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mr peabody

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Phage therapy: Beating superbugs at their own game

by Rana Samadfam | January 21, 2019

Is the centuries-old strategy of using bacteria-eating viruses to fight disease making a comeback?

The word “superbug” is a familiar term. We now know that the overuse of antibiotics has contributed to creating these tiny, life-threatening monsters. The hunt is on for new antibiotics to fight these superbugs, but it cannot completely resolve the issue of multi-drug-resistant (MDR) bacteria. Bacteria are dynamic systems; continuously evolving to survive by gaining resistance to new antibiotics almost as fast as we can create them. Alternative approaches are necessary to combat MDR, and phage therapy may be the top candidate.

A phage, also known as a bacteriophage, is a virus that infects and replicates within bacteria, and phage therapy is the therapeutic use of bacteriophages to treat infectious disease. The phage is often an obligate killer of its host, meaning the phage kills its host bacterial cell in order to reproduce. Bacteriophages are widely distributed in locations populated by their hosts, including the human body, seawater and soil. The largest population among different organisms on Earth belongs to phages, with an estimated number of viral SpeTarparticles around 10 31.

Phage therapy is not a new concept. It dates back to the pre-penicillin era, when French-Canadian microbiologist Félix d’Herelle used phages to cure four patients of dysentery in 1919. However, the interest in phage therapy was dampened with the discovery of conventional antibiotics and their effectiveness in treatment of infectious disease. It was revived decades later with the first scientific phage summit in 2004. The burgeoning interest in phages led to, among other things, the revolutionary gene editing tool known as CRISPR Cas-9.

Phage characteristics: Specific, adaptable and easy to find

What have we learned about phages? Well, they are relatively specific. They typically have a very narrow antibacterial spectrum limited to one single species of bacteria, or even a single strain within a species. Their specificity can make them tricky to use, but they have the benefit very low collateral damage outside of their chosen target. This stands in sharp contrast with common antimicrobial drugs that do not distinguish between infectious and harmless bacteria, resulting in a greater disturbance of microbiota.

Phages are also dynamic and highly adaptable, much like their hosts. If the bacteria mutates to resist the phage, the phage counters with mutations of its own. This enable us to beat the superbugs at their own game, fighting fire with fire. Although there is a potential for direct interaction with the human immune system, these concerns are no greater than those with biologics, viral vectors used to deliver in gene therapies or live-attenuated vaccines (vaccines that have been genetically modified to make then less virulent or harmless.

What’s more, phages are easy to discover and characterized due to the use of next-gen imaging and genetic tools. After the first phage was discovered, in 1915, only a handful of phages were studied in great detail. The recent renaissance seen in phage biology has been triggered due to a growing awareness of the number of phages in all bacterial dominated environments revealed by epiflourescent and electron microscopy, molecular studies and the genomes of bacteria following whole genome sequencing projects.
Phages and modern medicine

An ideal candidate for phage therapy is an obligately lytic phage with a high potential to reach and then kill bacteria. Like any other drug candidate, phages must display a good pharmacokinetic profile with optimal absorption, distribution, and half-life (survival in a live biologic system). In addition, they must have sufficient stability under typical storage conditions and temperatures. Most importantly, they need to be fully sequenced to ensure absence of undesirable genes and low ability for transduction (ability to transfer gene from one bacteria to other).

There is some resistance in modern medicine to phage therapy, which may stem from unfamiliarity or from guilt by association to the infectious viral family. After all, phages are viruses. The word can be frightening to consumers because it is reminds one of past mass casualties or present infectious diseases such as the flu and HIV. It may not be that easy, therefore, for a drug developer to convince the public to embrace phage therapies. Although there are several clinical trials currently ongoing, ListshieldTM is the only FDA approved phage therapy. It is used as a food additive and kills Listeria monocytogenes, one of the most virulent foodborne pathogens and a cause of meningitis. In the absence of urgent corrective actions to limit overuse of antibiotics and insufficient investment in research for new and innovative weapons against superbugs, the world is heading toward an era in which many common infections will have no cure. With advances in gene editing technology (including CRISPR–Cas9, the genome editing derived from a bacteriophage), we have the necessary tools to investigate the genetic relationship between phages and their bacterial hosts. These tools can also be used to characterize, optimize and select phages for relevant diseases.

 
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Genetically engineered phage therapy rescues teenager on the brink of death

MIT Technology Review | May 9, 2019

It’s a remarkable story of recovery, but it’s unclear how useful this sort of therapy could become.

The background: Isabelle Holdaway had been given less than a 1% chance of survival after a lung transplant, carried out to combat the symptoms of cystic fibrosis, left her with an antibiotic-resistant infection. She had been sent home and was in a terrible physical condition: underweight, with liver failure, and with lesions on her skin from the infection.

A breakthrough: Her consultant at Great Ormond Street Hospital in London worked with a team at the University of Pittsburgh to develop an untested phage therapy. This treatment used a cocktail of three phages, which are viruses that solely attack and kill bacteria. Two of the three phages, selected from a library of more more than 10,000 kept at the University of Pittsburgh, had been genetically engineered to be better at attacking the bacteria. The therapy was injected into her bloodstream twice daily and applied to the lesions on her skin, according to Nature Medicine.

Now: Holdaway is not fully cured, but her infection is under control. Virtually all her lesions have cleared. She is still having twice-daily injections of the therapy, and her treatment team is planning to add a fourth phage to try to clear her of the infection entirely.

The promise: Antibiotic resistance is a growing emergency, and phage therapy is being held up as a potential treatment for antibiotic-resistant superbugs. It’s a promising avenue, but it’s a deeply personalized form of therapy, and we must be careful extrapolating too much from this single case study alone, which was not a full clinical trial.

 
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mr peabody

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What are phages and how do they work?

Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulyuren GmbH

Bacteriophages, short form: phages are viruses in the wider biological sense. They exclusively attack bacteria and lyse them (“bacteria eaters”).

Phages cannot reproduce alone by themselves, they require the bacterial cell as a host to reproduce within the host.

A phage is much smaller than a bacterial cell and consists of its hereditary material (nucleic acid, mostly DNA) that is embedded in a protein envelope.

This envelope is the “head” of the phage and has a crystalline shape that is only visible in an electron microscope. Additionally, a phage has a protein “tail” with a morphologically delicate fine structure at the end, for adsorption to the bacterial cell surface, the receptor.

This receptor structure is so specific that a phage can only attack bacteria having a cell surface that exactly “matches”. After adsorption to the bacterial surface, the phage injects its nucleic acid into the bacterium that will now be forced to produce a new phage generation by using the bacterial enzyme equipment. One single bacterial cell produces such an enormous number of new phages that the pressure forces the bacterium to burst. The phages will immediately kill other bacteria with a surface matching with the phage. This effect is easily visible as lysis holes on densely grown bacterial layers.

As the number of young phages of a new phage generation is usually very high, the phages will quickly and completely attack bacteria in their proximity. This will happen as long as bacteria are available and receptors properly match.

Among the giant numbers of bacterial cells at a certain habitat a statistical number of bacterial mutations can lead to phage-resistant bacteria. But, these will remain a minority and the phages will “co-evolve” and develop mutations that target the resistant bacteria. As a consequence, the phages will further be able to attack the bacteria.

This phenomenon called “population dynamics” demonstrates in a simple manner what evolution of phages and their bacterial hosts in a habitat means. In natural environments, such a competition between bacteria and phages is mostly of scientific interest.

But, if the focus of interest is phage application or phage therapy in order to fight against a dense bacterial colonization, attention is drawn to these specific bacteria and a suitable phage: a special host-phage system.

The “habitat” would not be garden soil or a water pit but a wound of the human body, an abscess, a chronic inflammation, a bacterial biofilm within the airways, an infected burn wound etc. In such cases, the bacterial numbers are often an extreme challenge for the human immune system that in the worst case collapses: antibiotics are the last possibility to fight against the bacteria.

There are many different kinds of antibiotics, they can be allocated to some well-defined chemical substance classes. Antibiotics are more or less specific against bacteria, but never specific for a certain bacterial species and even less against certain strains of a certain species. This is in contrast to phages that attack almost always only one bacterial species and more typically, only few strains of a species.

This high specificity is typical for phages because of their biology: a phage-host system is like a key-lock function.

Therefore, phages are a completely different though biologically logic alternative to antibiotics: once a phage is found that attacks a pathogenic bacterium, the bacterium can be killed fast, specifically and without known side effects. The phage will leave other bacteria intact like the gut flora.

After having lysed all target bacteria, the phage will decay and disappear, its components will be metabolized by the human body: when the bacterial number has decreased dramatically, the phage will be target of the reticulo-endothelian system: the therapeutic phage effect is “self-limiting”.

While antibiotics are usually blocking bacteria during their fastest growth phase, phages are lysing bacteria independently of the growth phase.

As phages are the most abundant free living entities on earth (probably the tenfold number compared to bacteria), the human immune system knows phages from evolutionary history: we have a constant intake of phages via water, food and contact with natural materials, our gut flora contains enormous amounts of phages, like a complex ecosystem in balance.

This and the rather simple composition is probably the reason why phages are not known to cause allergic effects.

 
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Isabelle Carnell (second from right) with her doctor, Helen Spencer (left); phage researcher Graham Hatfull
(second from left); and her mother (right).


Viruses genetically engineered to kill bacteria rescue girl with antibiotic-resistant infection

by Alex Fox | 08 May 2019

One week after Helen Spencer's 15-year-old cystic fibrosis patient had a double lung transplant in September 2017, the incision wound turned bright red. For half her life, Isabelle Carnell had been battling a drug-resistant infection of Mycobacterium abscessus, and now it was rapidly spreading, erupting in weeping sores and swollen nodules across her frail body. "My heart sinks when I see that a [lung transplant] patient has got a wound infection, because I know what the trajectory is going to be," says Spencer, Isabelle's respiratory pediatrician at Great Ormond Street Hospital in London. "It's a torturous course that has ended in death for all those children."

With the standard treatments failing, Isabelle's mother asked Spencer about alternatives—adding that she had read something about using viruses to kill bacteria. Spencer decided to take a gamble on what seemed like a far-fetched idea: phages, viruses that can destroy bacteria and have a long—if checkered—history as medical treatments. She collaborated with leading phage researchers, who concocted a cocktail of the first genetically engineered phages ever used as a treatment—and the first directed at a Mycobacterium, a genus that includes tuberculosis (TB). After 6 months of the tailor-made phage infusions, Isabelle's wounds healed and her condition improved with no serious side effects, the authors report today in Nature Medicine.

"This is a convincing proof of concept, even though it's just a single case study," says infectious disease researcher Eric Rubin of the Harvard T.H. Chan School of Public Health in Boston. But, he adds, "This needs to be tested rigorously with a real clinical trial."

Phage therapy dates back a century, but until recently the idea was relegated to fringe medicine in most countries, mainly because of the advent of antibiotics. Unlike broad-spectrum antibiotics, individual phages typically kill a single bacterial strain, which means a treatment that works against one person's infection might fail in another person infected with a variant of the same bacterium. Phages can also be toxic. But a string of recent successes against antibiotic-resistant bacteria have revived interest in the idea, leading major U.S. universities to launch phage research centers. Drug-resistant TB strains are an especially tempting target for phage therapy.

M. abscessus and other bacteria often colonize the thick mucus that builds up in the lungs of people with cystic fibrosis, a genetic disease that afflicts some 80,000 people worldwide. The infections can lead to severe lung damage, for which a transplant is a last resort. Isabelle, for example, had lost two-thirds of her lung function. But her infection persisted after the transplant, threatening her life.

To help Isabelle, Spencer's team contacted phage researcher Graham Hatfull of the University of Pittsburgh in Pennsylvania. Hatfull and his team curate a collection of more than 15,000 phages, one of the world's largest, many found by undergraduates at more than 150 schools who take part in an educational phage hunting effort. Hatfull and his team spent 3 months searching for phages that could kill M. abscessus isolated from Isabelle's wounds and sputum. They found three.

