Bacteriophages (longish, more technical New Scientist article)

5 April 2003

Published in New Scientist  (This is my original draft - the New Scientist rewrite wasn't as good.)

The male patient, a diabetic in Tbilisi, Republic of Georgia, had been told his foot would be amputated. He no longer had any circulation to move antibiotics to the infection there. As a last resort, his doctor had sent the man to Tbilisi’s main hospital, where they still used a therapy dating back to the dawn of the Soviet era.

Dr Elizabeth Kutter, visiting from Washington State, was there a few days later, to watch Dr Guram Gvasalia, the hospital’s chief surgeon, unwrap the infected foot.

“There was literally no sign of purulence,” Kutter says, from her office at Washington’s Evergreen State College. “As was later confirmed, they had saved his foot. My reaction was: how incredible! Here in the United States we have no real way to treat diabetic feet.”

That was 1996, and Dr Gvasalia had been routinely saving diabetic feet for 25 years. His secret: the humble bacteriophage, probably the most common life-form on Earth.

Bacteriophages (better-known simply as “phages”) are viruses that parasitise and kill bacteria. They have been known about since the early 20th century, and now that our antibiotic arsenal is starting to fail us, interest in them is reviving. As biotechnologists learn to upgrade these viruses with extra molecular weaponry, phages could become a last line of defence against a host of infectious diseases.

Phages came to our attention as early as 1896, when British chemist EH Hankin found that something in the waters of India’s River Ganges could check the spread of Vibrio cholerae bacteria. The invisible agent was destroyed by boiling, so appeared to be a living entity. It was, Hankin speculated, keeping cholera epidemics at bay among those who drank the river water.

It wasn’t till 1915 that British bacteriologist Frederick Twort identified the likely agent as a virus, the bacteriophage - a name actually given to it by Canadian microbiologist Félix d’Hérelle, who discovered phages independently two years later. (The two are now co-credited with the discovery.)

The clinical efficacy of phages was first demonstrated in 1919, when d’Hérelle treated several children with severe dysentery at a Paris hospital. Lacking any safety data on the phage, d’Hérelle and his colleagues drank a substantial quantity of it the day before treatment. “It was their version of a phase one clinical trial,” says Elizabeth Kutter with amusement. “When nothing happened to them, they used it on some children, including several boys whose sister had just died - all of whom were close to death. And it cured all of them.”

D’Hérelle went on to investigate phages at the Pasteur Institutes in Paris and Saigon in the early 1920s. In 1925 he became a health officer to the League of Nations in Egypt, using phages to control infectious disease during the Muslim pilgrimages, and on ships passing through the Suez Canal. The fame of the new treatment spread, and soon Western drug companies such as Eli Lilly had put phages in their product lines.

Therapeutically useful phages are lytic - those which, in classical viral fashion, pierce the offending bacteria, multiply, kill the bacterial cell by bursting (or lysing) it open, and liberate hundreds more viral particles to infect further bacteria.

Like the early “natural” antibiotics such as penicillin, phages are cheap and easy to produce. Scientists first find a source, like local sewage. After taking the larger particles out of it, they mix it in a medium with the bacterial species they want phages against. A day later, lots of the required phages will usually have bred up. The bacterial cells are then broken open with chloroform, to kill them and release the phages.

After that the phages are put onto a plate of agar with the appropriate bacteria. Clear patches will form on the bacterial lawn: that’s where the phages are.

Dr Elizabeth Kutter says phages are often bred up through several generations to select for particular combinations of bacterial strains. “While it is very easy to isolate a phage against most bacteria, it is much more challenging to isolate one that will be effective, and to do all the characterisation and analysis that should be done before they are broadly used therapeutically.”

Before World War Two, phages were poured onto lesions, taken orally, or used in aerosols and enemas. On rare occasions they were injected. They treated typhoid and paratyphoid fevers, cholera, and pus-producing and urinary tract infections, among others. Hundreds of studies were written up, though many of them were anecdotal or poorly controlled. Doctors jumped onto the phage bandwagon with little comprehension of how phages worked. Writes one author: “Often, uncharacterised phages at unknown concentrations were given to patients without specific bacteriological diagnosis, and there is no mention of follow up, controls or placebos.” (“Viruses of Prokaryotes”, Hans Ackermann, CRC Press, 1987.)

In 1931, the American Medical Association commissioned a major study on phages, which concluded that evidence for the efficacy was “contradictory”.

