pCURE4Pigs

Endemic diseases in farm animals are one of the BBSRC priorities and with the Agriculture and Horticulture Development Board (AHDB) is an area where novel solutions are being sought. Pig farming provides a large proportion of the meat supply in many countries including the UK, Europe and USA. However, antibiotic resistance (AMR) is a major limitation on controlling infections and the imminent ban on the use of zinc oxide-medicated food in EU pig farming, which is currently used to control pathogenic bacteria which cause post-weaning-diarrhoea, is likely to exacerbate the situation, reducing even further the ever-decreasing range of antibiotics available for treatment of animal infections. Therefore, novel approaches to reducing this burden of resistance are required which could prevent the welfare, economic and societal impacts of infectious disease in pigs. Under a recent AHDB/BBSRC initiative, we are part of a funded consortium led by scientists at the Animal and Plant Health Agency (APHA) focusing on the pig industry and involving scientists, vets and pig producers. Much of the problem involves gut bacteria which often carry both antibiotic resistance genes and virulence factors on mobile genetic elements called plasmids that can facilitate spread between bacteria.
One of the possible solutions is "plasmid displacement" (also known as "plasmid-curing") that the Thomas lab has been developing over the last twenty years. This involves a "good"("pCURE") plasmid carrying a set of genes as a "gene cassette" that stops the "bad" plasmid from multiplying as well as then neutralising its "addiction" functions. Addiction functions prevent the bacteria from growing if they lose the plasmid because it leaves behind a toxin that is unmasked when the plasmid is lost. Thus, pCURE treatment allows the bacteria to survive the process of losing the plasmid, but they no longer carry the antibiotic resistance genes that were on the plasmid that was lost and so are less of a danger to their animal host. There is considerable enthusiasm from vets and others associated with the livestock industry in our network, that if this technology is effective in live animals, then it could represent a practicable and acceptable probiotic treatment option that could also be delivered economically to a variety of food-producing animals. The key challenges are to get a pCURE plasmid that can deliver the plasmid displacement cassette efficiently to essentially all relevant bacteria in the animal gut and to ensure that the "pCURE" plasmid does not escape into the environment.
Work at the University of Birmingham will carry out genetic manipulation on the pCURE plasmid to increase its ability to spread and on the "self-destruct" cassette to optimise its effectiveness. These plasmids will be tested at the University of Surrey for spread in a laboratory pig gut model as a key validation stage before a prototype is chosen for testing in live pigs. DNA sequencing and genomic analysis jointly at the APHA and the University of Surrey will uncover the changes in the antimicrobial resistance (AMR), diversity and abundance of the gut model bacteria caused by the spread of pCURE, including loss of target plasmids plus the genes they carry, and the diversity of bacteria the pCURE have transferred to. The Pig Unit at Harper Adams University along with APHA will provide pig microbiota and ensure that pCURE is validated against a setting of real-world criteria in animal husbandry.
The long-term plan is to develop a set of probiotic strains that can be administered as feed or water supplements to reduce the burden of antimicrobial resistance in pigs on farms where problems of significant resistance levels are identified. The benefit should be healthier pigs due to reduced levels of infection (by displacement of bacterial virulence functions) and increased effectiveness of currently available antibiotics for therapeutics (due to reduced levels of resistance).

It is well established that many animal health and welfare problems involve gut bacteria which often carry both antibiotic resistance genes and virulence factors on plasmids that facilitate spread between bacteria. The DNA sequencing of bacterial strains collected from pigs confirms this and shows that most of these plasmids belong to well established groups, allowing us to predict many of their properties.
One of the possible solutions is "plasmid displacement" (also known as "plasmid-curing") that involves a conjugative pCURE plasmid carrying a set of genes as a "gene cassette" chosen to stop the target plasmid from replicating, as well as then neutralising its "addiction" functions. Although this process works well in the lab, the challenge is turning it into a practicable probiotic treatment in animals: can we deliver the curing cassette to essentially all bacteria in the animal gut and ensure that the pCURE plasmid does not escape into the environment?
We will build new pCURE plasmids with increased ability to spread by exploiting transfer systems that encode both long flexible pili, that stabilise mating pairs between donor and recipient bacteria, and short stubby pili that create efficient mating bridges across which the plasmid can transfer. These plasmids will be tested for spread in an in vitro pig gut model seeded with faecal bacteria from commercial pig units. DNA sequencing and genomic analysis will uncover the changes in the antimicrobial resistance, diversity and abundance of the gut model bacteria caused by the spread of pCURE, including loss of target plasmids plus the genes they carry, and the diversity of bacteria the pCURE plasmids have transferred to. We will also develop and test a self-destruction system that based on CRISPR or similar technology that can be triggered by natural and artificial environmental signals to destroy the pCURE plasmid when it has performed its desired function.