Hatfull's group wanted to combine the phages into a cocktail to reduce the chances of M. abscessus developing resistance, but there was a catch. Two of the three are so-called temperate phages, which have repressor genes that limit their lethality. To turn those two into reliable bacteria killers, Hatfull removed the repressor genes with a gene-editing technique his lab developed to study phage genetics.

Isabelle first received an infusion of the phage cocktail in June 2018. Within 72 hours, her sores began to dry. After 6 weeks of intravenous treatment every 12 hours, the infection was all but gone. Traces remain, however, so she still receives infusions twice a day and applies the treatment directly to her remaining lesions. But she lives a more normal teen life, attending school, shopping with friends, and taking driving lessons. "We are optimistic that in time it can completely clear the infection," Spencer says.

Spencer, Hatfull, and co-authors stress that Isabelle might have improved without phage therapy. They also note that her tailor-made cocktail doesn't work against other M. abscessus isolates they have tested. Still, the apparent success has encouraged phage researchers. Other phages in Hatfull's library infect and kill M. tuberculosis in test tubes, and he thinks they might prove useful weapons against drug-resistant strains.

But William Jacobs, a TB specialist at Albert Einstein College of Medicine in New York City, has tested those phages in a mouse TB model and seen no effect. "TB lives inside cells and I don't think the phages are able to get inside," Jacobs says. (M. abscessus primarily lives outside cells.) Others say there could be ways to ferry phages into the infected cells.

Phage therapy companies have at least three trials underway to rigorously assess the worth of their potential products for several different bacterial infections. "Even if the treatments succeed, they face tall practical hurdles," says Madhukar Pai, an epidemiologist at McGill University in Montreal, Canada. "For this to become a real world therapy we need to find out if we can do this with less effort and cost."

 
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First phage therapy center in the U.S. signals growing acceptance

by Eric Boodman | June 21, 2018

When her husband was dying of a drug-resistant infection, Steffanie Strathdee had a last-ditch idea. They could try treating him with a virus that would kill the bacteria colonizing his insides. The method, called Phage Therapy, was popular in former Soviet republics, but had mostly been abandoned in the U.S. Researchers had to hunt for the right virus in Texas pigsties and sewage treatment plants.

That was 2016. Phage Therapy is still very much experimental — but it’s come a long way since then. New companies have popped up, hoping to get approval to sell these viruses as drugs. A phage directory has come together, lab by lab, helping doctors figure out who has which virus.

Now, the U.S. is getting its first Phage Therapy center, at the University of California, San Diego. Its mission is to run clinical trials, but also to streamline the mad dash to secure the right phage before a patient dies.

In some ways, the Center for Innovative Phage Applications and Therapeutics simply gives a name — and $1.2 million — to an institute that already exists. After word got out that Strathdee and her husband’s doctors had managed to save his life with a bacteriophage — literally, a bacteria-eater — her inbox filled with pleas for a repeat performance. They came from all over: the U.S., the U.K., Australia, India, China, Albania. In almost every case, there was someone dying because of antibiotic-resistant bacteria. Viruses that had specifically evolved to kill those microbes might be able to help.

Sometimes, Strathdee and her network couldn’t act fast enough, and the patient died. But occasionally, it worked out. “I’ve had a second job as a phage-wrangler,” said Strathdee, who is the associate dean of global health sciences at UCSD, and who has been named a co-director of the new center. “When we started to treat patients, each one was like reinventing the wheel all over again: The phone calls at the 11th hour, the paperwork.”

Getting phage therapy to a patient can be a bit a puzzle. These viruses are picky about the microbes they feast on, so you often need to take a swab of the patient’s bacteria, nurture it in a dish, and then test which phages are able to kill it off. You need to make sure that the phages in question will explode a bacterial cell, rather than settling comfortably inside like lice on a kindergartener’s scalp. And then you need to purify it before delivery, so there aren’t any bacterial leftovers that might poison the person instead of saving them.

There’s also plenty of bureaucracy, because phages have not been approved by the Food and Drug Administration.

It’s often a crazy rush to find the right phage with emails and calls and tweets, then getting emergency experimental approval — and that was largely what happened for the five other patients who’ve been treated with phages at UCSD since Strathdee’s husband was revived.

“We wanted to make it so it isn’t such a scramble,” said Dr. Robert “Chip” Schooley, an infectious disease specialist at UCSD, who administered the phage to Strathdee’s husband, and who is also co-director of the new center.

The announcement is also symbolic of a wider shift. With the rise of antibiotics in the 1930s and ’40s, phages went out of fashion in the U.S. But the person who had named them, a Canadian microbiologist named Felix d’Herelle, moved to Tbilisi, in the republic of Georgia, continuing his research at an institute that attracted the admiration of Joseph Stalin himself. Even after d’Herelle’s death, the Eliava Institute kept the flame of phage therapy alive.

That hardly helped the viruses’ reputation in America during the Cold War. “It was commie science; there was a taint to it,” explained Dr. William Summers, a phage biologist, historian, and professor emeritus at Yale University.

Even though phages continued to be an important part of lab science, the researchers who used them thought they were good for just that: research. The idea of using them for therapy was almost a joke.

Then, as antibiotic resistance grew into a worldwide crisis — one that kills some 23,000 Americans a year — that joke started sounding more and more appealing. The funding of a center to administer and collect data on phage therapy is a reversal, of sorts: An admission that this long-disparaged idea is worth a million-dollar second glance.

“Trust me, at the Eliava, we have tried to convince people that phages are a safe and good alternative to antibiotics for many years,” said Mzia Kutateladze, the director of the Eliava Institute. “Finally the people agreed to use it, and we are very happy, of course.” She estimated that Eliava’s phage therapy center gets around 15 to 20 Americans every year.

“There really is, thankfully, some momentum building … around these non-traditional therapies,” said Dr. Helen Boucher, an infectious disease specialist at Tufts Medical Center in Boston, who is not involved with the UCSD project. “I would think of this more in a high-risk, high-reward category. This is largely uncharted territory. … At the end of the day, if you have a product that can work against antibiotic-resistant organisms that isn’t antibiotics, that would be huge.”

The news that her brainchild had been funded took Strathdee by surprise in late May. She was at a ceremony for UCSD professors with endowed chairs, at which all of them received medals. “The chancellor’s literally putting the medal around my neck, and he said, ‘Hey, I just sent you some money today,’ ” she recalled.

The new center will collaborate with companies such as AmpliPhi Biosciences and Adaptive Phage Therapeutics to treat future patients. Some of them will have cystic fibrosis, which causes mucus in the lungs to be overly sticky, often allowing drug-resistant microbes to proliferate. Others might have long-term infections that are preventing them from getting organ transplants. Yet others will have implanted devices, which sometimes provide the nooks and crannies where bacteria can grow into a slimy film.

“The sad thing is that there is going to be no shortage of patients,” said Strathdee.

 
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Phage therapy could beat drug-resistant illnesses

by Charles Schmidt | Scientific American | Nov 1 2019

Treatment first used in the early 20th century is showing promise against deadly infections.

Bobby Burgholzer has cystic fibrosis, a genetic disease that throughout his life has made him vulnerable to bacterial infections in his lungs. Until a few years ago antibiotics held his symptoms mostly at bay, but then the drugs stopped working as well, leaving the 40-year-old medical device salesman easily winded and discouraged. He had always tried to keep fit and played hockey, but he was finding it harder by the day to climb hills or stairs. As his condition worsened, Burgholzer worried about having a disease with no cure. He had a wife and young daughter he wanted to live for. So he started looking into alternative treatments, and one captured his attention: a virus called a bacteriophage.

Phages, as they are known, are everywhere in nature. They replicate by invading bacteria and hijacking their reproductive machinery. Once inside a doomed cell, they multiply into the hundreds and then burst out, typically killing the cell in the process. Phages replicate only in bacteria. Microbiologists discovered phages in the 1910s, and physicians first used them therapeutically after World War I to treat patients with typhoid, dysentery, cholera and other bacterial illnesses. Later, during the 1939–1940 Winter War between the Soviet Union and Finland, use of the viruses reportedly reduced mortality from gangrene to a third among injured soldiers.

Treatments are still commercially available in former Eastern Bloc countries, but the approach fell out of favor in the West decades ago. In 1934 two Yale University physicians—Monroe Eaton and Stanhope Bayne-Jones—published an influential and dismissive review article claiming the clinical evidence that phages could cure bacterial infections was contradictory and inconclusive. They also accused companies that manufactured medicinal phages of deceiving the public. But the real end of phage therapy came in the 1940s as doctors widely adopted antibiotics, which were highly effective and inexpensive.

Phage therapy is not approved for use in humans in any Western market today. Research funding is meager. And although human studies in Eastern Europe have generated some encouraging results—particularly those from the Eliava Institute in Tbilisi, Georgia, the field's research epicenter—many Western scholars say the work does not meet their rigorous standards. Furthermore, a smattering of clinical trials in Western Europe and the U.S. have produced some high-profile failures.

Yet despite the historical skepticism, phage therapy is making a comeback. Attendance at scientific conferences on the treatment is skyrocketing. Regulators at the U.S. Food and Drug Administration and other health agencies are signaling renewed interest. More than a dozen Western companies are investing in the field. And a new wave of U.S. clinical trials launched this year. Why the excitement? Phage treatments have been curing patients with multidrug-resistant (MDR) infections that no longer respond to antibiotics. The FDA has allowed petitioning doctors to administer these experimental treatments on a “compassionate use” basis when they could show that their patients had no other options—exactly what Burgholzer was hoping to prove.

MDR infections are a rapidly growing public health nightmare. At least 700,000 people worldwide now die from these incurable maladies every year, and the United Nations predicts that number could rise to 10 million by 2050. In the meantime, the drug industry's antibiotic pipeline is running dry.

Like all viruses, phages are not really alive—they cannot grow, move or make energy. Instead they drift along until by chance they stick to bacteria. Unlike antibiotics, which kill a range of helpful bacteria as they kill the strains making a person sick, a phage attacks a single bacterial species, and perhaps a few of its closest relatives, and spares the rest of the microbiome. Most phages have an icosahedral head—like a die with 20 triangular faces. It contains the phage's genes and connects to a long neck that ends in a tail of fibers, which bind to receptors on a bacterium's cell wall. The phage then plunges a kind of syringe through the wall and injects its own genetic material, which co-opts the bacterium into making more phage copies. Other types of phages, not used medically, enter the same way but live dormantly, reproducing only when the cell divides.

Phages have co-evolved with bacteria for billions of years and are so widespread that they kill up to 40 percent of all the bacteria in the world's oceans every day, influencing marine oxygen production and perhaps even Earth's climate. The spotlight on phages as medical tools is getting brighter as technological advances make it possible to match the viruses to their targets with better accuracy. The few facilities that are technically able to provide phage therapy, under strict regulatory protocols, are being overwhelmed with requests.

Clinical trials underway are beginning to generate the high-quality data needed to convince regulators that phage therapy is viable, but considerable questions remain. The biggest is whether phage therapy can tackle infections on a large scale. Clinicians have to match phages to the specific pathogens in a patient's body; it is not clear whether they can do that cost-effectively and with the speed and efficiency needed to bring phages into routine use. Also problematic is a shortage of regulatory guidelines governing the production, testing and use of phage therapy. “But if it has the potential to save lives, then we as a society need to know whether it will work and how best to implement it,” says Jeremy J. Barr, a microbiologist at Monash University in Melbourne, Australia. “The antibiotic-resistance crisis is too dire to not embrace phage therapy now.”

Trading vulnerabilities

Burgholzer learned about phages by talking to other people with cystic fibrosis around the country. While scouring the Internet for more information, he came on a YouTube video made by phage researchers at Yale University. Soon he was corresponding with Benjamin Chan, a biologist in Yale's department of ecology and evolutionary biology. Since arriving there in 2013, Chan has accumulated a “library” of phages, harvested from sewage, soil and other natural sources, that he makes available to doctors at Yale New Haven Hospital and elsewhere.

