Will viruses save us from superbugs?

When antibiotics did not help a seriously ill patient, he was saved by a virus from a local pond.

[It is not recommended to read the text for food, as well as for impressionable people and pregnant women - approx. transl.]



For years, Ali Khodadoust existed with a literally open heart. In 2012, surgeons replaced the aortic arch and unintentionally planted a bacterium. The bacterium formed colonies in the form of biofilms and drilled a tunnel through its chest, which as a result went outside.
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It was a dangerous affinity. Antibiotics to fight infection, which the old man dutifully swallowed daily, did not kill the bacteria. Then the doctors inserted a plastic tube into his shoulder and fed antibiotics directly into the bloodstream. But antibiotics failed one after another. Three years later, Khodadust, an ophthalmologist from New Haven, Connecticut, was sent to Yale-New Haven Hospital for treatment. Brown pus oozed from a hole the diameter of a pencil on his chest. Sometimes it was stained with stripes of bright blood. At any time, the bacterium could enter the bloodstream, cause septic shock and kill it.

To eliminate the abominable bacterium, surgeons needed to cut off the infected tissues, wash the heart cavity, and replace the aortic arch again. But they were afraid to perform operations on elderly patients, especially in this situation. They decided that the operation was too risky and postponed it. Then he was rejected by another group in Texas. And after the refusal in the Zurich hospital, Khodoust’s last hope disappeared.

While Hodadoust struggled for life, microbiologist Benjamin Chan was a mile away from him, working in an experimental evolutionary laboratory. Chen studied bacteriophages in the laboratory of Paul Turner [Paul Turner], a professor of ecology and evolutionary biology at Yale University. Bacteriophages, from the Greek phagein, are viruses that devour the bacteria. Phages thrive in the same place where bacteria - that is, almost everywhere. Not a single organism on Earth is as widespread and diverse as phages. We touch them every time, swimming in the ocean, chewing cabbage salad or kissing. Billions of years of evolution have made phages the ideal killers of bacteria - quiet, secretive, effective. But what is interesting, no hospital in the USA is currently treating patients with phages.

Then Hodadoust did not know about it, but Chen was going to make an exception for him.


E. coli nightmare: T4 bacteriophage (red) and its arachnid filaments used to attach and infect E. coli.

One afternoon, I sat with Chen at his office in the laboratory of the Osborne Memorial, in the Science Hill-like building at Yale University (we are just friends with him). The sun was breaking through the large windows. A portable electric stove on a shelf was sandwiched between textbooks on microbiology. Sitting at his desk in a waistcoat, narrow tie, and pale purple checkered socks, peeking out of his oxfords, Chen was more like a member of the indie group than an enthusiastic microbiologist.

My face probably wriggled when Chen said he plans to infect the fragile body of an 80-year-old patient with an experimental virus. He assured me: "Phages only infect bacteria." Often, phages generally attack a single species of bacteria, or several varieties of it. Phage is the key of a complex construction, suitable for a single keyhole, to the receptor on the bacterial cell wall. When a phage opens its victim, it pushes its genome inside and turns the bacteria into a phage copying machine. As a result, the bacterium breaks, and hundreds of phage clones come out, leaving the remains of bacteria. Some phages act less audaciously, inserting their code into the DNA of the bacterium so that with each reproduction of the bacterium the phage is also copied.

This is very different from the work of antibiotics that destroy any bacteria, including the micro community that supports our health. Phages are refined and reserved gourmets. They penetrate into biofilms, infect their victims, and neatly destroy bacteria, leaving the patient's microflora intact.

Their unique method of work can allow them to become a formidable weapon in the war against superbugs — such as MRSA, C. diff, and CRE, which, as in Hodadust’s case, can elude most antibiotics. Superbugs lurk in chicken packaging at the grocery store, on the train seat, on fresh laundry in hospitals, and every year 2 million Americans are infected, 20,000 of whom die. If antibiotics prove to be powerless and superbugs take up, millions of routine procedures — organ transplantation, chemotherapy, even the simplest visit to the dentist — will become potentially dangerous. Phages can open a second front in this war. But, for many reasons, they have not done this yet.

Phage therapy began at the beginning of the 20th century. Microbiologist Felix D'Herrell served at the Pasteur Institute in Paris to investigate the dysentery epidemic that afflicted French soldiers in the First World War. Curious about why some people became deadly ill, while others were relatively easy to get sick, D'Herrelle grew bacteria obtained from soldiers' feces in the laboratory. Some colonies of bacteria in a Petri dish (“bacterial lawns,” as biologists affectionately call them) turned out to be spotty. In these places, something invisible was killing bacteria. And in the colonies of bacteria from the recovering soldiers, these points increased. He suggested that these invisible “microbes of immunity” may help the recovery of the patient.



