Phages on the farm: Can these tiny viruses help us overcome antibiotic resistance?

In 2013, Tobi Nagel ended a decade-long search in the country of Georgia. Local scientists she was teaching a workshop with spoke wonders about a ubiquitous, over-the-counter medicine. In pharmacies, alongside conventional antibiotics, consumers in Georgia had another option: solutions of bacteriophages, tiny viruses that can infiltrate bacteria and destroy them from the inside.

At the time, Nagel consulted in lower-income countries—a side gig from her work as a drug development scientist in the pharmaceutical industry—and was constantly looking for cost-effective ways to produce drugs. “It was unsettling living in two worlds, developing a drug in the US that might cost a billion dollars to make, while working with people in developing countries who would never be able to access that drug,” Nagel says. “When I learned about phages, I instantly realized, this is a cheap technology.”

The viruses, considered to be the most prevalent biological entity on Earth, have been used as treatments for bacterial infections in eastern Europe since World War II. More important for Nagel, they were an affordable alternative to run-of-the-mill antibiotics.

So in 2014, Nagel started work on what would eventually become Phages for Global Health (PGH), a nonprofit that aims to bring phages, and the know-how to use them, to Africa and Asia.

Organizations like PGH and some start-ups see an inroad for phages in livestock and food production, sectors in which the relatively low cost of getting approval for and manufacturing phage treatments would keep prices competitive with those of antibiotics and could bring new bacteria-fighting tools to farmers quickly.

The viruses could make farmers around the world less dependent on small-molecule antimicrobials and give humans a much-needed weapon against antibiotic-resistant infections. Chemists and engineers are still tinkering with different formulations, trying to figure out what a phage product might look like and how to extend phages’ shelf life—and after all that, farmers accustomed to conventional antibiotics have to be convinced to switch to the new technology.

A needed alternative

Campylobacter, a corkscrew-shaped bacterium, measures just a few micrometers long, but it looms large in Kenya. Along with Shigella and Salmonella, the foodborne bacterium is one of the country’s leading causes of diarrheal diseases, which in 2019 killed more than one in 1,000 children under the age of 5, according to estimates by Global Burden of Disease.

Infections of Campylobacter can often be traced back to live chickens. If, between slaughter and sale, bird carcasses sit unrefrigerated, they become an excellent breeding ground for the bacteria. And when an unlucky consumer eats Campylobacter-containing meat and develops an infection, that person has a dwindling choice of effective antibiotics, because resistant strains of the bacterium are prevalent in Kenya. A 2016 study in the country found Campylobacter on more than 90% of chickens and their manure, and at least 61% of cultured samples were multidrug resistant.

One culprit in that resistance: farmers, who use antibiotics liberally to prevent infection or to futilely treat undiagnosed illnesses that aren’t even of microbial origin. They also use the drugs to promote growth—rather than expending calories to stave off an infection, the animal can use its energy to build profitable bone and muscle.

“When a farmer looks at the monetary value of the animal, they will do anything to see that animal survive,” says Jesca Nakavuma, a microbiologist and self-described phage hunter at Makerere University in Uganda. The temptation to overuse antibiotics has long existed worldwide, so much so that the European Union and US have banned farms’ use of the drugs for preventing disease and promoting growth.

But antibiotics are a blunt tool, eradicating all sorts of bad as well as good bacteria. Long term, their overuse causes bacteria to get stronger and more difficult to kill with existing drugs, creating a therapeutic void that pharmaceutical companies are struggling to fill.

That’s why Nakavuma collaborates with PGH to identify useful phages and eventually get farmers and doctors to use them as a complementary treatment to antibiotics.

In contrast to antibiotics, phages are like a scalpel. They’ve evolved alongside bacteria in their region and are tailored to attack specific microbes, allowing others to thrive. According to phage researchers, overuse of phages in agriculture shouldn’t be an issue because in theory they’re self-dosing and self-limiting: a farmer would need to provide just enough of the viruses to allow them to reach their target bacteria inside, say, an animal’s small intestine. Once there, the viruses can reproduce and create a self-sustaining population. That population infects, multiplies inside of, and ultimately ruptures bacteria until they largely die out. Then, the phage population peters out too.