Chan's first case, in 2016, was a resounding success. He isolated a phage from pond water, and doctors used it to cure Ali Khodadoust, a prominent eye surgeon. Khodadoust had been suffering from a raging MDR infection in his chest, a complication from open-heart surgery four years earlier. He was taking massive daily doses of antibiotics to try to fight his invading pathogen, the tenacious bacterium Pseudomonas aeruginosa. The virus Chan selected latches on to what is known as an efflux pump on the bacterial cell wall. The pumps expel antibiotics and are frequently found in drug-resistant bacteria. Most of the P. aeruginosa in Khodadoust's body had the pumps, and the phage killed them. The relatively few remaining P. aeruginosa faced an evolutionary trade-off: their lack of efflux pumps meant they survived the virus attack, but it made them defenseless against antibiotics. By taking the phages and antibiotics together, Khodadoust gradually recovered in just a few weeks. He died two years later, at age 82, from noninfectious illnesses.

After that first case, Chan supplied phages for nearly a dozen more experimental treatments at Yale, most involving cystic fibrosis patients with P. aeruginosa lung infections. He asked Burgholzer to send a sputum sample by overnight delivery so he could identify phages that might help.



I visited Chan at Yale last December, after the screening had begun. He was wearing a checkered oxford shirt, khakis and loafers, and before long he was calling me “dude,” his preferred moniker. After chatting briefly in his office, we headed for an adjacent laboratory, where Chan showed me a petri dish. Burgholzer's bacteria had developed into a gray lawn spanning the dish, but two thin, clear rows cut across it. "The bacteria that had been in those rows were all dead," Chan told me, killed by drips of a phage solution Burgholzer would soon be treated with. Burgholzer's infection was caused by three species of the bacterial genus Achromobacter, and Chan planned to select individual phages that could pick them off one by one—an approach known as sequential monophage therapy. “We're essentially playing chess in an antimicrobial war,” Chan said. “We need to make calculated moves.”

Chan hoped to induce an evolutionary trade-off similar to the one he believes worked for Khodadoust. Unable to find a phage that targets efflux pumps on Achromobacter bacteria, he instead selected one that targets a large protein called lipopolysaccharide (LPS) in the microbe's cell wall. LPS has side chains of molecules known as O antigens, which vary in length. The longer the chain, the better the bacteria's ability to resist not only antibiotics but also the host's immune system. Chan planned to kill the hardy long-chain strains with phages, leaving the weaker short-chain pathogens behind. "In the best scenario," he said, "a succession of phages would shift the bacterial population toward short-chain strains that might be more easily controlled by drugs and Burgholzer's own immune defenses. Bacteria compete for real estate in the body,” Chan said. “After large numbers of one species are suddenly killed by phage, in many cases, others move in.” He wanted the new occupants to be less virulent than their predecessors.

Chan's boss, Paul Turner, has devoted his career to studying evolutionary trade-offs in the microbial world. A professor in Chan's department, he explained later on the day of my visit that "phage treatments can work without completely ridding the body of a disease-causing bacteria. Especially when treating chronic conditions, doctors can use phages to selectively shape the population of the bad bacteria so it develops other vulnerabilities. Should those vulnerabilities be toward antibiotics, then so much the better,” he told me. "Combining antibiotics with phages to achieve optimal effects for patients," he says, “makes it easier to move forward with phage therapy quickly.”

I drove with Chan to Yale New Haven Hospital to watch as Burgholzer's phage treatment got underway. We took an elevator to the second floor, where we waited for Chan's clinical collaborator, Jonathan Koff, to arrive. A pulmonologist and director of the Adult Cystic Fibrosis Program, Koff soon came bounding in, a knapsack slung over his shoulder. Burgholzer met the three of us in a treatment room and spoke with a rasp—the only outward sign of his disease. As Koff and Chan compared notes, he told me he wanted to stay healthy for his three-year-old daughter. When treatment time arrived, he tossed his cell phone to his wife. “Here, take a photo for my mother,” he said with a grin. Then he raised a nebulizer over his mouth and nose and began inhaling a vaporized phage solution into his lungs.

Phage cocktails

According to Koff, sequential monophage therapy makes sense for treating cystic fibrosis and certain other chronic diseases that sequester bad bacteria in the body. When there is no proven way to eliminate the pathogens completely, he says, the tactic is to chip away at the harmful strains.

Some clinicians are choosing a different approach: They give patients multiple phages in a therapeutic cocktail, trying to knock out an infection completely by targeting a variety of bacterial resistance mechanisms simultaneously. Ideally, each phage in a cocktail will glom on to a different receptor, so if bacteria evolve resistance to one virus in the mixture, other viruses will keep up the attack.

Chan and Koff argue that phage interactions with bacteria are unpredictable and that when exposed to cocktails, pathogens might develop resistance to all the viruses in the mixture at once, which could limit future treatment options. “Splitting the cocktail into sequential treatments allows you to treat patients for longer durations,” Koff says.

Jessica Sacher, co-founder of the Phage Directory, an independent platform for improving access to phages and phage expertise, says "convincing arguments can be made for either method. The science isn't there yet to say one is necessarily better than the other.” She notes that "cocktails might be more appropriate for acutely ill patients, who cannot always wait for doctors to develop a sequential strategy."

"Experts cannot say which phage therapies may win out. What is needed now are results from clinical trials that can help overcome residual skepticism."


Urgency was paramount in the now famous case of Tom Patterson, a professor at the University of California, San Diego, who in 2016 was saved by phage cocktails after being stricken by an MDR infection during a trip to Egypt. The invader was Acinetobacter baumannii, a notoriously drug-resistant microbe that is common in Asia and is spreading steadily toward the West. Patterson was in multiorgan failure by the time doctors delivered mixtures of four viruses through a catheter into his abdomen and a fifth intravenously. The physicians treated him twice a day for four weeks, and he was cleared of infection within three months. He still needed extensive rehabilitation, but he remains healthy today.

The case drew worldwide media attention. The treating physicians were Robert Schooley, a friend of Patterson's and chief of infectious diseases at U.C. San Diego, and Patterson's wife, Steffanie Strathdee, then director of the university's Global Health Institute. Two years later, with an initial investment of $1.2 million, Schooley and Strathdee launched the Center for Innovative Phage Applications and Therapeutics at U.C. San Diego to fund clinical research and promote the field.

Each phage Patterson was treated with was screened for its ability to kill A. baumannii in infectious samples obtained from his body, using assays at the Naval Medical Research Center at Fort Detrick, Md., and at Texas A&M University. "The assays can test hundreds of phages against bacterial pathogens simultaneously in just eight to 12 hours," according to Biswajit Biswas, chief of the bacteriophage division at the center, which supplied some of the phages used in Patterson's treatment. Biswas, who developed the assay and created the center's phage bank, says "the assay allows new viruses to be easily swapped in to counter the onset of resistance." Patterson did develop resistance to his first cocktail within two weeks, prompting the navy to prepare a second one with longer-lasting effects. A company called Adaptive Phage Therapeutics in Gaithersburg, Md., has since licensed the navy's assay and its phage bank and will soon take them both into clinical trials in patients with urinary tract infections.

The navy assay checks only for bacterial cell death; it does not reveal which receptors are targeted. Whether cocktails should target known receptors is in debate. Ry Young, a phage geneticist at Texas A&M, who supplied viruses for Patterson, argues they should. “We don't even know if phages were responsible for his successful outcome,” he says. “Our best guess is that phage treatment lowered his infectious load to a level where his immune system took over. The better approach to cocktails," Young says, "is to combine three or four viruses targeting distinct receptors on the same bacterial strain. The odds of a bacterium evolving resistance to a single phage are about a million to one," he says, "whereas the odds of it losing or developing mutant forms of receptors targeted by all the phages in a cocktail are essentially zero.” Furthermore, the identification of important receptors is critical if clinicians hope to make bacteria sensitive to antibiotics again.

Barr says scientists are working to identify the receptors targeted by Patterson's cocktails, but he disagrees on the need to identify the receptors prior to use. “It's an understandable viewpoint and a hot topic in the field,” he says. “We know very little about these phages, and we need checks and balances before using them in therapy. Does that mean we need to identify host receptors? That is a huge amount of work currently, so I would say it's not required but definitely desirable.”

Engineered phages

Given the vagary of cocktails, some researchers say phages should be genetically engineered to bind to specific receptors and also to kill bacteria in novel ways. The vast majority of phages used thus far have been natural, harvested from the environment, but phage engineering is an emerging frontier with a new success story under its belt. Isabelle Carnell, a British teenager with cystic fibrosis, was suffering from life-threatening infections in her liver, limbs and torso after undergoing a double lung transplant in 2017. Her bacterial nemesis—Mycobacterium abscessus—was not responding to any antibiotics. Yet this year, in a first for the field, researchers from several institutions successfully treated the girl with an engineered cocktail of three phages. One naturally rips apart M. abscessus as it replicates. The other two also kill bacteria but not as completely, leaving 10 to 20 percent surviving the process. So the team, led by Graham Hatfull, a professor of biological sciences at the University of Pittsburgh, deleted a single gene from each of those two phages, turning them into engineered assassins. The cocktail of three phages cleared Carnell's infection within six months.

Researchers at Boston University first developed engineered phages in 2007. They coaxed one into producing an enzyme that more effectively degrades the sticky biofilms secreted by certain infectious bacteria for protection. Scientists have since modified phages to kill broader ranges of harmful bacteria or potentially to deliver drugs and vaccines to specific cells. These lab-designed viruses are also more patentable than natural phages, which makes them more desirable to drug companies. As if to underscore that point, a division of the pharmaceutical giant Johnson & Johnson struck a deal in January with Locus Biosciences, worth up to $818 million, to develop phages engineered with the gene-editing tool CRISPR.

Developing a phage therapy that is commercially viable will not be easy. Barr and other scientists point out that it takes a tremendous amount of time, money and effort to engineer a phage, and after all that the target bacteria might soon evolve resistance to it. Furthermore, "regulatory approval for an engineered phage could be a tough sell,” says Barr, echoing the view of several scientists interviewed for this story. But FDA spokesperson Megan McSeveney, in an e-mail, claimed "the agency does not distinguish between natural and engineered phages as long as therapeutic preparations are deemed safe."

Future prospects

Companies are now testing different ways to bring phages to broader markets. Some companies want to supply patients with personalized therapies matched specifically to their infections. That is the strategy at Adaptive Phage Therapeutics. The company's chief executive officer, Greg Merril, says "assays used to screen the navy's phages against infectious samples could be offered at diagnostic labs and major medical centers worldwide." Phages effective against locally prevalent bacteria in each region could be supplied in kiosks, bottled in FDA-approved, ready-to-use vials. Merril says "doctors could continually monitor treated patients for resistance, swapping in new phages as needed until the infections are under control." He estimates that the per-patient cost under the current compassionate-use system is approximately $50,000, an expense that should fall with economies of scale.

Other companies reject this personalized strategy in favor of fixed phage products more akin to commercial antibiotics. Armata Pharmaceuticals' lead product is a cocktail of three natural phages targeted at Staphylococcus aureus bacteria, the cause of common staph infections often contracted at hospitals. It is in clinical trials in patients who have infected mechanical heart pumps. Armata's plan is to monitor for treatment-resistant staph in the general population, then introduce new cocktails as needed, in much the same way that influenza vaccines are tuned every year to match the latest circulating strains. Pharmaceutical executives say it's too soon to estimate what the costs would be.

Experts still cannot say which of the current strategies—sequential monotherapy, cocktails, engineered phages, and general or personalized treatments—may ultimately win out, assuming any do. "An optimal approach might not even exist,” says Barr, "considering that phage treatments in each case could depend on complicating issues, such as the target pathogen, the disease and the patient's medical history.”

