Natural Born KILLER

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By Amber Lepage-Monette

Due in large part to the growing concern surrounding antibiotic resistance, many researchers are looking for alternative ways to treat bacterial infections in both animals and humans. Rather than looking at completely new research areas in bacterial infection, some companies and researchers are looking to nature for solutions.

Enter bacteriophage, or phage, as it is also known. Phages are naturally occurring viruses that target and attack bacteria. They can be found anywhere bacteria are found, most often in water and fecal matter — environments that are typically intertwined. And there is no short supply of them.

“The place is teeming with them,” says Roger Johnson, PhD, a research scientist with Health Canada’s Laboratory for Foodborne Zoonoses (Guelph, ON).

As Johnson explains, phages have two kinds of life cycles: lytic and lysogenic. In the lytic life cycle, a phage works by locating its target bacterium, attaching to the receptor on the outside of that bacterium and then injecting its DNA into it. The phage contains only DNA and proteins, and so it uses the bacterium’s machinery to replicate itself, and at the same time produces proteins that break down the bacterial cell wall.

“The bacterium bursts and releases all of the progeny phages that have replicated inside of the bacterium — so this can be anywhere from 10 to several hundred or thousand new virus particles, or phages, for each bacterial cell that’s infected,” Johnson says. “That’s the basis of phage therapy.”

The second life cycle — lysogenic — is considered more virulent.

“In this situation, once the phage actually binds to the bacterial cell and injects its DNA into the bacterium, the DNA from the phage becomes incorporated into the DNA of the bacterium,” Johnson says. When the bacterial cell divides, it carries copies of the integrated phage DNA with it.

Johnson is part of a Health Canada project, funded by the Canada Alberta Beef Industry Development Fund (Calgary, AB), that is using phage therapy to treat E. coli O157:H7 in cattle. So far, he says, they have had success in treating experimentally infected cattle for E. coli O157, and are currently characterizing the phages in greater detail in order to move forward into field studies testing naturally infected cattle.

Treating E. coli in animals is an obvious place to start, Johnson says, because animals are a natural reservoir of the bacterium and the point at which the bacterium multiplies, and because humans acquire infection from the bacterium through a variety of ways.

“We know that a lot of infections, human infections caused by E. coli O157:H7, are not necessarily arising from consumption of hamburger meat, for example,” he says. “There have been a number of outbreaks, and many sporadic cases, which are associated with fresh produce, for example, that’s been irrigated with water that contains E. coli O157.

“There (are) many, many instances in which people, and particularly children, attending country fairs, or animal or livestock shows have acquired infection by direct contact with animals.”

Health Canada isn’t the only organization looking at combating foodborne bacterial illness with phage. Several Canadian companies are also looking into phage therapy as a means of treating several bacterial illnesses, which include E. coli and salmonella, among others.

The Numbers Game
GangaGen Life Sciences Inc. (Ottawa, ON) — a subsidiary of San Francisco, Calif.-based GangaGen Inc. — is working with phage therapy in a number of areas. The company is in the early stages of research with Salmonella and Campylobacter, isolating phages for these bacteria. All current projects are focused on treating E. coli O157 in animals.

As GangaGen Life Sciences president and CSO Kishore Murthy, PhD explains, E. coli O157 is found naturally in the gut of cattle. The animals act as carriers for the illness, which only poses a threat to human health. E. coli O157 infection in humans causes bloody diarrhea and severe abdominal cramps.

“What happens is, normally (E. coli bacteria) are carried in the gut and they’re shed, like in the cattle droppings . . . if there are heavy rains and all that, they could get washed into the surface water, and if that is not properly treated, people could get sick,” Murthy says.

Murthy explains that sometimes during slaughter, an animal’s gut is inadvertently cut, exposing meat to E. coli. When E. coli gets onto the surface of meat, such as a steak, there is a good chance cooking with sufficient heat will kill it, Murthy says. But when meat is ground together, as is the case with hamburger meat, E. coli is spread throughout the meat product, and is much harder to kill.

GangaGen Life Sciences’ technology involves isolating phages from the natural environment — either manure or groundwater from farms — purifying the phage and increasing their numbers. Because phages are located wherever there are bacteria, they are not foreign organisms to animals. It is putting them to use in larger numbers that the company is taking advantage of.

“These animals that have seen phages, they see phages all the time,” Murthy says. “But the numbers are so small that the possibility of the bacteria and the phage coming together is very low. So we are putting that in our favour by increasing the number.”

GangaGen Life Sciences is working to place phages into cattle feed to treat animals before slaughter, and thereby reduce the chances of E. coli getting into either the water system or the food chain.

The company plans to conduct studies in target animals shortly, and hopes to move into field studies by the end of this year or early next year.

A Strong Defence
Biophage Pharma Inc. (Montreal, QC) is also working in areas of food safety, targeting both E. coli and Salmonella.

When it comes to dealing with the issue of antibiotic resistance, two approaches are often taken, says T. Toney Ilenchuk, PhD, Biophage’s vice-president and chief development officer. One approach is to treat humans, but the other, the company realized, was to deal with antibiotic use on farms and its contribution to antibiotic resistance in man.