Meanwhile, an entirely new sphere of bacteriology was taking shape. In 1928 Alexander Fleming had discovered penicillin. By 1939, Howard Florey began small-scale manufacture of the drug, and in 1940 penicillin’s success against infection became widely known. With World War Two escalating, penicillin use became widespread in the Allied military. In the post-War West, phages were increasingly discarded as antibiotics became more available.

But behind the Iron Curtain it was a different story. In the early 1920s, Félix d’Hérelle had collaborated at the Pasteur Institute in Paris with a talented Georgian microbiologist named George Eliava. In 1923 Eliava had founded an Institute of Bacteriology in Tbilisi, the Georgian capital. D’Hérelle spent a total of 16 months at the Institute between 1933 and 1935, purchasing it books, materials and equipment with his own money. In 1936 Eliava embarked on a building program, and by 1938 a much larger Institute of Microbiology, Bacteriology and Bacteriophages had arisen on the site.

But in 1937 Eliava had been executed by Lavrenti Beria - then head of the Georgian KGB - as “an enemy of the people”. (Reportedly, the two fancied the same woman.) Disillusioned, d’Hérelle cut his ties. But the Institute plugged on, in the phlegmatic Georgian way, till the present day. It is now generally known as the Eliava Institute.

Today, says Zemphira Alavidze, who heads Eliava’s Phage Morphology and Biology lab, “our phage preparations are used successfully in almost every hospital in Tbilisi and other regions of Georgia”.

In recent years the Institute has studied thousands of strains of Staphylococcus, Streptococcus, Proteus, Clostridium and other bacteria for sensitivity to phages. Phage strains which inhibit most of them have been isolated, and phages are routinely used in Georgian hospitals and clinics against the above bacteria as well as others such as Pseudomonas, E. coli, Shigella and Enterococcus.

Until the collapse of the Soviet Union, the Institute exported phages throughout the eastern bloc. Each year it produced 3-5 tonnes of phage tablets against dysentery alone. For the moment, though, its 800 production workers are gone, its pill-stampers and bottling machines are silent, and its fermentation vats are empty.

Eliava’s industrial production has been privatised, though not all the companies which have taken it over are thriving. The Institute itself presently produces two phage “cocktails” - one for intestinal infections, the other for purulent wound infections. But at 200 litres a year each, this production is less than a tenth of what it was. Russia and Poland still have some phage production.

“My children,” says Zemphira Alavidze, “have never used antibiotics. My ten-year-old grandson also uses only phages.” And now Alavidze is reversing the historical trend, by reconstructing the Institute’s production facility. Alavidze plans to soon produce fifteen phage products in sufficient quantity to supply Georgia.

The Institute has Georgian patents for numerous products including a phage cocktail against purulent infection which attacks Staphylococcus, Streptococcus, E. coli, Proteus, P. aeruginosa, Enterobacter and Acinetobacter.

Whilst the immediate post-Soviet period was grim (mounting debt, unpaid staff, on-and-off water and power), the Institute’s debts are paid off now, and Eliava’s power and water are back on - due in part to several international grants.

Eliava has a small clinic, headed by Dr Inga Georgadze, whose father headed the Institute for many years. The products it dispenses include phage suppositories for small children, creams for skin complaints, and liquids for gastrointestinal disorders.

If it weren’t for the decline of antibiotics, the story of phages might have remained confined to the crumbling healthcare system of a former Communist state. But the proliferation of antibiotic-resistant “superbugs” has accelerated sharply over the last decade or so ago - mostly the result of the over-use of antibiotics in food-producing animals, and over-prescribing by doctors. Thousands have now died from untreatable infections - and it’s getting worse. Dr John Tapsall from the World Health Organization’s Drug Resistance Surveillance Group, says: “We have just had reports from Japan of treatment failure, due to resistance, of the last remaining group of antibiotics active against gonorrhoea.”

Citing the Central Intelligence Agency, a WHO 2001 “Global Strategy” document states: “The emergence of antimicrobial resistance is regarded as a major future threat to the security and political stability of some regions.”

Because of the mounting crisis, phages are once again starting to attract the attention of idealistic grant foundations and concerned governments. The Eliava Institute recently received $US315,000 from the Civilian Research and Development Foundation in the US, and $US800,000 over three years from the International Science and Technology Centers. Another senior scientist at Eliava, Liana Gachechiladze, has also received an ISTC grant - for developing new phage products in collaboration with one of the small companies that arose from the Institute’s privatised production.

But regardless of the antibiotic resistance crisis, it’s believed by phage aficionados that bacteriophage therapy has several advantages over antibiotics in the first place:

Whilst antibiotics decrease in concentration from the moment they’re administered, phages typically do the reverse, by breeding up rapidly into a formidable army. And phages offer the best of both worlds: they’re are also self-limiting. Once their job is done and the “bad” bacteria are dead, they too die away.