"Phage therapy is still saddled by geopolitical biases, too,"
says Strathdee. "What is really needed now," she says, "are positive results from well-controlled clinical trials that can help overcome residual skepticism." Alan Davidson, a biochemist at the University of Toronto, speculates that "within a decade phage therapy might be cheaper, easier and faster than it is today. He leans toward the engineering approach," saying "sequencing the whole genome of a patient's bacteria and then synthesizing a phage to cure an infection could be quicker and less expensive than screening the pathogens against a battery of viruses drawn from nature.”

Meanwhile Burgholzer, who was self-administering phage therapy with a nebulizer at home until March 2019, has not yet experienced the clinical improvements he was hoping for. In March, Chan and Koff introduced a second phage targeted at another Achromobacter strain. By April the bacterial counts in Burgholzer's lungs had fallen by more than two orders of magnitude since the initial treatment began. “So it does appear we can pick off those strains successively,” Koff told me. Yet Koff acknowledged that Burgholzer was not noticing a dramatic change in lung function. When I asked why, Koff responded, “We know a lot more about the phage we use against P. aeruginosa than we do about phages targeting Achromobacter.” The ability to manipulate the infection “is less informed.”

"The next step," Koff says, "will be to genetically sequence mucus samples from Burgholzer's lungs. We really need to understand what's happening with his bacteria so we can get to the high level of sophistication we have with P. aeruginosa. Bobby is letting us take a chance to see if, at a minimum, we can help.” Frustrated but still eager, Koff says, “Some patients respond better than others. We need to understand those dynamics.”

 
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San Diego man saved by therapeutic use of viruses

by Kenny Goldberg | April 25, 2017

Viruses can kill, but they can also cure. Just ask Tom Patterson.

He and his wife, Steffanie Strathdee, were on vacation in Egypt in late 2015.

One day, Patterson became violently ill. He was treated at a local clinic for an inflamed pancreas.

Standard treatments did not help, and Patterson became sicker. He was medevaced to Germany, where doctors discovered that Patterson was infected with a multidrug resistant pathogen.

He was flown to UC San Diego’s Thornton Hospital. Patterson went into septic shock and fell into a coma.

"By then, I realized that all the antibiotic bags that were hanging on his IV pole were just there to make us feel better, and that there was nothing else that they could do," Strathdee said.

But Strathdee, an infectious disease epidemiologist at UCSD, started to research alternative treatments.

Then she discovered phage therapy. Phages are viruses that can destroy bacteria. They were used therapeutically before the age of antibiotics.

Strathdee sent out inquiries to the phage research community. She got a response from the head phage research at Texas A&M University.

He was moved by Patterson's plight, and told Strathdee that if she sent his bacterial isolate and he could find a match, he would help her.

Eventually, three different teams of researchers found suitable phages for Patterson's infection.

The FDA gave its OK, and on March 13, 2016, a cocktail of phages was pumped into Patterson's body.

Three days later, Patterson woke up.

He said at first, he could not see clearly, and his mind was barely functioning.

"But when people began to speak to me, it was as if somebody had plugged in one of the strings of lights, and my memories came flooding back," he said.

Patterson was finally released from the hospital last August. He would like to think that his bout with multidrug resistant bacteria will be instructive.

"My hope is my experience is going to really lead to potentially saving millions of lives," Patterson said.

Phage therapy is still considered experimental. A 2016 report predicts antimicrobial resistance could kill 300 million people worldwide by 2050.

 
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Phage therapy

Phage therapy or viral phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture. If the target host of a phage therapy treatment is not an animal, the term "biocontrol" (as in phage-mediated biocontrol of bacteria) is usually employed, rather than "phage therapy".

Bacteriophages are much more specific than antibiotics. They are typically harmless not only to the host organism, but also to other beneficial bacteria, such as the gut flora, reducing the chances of opportunistic infections. They have a high therapeutic index, that is, phage therapy would be expected to give rise to few side effects. Because phages replicate in vivo (in cells of living organism), a smaller effective dose can be used.

This specificity is also a disadvantage: a phage will kill a bacterium only if it matches the specific strain. Consequently, phage mixtures ("cocktails") are often used to improve the chances of success. Alternatively, samples taken from recovering patients sometimes contain appropriate phages that can be grown to cure other patients infected with the same strain.

Phages tend to be more successful than antibiotics where there is a biofilm covered by a polysaccharide layer, which antibiotics typically cannot penetrate. In the West, no therapies are currently authorized for use on humans.

Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics, particularly in Russia and Georgia. There is also a phage therapy unit in Wrocław, Poland, established 2005, the only such centre in a European Union country.

History

The discovery of bacteriophages was reported by the Englishman Frederick Twort in 1915 and the French-Canadian Felix d'Hérelle in 1917. D'Hérelle said that the phages always appeared in the stools of Shigella dysentery patients shortly before they began to recover. He "quickly learned that bacteriophages are found wherever bacteria thrive: in sewers, in rivers that catch waste runoff from pipes, and in the stools of convalescent patients". Phage therapy was immediately recognized by many to be a key way forward for the eradication of pathogenic bacterial infections. A Georgian, George Eliava, was making similar discoveries. He travelled to the Pasteur Institute in Paris where he met d'Hérelle, and in 1923 he founded the Eliava Institute in Tbilisi, Georgia, devoted to the development of phage therapy. Phage therapy is used in Russia, Georgia and Poland.

In Russia, extensive research and development soon began in this field. In the United States during the 1940s commercialization of phage therapy was undertaken by Eli Lilly and Company.

While knowledge was being accumulated regarding the biology of phages and how to use phage cocktails correctly, early uses of phage therapy were often unreliable. Since the early 20th century, research into the development of viable therapeutic antibiotics had also been underway, and by 1942 the antibiotic penicillin G had been successfully purified and saw use during the Second World War. The drug proved to be extraordinarily effective in the treatment of injured Allied soldiers whose wounds had become infected. By 1944, large-scale production of Penicillin had been made possible, and in 1945 it became publicly available in pharmacies. Due to the drug's success, it was marketed widely in the U.S. and Europe, leading Western scientists to mostly lose interest in further use and study of phage therapy for some time.

Isolated from Western advances in antibiotic production in the 1940s, Russian scientists continued to develop already successful phage therapy to treat the wounds of soldiers in field hospitals. During World War II, the Soviet Union used bacteriophages to treat many soldiers infected with various bacterial diseases e.g. dysentery and gangrene. Russian researchers continued to develop and to refine their treatments and to publish their research and results. However, due to the scientific barriers of the Cold War, this knowledge was not translated and did not proliferate across the world. A summary of these publications was published in English in 2009 in "A Literature Review of the Practical Application of Bacteriophage Research".

There is an extensive library and research center at the George Eliava Institute in Tbilisi, Georgia. Phage therapy is today a widespread form of treatment in that region.

As a result of the development of antibiotic resistance since the 1950s and an advancement of scientific knowledge, there has been renewed interest worldwide in the ability of phage therapy to eradicate bacterial infections and chronic polymicrobial biofilm (including in industrial situations).

Phages have been investigated as a potential means to eliminate pathogens like Campylobacter in raw food and Listeria in fresh food or to reduce food spoilage bacteria. In agricultural practice phages were used to fight pathogens like Campylobacter, Escherichia and Salmonella in farm animals, Lactococcus and Vibrio pathogens in fish from aquaculture and Erwinia and Xanthomonas in plants of agricultural importance. The oldest use was, however, in human medicine. Phages have been used against diarrheal diseases caused by E. coli, Shigella or Vibrio and against wound infections caused by facultative pathogens of the skin like staphylococci and streptococci. Recently the phage therapy approach has been applied to systemic and even intracellular infections and the addition of non-replicating phage and isolated phage enzymes like lysins to the antimicrobial arsenal. However, actual proof for the efficacy of these phage approaches in the field or the hospital is not available.

Some of the interest in the West can be traced back to 1994, when Soothill demonstrated (in an animal model) that the use of phages could improve the success of skin grafts by reducing the underlying Pseudomonas aeruginosa infection.[25] Recent studies have provided additional support for these findings in the model system.

Although not "phage therapy" in the original sense, the use of phages as delivery mechanisms for traditional antibiotics constitutes another possible therapeutic use. The use of phages to deliver antitumor agents has also been described in preliminary in vitro experiments for cells in tissue culture.

In June 2015 the European Medicines Agency hosted a one-day workshop on the therapeutic use of bacteriophages and in July 2015 the National Institutes of Health (USA) hosted a two-day workshop "Bacteriophage Therapy: An Alternative Strategy to Combat Drug Resistance."

In 2017, a pair of genetically engineered phages along with one naturally occuring (so-called "phage Muddy") each from among those catalogued by Science Education Alliance-Phages Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) at the Howard Hughes Medical Institute by Graham Hatfull and colleagues, was used by microbiologist James Soothill at Great Ormond Street Hospital for Children in London to treat an antibiotic-resistant bacterial (Mycobacterium abscessus) infection in a young woman with cystic fibrosis.

Potential benefits

Phage therapy is the use of bacteriophages to treat bacterial infections. This could be used as an alternative to antibiotics when bacteria develops resistance. Superbugs that are immune to multiple types of drugs are becoming a concern with the more frequent use of antibiotics. Phages can target these dangerous microbes without harming human cells due to how specific they are.

Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. It is conceivable that, although bacteria can develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics. Just as bacteria can evolve resistance, viruses can evolve to overcome resistance.

Bacteriophages are very specific, targeting only one or a few strains of bacteria. Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The species and strain specificity of bacteriophages makes it unlikely that harmless or useful bacteria will be killed when fighting an infection.

A few research groups in the West are engineering a broader spectrum phage, and also a variety of forms of MRSA treatments, including impregnated wound dressings, preventative treatment for burn victims, phage-impregnated sutures. Enzybiotics are a new development at Rockefeller University that create enzymes from phage. Purified recombinant phage enzymes can be used as separate antibacterial agents in their own right.

Phage Therapy also has the potential of preventing or treating infectious diseases of corals. This could assist with decline of coral around the world.

 
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Phage Therapy: Beating superbugs at their own game

by Rana Samadfam | January 21, 2019

Is the centuries-old strategy of using bacteria-eating viruses to fight disease making a comeback?

The word “superbug” is a familiar term. We now know that the overuse of antibiotics has contributed to creating these tiny, life-threatening monsters. The hunt is on for new antibiotics to fight these superbugs, but it cannot completely resolve the issue of multi-drug-resistant (MDR) bacteria. Bacteria are dynamic systems; continuously evolving to survive by gaining resistance to new antibiotics almost as fast as we can create them. Alternative approaches are necessary to combat MDR, and phage therapy may be the top candidate.

A phage, also known as a bacteriophage, is a virus that infects and replicates within bacteria, and phage therapy is the therapeutic use of bacteriophages to treat infectious disease. The phage is often an obligate killer of its host, meaning the phage kills its host bacterial cell in order to reproduce. Bacteriophages are widely distributed in locations populated by their hosts, including the human body, seawater and soil. The largest population among different organisms on Earth belongs to phages, with an estimated number of viral SpeTarparticles around 10 31.

Phage therapy is not a new concept. It dates back to the pre-penicillin era, when French-Canadian microbiologist Félix d’Herelle used phages to cure four patients of dysentery in 1919. However, the interest in phage therapy was dampened with the discovery of conventional antibiotics and their effectiveness in treatment of infectious disease. It was revived decades later with the first scientific phage summit in 2004. The burgeoning interest in phages led to, among other things, the revolutionary gene editing tool known as CRISPR Cas-9.

Phage characteristics: Specific, adaptable and easy to find

What have we learned about phages? Well, they are relatively specific. They typically have a very narrow antibacterial spectrum limited to one single species of bacteria, or even a single strain within a species. Their specificity can make them tricky to use, but they have the benefit very low collateral damage outside of their chosen target. This stands in sharp contrast with common antimicrobial drugs that do not distinguish between infectious and harmless bacteria, resulting in a greater disturbance of microbiota.