And although phages and antibiotics both kill bacteria, the way the bacteria develop resistance to the two differs. Antibiotic resistance happens in a few ways: bacteria evolve proteins to pump antibiotics out of their cells, or they evolve an enzyme that can chemically deactivate the antibiotic molecule among other strategies. Resistance then gets amplified as the surviving bacteria meet and share DNA that they used to evade the antibiotic.

Phages, on the other hand, are moving targets. They mutate in step with bacteria, changing too fast for bacteria to eliminate the threat simply by sharing their defense mechanisms.

Some defenses against phages do provide more permanent resistance, however. For example, certain bacteria save snippets of phage DNA in their genome so that they can later recognize and attack a virus based on key characteristics. But because those DNA records take a long time to develop, a virus might have evolved away from that genetic sequence by the time the bacteria share it among themselves. On top of that, the string of records is not infinitely long, so to keep picking up new sequences, the bacteria need to forget others. “Bacteria do not evolve phage resistance and share with one another the same way” as when they develop antibiotic resistance, says Paul Turner, a biologist at Yale University and cofounder of phage therapeutic start-up Felix Biotechnology. “It just doesn’t happen.”

Phage hunters, phage guardians

Novel antibiotics don’t grow on trees. They require hundreds of millions of dollars to discover, tweak, and ultimately manufacture in facilities that are up to code. But phages grow wherever bacteria are found: in soil and water and inside animals. That’s where phage researchers hunt for their candidates.

To develop phage treatments that will work best, experts try to harvest viruses from the local environment, where there’s plenty of bacteria to bait them—such as in sewage and farmyard manure and runoff.

By collecting these wild viruses, Nakavuma hopes to assemble a regional phage library. Specific types of phages have evolved to kill only a subset of the species of bacteria that they encounter in the wild. Once biologists figure out which viruses grow alongside problematic bacteria in culture, they sequence that virus’s genome and add it to a library, where it will be maintained. Later when livestock in a region are plagued with an emerging pathogen, the expertise and infrastructure will be in place to deploy a tailored phage cocktail—an approach akin to tweaking the annual flu vaccine to target circulating viral strains. In this model, phage treatments work best when the viruses are centrally controlled and curated.

But once removed from their habitat, phages get a bit finicky. A big reason antibiotics still rule the roost on farms is that they are cheap and shelf stable. Phages can be more fragile—without bacteria to feed on, they become inactive at room temperature. That’s an issue for farmers who don’t have fridges that can maintain liters and liters of phage solution at 4 °C.

So the search is on to find a way to dry those solutions and lock the phages in a solid state that doesn’t require cold storage. PGH has teamed up with engineers to devise ways to do just this. Reinhard Vehring of the University of Alberta is working with PGH to perfect spray-drying processes to make powders loaded with anti-Campylobacter phages (Microorganisms 2020, DOI: 10.3390/microorganisms8020282).

When it comes to making powders out of liquids, spray-drying—spraying an aerosol and rapidly drying it with a hot gas—has long been a go-to method for cranking out tons of product per hour. It’s how you make powdered milk at scale. But phages aren’t milk; they’re constructed out of protein complexes that need to stay intact, or else the viruses won’t be able to replicate once rehydrated.

To keep powdered phages safe, Vehring’s lab borrowed a strategy from the resurrection plant Selaginella lepidophylla, which can go without water for several years. As it dries out, the plant puts sugar molecules in place of the hydrogen-bonded water molecules that maintain the structure of its cellular machinery, creating so-called sugar glass. When it rains, the plant comes back to life as water fills its cells again.

To mimic that process, the researchers add the sugar trehalose to their phage suspensions before spray-drying them. One theory, Vehring says, is that the trehalose molecules encase and stabilize the phages. The trehalose-treated viruses can be kept cozy and shelf stable at room temperature for months, possibly a year. Just add water, and the phages will be ready to attack strains of Campylobacter again.