Unlike antibiotics, phages are “smart weapons” specific to bacterial species and strains. “Phages’ tail-fibres interact with molecules on the surface of a particular bacterium, sort of like a lock and key might interact,” says Elizabeth Kutter. “So those phages will only infect bacteria that have those correct receptors on the surface. In fact, phages will generally infect a subset of a particular species of bacteria.”

Phages trigger no allergies, and few side-effects, and are cheap and easy to produce. Nature, indeed, produces them effortlessly: there are an estimated 10/32 [10 to the power of 32] on Earth.

There are some sceptics. James Bull, Miescher Regents Professor in Integrative Biology at the University of Texas, says “One can either take a stance of ‘cautious until proven effective’, or of ‘worthy until proven ineffective’. I'm in the former camp.” Bull says that infections are difficult to treat once established - by any method. And “virtually all experimental phage therapy applies phages at the same time as the bacteria, so one cannot assume that the current experimental work will reflect phage treatment of an established infection.” And Bull suggests another potential problem: “There may be a major regulatory impediment, depending on how the Food and Drug Administration approves phages for clinical use. If each phage needs to be tested separately, then the cost will be prohibitive, because individual phages tend to have too narrow a host range to justify seeking approval one at a time.”

Professor Bull also points out that some bacteria invade cells, to help them evade the immune system, and that it remains unproven that phages would be able to attack such bacteria.

“By and large,” he says, “there has been so much hype about phage therapy that the field really needs some objective and quantitative studies to explore where it works, where it doesn’t, and why.”

To this end, Dr Elizabeth Kutter was inspired by her experience in Tbilisi to found the non-profit PhageBiotics Foundation. The Foundation promotes and supports phage research in the West, and provides moral and financial support to phage researchers in Poland and the former Soviet lands.

The healing of the Georgian man’s “diabetic foot” helped Kutter to see understand another advantage phages have over antibiotics: their ability to replicate allows them to get into local infections where there is poor circulation. “Things like diabetic ulcers or infections from osteomyelitis,” she says, “which antibiotics can’t get into. But the phage just goes in, and multiplies. Then the daughter phages go deeper, and find more bacteria, and multiply further. Phages just keep expanding, and going deeper, until they get most of the bacteria that are there.”

One phage will parasitise several - and often most - strains in a bacterial species, says Nina Chanishvili, head of Eliava’s Genetics of Microorganisms and Bacteriophages lab. “And there is sometimes a ‘cross-lyse’ - for example between E. coli and Shigella, because those two bacteria are evolutionarily very close to each other.”

On the other hand, phages’ high specificity can present difficulties if a patient’s bacterial strain is unknown, and there is no time to test for it.

And just as they can develop resistance to antibiotics, bacteria can also develop resistance to phages. Thus Kutter believes “phage cocktails” are the best approach to killing pathogenic bacteria. “That way,” she says, “if any bacteria start to develop resistance to one of the phages, they’re likely to be hit by another one” - before they can proliferate.

The Eliava Institute’s researchers, from the days of d’Hérelle and Eliava till now, also believed in “cocktails” for maximum impact. “For example the bacteria usually associated with wounds are Pseudomonas, E. coli, Streptococci, Staphylococci, and so on,” says Chanishvili. “Phages against all these bacteria have been made into a cocktail we name Pio-phage.”

The “cocktail” approach can also solve the problem of an unidentified pathogen: cocktails are designed to knock out all likely culprits.

Phages won’t be widely used in the West unless standardised formulations are tested in modern clinical trials, and licensed by regulatory bodies such as the British Medicines Control Agency and the US Food and Drug Administration.

“But you’re not going to find double-blind studies done in Tbilisi,” says Elizabeth Kutter. “That’s because phages so clearly make such a difference that it would be considered malpractice not to give them to subjects in a control group.”

In Poland phages were used by researchers from the Immunology Institute of the Academy of Sciences in 550 patients with infections such as septicaemia, abscesses and bronchopneumonia, between 1981 and 1986. Antibiotics had failed to help 518 of them. The phages had a 92 percent success rate in clearing up the patients’ infections. (Slopek et al (1981). Results of Bacteriophage Treatment of Suppurative Bacterial Infections I. General Evaluation of the Results. Arch. Immunol. Ther. Exp. 31:267-291.)

Large pharmaceutical companies will only take interest in phages if they can develop profitable, proprietary formulations. But how, exactly, can one patent something from Nature? The answer seems to be in developing innovative methods of delivering them.