Phages are also dynamic and highly adaptable, much like their hosts. If the bacteria mutates to resist the phage, the phage counters with mutations of its own. This enable us to beat the superbugs at their own game, fighting fire with fire. Although there is a potential for direct interaction with the human immune system, these concerns are no greater than those with biologics, viral vectors used to deliver in gene therapies or live-attenuated vaccines (vaccines that have been genetically modified to make then less virulent or harmless.

What’s more, phages are easy to discover and characterized due to the use of next-gen imaging and genetic tools. After the first phage was discovered, in 1915, only a handful of phages were studied in great detail. The recent renaissance seen in phage biology has been triggered due to a growing awareness of the number of phages in all bacterial dominated environments revealed by epiflourescent and electron microscopy, molecular studies and the genomes of bacteria following whole genome sequencing projects.
Phages and modern medicine

An ideal candidate for phage therapy is an obligately lytic phage with a high potential to reach and then kill bacteria. Like any other drug candidate, phages must display a good pharmacokinetic profile with optimal absorption, distribution, and half-life (survival in a live biologic system). In addition, they must have sufficient stability under typical storage conditions and temperatures. Most importantly, they need to be fully sequenced to ensure absence of undesirable genes and low ability for transduction (ability to transfer gene from one bacteria to other).

There is some resistance in modern medicine to phage therapy, which may stem from unfamiliarity or from guilt by association to the infectious viral family. After all, phages are viruses. The word can be frightening to consumers because it is reminds one of past mass casualties or present infectious diseases such as the flu and HIV. It may not be that easy, therefore, for a drug developer to convince the public to embrace phage therapies. Although there are several clinical trials currently ongoing, ListshieldTM is the only FDA approved phage therapy. It is used as a food additive and kills Listeria monocytogenes, one of the most virulent foodborne pathogens and a cause of meningitis. In the absence of urgent corrective actions to limit overuse of antibiotics and insufficient investment in research for new and innovative weapons against superbugs, the world is heading toward an era in which many common infections will have no cure. With advances in gene editing technology (including CRISPR–Cas9, the genome editing derived from a bacteriophage), we have the necessary tools to investigate the genetic relationship between phages and their bacterial hosts. These tools can also be used to characterize, optimize and select phages for relevant diseases.

 
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The virus that could cure Alzheimer’s, Parkinson’s, and more

by Jon Palfremen | NOVA | March 23, 2016

A humble phage could hold the key to unraveling the misfolded proteins that underlie Alzheimer’s and other diseases.

In 2004, the British chemist Chris Dobson speculated that there might be a universal elixir out there that could combat not just alpha-synuclein for Parkinson’s but the amyloids caused by many protein-misfolding diseases at once. Remarkably, in that same year an Israeli scientist named Beka Solomon discovered an unlikely candidate for this elixir, a naturally occurring microorganism called a phage.

Solomon, a professor at Tel Aviv University, made a serendipitous discovery one day when she was testing a new class of agents against Alzheimer’s disease. If it pans out, it might mark the beginning of the end of Alzheimer’s, Parkinson’s, and many other neurodegenerative diseases. It’s a remarkable story, and the main character isn’t Solomon or any other scientist but a humble virus that scientists refer to as M13.

Among the many varieties of viruses, there is a kind that only infects bacteria. Known as bacteriophages, or just phages, these microbes are ancient (over three billion years old) and ubiquitous: they’re found everywhere from the ocean floor to human stomachs. The phage M13’s goal is to infect just one type of bacteria, Escherichia coli , or E. coli , which can be found in copious amounts in the intestines of mammals. Like other microorganisms, phages such as M13 have only one purpose: to pass on their genes. In order to do this, they have developed weapons to enable them to invade, take over, and even kill their bacterial hosts. Before the advent of antibiotics, in fact, doctors occasionally used phages to fight otherwise incurable bacterial infections.

To understand Solomon’s interest in M13 requires a little background about her research. Solomon is a leading Alzheimer’s researcher, renowned for pioneering so-called immunotherapy treatments for the disease. Immunotherapy employs specially made antibodies, rather than small molecule drugs, to target the disease’s plaques and tangles. As high school students learn in biology class, antibodies are Y-shaped proteins that are part of the body’s natural defense against infection. These proteins are designed to latch onto invaders and hold them so that they can be destroyed by the immune system. But since the 1970s, molecular biologists have been able to genetically engineer human-made antibodies, fashioned to attack undesirable interlopers like cancer cells. In the 1990s, Solomon set out to prove that such engineered antibodies could be effective in attacking amyloid-beta plaques in Alzheimer’s as well.

In 2004, she was running an experiment on a group of mice that had been genetically engineered to develop Alzheimer’s disease plaques in their brains. She wanted to see if human-made antibodies delivered through the animals’ nasal passages would penetrate the blood-brain barrier and dissolve the amyloid-beta plaques in their brains. Seeking a way to get more antibodies into the brain, she decided to attach them to M13 phages in the hope that the two acting in concert would better penetrate the blood-brain barrier, dissolve more of the plaques, and improve the symptoms in the mice—as measured by their ability to run mazes and perform similar tasks.

At the time, Solomon had no clear idea how a simple phage could dissolve Alzheimer’s plaques.

Solomon divided the rodents into three groups. She gave the antibody to one group. The second group got the phage-antibody combination, which she hoped would have an enhanced effect in dissolving the plaques. And as a scientific control, the third group received the plain phage M13.

Because M13 cannot infect any organism except E. coli , she expected that the control group of mice would get absolutely no benefit from the phage. But, surprisingly, the phage by itself proved highly effective at dissolving amyloid-beta plaques and in laboratory tests improved the cognition and sense of smell of the mice. She repeated the experiment again and again, and the same thing happened. “The mice showed very nice recovery of their cognitive function,” Solomon says. And when Solomon and her team examined the brains of the mice, the plaques had been largely dissolved. She ran the experiment for a year and found that the phage-treated mice had 80% fewer plaques than untreated ones. Solomon had no clear idea how a simple phage could dissolve Alzheimer’s plaques, but given even a remote chance that she had stumbled across something important, she decided to patent M13’s therapeutic properties for the University of Tel Aviv. According to her son Jonathan, she even “joked about launching a new company around the phage called NeuroPhage. But she wasn’t really serious about it.”

The following year, Jonathan Solomon—who’d just completed more than a decade in Israel’s special forces, during which time he got a BS in physics and an MS in electrical engineering—traveled to Boston to enroll at the Harvard Business School. While he studied for his MBA, Jonathan kept thinking about the phage his mother had investigated and its potential to treat terrible diseases like Alzheimer’s. At Harvard, he met many brilliant would-be entrepreneurs, including the Swiss-educated Hampus Hillerstrom, who, after studying at the University of St. Gallen near Zurich, had worked for a European biotech venture capital firm called HealthCap.

Following the first year of business school, both students won summer internships: Solomon at the medical device manufacturer Medtronic and Hillerstrom at the pharmaceutical giant AstraZeneca. But as Hillerstrom recalls, they returned to Harvard wanting more: “We had both spent…I would call them ‘weird summers’ in large companies, and we said to each other, ‘Well, we have to do something more dynamic and more interesting.’ ”

In their second year of the MBA, Solomon and Hillerstrom took a class together in which students were tasked with creating a new company on paper. The class, Solomon says, “was called a field study, and the idea was you explore a technology or a new business idea by yourself while being mentored by a Harvard Business School professor. So, I raised the idea with Hampus of starting a new company around the M13 phage as a class project. At the end of that semester, we developed a mini business plan. And we got on so well that we decided that it was worth a shot to do this for real.”

In 2007, with $150,000 in seed money contributed by family members, a new venture, NeuroPhage Pharmaceuticals, was born. After negotiating a license with the University of Tel Aviv to explore M13’s therapeutic properties, Solomon and Hillerstrom reached out to investors willing to bet on M13’s potential therapeutic powers. By January 2008, they had raised over $7 million and started hiring staff.

Their first employee—NeuroPhage’s chief scientific officer—was Richard Fisher, a veteran of five biotech start-ups. Fisher recalls feeling unconvinced when he first heard about the miraculous phage. “But the way it’s been in my life is that it’s really all about the people, and so first I met Jonathan and Hampus and I really liked them. And I thought that within a year or so we could probably figure out if it was an artifact or whether there was something really to it, but I was extremely skeptical.”

“Why would a phage do this to amyloid fibers?”


Fisher set out to repeat Beka Solomon’s mouse experiments and found that with some difficulty he was able to show the M13 phage dissolved amyloid-beta plaques when the phage was delivered through the rodents’ nasal passages. Over the next two years, Fisher and his colleagues then discovered something totally unexpected: that the humble M13 virus could also dissolve other amyloid aggregates—the tau tangles found in Alzheimer’s and also the amyloid plaques associated with other diseases, including alpha-synuclein (Parkinson’s), huntingtin (Huntington’s disease), and superoxide dismutase (amyotrophic lateral sclerosis). The phage even worked against the amyloids in prion diseases (a class that includes Creutzfeldt-Jakob disease). Fisher and his colleagues demonstrated this first in test tubes and then in a series of animal experiments. Astonishingly, the simple M13 virus appeared in principle to possess the properties of a “pan therapy,” a universal elixir of the kind the chemist Chris Dobson had imagined.

This phage’s unique capacity to attack multiple targets attracted new investors in a second round of financing in 2010. Solomon recalls feeling a mix of exuberance and doubt: “We had something interesting that attacks multiple targets, and that was exciting. On the other hand, we had no idea how the phage worked.”

The key

That wasn’t their only problem. Their therapeutic product, a live virus, it turned out, was very difficult to manufacture. It was also not clear how sufficient quantities of viral particles could be delivered to human beings. The methods used in animal experiments—inhaled through the nose or injected directly into the brain—were unacceptable, so the best option available appeared to be a so-called intrathecal injection into the spinal canal. As Hillerstrom says, “It was similar to an epidural; this was the route we had decided to deliver our virus with.”

While Solomon and Hillerstrom worried about finding an acceptable route of administration, Fisher spent long hours trying to figure out the phage’s underlying mechanism of action. “Why would a phage do this to amyloid fibers? And we really didn’t have a very good idea, except that under an electron microscope the phage looked a lot like an amyloid fiber; it had the same dimensions.”

Boston is a town with enormous scientific resources. Less than a mile away from NeuroPhage’s offices was MIT, a world center of science and technology. In 2010, Fisher recruited Rajaraman Krishnan—an Indian postdoctoral student working in an MIT laboratory devoted to protein misfolding—to investigate the M13 puzzle. Krishnan says he was immediately intrigued. The young scientist set about developing some new biochemical tools to investigate how the virus worked and also devoured the scientific literature about phages. It turned out that scientists knew quite a lot about the lowly M13 phage. Virologists had even created libraries of mutant forms of M13. By running a series of experiments to test which mutants bound to the amyloid and which ones didn’t, Krishnan was able to figure out that the phage’s special abilities involved a set of proteins displayed on the tip of the virus, called GP3. “We tested the different variants for examples of phages with or without tip proteins, and we found that every time we messed around with the tip proteins, it lowered the phage’s ability to attach to amyloids,” Krishnan says.

Virologists, it turned out, had also visualized the phage’s structure using X-ray crystallography and nuclear magnetic resonance imaging. Based on this analysis, those microbiologists had predicted that the phage’s normal mode of operation in nature was to deploy the tip proteins as molecular keys; the keys in effect enabled the parasite to “unlock” E. coli bacteria and inject its DNA. Sometime in 2011, Krishnan became convinced that the phage was doing something similar when it bound to toxic amyloid aggregates. The secret of the phage’s extraordinary powers, he surmised, lay entirely in the GP3 protein.

