Researchers headed by a team at the University of Birmingham in the U.K. have developed a probiotic drink containing genetic elements that are designed to thwart antimicrobial resistance (AMR) in gut bacteria at the genetic level. The drink targets small DNA elements called plasmids that carry antibiotic resistance genes, and which are able to replicate independently and spread between bacteria. By preventing these plasmids from replicating, the antibiotic resistance genes are displaced, effectively resensitizing the bacteria to antibiotics.
Research head Christopher Thomas, Ph.D., explained: “We were able to show that if you can stop the plasmid from replicating, then most of the bacteria lose the plasmid as the bacteria grow and divide. This means that infections that might otherwise be hard to control, even with the most powerful antibiotics available, are more likely to be treatable with standard antibiotics.”
The researchers report their developments in PLOS One, in a paper titled, “Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR.”
Antibiotic resistance in bacteria is an increasingly global health issue and represents an urgent challenge, the authors stated. “The rise of resistance is due to the selective pressure from widespread use of antimicrobial agents, combined with the genetic plasticity of bacteria, allowing resistance mechanisms to evolve and spread rapidly between bacteria. Once such resistance mechanisms exist, it is very difficult to get rid of them.”
Plasmids represent a key component of the bacterial “genetic arsenal,” the team continued Many plasmids can transfer between bacteria via conjugation, which promotes the spread of resistance-related genes. “In clinical contexts plasmids have often accumulated resistance determinants to all the antimicrobial agents that their hosts have been exposed to.” Some Gram-negative bacteria, for example, represent a major challenge because they are now resistant to key antibiotics due to plasmid-encoded enzymes.
One potential approach to tackling the problem is to displace these plasmids from bacteria—an approach known as curing—in major reservoirs of resistance, such as the gut. “Much of the resistance gene load carried in otherwise healthy individuals is within the gut microbiota and plasmids carrying the resistance genes facilitate their dissemination throughout human communities and the global population as a whole,” the investigators stated. “Being able to displace plasmids from a target population was therefore a key goal of this work.” It’s a promising strategy for tackling antibiotic resistance, they suggested, because selectively removing plasmids that carry resistance genes shouldn’t disrupt the natural microbiome mix.
Closely related plasmids introduced in the same cell will result in segregation of the cells into different lineages. It’s a phenomenon known as incompatibility, and represents one way of displacing plasmids from bacteria. Building on an adaptation of their previous exploitation of this phenomenon, the researchers have now developed a technology that uses a reproductively unrelated vector to displace the target plasmid because it carries genetic regions that block all replication and addiction systems encoded by the target plasmid.
The curing approach developed by Thomas and colleagues harnesses bacteria carrying a type of plasmid construct dubbed pCURE plasmids, which prevent the resistance plasmids from replicating and also block the “addiction,” or post-segregation killing (PSK) systems that are used by plasmids to kill bacteria that lose them.
In this new system, the resistance plasmid carries a stable toxin and an unstable antidote into the host cell. If the plasmid is lost from the cell, the antidote breaks down, leaving the harmful toxin to attack its host. pCURE plasmids also carry the antidote, ensuring that cells that lose the resistance plasmid survive and take over the gut. “PSK systems rely on expression from the plasmid of both an unstable antitoxin or regulatory RNA and a stable toxin or metastable RNA encoding delayed toxin expression: the toxin becomes active in the cell after plasmid loss,” the authors stated.
Thomas further explained: “We manipulated our pCURE plasmids to incorporate genes that block the replication of the resistance plasmid. We also target the plasmid’s addiction system by designing our pCURE plasmids to ensure the antidote is still available to the host.”
The Birmingham team demonstrated that by doubling the number of copies of the pCURE plasmid in each bacterium it became very effective at displacing different types of resistance plasmids, and would spread through laboratory cultures unaided, to clear out resistance. Given the findings in lab-grown cells, the researchers then collaborated with colleagues at the University of Sydney, to test the system in mice. They found that pCURE plasmids worked effectively, after they were “primed” by giving the mice an initial dose of antibiotic to reduce the number of competing bacteria.
“While the need for a period of antibiotic selection is not ideal, these results do demonstrate that curing plasmids are able to be delivered to the mouse gut and can transfer into target cells,” the authors noted. “The overall result is a gut that is free of the target bacteria and contains plasmid-free derivatives of the original plasmid-carrying bacteria. The use of a short selection with antibiotics allows endogenous microbiota to survive, leaving the potential to recolonize the gut.”
The next step will be to see if plasmids can spread fast enough in human volunteers to get rid of resistance plasmids. The concept is to develop a probiotic drink that will contain bacteria carrying the pCURE plasmids. “This is a promising start,” Thomas stated. “We aim to make modifications to further improve the efficacy of our pCURE plasmids before moving towards a first clinical trial … Antibiotic resistance is one of the biggest medical challenges of our time. We need to be tackling this on a number of different fronts including by reducing our use of antibiotics and searching for new, more effective drugs. Our approach, which tackles one of the causes of antimicrobial resistance at a genetic level, could be an important new weapon in this battle.”
The team is looking for the funding to start a clinical trial with the drink, which could feasibly be effective against common drug-resistant gut bacteria, including E. coli, Salmonella, and Klebsiella pneumoniae.