Exponential

Dr Richard Carlton, President of Exponential Biotherapies, Inc., in Port Washington, New York State, has developed a phage against Vancomycin-Resistant Enterococci (VRE) - the mutant bacterium that famously kicked off the world’s “superbug” anxieties, in US hospitals in 1989. Safety trials have been successful, though results won’t be published till full clinical trials have been completed: the company is presently gearing up for these.

Exponential has patented a technique pioneered in a 1995 experiment by Carlton, and Carl Merril and Sankar Adhya from the NIH, among others, where powerful, mutant lambda phages were “selected” from “wild” strains that had been injected into a series of mice. (Long-circulating bacteriophage as antibacterial agents, Merril et al; Proceedings of the National Academy of Sciences, vol 93, April 1996.)

In a second study, Carlton and colleagues experimented with both these new “long-circulating” lambda phages and “wild-type” lambda phages - in addition to a placebo - in three sets of experimental mice.

The long-circulating phages were far more potent as anti-bacterial agents than the wild-type phages, Carlton says. “The animals that got placebo were all dead within 48 hours. The animals that received a single injection of the wild-type phage became critically ill, but eventually recovered. But the animals that received a single injection of the long-circulating phage were only a little bit lethargic for 24 hours, and then recovered completely.”

By examining the new phage’s genetic structure, Carlton and colleagues were able to identify which specific mutation had occurred. “The mutation was in the phage’s major head protein,” Carlton says, “of which there are many copies - and was situated in a segment of that protein that sticks out in space where it can interact with the surrounding environment.” Based on that finding, “the patent office granted us the patent”.

Exponential now has long-circulating phages against several bacteria, including Pseudomonas aeruginosa (multi-drug-resistant strains of which exist in many hospitals) and Staphylococcus aureus, the well-known “staph” infection also often acquired in hospitals. The company has additionally developed potent (non-long-circulating) phages against Campylobacter jejuni and Listeria moncytogenes, for use in food preparation rooms.

Exponential Biotherapies has patented all its products hitherto, Carlton says.


Intralytix

A Baltimore company, Intralytix, Inc., is making an American version of a  product pioneered by Ramaz Katsarava at the Georgian Technical University, called PhageBioDerm.

“PhageBioDerm is a phage-impregnated slow release polymer that is used in management of hard-to-heal wounds such as stasis ulcers and bedsores,” says Intralytix’s Dr Glen Morris, who is also Professor of Epidemiology and Medicine at the University of Maryland. The product is applied directly to wounds, where it acts as an artificial skin, and gradually releases phages to combat infection. In Georgia it is patented to Ramaz Katsarava - and Zemphira Alavidze of the Eliava Institute, who has worked privately to further develop the product. PhageBioDerm is licensed for sale in Georgia, with a US patent pending on an American version, made with phages isolated in the US. The patent will be “on the patch and on the combination of patch and phages,” says Intralytix’s Alexander Sulakvelidze. “Currently, no specific phages in PhageBioDerm are patented.”

In a Georgian study in 2000, PhageBioDerm cured 70% of patients of wound and ulcer infections which had failed to respond to conventional treatments. (A novel sustained-release matrix based on biodegradable poly(ester amide)s and impregnated with bacteriophages and an antibiotic shows promise in management of infected venous stasis ulcers and other poorly healing wounds. Markoishvili et al. Pharmacology and Therapeutics.)

Otherwise, Intralytix is moving into phage products for use in food safety.

A phage product active against Listeria, to be used as a secondary food additive, will be on the market in the US by the end of the year, and the company hopes to have a similar product active against Salmonella available soon thereafter.


GangaGen

An emerging player in the global phage game is GangaGen, based in Palo Alto, California, and Bangalore, India. (The company’s name, meaning “born of the Ganges”, recalls EH Hankin’s original discovery of a bacteria-killing principle in the river in 1896.)

GangaGen founder Dr Ram Ramachandran, who has a long history in biotech research in both India and the US, is aiming for products for both developed and developing worlds. “Antibiotic-resistant infection is a global problem,” Ramachandran says. “People in the West go to hospitals to have surgery for broken bones, and die of hospital-acquired infection. In the developing world, children die of Shigella dysentery and other bacterial infections including drug-resistant tuberculosis.”

For starters, the company is developing phages against a broad range of strains of antibiotic-resistant Pseudomonas aeruginosa (a bacterium Exponential has also targeted). “Stability and safety” studies in animals are in preparation for one of these, as a prelude to human clinical trials. The company is presently filing several patents including one for the Pseudomonas phage. “We also have an operation in Ottawa - GangaGen Life Sciences Inc,” says Ramachandran, “focusing on veterinary, agricultural and environmental applications of phages.” Indeed the first GangaGen product is likely to come from Ottawa because Canada’s regulatory processes are shorter than those in the US.


