Phage

Electron micrograph courtesy of the University of Nottingham

Bacteriophages, a type of virus, resemble tadpoles, with distinctive heads and tails, as shown in this image captured by an electron microscope.

Jeroen De Buck's years-long effort to find a treatment for a fatal affliction in cattle called Johne's disease recently took a twist. The veterinary microbiologist and his team spent months, partly through the harsh Canadian winter, collecting dung, soil, wastewater, milk and calf bedding at a dozen dairy farms in Alberta.

Johne's disease is widely considered to be untreatable, in part because the pathogen that causes it, Mycobacterium avium subspecies paratuberculosis, has thick, waxy walls that are hard for antibiotic drugs to penetrate. Vaccines are available but far from perfect.

"I've been studying Johne's disease since 2006, and we've been developing a vaccine, which is still not satisfactory in terms of the protection it gives," he said. "Other labs have found the same."

Hence to the dairy farms, where De Buck was hunting for a type of organism that, it turned out, has provided a breakthrough. The discovery also puts him in the middle of a broader effort to develop alternatives to antibiotic drugs, many of which are becoming ineffective due to pathogens' evolving resistance.

The organisms he sought are bacteriophages — or phages for short —  a subset of viruses that infect and kill bacteria.

An assortment of phages isolated in Alberta by De Buck and his team at the University of Calgary Faculty of Veterinary Medicine have formed the basis, outlined in a paper published last summer, of what promises to be a new preventive treatment to defend cattle against Johne's disease.

Phages also have cropped up recently in a limited number of approved treatments in Brazil, Europe and the United States designed to prevent the spread of bacteria like Listeria and Salmonella in live food animals and their meat. Phage-based treatment has emerged in the realm of companion animal medicine, too. Last year, microbiologists and veterinarians in Israel successfully treated a cat that had a drug-resistant bacterial infection with what they believe is the first-ever application of phages in a feline patient.

Still, phages are little understood compared with antibiotics and have drawbacks related to efficacy, availability and safety.

Comprehensive regulatory frameworks haven't been established for phage therapies, stymieing the advance of commercially available treatments. In human medicine, for instance, phage therapy largely has been confined to compassionate use, under which patients facing death may be given an unapproved product if all other options fail.

What are phages?

Phages are viruses that specifically kill bacteria. They do so by injecting their DNA into a bacterial cell and hijacking its internal machinery to replicate. The new phages then burst out and repeat the process. Phages are found just about everywhere on Earth, in the deepest ocean trench and the hottest thermal spring. They are literally under our noses: Billions of phages live in and on each human body.

They tend to prefer places where there are plenty of bacteria to feast upon. "I know it doesn't sound very nice, but sewage is a very, very good source of phage," said Robert Atterbury, a microbiologist at the University of Nottingham's School of Veterinary Medicine and Science in England.

Phage therapy is nothing new: Their use to treat bacterial diseases in humans and other animals dates back about a century. But early understanding of their efficacy was limited, and interest in phages waned when antibiotics took hold in the 1940s. Still, phages continued to be studied and administered in the former Soviet Union and Eastern Europe.

Applications began reemerging in the West as concerns about antimicrobial resistance took root. In the 1980s, the English veterinarian Dr. Herbert Williams Smith and colleagues successfully used phages to treat Escherichia coli infections, first in mice, then in calves, lambs and piglets.

Interest in phages has picked up in the realms of veterinary and human medicine, especially in the last decade, as concerns about antimicrobial drug resistance intensify.

"And we know a lot more about phages now," Atterbury said. "We're able to design more effective treatments. We understand the biology a lot better."

Scientists are learning that phages are superior to antibiotics in some ways and less so in others. One advantage is that, unlike antibiotics, each type of phage tends to target a specific pathogen — invoking, Atterbury suggests, the steady hand of an accomplished surgeon.