Other groups are trying to seal their phages in a different way. Nicholas Svitek, a senior scientist at the International Livestock Research Institute (ILRI), works in Kenya combating Salmonella in animals. One of ILRI’s initiatives is the development and promotion of sustainable and scalable livestock practices that could limit antibiotic resistance. The institute’s team is initially exploring approaches to phage encapsulation, in which the viruses are corralled into or onto micro- and nanoscale particles of polysaccharides or silica, Svitek says.

Encapsulation materials like alginate mixed with the basic polysaccharide chitosan can protect phages not just from the outside environment during storage but also from animals’ acidic stomachs, which comes in handy if the phages need to navigate through the stomach to get to bacteria that thrive in the gut. The plan is for these dried powders and encapsulations to be given as a feed additive or in water.

Going a step further, the Scotland-based start-up Fixed Phage is putting phages on the food itself. The firm is currently fine-tuning fish-feed pellets that have phages stuck to their surfaces.

Fixed Phage sees the pretreated feed as a drop-in product that could be simpler for farmers to use. “In terms of compliance, that is much better than having a jug that we keep at 4 degrees that they have to get out of the fridge, use immediately, remember to use the right amount, and put back in the fridge without leaving it out for too long,” says Jason Clark, the company’s chief scientific officer.

To anchor viruses onto food pellets, Fixed Phage leverages a process that’s routinely used to make ink stick to plastic. The firm runs the pellets under corona discharge—little spurts of plasma that ionize a surface—and promptly sprays them with solutions of phages. As the solution dries, the viruses stick to the ionized pellets and can be stored for months at room temperature without losing infectivity. The company is conducting field trials of its feed to see how the phages manage in the real world.

Reaching out locally

Last October, Evans Agbemafle stood masked in front of a room full of fish farmers in eastern Ghana. He was detailing the benefits of phages and attempting to dispel the farmers’ concerns.

Agbemafle, a research scientist at the Ghanaian government’s Council for Scientific Industrial Research (CSIR), is one of the coprincipal investigators contributing to SafeFish, which aims to solve the problem of fish death in Uganda and Ghana’s booming aquaculture sector. With $1 million in funding from the African Union and the European Commission last year, SafeFish scientists are just starting up the slow process of going through local soil, manure, run-off, and fish’s intestinal contents so that they can eventually purify and characterize the area’s phage population.

The researchers would prefer to use local phages, but Ghana’s lack of sophisticated gene-sequencing equipment and bioinformaticians to comb through genetic data are major obstacles. So Agbemafle and Makerere University’s Nakavuma want to team up with other institutions to assemble equipment and expertise to identify and catalog phages. Their larger goal is to build a regional consortium for phage research and a library that African nations could dip into when faced with resistant bacteria. But that’s a long way off. “We’re starting from scratch,” Agbemafle says. “We have to improvise.”

The technological issues can be profound. PGH’s Nagel recalls leading a workshop in Tanzania in early 2020 during which she wanted to show local biochemists how to use Oxford Nanopore Technologies’ MinION handheld gene sequencer. Though a dedicated router had been set up for the device, there wasn’t enough bandwidth to sequence the genome in the sample. The PGH scientists resorted to a mobile hot spot to finish the training. “So it’s things like that,” Nagel says. “Or the power goes out, or there’s no refrigeration.”

PGH, the SafeFish team, and ILRI say interactions with farmers are vital for the eventual adoption of new agricultural products. Feedback from workshops and focus groups, along with staff from the area, guide their design processes.

“When you understand the dynamics of the farm system, then you understand the dynamics of product development,” ILRI postdoc Angela Makumi says. “It’s not one size fits all.”

Makumi, who is from Kenya and trained as a phage biologist in Europe before returning to the country, brings cultural understanding that has helped the research team discern what farmers would want in a phage product. To reduce costs, for instance, they prefer not to have to buy a separate item; they would want phages already added to feed or combined with a vaccine that they already use.

“If we get a product without prior sensitization of the farmers, you have an uphill task to have it adopted,” Nakavuma says. “So I hope that by the time we have a product, we will have won over their trust.”