes

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Antibiotic resistance in bacteria is becoming an increasingly urgent problem that is recognised as one of the key global challenges to public health. The rise of resistance is due to the selective pressure imposed by the use of antibiotics and other antimicrobial agents to control infection combined with the genetic plasticity of the bacteria themselves. This allows resistance mechanisms to evolve and spread rapidly between bacteria. Once such resistance mechanisms exist it is very difficult to get rid of them. A key part of this genetic arsenal possessed by bacteria are plasmids that are characteristically small relative to the chromosome, often circular, DNA elements capable of replication within bacterial cells independently of the host chromosome. Many plasmids possess the ability to transfer between bacteria via specialised nano-molecular machinery that creates a fusion and a tunnel between bacteria and a docking process that allows a copy of the plasmid to be transported from one bacterium to another. This process, called conjugation or bacterial sex, provides a powerful mechanism for bacteria to acquire advantageous genes from elsewhere in a bacterial community. Indeed, many plasmids are able to transfer between and multiply in many different bacterial types, thus allowing resistance genes to spread rapidly between different species of bacteria and different ecological niches. In a selective environment the dominant plasmids tend to carry antibiotic resistance. Principally in clinical contexts where new antimicrobial agents are used to treat infections, plasmids accumulate resistance determinants to the multiple antibiotics that their host has been exposed to so that when they move their new host becomes resistant to many antibiotics simultaneously. Thus, in situations where strains have become untreatable due to the accumulation of resistance genes on a self-transmissible plasmid, a possible way to reverse the situation might be to displace the resistance plasmids themselves. Thus if the plasmid carrying multiple resistance genes can be displaced then all the resistance genes would be lost, allowing the re-use of antibiotics that would otherwise be ineffective. We have developed a way of doing this using a broad host range plasmid to carry a cassette of genetic functions that stop the target plasmids from multiplying and blocking their survival mechanisms. We know a lot about the plasmid we have used to carry this anti-plasmid cassette but we discovered that its ability to promote plasmid displacement depends on a specific gene that belongs to a gene family that is widespread on different plasmids. Understanding how this potentiation works may help us to design better ways to displace plasmids and this forms the first work package. We will first create mutations in the gene and see which ones affect this potentiation. This will be followed by biochemical analysis to see what properties of the plasmid are affected by these mutations. The output of this work package will underpin further curing plasmid development.The second work package focuses on the speed with which the plasmid can spread from one bacterium to another in the gut. The spread depends on a sort of protein "hair" on the bacterial surface called a "pilus". Different sorts of plasmids have different sort of pilus but the long flexible ones are thought to be better at stabilising the pairings that allow plasmid transfer. The plasmid we chose has a short rigid pilus that is not so good in liquid. Some plasmids have both sorts of pilus and so using this as a model we will engineer our chosen plasmid to encode a long flexible pilus so it has one of each kind. We will then mutate this new hybrid and put it into situations where we can isolate mutants that spread more rapidly. Such plasmids will form the basis of further work involving animal trials and we hope eventually clinical trials.