As Fisher notes, this is serendipitous. Just by “sheer luck, M13’s keys not only unlock E. coli ; they also work on clumps of misfolded proteins.” The odds of this happening by chance, Fisher says, are very small. “Viruses have exquisite specificity in their molecular mechanisms, because they’re competing with each other…and you need to have everything right, and the two locks need to work exactly the way they are designed. And this one way of getting into bacteria also works for binding to the amyloid plaques that cause many chronic diseases of our day.”

Having proved the virus’s secret lay in a few proteins at the tip, Fisher, Krishnan, and their colleagues wondered if they could capture the phage’s amyloid-busting power in a more patient friendly medicine that did not have to be delivered by epidural. So over the next two years, NeuroPhage’s scientists engineered a new antibody (a so-called fusion protein because it is made up of genetic material from different sources) that displayed the critical GP3 protein on its surface so that, like the phage, it could dissolve amyloid plaques. Fisher hoped this novel manufactured product would stick to toxic aggregates just like the phage.

By 2013, NeuroPhage’s researchers had tested the new compound, which they called NPT088, in test tubes and in animals, including nonhuman primates. It performed spectacularly, simultaneously targeting multiple misfolded proteins such as amyloid beta, tau, and alpha-synuclein at various stages of amyloid assembly. According to Fisher, NPT088 didn’t stick to normally folded individual proteins; it left normal alpha-synuclein alone. It stuck only to misfolded proteins, not just dissolving them directly, but also blocking their prion-like transmission from cell to cell: “It targets small aggregates, those oligomers, which some scientists consider to be toxic. And it targets amyloid fibers that form aggregates. But it doesn’t stick to normally folded individual proteins.” And as a bonus, it could be delivered by intravenous infusion.

The trials

There was a buzz of excitement in the air when I visited NeuroPhage’s offices in Cambridge, Massachusetts, in the summer of 2014. The 18 staff, including Solomon, Hillerstrom, Fisher, and Krishnan, were hopeful that their new discovery, which they called the general amyloid interaction motif, or GAIM, platform, might change history. A decade after his mother had made her serendipitous discovery, Jonathan Solomon was finalizing a plan to get the product into the clinic. As Solomon says, “We now potentially have a drug that does everything that the phage could do, which can be delivered systemically and is easy to manufacture.”

Will it work in humans? While NPT088, being made up of large molecules, is relatively poor at penetrating the blood-brain barrier, the medicine persists in the body for several weeks, and so Fisher estimates that over time enough gets into the brain to effectively take out plaques. The concept is that this antibody could be administered to patients once or twice a month by intravenous infusion for as long as necessary.

“If our drug works, we will see it working in this trial.”

NeuroPhage must now navigate the FDA’s regulatory system and demonstrate that its product is safe and effective. So far, NPT088 has proved safe in nonhuman primates. But the big test will be the phase 1A trial expected to be under way this year. This first human study proposed is a single-dose trial to look for any adverse effects in healthy volunteers. If all goes well, NeuroPhage will launch a phase 1B study involving some 50 patients with Alzheimer’s to demonstrate proof of the drug’s activity. Patients will have their brains imaged at the start to determine the amount of amyloid-beta and tau. Then, after taking the drug for six months, they will be re-imaged to see if the drug has reduced the aggregates below the baseline.

“If our drug works, we will see it working in this trial,” Hillerstrom says. “And then we may be able to go straight to phase 2 trials for both Alzheimer’s and Parkinson’s.” There is as yet no imaging test for alpha-synuclein, but because their drug simultaneously lowers amyloid-beta, tau, and alpha-synuclein levels in animals, a successful phase 1B test in Alzheimer’s may be acceptable to the FDA. “In mice, the same drug lowers amyloid beta, tau, and alpha-synuclein,” Hillerstrom says. “Therefore, we can say if we can reduce in humans the tau and amyloid-beta, then based on the animal data, we can expect to see a reduction in humans in alpha-synuclein as well.”

Along the way, the company will have to prove its GAIM system is superior to the competition. Currently, there are several drug and biotech companies testing products in clinical trials for Alzheimer’s disease, against both amyloid-beta and tau and also corporations with products against alpha-synuclein for Parkinson’s disease. But Solomon and Hillerstrom think they have two advantages: multi-target flexibility (their product is the only one that can target multiple amyloids at once) and potency (they believe that NPT088 eliminates more toxic aggregates than their competitors’ products). Potency is a big issue. PET imaging has shown that existing Alzheimer’s drugs like crenezumab reduce amyloid loads only modestly, by around 10%. “One weakness of existing products,” Solomon says, “is that they tend to only prevent new aggregates. You need a product potent enough to dissolve existing aggregates as well. You need a potent product because there’s a lot of pathology in the brain and a relatively short space of time in which to treat it.”

Future targets

NeuroPhage’s rise is an extraordinary example of scientific entrepreneurship. While I am rooting for Solomon, Hillerstrom, and their colleagues, and would be happy to volunteer for one of their trials (I was diagnosed with Parkinson’s in 2011), there are still many reasons why NeuroPhage has a challenging road ahead. Biotech is a brutally risky business. At the end of the day, NPT088 may prove unsafe. And it may still not be potent enough. Even if NPT088 significantly reduces amyloid beta, tau, and alpha-synuclein, it’s possible that this may not lead to measurable clinical benefits in human patients, as it has done in animal models.

But if it works, then, according to Solomon, this medicine will indeed change the world: “A single compound that effectively treats Alzheimer’s and Parkinson’s could be a twenty billion-dollar-a-year blockbuster drug.” And in the future, a modified version might also work for Huntington’s, ALS, prion diseases like Creutzfeldt-Jakob disease, and more.

I asked Jonathan about his mother, who launched this remarkable story in 2004. According to him, she has gone on to other things. “My mother, Beka Solomon, remains a true scientist. Having made the exciting scientific discovery, she was happy to leave the less interesting stuff—the engineering and marketing things for bringing it to the clinic—to us. She is off looking for the next big discovery.”

 
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Can phages defeat antibiotic-resistant infections?

by Kelly Servick | Jun 21, 2018

One piece of good news can make all the difference. In the fight against antibiotic-resistant infections, a decades-old approach based on bacteria-slaying viruses called phages has been sidelined by technical hurdles, dogged by regulatory confusion, and largely ignored by drug developers in the West. But 2 years ago, researchers at the University of California, San Diego (UCSD), used phages to knock out an infection that nearly killed a colleague. Propelled by that success and a handful of others since, UCSD is now launching a clinical center to refine phage treatments and help companies bring them to market.

A first in North America, the center will initially consist of 16 UCSD researchers and physicians. It aims to be a proving ground for a treatment that has long been available in parts of Eastern Europe, but that still lacks the support of rigorous clinical trials. "There have been just a ton of failures and false starts," says Paul Bollyky, a microbiologist and physician at Stanford University Medical Center in Palo Alto, California, who studies phages. "The fact that a major American medical center is going to set up an ongoing enterprise around phage therapy … that's kind of a game changer for the field, at least in the United States."

Turning phages—found in soil, water, and sewage—into treatments isn't straightforward. Because each of the millions of phage strains in nature targets a specific bacterium, putting them to use means finding the specific phages that attack the menace at hand. Still, clinical centers overseas, in Georgia and Poland, have reported encouraging results with phages over the years. And the rise of antibiotic-resistant infections has prompted a handful of U.S. companies and research centers to reconsider the approach.

The case that mobilized the UCSD team hit close to home. In 2015, UCSD psychologist Tom Patterson was airlifted home after a vacation in Egypt when a drug-resistant strain of the bacterium Acinetobacter baumannii invaded his pancreas. As available antibiotics failed and Patterson fell into a coma, his wife, UCSD epidemiologist Steffanie Strathdee, launched an international effort to find strains of phage that might save him. After treatment with a variety of phages donated by San Diego–based biotech AmpliPhi Biosciences, Texas A&M University, and the U.S. Navy, Patterson made a dramatic recovery.

"Everybody's been talking about this case," Bollyky says. "Not only did he survive the treatment, which can't be taken for granted, but he also got better, and miraculously so." Patterson received some of the phages intravenously—an approach considered risky because toxins from bacteria used to grow the phages could linger in the mixture. His recovery helped allay safety fears, and it turned Strathdee into a self-described "phage wrangler," who helped match other patients with the right mixture of experimental phages. Since her husband's recovery, the UCSD team has successfully cleared infections in five more people with phage cocktails, under a U.S. Food and Drug Administration (FDA) process designed for emergencies where no approved treatments are available.

But a string of anecdotes does little to answer key scientific questions: What is the safest and most effective way to administer phages? How well do phages target the site of infection? How quickly are bacteria likely to develop resistance? "Those are the kinds of things you have to ask in structured clinical trials," says Robert Schooley, a UCSD physician and infectious disease researcher who treated Patterson and oversaw the other recent cases.

So he and Strathdee proposed the new clinical center, which will launch with a 3-year, $1.2 million grant from UCSD. The Center for Innovative Phage Applications and Therapeutics (IPATH) won't manufacture any phage treatments itself, but it will collaborate with companies and academic groups outside UCSD on multi-center clinical trials. IPATH will initially focus on treating patients with chronic, drug-resistant infections related to organ transplants, implanted devices such as pacemakers or joint replacements, and cystic fibrosis. Schooley is discussing possible trials with a team at the National Institute of Allergy and Infectious Diseases, and with two companies that have provided phages to patients at UCSD: AmpliPhi and Adaptive Phage Therapeutics (APT), based in Gaithersburg, Maryland, which has licensed the Navy's phage collection.

Running phages through modern clinical testing has proved difficult in the past. A European Union–sponsored trial known as PhagoBurn was all but derailed by a series of setbacks. "It was not an ideal trial, let me say it like that," says Jean-Paul Pirnay, a bioengineer at Queen Astrid Military Hospital in Brussels, one of the partners in PhagoBurn. A key obstacle was the fact that the trial targeted burn wounds, which often harbor multiple bacterial infections. That made it hard to test the effects of a phage therapy aimed at just one species. Designed to include 220 patients, the trial ultimately recruited only 27, and it has not yet published its results.

The anticipated trials at UCSD, on the other hand, will focus on patients with a single, known bacterial infection, Schooley says. But he admits it will still be tricky to design trials that isolate the effect of phages without withholding other potentially beneficial treatments, including antibiotics. (Ultimately, Schooley and many others expect phages to work in tandem with antibiotics—not to replace them.)

IPATH collaborators will also have to navigate a drug approval system suited to more conventional treatments. Because a phage cocktail will often have to be custom-designed for an individual, regulatory agencies may not have a single product to evaluate for safety and efficacy. But after initial talks with FDA, Greg Merril, APT's CEO, is confident the agency will be flexible. He plans to seek approval for an entire library of phages—about 100 for each bacterial species—from which doctors could create a cocktail of one to five phages for a patient.

In the meantime, Strathdee says the UCSD team plans to keep securing phages for individual cases under FDA's emergency pathway. She and Schooley already get several inquiries a week from patients and families fighting drug-resistant infections. "We hope to not send people with superbugs away, but to welcome them with open arms," she says. "Right now, they don't have anywhere to go."

Pirnay, whose team finds and formulates phages to treat infections related to battlefield injuries, has a piece of advice for the UCSD group: "Be careful not to create too high an expectancy with the public," he says. "Even when you do not say that you will be able to treat everything, you create a demand with desperate patients."

 
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Bonnie Bassler

Viruses that kill superbugs when antibiotics don't work

CBC Radio | Jan 12, 2019

Antibiotic resistant bacteria could be treated with the viruses that attack them in nature.

Fighting terrible and potentially lethal bacterial infections with viruses may sound like a bad idea, but it may just be the best strategy we have for battling antibiotic resistant infections in patients who have little other hope.

Phage therapy, the use of bacteria-killing viruses called bacteriophages against superbugs that no longer respond to antibiotics, is currently a last-resort, experimental therapy available only to those for whom traditional treatments aren't working. But the success of these experimental treatments is providing evidence that could open the door to larger clinical trials which could prove the efficacy and safety of phage therapy.