Biophage realized that antibiotic use on the farm was one area worth looking at, says Ilenchuk. “We thought the more appropriate target then would be to go after animals that were part of our food system — swine, poultry and beef — and start looking at two issues there: treating infections that might occur, but more to the point, to treat, to decontaminate animals so that the quality of the meat that ends up at the slaughterhouse, and therefore ends up at the retail shop, and the meat that you buy, has a better chance of not being contaminated.”

Biophage is currently in the developmental stages with both its Salmo-Pro and Coli-Pro products. Salmo-Pro is targeted for Salmonella DT108. The company is looking to place the product into animal drinking water between 24 and 48 hours before the animals are sent to slaughter to reduce the amount of Salmonella shed that could cross- contaminate the animals during shipping and processing.

Coli-Pro is designed to treat E. coli O64 in piglets. Farmers could treat the animals upon seeing symptoms of illness, thus preventing the condition from spreading. Additional Coli-Pro lines for other strains of E. coli are planned for the future.

Ilenchuk says Biophage studies have shown that phages are no longer present in experimentally infected animals within five days of treatment.

Biophage also has an ongoing project with Defence R&D Canada – Suffield’s (Medicine Hat, AB) Level III Biocontainment facilities researching phage therapy for Brucella and Bacillus anthracis, which causes anthrax infections. Ilenchuk says to date they have demonstrated, in situ, that the level of anthrax pathogenicity can be controlled.

B. anthracis produces spores, however, and therefore poses some particular problems when it comes to phage therapy, Ilenchuk explains. Phages do not target spores, but rather they require the cell membrane of the vegetative form of the bacterium.

“The broader question now is, we’re looking at combining antibodies against the spore to deal with what one does with anthrax that’s sitting around in spore form.”

Though anthrax can be a health issue in some animals, Biophage is currently focusing its efforts in areas for human use, Ilenchuk says, particularly for first responders. When it comes to an end product, the company’s anthrax program works a bit differently than traditional therapeutic programs.

“I don’t know that we’re looking at a product here . . . because who’s our customer here?” he says.

“I think we’re happy to be working with National Defence. We believe that dealing with first-responder issues is a key issue for us with our know-how, and I’m sure that at the end of the day, the armed forces will be dealing with purchasing any material if there is any material to be purchased.”

The Vaccine Approach
Montreal, Que.-based PhageTech Inc. is another company that is using phages to combat bacterial infection, though in different pathogens and through different means. The company is not using phage therapy, but rather developing novel antibiotics in the areas of Staphylococcus aureus, Streptococcus pneumoniae and Pseudomonas aeruginosa.

“The reason we chose these three major pathogens (is) because we believe the clinical need is more relevant,” says Dr. Jinzi Wu, PhD, PhageTech’s vice-president of R&D, Biology. The company, he says, also wanted to cover both gram-positive and gram-negative bacteria. PhageTech uses phage genomics to identify growth inhibitory proteins, which the company calls ORFs. The firm then uses these ORFs to identify bacterial targets that these proteins interact with.

“Basically, we believe these proteins derived from phage can lead us to identify the weak spots in bacteria,” Wu says. “So once these weak spots are identified, we develop small molecule drugs, just like any traditional antibiotics, which can do the same job as phage proteins do.”

Their approach has proven successful in discovering novel bacterial targets that are essential to bacterial growth and have been validated by phage.

Wu says that PhageTech’s approach has the advantage of being able to identify a smaller number of genes. To explain, Wu offers the example that in each bacterium, there may be thousands of genes, and among those, several hundred may be essential genes that may be targets for phage proteins. Rather than identifying the hundreds of essential genes, PhageTech’s work allows the company to identify and prioritize the handful of genes that are preferred, or tagged, by phage proteins.

“Via genomic efforts, all essential genes are identified for major pathogens,” Wu says. “However, as I said, people don’t know which top five they should work with. And our technology helps us identify (the) top five or top 10 genes we can work with. So in that sense, they are novel targets for developing new antibiotics.”

To date, PhageTech has sequenced the genomes of over 40 phages for the three pathogens they are investigating. The company’s next milestone is to demonstrate that the small molecules they have identified can treat bacteria in animal models, a goal they hope to meet sometime next year, Wu says.

Fighting Resistance
Developing new antibiotics and developing phage therapy are two differing ways companies are approaching the growing problem of antibiotic resistance. Differing approaches may be what’s needed, however, as phage therapy itself may not offer the be-all, end-all solution.

“(Bacteria) could become resistant to the phages,” Murthy says, noting that GangaGen Life Sciences is already considering this possibility, and therefore has an approach in which it is developing a cocktail of phages that will target multiple components on the bacterial surface.

For Ilenchuk, phage therapy simply offers a new alternative. “We’re not saying immediately get rid of antibiotics, but what we’re saying is that this is also a practical way . . . to manage the amount of antibiotic resistance that’s evolving from the farm,” he says.

“Because don’t forget, a lot of antibiotics that are used in the animals that we’re going to eat, are also antibiotics that we may one day be using to take care of our pneumonia,” he says. “And if we’re already preconditioned to these antibiotics, they are not going to work very well in us.”

Ultimately, despite differing approaches and target pathogens, the common denominator in all phage research comes down to the need to outsmart some of our most virulent bacteria.