Phage editing technology could lead to alternative treatments for antibiotic-resistant bacteria

Antibiotic resistance is a growing threat to our health. The scientific and medical communities are searching for new drugs to fight infections. Researchers at the Gladstone Institutes have come a step closer to achieving this goal with a new technique that harnesses the power of bacteriophages.

Bacteriophages, or phages for short, are viruses that naturally take over and kill bacteria. There are thousands of phages, but using them as treatments to fight specific bacteria has so far proven difficult. To optimize phage therapy and make it applicable to human diseases, scientists must find ways to turn phages into efficient bacteria-killing machines. This would also offer an alternative solution for treating bacterial infections that are resistant to conventional antibiotics.

Today, Gladstone scientists have developed technology that allows them to modify phage genomes in a simplified and highly efficient way, giving them the ability to design new phages and study how viruses can be used to target specific bacteria.

“Ultimately, if we want to use phages to save the lives of people with infections that are resistant to multiple drugs, we need to find a way to make and test many phage variants to find the best ones,” says Seth Shipman, Ph.D., a research associate at Gladstone and lead author of a study published in Biotechnology of nature“This new technique allows us to successfully and rapidly introduce different modifications into the phage genome so that we can create many variants.”

The new approach relies on molecules called retrons, which originate in the bacteria’s immune system and act as DNA-making factories inside bacterial cells. Shipman’s team has found ways to program retrons to make copies of a desired DNA sequence. When phages infect a bacterial colony containing retrons, using the technique described in the team’s new study, the phages integrate the DNA sequences produced by the retrons into their own genomes.

The enemy of your enemy

Unlike antibiotics, which kill many types of bacteria at once, phages are highly specific to certain strains of bacteria. As rates of antibiotic-resistant bacterial infections rise (an estimated 2.8 million such infections occur in the United States each year), researchers are increasingly interested in the potential of phage therapy as an alternative to combating these infections.

“They say the enemy of your enemy is your friend,” says Shipman, who is also an associate professor in UCSF’s Department of Bioengineering and Therapeutic Sciences and a researcher at the Chan Zuckerberg Biohub. “Our enemies are these pathogenic bacteria, and their enemies are phages.”

Phages have already been used successfully in the clinic to treat a small number of patients with potentially life-threatening infections that are resistant to antibiotics, but developing these therapies is complex, time-consuming, and difficult to replicate on a large scale. Doctors must screen collections of natural phages to see if any of them might work against the specific bacteria isolated from a given patient.

Shipman’s group wanted to find a way to modify phage genomes to create larger collections of phages that can be screened for therapeutic use, as well as to collect data on what makes certain phages more effective or what makes them more or less specific to bacterial targets.

“As natural predators of bacteria, phages play an important role in shaping microbial communities,” says Chloe Fishman, a former Gladstone research associate and co-senior author of the new study who is now a graduate student at The Rockefeller University. “Having tools to edit their genomes is important to better study them. It’s also important if we want to manipulate them in ways that can shape microbial communities to our advantage, such as killing antibiotic-resistant bacteria.”

Continuous editing of phages

To precisely edit phage genomes, scientists have turned to retons. In recent years, Shipman and his group have pioneered the development and use of retons to edit the DNA of human cells, yeast, and other organisms.

Shipman and his colleagues started by creating retrons that produce DNA sequences specifically designed to modify invading phages—a system the team dubbed “recombitrons.” They then placed these retrons into colonies of bacteria. Finally, they allowed the phages to infect the bacterial colonies. As the phages infected the bacteria, they continually acquired and integrated new DNA from the recombitrons, modifying their own genomes as they went.

The research team showed that the more phages infected a bacterial colony containing recombitrons, the greater the number of modified phage genomes. Additionally, the researchers were able to program different bacteria within the colony with different recombitrons, and the phages acquired multiple modifications as they infected the colony.

“When a phage goes from one bacterium to another, it gets a lot of modifications,” Shipman says. “Making multiple modifications to phages used to be incredibly difficult to do, so much so that most of the time, scientists just didn’t do it. Now, you just put a few phages in these cultures, wait a while, and you get phages that have been modified multiple times.”

A platform for screening phages

If scientists already knew exactly what modifications they wanted to make to a given phage to optimize its therapeutic potential, the new platform would allow them to make those modifications easily and efficiently. However, before they can predict the consequences of a genetic modification, researchers first need to better understand what makes phages work and how variations in their genome affect their effectiveness. The recombitron system is also helping to advance this area.

If several recombitrons are introduced into a bacterial colony and the phages are allowed to infect the colony for a short period of time, different phages will acquire different combinations of modifications. These diverse collections of phages could then be compared.

“Scientists now have a way to edit multiple genes at once if they want to study how those genes interact or introduce modifications that might make the phage a more powerful bacterial killer,” says Kate Crawford, a graduate student in the Shipman lab and co-first author of the new study.

Shipman’s team is working to increase the number of different recombitrons that can be placed in a single bacterial colony and then fed to phages. They hope that eventually millions of combinations of modifications could be introduced into phages to create huge screening libraries.

“We want to scale this project up to a high enough level, with enough phage variants, that we can start to predict which phage variants will work against which bacterial infections,” Shipman says.