To test his theory, D'Herrel collected these microbes — he and his wife called them phages — and planted them on a 12-year-old patient with dysentery. The patient recovered quickly. Inspired by this, D'Herrell opened the Laboratory of Bacteriophages in Paris. Like a perfumer who mixes smells for different occasions, he mixed and sold phages against diarrhea, skin diseases and colds. This occupation went beyond the borders of France - D'Herrelled traveled to Georgia and helped establish the Eliava Institute, which carried out phagotherapy in 1923, and in the 1940s, Eli Lilly sold various phage-based drugs in the United States.

It was a good start to a promising therapy. But another scientific discovery suppressed interest in phages. In 1928, Alexander Fleming accidentally found penicillin in the mold due to the confusion prevailing in his laboratory. Penicillin became a magic medicine: it did not have to be made as phages, it worked stably, it could be produced in large quantities, and it could be stored for months. He also appeared just in time: for World War II. Phages seemed invisible and capricious byproduct of bacteria, sometimes treating infections, but often not working. Before the era of DNA and molecular biology, it was even unknown what they were. By 1940, antibiotics had already been produced commercially in the United States and Europe. Phages were in a dusty closet among medical rogue.

But antibiotics have not met the same enthusiasm everywhere. In the USSR, they were expensive. And while the study of phages in Western Europe had subsided, it continued in the network of Soviet laboratories under the leadership of the Institute of Eliab. By the 1980s, the Georgian laboratories distributed weekly two tons of sprays, powders and patches with phages. Most of the products entered the commissary. But research was published in Russian and Polish, and did not penetrate into the United States.

But in the western laboratories of molecular biology of phages studied in detail. They found that, as with the "classical" living organisms, the hereditary information of phages is transmitted using DNA. Biologists have extracted phages for many enzymes used in modern laboratories. And in 1976, the first genome decoded was the phage genome. An effective and controversial technology for editing the CRISPR-Cas genes was invented on the basis of protecting bacteria against phages. As a result, interest in phages began in the United States, in particular because of the beginning of a scientific exchange between scientists in the United States and Russia.

Chen began hunting phages at OmniLytics, one of the companies that sold phages to farmers. There Chen was looking for phages to protect livestock from Escherichia coli O157: H7, and tomato plantations from plant diseases. According to Chen, we already use phage-containing products all the time. Sausages and meat for buters are treated with Listex, a multi-phage cocktail approved by the FDA in 2006, which protects against listeria . A competitor, Intralytix, offers a SalmoFresh spray that attacks Salmonella and infects poultry, fruits, and vegetables. Phages, unlike food additives, do not have to be mentioned on the packaging - so it’s hard for the buyer to know if he consumes the viral spray. Phage sprays are organic, and Omnilytics advertises phages as a tasty and natural alternative to pesticides.

When Chen moved to New Haven in 2013, he wanted to explore the potential of phages as a medicine for humans. He wrote to the president of Yale-New Haven Hospital that he was hunting for phages and he needed a patient. Quite quickly, Chen met Dr. Hodadusta, Deepak Narayan [Deepak Narayan], took his prize (a test tube with frozen pus) and got acquainted with his bacterial target. He planted it in agar broth mixture. The Hodadusta bacterium, Pseudomonas aeruginosa, grew violently and quickly. “It smells pretty good, like grape flavoring,” Chen told me. He filled the refrigerator with test tubes with a bacterium and tested various antibiotics on it. The bacterium showed its resilience, and then Chen began searching for a phage that could kill her.

A variety of phages blocks the diversity of the rest of life on Earth. Everywhere, wherever scientists are looking for - in the soil, in caves, in the depths of the ocean - they find millions of new phages. They do not exceed 100 nm in size, and about 10 ^{32 of} them live on the planet. If the phages were the size of a grain of sand, 1000 such planets as the Earth could have been filled with them. For phage hunters there are no maps where the location of the viral treasure would be indicated. The phage searched for by Chen could end up everywhere where Pseudomonas live - in people, in hospitals, in nature.

Chen searched everywhere, and did not disdain to ask friends and colleagues to share samples of their own feces. Other biologists of the institute helped Chen. Fish farmers from the top floor — environmentalists who have investigated freshwater trout — have shared samples from New England rivers with Chen. Chen collected dozens of samples from lakes, puddles, sewers, compost and soil. In each tiny test tube there were many bacteria and phages.

In the lab, Chen mixed each sample with a few drops of Pseudomonas bacteria. Only a suitable phage could infect their cells and multiply. Like a winemaker, filtering the skins, seeds and leaves of grapes from wine, Chen let the mixture through the filter 100 times smaller than the thickness of a human hair. There remained only purified "nectar" - a set of phages.

And to test the nectar's ability to kill Pseudomonas, and not just to multiply in them, he mixed Pseudomonas from Hodadusta with purified phages, laid them out in Petri dishes and grew cultures. If the phage were successfully introduced into the cells of the bacterium, a characteristic stain would form on the bacterial lawn.