Merril’s work: Enzyme on tail

Phage research trailblazer Dr Carl Merril is the chief of biochemical genetics at the US National Institute of Mental Health. Merril has been intrigued by phages since the 1960s, but along the way he’s had many reservations to overcome: “After all, it was a bacterial virus,” he says, “that probably killed one-third of the Europeans, and came to North America in the 1770s - diphtheria.”

Did that have relevance to the safety of today’s phages?

“Well, we certainly have to be aware of it. One of the problems with phages is that they can carry these toxic genes - people weren’t even aware of that before.”

Many phage researchers invoke cautions about “toxic genes”. As Merril explains them, they can cause phages to play a role in bacterial pathogenesis: “A number of phage genes have been discovered that encode toxins, or factors that enhance bacterial virulence. They may also contribute, through transduction, to the transmission of antibiotic resistance genes.”

However it is possible to reduce such occurrences by sequencing the genomes  of therapeutic phages, and using that information to seek out problem areas in these genes and identify them. “If potentially detrimental genes are identified,” Merril says, “they can be deleted from the genome of phage that are being developed for therapeutic use as antibacterial agents.”

But Merril has more exciting news than that: “I have a post-doctoral fellow in my lab, Dr Dean Scholl, and he’s discovered how to put extra enzymes on the tail of a phage. The ‘tail’ is the phage-part which both recognises a bacterium, and punches a hole through the bacterial protective capsule to get the phage genome into the bacteria. So you can extend the host range that way.”

Dr Scholl has found a “dual specificity” phage which encodes two different tail proteins, allowing it to attack both K1 and K5 strains of E. coli, and possibly the Salmonella bacterium as well. (Scholl, D., Rogers, S., Adhya, S., and Merril, C., 2001, Bacteriophage K1-5 encodes two different tail fibre proteins allowing it to infect and replicate on both K1 and K5 strains of E. coli.,¬† J. Virol. 75: 2509-2515.) The versatility of these newly found phage proteins suggests that their “genome motifs” might serve as a model for creating - as Merril puts it - “a modular phage platform that could operate over a wide bacterial host range”.

NIH researchers Scholl, Merril and their colleague Sankar Adhya have applied for a patent for this idea of extending the phage host range by adding this new information to the phage genome. The NIH has also patented the “long-circulating” phage which Exponential Biotherapies now has an exclusive licence on, as well as so-called “reporter” genes - one of which employs luciferase.


Luciferase

Merril appears to have overcome a long-standing objection to the use of phages in severe infections such as meningitis. By the time you take 48 hours to culture the responsible bacterium, critics have said, then maybe another 48 hours to find the right phage, the patient may be dead. But what if bacteria could be identified in three hours instead of 48?

As a first step to speeding identification, Merril says, “we’ve put luciferase gene into our phages. That’s the enzyme which gives off the light in the firefly.”

If a patient comes in with a cough, for example, his sputum is put into a multiwell plate in which each “well” contains a different species of luciferase-added phage. Phages then infect the bacteria, Merril says, “and now only have to express the luciferase gene to provide a signal”.

Instead of waiting 48 hours for bacteria to breed up, now Merril waits a mere three hours to see which phage infects - and thus identifies - a bacterium. Because of the luciferase gene, Merril says, the activated phage “gives off a burst of light”. The researchers simply “wait three hours and see which wells light up”.


More biotech innovations to come?

The profile of therapeutic phages received a boost last November when James Watson (of double helix fame) hosted an invitation-only international conference on phage therapy. The conference was at the Banbury Center at Cold Spring Harbor Laboratory, of which Watson is President, in New York State.

And what if the present exctement about phage research leads to successful trials and regulatory approval in the West? Will this kick-start the fledgling US phage industry, and bring “big pharma” onboard?

“I think that’s probably right,” says Exponential’s Richard Carlton. “That will be the signal to them.”

“What we have now,” says Carl Merril, “that we didn’t have in the 1920s, 1930s and 1940s when the eastern Europeans got into phages, is that we’ve invented a whole world of molecular biology. And we can use that to engineer these phages so they’re more efficient, and to make sure they don’t carry the toxin genes, and to purify them, and to figure out which ones to use.

“The point is, we can do this scientifically now. We can make phages into a very effective agent. The only questions are, do we have the will to do it, and do we have the funds to do it?

“I think that we’ve only sliced the tip of the iceberg when it comes to phages.”