The superbug crisis

"Superbugs are dangerous," said Jonathan Dennis, a phage therapy researcher at the University of Alberta. "These bacteria have been exposed to high levels of antibiotics and have developed resistance mechanisms against them."

The overuse of antibiotics in the past few decades has led to the antimicrobial resistance crisis. Antimicrobial resistance has been increasing year by year, and scientists estimate that approximately 10 million people could die every year as a result of antimicrobial resistance by 2050.

"At some point, we're going to have to discover alternatives to the current antibiotics we have, whether that's discovering new classes of antibiotics, or developing completely new therapies such as bacteriophage therapy," said Dennis.

Bacteriophages were discovered around 1915, and there was a short burst of interest in using them to fight disease. But research on them was later abandoned in Western medicine once antibiotics were discovered.

However, in the last decade or so, with concerns about superbugs on the rise, there's been new research interest in bacteriophages in North America and Western Europe.

Paige's story

Paige Rogers lives in Lubbock, Texas. Since the age of two, she's spent a good part of her life in the hospital.

The 23-year-old was born with cystic fibrosis, a genetic disorder that causes her lungs to fill with thick mucus. This creates the perfect conditions for bacteria to grow, and she's suffered from chronic lung infections with a bacteria called Pseudomonas that antibiotic treatments controlled, but could never wipe out completely.

In 2015, the bacteria had flared up again in her lungs. She did her regular two week round of antibiotics, but there was no improvement. The Pseudomonas bacteria had evolved antibiotic resistance, and the powerful intravenous antibiotics she depended on for most of her life could no longer effectively fight her infection.

Rogers was facing a very dangerous situation. Desperate, her father searched online for a way to save his daughter and stumbled upon phage therapy.

Bacteriophages, or phages for short, are viruses that have evolved to attack specific strains of bacteria cell. They are the most numerous biological entity on Earth, and can be found everywhere including the soil, on our skin, on animals and plants. They've coexisted with bacterial cells for billions of years — long before animals appeared.

Treating bacterial infections with phages is still considered an experimental therapy in the United States and Canada.

Rogers was hesitant to try it at first. But with few options left, she agreed.

After several rounds of phage treatments, she started to feel better. Her pulmonary function improved and she was more energetic.

"Now that the phages have started working, I have had way more energy. I can actually take trips, go places on weekends, I can work more and actually have a day without having to have a nap," said Rogers. "I can just do simple things most people are used to doing and I never got to. I feel like the future is only going to get brighter and brighter because the phages are actually helping me live a better life."

The demand for phage therapy

Dr. Benjamin Chan is a phage researcher at Yale University and the doctor who supplied the phages that appear to have worked well for Rogers.

He's one of a handful of researchers in the United States treating patients using phages and investigating whether phage therapy can be practical in the wider population.

"We've treated seven patients so far and an eighth one is very near," said Chan. "I get a lot of inquiries about phage therapy everyday, so we've got a pretty big and growing list of people that are interested in doing this just because antibiotic resistance is a big problem."

Push backs and safety concerns

Chan's work has met with some skepticism from the medical community, but he thinks that could change if larger scale clinical trials show that phage treatment is as effective and safe as these experimental treatments suggest.

"We're getting with these one off cases — seven so far — some really encouraging data," he said. "It looks like we're having a real impact on these infections, so I think when we show people strong data, they will slowly get convinced that this is something that could be a really useful tool in treating these infections."

Safety is a significant public concern with phages, as they're viruses. But Chan explained that they can't infect humans because phages only target specific bacteria strains. Safety studies, so far, haven't shown any major problems.

Phage research in Canada

In Canada, Jonathan Dennis is one of only six scientists conducting phage research, and the only one to focus on phage therapy.

He's been building a catalogue of different phages that work against specific strains of antibiotic resistant bacteria so he'll have the right viruses on hand to help fight any patient's specific infection.

He has worked with clinicians in the U.S. to treat a few patients on an experimental basis, and may be providing phages for an experimental treatment on a Canadian patient soon.

That would be an important step, but he says that compared to the U.S. and Europe, the speed of research is much slower in Canada due to the lack of funding for phage research, which has forced many Canadian scientists who were interested in phage therapy to move on to new research topics.

Going mainstream

There's still a long road of further investigation before phage therapy can become mainstream, but both Chan and Dennis are optimistic.

"It'll just take a bit more work," said Chan. "We'll just have to get these phages through clinical trials and show that they are actually doing what we think they're doing."

"I think as antimicrobial resistance increases over time, more people will realize that phage therapy is our best option for treating bacterial infections other than chemical antibiotics,"
said Dennis, "and more money will be funneled into funding phage therapy and phage biology research."

 
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How bacteria become drug-resistant when exposed to antibiotics

Katarina Zimmer | May 23, 2019

Escherichia coli is capable of synthesizing drug-resistant proteins even in the presence of antibiotics designed to cripple cell growth. That’s the finding by a group of French researchers reporting today (May 23) in Science. They also discovered how the bacteria manage this feat: a well-conserved membrane pump shuttles antibiotics out of the cell—just long enough to buy the cells time to receive DNA from neighbor cells that codes for a drug-resistant protein.

“This is a key discovery,” microbiologist Manuel Varela of Eastern New Mexico University who wasn’t involved in the study says to The Scientist in an email. “This finding will help explain how bacteria manage to spread antimicrobial resistance as they encounter toxic levels of antibiotic.”

The discovery was a surprise to Christian Lesterlin, a bacterial geneticist at the University of Lyon and the senior author of the study. He and his colleagues had initially started the project to develop a real-time microscopy system to observe in detail plasmid transfer—a process by which bacterial cells share DNA with one another. Using carefully designed fluorescent proteins, they could track plasmids shuttling DNA from donor cells to recipient microbes as well as the resulting proteins once they had been translated inside the new hosts.

Using E. coli’s habitual sharing of antibiotic resistance genes as a case study, they watched as bacteria passed around DNA encoding the TetA protein—a pump that makes cells resistant to tetracycline by shunting it out of the cell. Shortly after, they observed plasmid DNA arriving in non-resistant cells, and some time later, red fluorescent spots appearing in the membranes of recipient cells, indicating the TetA protein was translated and the cells proved resistant to tetracycline.

The antibiotic, which is commonly used in livestock, but sometimes also to treat people for pneumonia, respiratory tract infections, and other conditions, ordinarily stunts the growth of bacteria that don’t have TetA, but numerous bacteria strains are becoming resistant through the adoption of such mechanisms. Tetracycline wasn’t present for this initial experiment, so to see how this process is influenced by the drug itself, the researchers exposed the cells to high concentrations of tetracycline and once again put them under the microscope.

As expected, they observed plasmid DNA arriving in new, non-resistant cells. This was expected, because tetracycline does nothing to hinder that process. Instead, it’s designed to stall protein production. And surprisingly, the researchers saw the red fluorescence appearing in the some of the new recipient cells that didn’t previously have the TetA protein: evidently, they were still able to synthesize proteins, including TetA, despite being exposed to tetracycline. “We spent many, many weeks just confirming this result, which was very counterintuitive, and we had a hard time being convinced that it was actually happening,” recalls Lesterlin.

The team made an educated guess as to why the cells were capable of this: many bacterial membranes are known to harbor a multidrug efflux pump known as AcrAB-TolC, which is capable of shuttling a wide range of antibiotics out of cells, and the scientists figured that it was getting tetracycline out of the cell before it could stop protein synthesis and cell growth. To test that idea, the researchers engineered several mutants with a genetic mutation in one of the genes that encodes the different proteins that make up the pump.

They found that the mutants, although they received the plasmid bearing the genetic code for TetA from neighboring cells, weren’t capable of synthesizing TetA protein. Without the functional efflux pump, the mutants can’t shuttle the tetracycline out of the cells. As levels of the antibiotic surged inside the cells, they could no longer make proteins or grow.

When functional, the AcrAB-TolC pump buys the bacteria time by keeping antibiotic concentrations just low enough for the cells to synthesize the resistance proteins encoded in the plasmid DNA, according to the researchers. In this case, it allows for the production of the TetA protein, which then shunts more tetracycline out of the cell. Ultimately, bacteria can become resistant while still under the influence of antibiotics. As Lesterlin puts it, “better news for bacteria than for human health.”

“The multidrug efflux pump AcrAB-TolC has been known in the field for quite some time,”
notes Anushree Chatterjee, a chemical engineer and microbiologist at the University of Colorado Boulder who wasn’t involved in the study. But the fact that it helps bacteria acquire drug resistance while they are simultaneously exposed to antibiotics is news, she says. “It’s always fascinating to see how there’s so many things bugs can do.”

The findings are widely relevant, she says—for one, because AcrAB-TolC is so broadly conserved across bacteria, and also because the mechanism is not limited to tetracycline. Lesterlin and his colleagues demonstrated that the pump also allows bacteria to produce drug-resistant proteins in the presence of other antibiotics designed to stifle gene expression, such as the translation-inhibiting chloramphenicol, and the transcription-inhibiting rifampicin. This mechanism is relevant for so-called bacteriostatic antibiotics, which don’t kill but only stifle bacterial growth, Lesterlin adds. He doubts it will work for bacteriolytic antibiotics, which destroy bacteria outright before they can become resistant.

Both Chatterjee and Varela find the new study thorough and its findings robust, Varela being particularly impressed by the technique the team developed to visualize the transfer of plasmid DNA between cells while watching TetA protein synthesis at the same time.

“The authors have [also] shed light on identifying key bacterial machinery that could serve as new targets for developing new anti-bacterial agents,” Varela adds in an email. For instance, one could build antibiotics by targeting the AcrAB-TolC pump—an approach some labs are already working on. Alternatively, one could target genes that regulate its production—an angle that appeals to Chatterjee. Traditional approaches of designing antibiotics have largely relied on small molecules that target specific proteins, many of which bacteria have seen for many years and ultimately select for more resistance mechanisms.

“We need to look at non-traditional pathways,” Chatterjee says. “What are the regulatory mechanisms that allow cells to navigate these stressful situations? I think targeting those processes seem to be a pathway towards building smarter therapies that can hopefully thwart resistance from the very beginning.”

 
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Killing antibiotic resistant bacteria with viruses

by Ben Chan | Yale | Feb 8, 2019

Since bacteria, such as those that cause gonorrhea or gastrointestinal infections, are becoming increasingly resistant to antibiotics, scientists are searching for outside-the-box ideas to tackle the problem. They may have found in one in bacteriophage: viruses that exclusively infect and kill bacteria.

Bacteriophage (abbreviated “phage”) therapy isn’t new. It was originally used by Felix d’Herelle in the 1910’s to treat Shigella, a cause of dysentery. However, its clinical development all but stopped due to the discovery of antibiotics. The major reason is because antibiotics can kill a wide variety of bacteria, while phage are extremely specific. There were also issues with production, perceived inefficacy, and geopolitical concerns since phage therapy continued to be developed in what was then the Soviet Union.

But today, as our repertoire of effective antibiotics continues to dwindle, scientists are returning to their old friend, the bacteriophage. Are phage a potential solution to the global antibiotic resistance problem? Yes, quite possibly.

The single biggest benefit of phage therapy is that, unlike antibiotics which are just chemicals, phage are viruses. Though biologists disagree if viruses are technically “alive,” what is not doubted is that viruses can evolve. That means, in theory, if bacteria become resistant to phage, the phage will naturally evolve with the bacteria. How?

Bacteria and phage have been in an evolutionary arms race for billions of years. Phage cannot reproduce unless they have infected a bacterial cell. Many infections begin when a phage particle binds a bacterium and injects its own DNA into the cell, which essentially hijacks it. Under the command of enemy instructions, the bacterium turns into a virus-making factory, after which it explodes and dies.