One day, late in the evening, a few months later, Chen found the right phages. Standing alone in his Gothic-looking laboratory with high ceilings, he looked with disbelief at the Petri dish, which he held in gloved hands. Clearly visible circles stood out against the emerald cup background, like planets of different colors on a dark sky. He was holding a bacterial cemetery. After preparing hundreds of cups with Pseudomonas and adding a carefully crafted sample, he found a phage key capable of unlocking the Hodadust bacterium, breaking into it and causing chaos.

He looked at the markings on the Petri dish. Pond Dodge. All thanks to "fish scholars from above." Water from a pastoral pond in Connecticut has raised killer bacteria that are more capable than the strongest antibiotics. Chen quickly became convinced that his killer phage could also penetrate the protective biofilm erected by the Pseudomonas bacteria on the Hodadoust artificial aorta.

The time has come for the most difficult test - will not Pseudomonas evolve and resist phage? There are no evolution-protected therapies. One of the main arguments against phages is that this therapy will fail in the same way as antibiotics. Bacteria can develop resistance to phages. Therefore, phages are often used in mixtures. Ideally, if the bacteria develop resistance to one of them, the others will do their job. As expected, the bacterium developed immunity overnight. But did the bacterium that resisted phages have a weak spot? Chen sowed a new, resistant bacterium and added the antibiotic ceftazidime. The next morning a beautiful spot of death formed in this place.

Pseudomonas have developed resistance by eliminating the receptors through which phages have penetrated from the pond. But without them, the bacteria became vulnerable. These receptors play another important role - they pump antibiotics out of the cell. When Chen added antibiotics, the previously ineffective drug easily penetrated the cell wall. And without the ability to pump them out, Pseudomonas were poisoned from the inside. A combination of phages from the pond and ceftazidime staged an evolution of the mat.

Reappearing in the US, phages face new obstacles to their use in medicine, not the least of which is FDA approval. “The phages seem to work,” says Randall Kincaid, chief science officer at the United States National Institutes of Health. - But we do not have good statistics on accurate scientific research, which would say that it was phages that caused recovery. We need to think about it from the point of view of doctors. When a patient comes to a doctor with a bacterial infection, it is customary to treat him with antibiotics. We need unequivocal evidence of the benefits of phages to accept them. ”

There have been several successful clinical trials. Phase I and II trials conducted by AmpliPhi Biosciences Corporation revealed that a single dose of a phage cocktail helped well against antibiotic-resistant Pseudomonas aeruginosa ear infections. Another Phase I trial found that the phage cocktail for treating leg ulcers was safe, but could not significantly speed up the patient's recovery (during phase I, the safety of the drugs is checked, and during phase II, the effectiveness).

But so far no phase III trials have been carried out - the final stage confirming the efficacy of medicine, for which at least 1000 patients are required. Since the principle of operation of phages is fundamentally different from antibiotics, it is unclear how to evaluate their effectiveness. Kevin Otterson, a law professor at Boston University Law School, and the executive director of CARB-X accelerator, who is going to inject $ 350 million into developing new products to combat superbugs, says that “antibiotics are clear what evidence you need to get in animal experiments, and what research to do in humans. How an experiment with phages should look like is a very open question. ”

Some of the benefits of phages cause problems for regulators and critics. Home - specialization, which makes them accurate tools, but it means that clinical trials must confirm the safety and effectiveness for each type of bacteria. Broad-spectrum antibiotics are often prescribed without specifying the diagnosis, and treatment with phages requires precise identification of the type of bacteria. This may change the course of the examination procedure. The issue with the dosage in the case of phages is also complicated. Antibiotics pass through the body and are metabolized in predictable ways. Phages are not dead substances. If they find their bacterium, they will begin to multiply. This means that sometimes a single dose of phages will suffice, but it also means that their activity is difficult to predict. How many phages to give to the patient? How fast can bacteria develop resistance? Even when phages behave beautifully in vitro, it is difficult to predict how they will act in vivo, in the complex and difficult for navigation environment of the human body.

Another unpleasant question: who will pay for the clinical tests? They often cost hundreds of millions of dollars. Compared to cancer drugs, Kincaid says, “for the treatment of infectious diseases there are not so much investment returns.” Now it is difficult to sell the idea of ​​phages to investors who want a guarantee of success. Phages collected from sewage and compost are natural products and cannot be patented. Companies can get around this point by patenting cocktails or creating phages in the lab. “The precedent plays a big role here,” says Kincaid, who for years managed the biotechnology company prior to current work at NIH. "Investors do not like the possible vagaries of unknown things." He believes that the government needs to intervene in the acceleration and funding of phage research.