"They can kind of surgically remove particular pathogenic species from a mixture of species," he said, "whereas if you're looking at antibiotics, particularly broad-spectrum ones, they're like a sledgehammer. You might take out your pathogenic species, but you also cause dysbiosis, this potentially harmful disturbance of the microbiota." (Microbiota are the organisms that live inside a particular environment, such as an animal's gut. They play an important health role, affecting factors like immune system function and digestion).

Moreover, phages typically need be administered only once because they replicate inside a patient's body until they run out of target bacteria to kill.

Still, a key strength of phages — their specificity — also is one of their weaknesses. A certain phage usually will be effective only against a single type of pathogen, even a single strain, making a suitable phage tough to identify. A potential solution to that problem is the establishment of phage banks, which already exist in some countries, notably at the Eliava Institute in Georgia and the Hirszfeld Institute in Poland. (Why research into phage therapy has persisted more predominantly in Eastern Europe is complicated and explored in a paper entitled "The strange history of phage therapy.")

The downside also can be managed by administering phage cocktails, on the basis that at least one in a mixture of phages will have the desired effect on the targeted pathogen. Using cocktails can also address another problematic feature of phages: Bacteria can develop resistance to them, too.

Phages

Electron micrograph courtesy of the University of Nottingham

These phages are attached to the end of an E. coli bacterium. The phages with dark (empty) heads have injected their DNA into the cell.

Tackling pathogens' 'anti-phage defense'

In phages, the resistance often is described as "anti-phage defense," according to Atterbury. Unlike antibiotics, though, almost countless varieties of phages exist on the planet, which means there's a greater chance of finding another phage that works.

Also, pathogens that have developed resistance to a phage can still be vulnerable to that same phage through a phenomenon known as the "fitness cost" of resistance. This occurs when a defense mechanism that's evolved in the bacterium — such as a change in the cell surface that makes it harder for phages to latch on — in turn, makes it harder for the cell to reproduce or survive inside a patient.

Nevertheless, pathogens' potential to evolve to resist phages presents a clear challenge.

For their part, De Buck's team at the University of Calgary made a cocktail of nine phages for their Johne's disease prophylactic. Eight had been collected from the farms they visited in Alberta; the ninth was previously identified in the United Kingdom.

"What you're looking for is a cocktail of many different phages that basically don't show cross-resistance, meaning that if the bacterium becomes resistant against one phage, it is not resistant against the next one," he said.

They're hoping to scale down their therapy to a more tailor-made cocktail of three or four phages.

Indeed, there is some debate within the phage community about the appropriateness of cocktails because throwing a large number of phages at a single pathogen might hasten the development of resistance to multiple phages.

With that in mind, the scientists in Israel who successfully treated a cat with phages decided to take what the lead author, Ron Braunstein, describes as a "personalized therapy approach."

Combining with antibiotics

The researchers at the Hebrew University of Jerusalem's Phage Therapy Lab, working in collaboration with a veterinary hospital, collected 34 phage types from the Israeli Phage Bank and tested each against a sample of the pathogen Pseudomonas aeruginosa.

"We had around 20 phages that showed some sort of activity against this pathogen," Braunstein, a doctoral student in microbiology, said. "And then from them, we tested the best one, and we found one that was really, really good."

Squeaks

Photo courtesy of the Hebrew University of Jerusalem

Squeaks is thought to be the first feline patient to be treated successfully with phage therapy.

The feline patient was a 5-year-old Siamese that fell from a high-rise building, breaking bones in both hind legs. The cat underwent surgery to insert metal implants. The left hind leg developed necrosis and had to be amputated two weeks later, while an implant-associated P. aeruginosa infection developed in the right hind leg.

The infection turned out to be resistant to multiple antibiotics — amoxicillin, azithromycin, cefovecin, doxycycline and marbofloxacin — making the cat a candidate for the compassionate use of phage therapy. Notably, the phage was administered in combination with another antibiotic, ceftazidime.

The cat’s wound closed completely after 115 days of the combined treatment. One month later, however, the wound began expanding again, and ceftazidime alone was reintroduced. After three weeks without satisfying results, the phage was added back in. Ten days later, the wound had closed completely, and it stayed closed.