PhagoBurn became the largest test for phage therapy today, which cost the European Commission € 3.8 million, and it ran into several problems. Initially, they wanted to attract 220 people with burns and infected wounds from 11 different hospitals in France, Belgium and Switzerland.It was planned to test the effectiveness of two different phage cocktails compared to the usual antibiotic, silver sulfadiazine. After scientists barely managed to prove the stability of phages in cocktails, they encountered difficulties in finding patients. The tests required people infected with either Escherichia coli or Pseudomonas aeruginosa, but not both. But burn victims often become infected with many pathogens. There were only 15 patients for the Pseudomonas aeruginosa study. And the variant with E. coli had to be dropped altogether. The reduced version of the test continues, the results will be known next spring - four years after it began.

There are those who reject phagotherapy. Steve Projan [Steve Projan], senior vice president of research and development, and head of the department of infectious diseases and vaccines at the biotechnology company MedImmune, who previously worked at Novartis and Wyeth, wrote about this in a 2004 article: “Personal and anecdotal stories of former patients who are helped by phagotherapy are funny and sad at the same time - for obvious reasons, we will not hear anything from those patients whose infections could not be cured. ” Proyan writes that instead of trying to overcome all the obstacles of phagotherapy, money could be spent better - for example, to develop "therapies with small molecules", including antibiotics. He refused to be interviewed.

Others are more optimistic. Several countries in Eastern Europe regularly use phagotherapy. Institute. Ludwig Hirtsfeld in Warsaw, Poland, uses phage as a last resort for patients who have not been helped by antibiotics. Since 1980, more than 1,500 patients infected with drug-resistant bacteria have been treated there, and the institute reports that "most of them were cured." In Georgia, phages are even more widely used. Doctors treat about 20% of incoming patients with phages. Phage Therapy Center in Tbilisi attracts patients from all over the world who suffer from incurable urinary tract infections, acne, cystic fibrosis, and intestinal infections. The center claims about 95% of treatment success. But many Western scientists doubt this, since they have not been tested by either the FDA or the European Medical Agency.

And there is also Hodadoust and Chen.

A few months after Chen's success in the laboratory, it was time to try the treatment at Hodaduste. He was taken to a treatment room at Yale-New Haven Hospital, and two doctors rolled a cart with resuscitation equipment. Chen said that he was then covered with a wave of panic. In front of a small audience of surgeons and students, the radiologist injected a small amount of a mixture of salt water, phages and antibiotics into the chest cavity of Hodadust. Chen kept a close eye on the zigzags of the apparatus that marked the patient's heartbeat. Each jagged signal said that the delivery of the virus directly to the patient's heart did not kill him. In a day, Khodustus was discharged. No obvious changes in his condition were observed.

Chan did not know whether he was upset or calmed down. “I was worried that I would kill a man. Either phagotherapy will work — incredibly — or everything will go bad — terribly bad. ”

For several weeks, Chen received no reports. A month later, Narayan informed Chen that Hodadoust had gone by plane to visit relatives abroad. Did he feel so good to travel, or did he decide to see his family before he died?

Then, six months after the procedure, without warning, Hodadoute came to the clinic of Narayana. His chest was completely healed, instead of a hole there was a flat surface. Usually the silent Narayan told Chen that his patient looked like a million dollars. For the first time in three years, he did not take antibiotics. No side effects were noticed.

Narayan cannot be sure that Khodustus recovered through phagotherapy. In an ideal world, he would study it after the operation, monitor the condition of the cavity, test everything that oozed out for the presence of Pseudomonas. But he is sure that phages from Dodge pond helped his patient. He learned that five weeks after the treatment, a certain group of surgeons had taken Khodoust to remove the part of the transplant holding him in the new aorta. They tested the transplant, and it did not contain Pseudomonas. He stopped taking antibiotics for the first time in three years, and the infection never returned. The virus from the pond, located just 60 kilometers from his house, gave him a new chance - and a new meaning to the term “local medicine”.

Chen, Narayan and Turner noted this case, immersed in writing scientific papers. They hope to plan a clinical trial of a mixture of phages from Dodge's pond with ceftazidime. But before testing on humans, phages need to be tested on rats. The team uses the NIH preclinical services for in vivo animal testing.

Meanwhile, Chen is collecting a phage library. If you live in New Haven, or have recently used toilets there, you can become an unwitting donor. He visits the Newha Haven water treatment station weekly, takes samples of substances coming from toilets from all over the city, filters phages from them and checks them on many Petri dishes containing potential superbugs. The hunt has already brought success. Chen isolated phage for Klebsiella pneumoniae and Enterococcus faecalis, resistant to antibiotics and causing urinary tract infection. On his last phage trip to Haiti, Chen found a phage attacking a cholera bacterium, Vibrio cholerae.

Sometimes from the excrement of one person you can prepare a phage cocktail for another.