The researchers believe it unlikely the antibiotic was solely responsible for the cat's recovery, in part since decreased sensitivity to ceftazidime was observed during the first course of treatment, and it appeared to be ineffective during the second course when administered by itself. All considered, they believe their tests indicated that neither the antibiotic nor the phage alone could have effectively eradicated the bacteria, but worked well in combination.

"This is something we really strongly believe in," Braunstein said. "You need to give phages together with antibiotics because it's like you're attacking the bacteria from two angles. And a lot of the time you get, as our case demonstrates, a really strong synergistic effect."

One reason Braunstein is wary of relying heavily on phages alone is that, at least from his own anecdotal observations, they tend to develop resistance faster than antibiotics.

"Phage therapy is not magic," he said.

Bumpy road to commercial availability

Phages also have a dark side.

Some can transfer their genes to the bacteria they infect, potentially conferring the blueprint for traits related to virulence, resistance and toxicity. In other words, using those phages could inadvertently make a targeted pathogen stronger.

Phages that aren't prone to this complication are called lytic phages. The more potentially problematic ones are known as temperate or lysogenic phages.

Even lytic phages, though, could pose problems because when they break bacteria apart, they leave bacterial DNA fragments hanging around that could be incorporated into other bacterial species. "That happens naturally in some bacterial species, anyway," Atterbury said. "This is not an exclusive process to bacteriophage. This happens in your gut, on your skin, in the environment. It happens everywhere."

To reduce safety risks, researchers can first sequence the genes in a candidate phage to check for problematic genes, Braunstein said. "If we find any, we just say, 'OK, this phage is not suitable for patients.' "

At a broader level, there also are concerns phages could, like antibiotics, contaminate the environment, should they be propagated in large volume for medical use and find their way into rivers and other ecosystems.

Regulators also must grapple with overseeing dosing and delivery protocols for a vast number of different organisms. Moreover, establishing intellectual property protections for naturally occurring viruses presents a challenge, Atterbury said. "Now, you can change that by genetically modifying a virus, but then that adds more regulatory problems. …"

Despite such barriers, a small number of phage-based products have made their way onto the market, according to Adriano Gigante, a pharmacist and colleague of Atterbury's in Nottingham.

The product approved in the U.S., he said, is a spray that is applied to packaging to prevent food poisoning. In Europe, Gigante noted, the European Food Safety Authority in December found that a phage-based feed additive to protect live poultry from Salmonella was safe for the target species, consumers and the environment. Brazil has approved two similar phage-based products to combat Salmonella and E. coli infections in chickens.

"Bacteriophages as a product are safe to use if you use them properly, just like antibiotics," Gigante said. "But what makes for proper use of bacteriophages is not well understood yet by science."

As for the Johne's disease-prevention treatment De Buck and his colleagues developed using phages, its future is looking promising. De Buck said a company is helping to fund new research into its commercial potential. 

Not a panacea

Assuming the understanding of phages progresses rapidly, could they eventually replace antibiotics entirely?

That's unlikely, according to all the sources contacted by the VIN News Service.

"I think the future will probably be a combination of approaches, where antibiotics, I'm sure, will still have a role, but you may use them in combination with other things like phages, immunotherapies and antimicrobial peptide treatments," Atterbury said.

Braunstein contends that antiboics, including new classes of antibiotics, like one called lariocidin discovered earlier this year, will play a more important role than phages.

"I personally believe, and our lab's approach is, that we are not going to replace antibiotics," he said. "And I'm a phage therapist, you could say, and I still say that antibiotics are still the best drug we have against bacteria."

For his part, De Buck is reluctant to predict what will happen across the board, noting that some infections are more sensitive to the use of antibiotics than others. He agrees antibiotics and phages could work well together but posits phages alone might prove critical for fighting pathogens that are highly resistant to antibiotics.

Take Johne's disease. "As I explained, we are not using antibiotics for that, anyway," he said.

"I cannot just say, 'Yes, antibiotics are in the past, and phages are the new thing.' It depends. I would assess each disease and the specific circumstances to decide to actually use phages or not."