Bacteria can become resistant to viruses in a variety of ways. One common mechanism is to alter the cell surface so that the phage can no longer bind. But the viruses are constantly changing, too. There’s a very good chance that if a mutant, phage-resistant bacterium evolves, a mutant virus that can still bind the bacterium also will evolve. In other words, as the bacteria try to evade the virus, the virus will keep trying new ways to infect the bacteria. Antibiotics cannot do this.

Another strategy to avoid the development of resistance is to use a phage “cocktail” – that is, a mixture of phage that each target a bacterium in different ways. (Similarly, we treat HIV infections by giving patients a “cocktail” of drugs that target the virus in various ways.) This strategy makes it difficult for bacteria to evolve resistance in the first place.

Another benefit of phage therapy is that prescribing the correct “dose” may not be much of an issue. With antibiotics, the correct dose is key. Too much, and the patient can suffer side effects; too little, and the bacteria survive and become antibiotic-resistant. But viruses can essentially dose themselves. If there are bacteria present, the viruses will infect them and multiply. If the bacteria are gone, the viruses soon will be gone from the body, too.




So why isn’t phage therapy conveniently available at any hospital or pharmacy? The short answer is that bacteriophage therapy isn’t approved by the FDA… yet. But clinical trials are now underway.

Phage exposure is believed to be safe. Just like bacteria, phage particles are absolutely everywhere. It is estimated that there are 1031 (ten million trillion trillion) phage particles on Earth, ten times more than the number of bacteria. The FDA, in fact, has given a product that consists of phage that kill Salmonella the “Generally Regarded as Safe” label.

It is easy to imagine that, in the not-too-distant future, we will greatly reduce our reliance on broad spectrum antibiotics and focus on personalized infection management. With the help of rapid DNA sequencing and Big Data, it may be possible to administer precision phage cocktails to treat bacterial infections.

 
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Diabetic foot, a severe complication of diabetes with potential development of gangrene, foot loss, and disablement, is experimentally treated in a Novosibirsk clinic. Bacterial infection is one of the factors underlying this pathology. Phage therapy comprises the following stages: swabbing the affected tissues to isolate the pathogenic bacterium; selecting the bacteriophage that can lyse the target bacterium from a phage collection; and applying the bacteriophage preparation (on a sterile pad) to the wound. The treatment takes about a week.

The truth about Phage Therapy

by Nikolay Dobretsov | SCIENCE First Hand

Bacteriophages are not just common drugs. They are neither simple chemicals, like antibiotics and most other medicines, nor true living organisms since they, like all the other viruses, are able to reproduce only in the cell of their host. Actually, bacteriophages are nano-automatons with their own genetic program, which can penetrate into a bacterial cell and reproduce there causing its destruction.

Therefore, standard pharmacological approaches to bacteriophages are not always satisfactory. Although phage preparations are now produced and applied in medicine, our knowledge about the diversity of these viruses, about mechanisms of their interaction with bacteria, and about competition with their cognates is still insufficient to use their full therapeutic potential.

Safe and efficient

Phage therapy emerged almost immediately after bacteriophages were discovered; however, expanded trials of these antibacterial tools were launched in the Soviet Union only in the late 1930s. The trials proved the efficiency of bacteriophages as preventive measures against dysentery and cholera epidemics, and using them for healing wounds and curing pyoinflammatory diseases demonstrated their potential as an alternative to antibiotics.

However, the results achieved at that time were often contradictory: in some cases the phages immediately inhibited the progression of infection but sometimes they were of no use. The specialists grasped the reason right away: the treatment was successful only when they used phages that were able to infect the particular bacterial strain that had caused the target infection. Thus, it was necessary to isolate the pathogen that caused the epidemic, assay the available phages for their ability to inhibit this agent, and produce the most efficient bacteriophage as a drug.

Unfortunately, the results of the studies performed in the Soviet Union were not properly documented and described in scientific literature; moreover, they were conducted not in compliance with the currently recognized protocols for clinical trials. Nonetheless, the major results of that work were undoubted: phages demonstrated their safety and high efficiency in real situations. Since then they have been used in clinical practice in this country along with common therapeutic tools.




Come to know fecal microbiota transplantation

A common aftereffect of antibiotic therapy is rapid propagation of an aggressive bacterium, Clostridium difficile, which causes severe diarrhea and resistance to drugs. This is a very serious problem: not long ago it caused about a thousand lethal outcomes in the United States annually. A very simple means of curing diarrhea was found quite recently: the fecal microflora of a healthy donor is administered to the patient’s intestine. The recovery is almost immediate, literally on the following day. Evidently, the “transplantation” of feces gives the patient a complete set of “proper” microorganisms that had been killed by antibiotics and bacteriophages that control the abundance of pathogenic strains.

Initially, the FDA restrained the spread of this approach, trying to apply the regularity rules approved for ordinary drugs. However, the protests of both therapists and patients came into play, and the method was approved with common precautions, i. e. selection of healthy donors and performance of the procedure by specialists and in healthcare facilities. The method has recently become widespread in the United States and shows good results. It is likely that only the prejudice of physicians still hinders the use of fecal microbiota transplantation in several European countries; in Russia, this treatment is available only at the Center for New Medical Technologies in Novosibirsk Akademgorodok.

It is believed that the overwhelming majority of microorganisms in their natural environment (flow conditions) exist at the interface of two media as “biofilms,” a sort of “colonies” with a specific spatial and metabolic structure. The bacterial cells in such “microbial cities” are submerged into an extracellular mucous matrix formed of polymeric substances secreted by cells. The biofilm can be attached to the surface of either inanimate objects (for example, calculi, catheters, or joint implants) or to a part of an animate object (intestinal walls, teeth, or skin). This mode of existence provides many advantages for microorganisms; in particular, the biofilm microflora, owing to the mucous matrix, is much less affected by various adverse factors, such as ultraviolet radiation, dehydration, or antibiotics. The matrix also protects the bacteria against the attacks of bacteriophages and host immune cells.

With the advent of antibiotics, western countries lost interest in phages; however, the emergence of antibiotic-resistant bacterial strains made several countries begin to elaborate phage preparations and conduct clinical trials, which in fact were the same as the ones that had been performed in the Soviet Union. The new results confirmed the safety of bacteriophage preparations, which was confirmed by the Food and Drug Administration (FDA).

In the United Kingdom, experiments on treating chronic otitis caused by have proved to be successful. Under the Phagoburn project, seven medical centers in France, Belgium, and Switzerland are involved in the clinical trials of a phage cocktail for preventing infections in burn injuries. Several United States companies (Intralytix, Enbiotix, and AmpliPhi) report testing their original phage cocktails for a wide range of diseases, though none of these large-scale clinical trials has been completed yet.

What is a “medicinal bacteriophage?”

In Russia, bacteriophage preparations are available in pharmacies, but unlike other drugs with their precise chemical formula and concentration of the components, a bacteriophage preparation is a nonstandard solution containing live virus particles. Preparations that have the same name but were manufactured at different facilities or at different times may differ in their composition and/or ratio of phages.

All the differences are determined by the specificity of the phage selection procedure and their production. Bacteriophages are selected according to their ability to lyse an individual bacterial isolate; then a mixture of phages is grown on a specified bacterial culture, and goes into production, i. e. bacteriophages are grown in voluminous reactors (fermenters) with the help of bacterial strains.

As a result, a drug that can kill the necessary bacterial strain is created. For example, the Pseudomonas aeruginosa bacteriophage contains the phages that kill P. aeruginosa, but the physician does not know either the number of phages in the preparation or what phages it contains, what P. aeruginosa strains it can kill, and whether it is appropriate for a particular patient. The preparation will have an excellent effect if the patient is infected with the same bacterial strain as was used for phage production; otherwise, the only hope is that since the phage cocktail contains many components, one of the bacteriophages may be specific to the target pathogen.

Thus, it does not pay to buy a bacteriophage in a drugstore for self-treatment. It is up to the doctor to prescribe the treatment and drugs. The range of diseases susceptible to bacteriophage therapy is wide, including trophic ulcers, burn and wound infections, as well as various infections of respiratory, urogenital, gastrointestinal organs, and bones. In these cases, the causative agents are notorious bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, pathogenic Escherichia coli strains, salmonellas, Proteus, and streptococci, including their drug-resistant variants. In fact, it is possible to find naturally occurring bacteriophages against any bacteria, including those that cause plague and anthrax. Bacteriophages can also be used to prevent communicable bacterial diseases; for example, they were successfully used in kindergartens to prevent a dysentery epidemic.

Bacteriophage preparations are administered either locally, to the lesion, or orally. Advertisements allege that phages can spread within the human body and pass from the stomach to bloodstream; however, there is no clear and unambiguous scientific proof yet. Note that a bacteriophage preparation may contain most different bacterial viruses with most diverse fates in the human body.

In certain situations, it is difficult for bacteriophages to hit their “victims.” For example, tuberculosis bacteria reside within the body cells where phages cannot get, while some bacteria form biofilms which are impenetrable for both phages and antibiotics. Then, to destroy the biofilms it is necessary to use enzymes synthesized by specially constructed phages.

In an infected organism, phages reproduce until the majority of sensitive bacterial cells are killed. The patient in whose body bacteriophages fight with bacteria will finally get well when his/her immune system starts to work in full force, and will further protect the body for a long time independently of whether the pathogen is present or not.

Besides, thanks to their specificity, bacteriophages do not kill “good” microorganisms, i.e. unlike antibiotics, they do not damage the human microbiome. It is now known that the intestinal flora disturbance may cause severe consequences, namely certain problems with the gastrointestinal tract, various allergies, and functional impairments of the central nervous system. Another advantage is that bacteriophages do not interfere with the effects of other therapeutics, and are not influenced by them either.
An individual cocktail for everyone

Why have bacteriophages failed to become the main tool for controlling infections, and there are frequent complaints of unsuccessful treatment in the social networks? This is explained in part by the administering of inadequate preparations. A decade ago, numerous “healthcare facilities” advertised stem cell preparations as drugs to cure any disease; now, extracts of “bacteria isolated from permafrost” and “preparations involving bacteriophages” (with no bacteriophages detectable) are intensively advertised. When buying a preparation, you should be sure that it was produced by a reliable manufacturer.

The main cause of treatment failures is an awkward selection of phages for individual patients. Each particular phage is efficient against one or a few bacterial strains, while an infection similar in its appearance, for example, angina, can be caused in individual patients by different streptococcal strains. To cure a patient, it is necessary to isolate the culture of the pathogen and test it for sensitivity to different phages, i.e. bacteriophage therapy should follow the pattern of personalized medicine. Alas, current medicine is not ready to do this yet.




On the way to personalized phage therapy

In the Novosibirsk Science Center, the consortium of the Institute of Chemical Biology and Fundamental Medicine (Russian Academy of Sciences) and Center for New Medical Technologies is involved in developing the technologies for personalized bacteriophage therapy; this is done in collaboration with a team of clinicians from the Tsiv’yan Novosibirsk Institute of Traumatology and the Railroad Clinical Hospital.

The large collection of bacteriophages at the Institute of Chemical Biology and Fundamental Medicine contains unique strains that can fight the newly emerged but already widespread agents of hospital infections, such as the gram-negative bacteria Acinetobacter baumanii and Stenotrophomonas maltophilia. The phages that can lyse a broader range of bacterial strains, including drug-resistant variants, more efficiently than the bacteriophages available as commercial preparations have already been isolated and characterized. This set includes the bacteriophages of the Pseudomonas aureginosa, Proteus, Staphylococcus aureus, S. epidermidis, Klebsiella, and pathogenic Enterococcus. Genome sequencing of the most promising bacteriophages has allowed the researchers to find strains that differ from the known ones.

Clinicians participated in accumulating the expertise to use bacteriophages for treating infections that accompany the diabetic foot syndrome, osteomyelitis, and surgical wound infections. A few cases of curing respiratory infections caused by drug-resistant pathogens and urogenital diseases have been reported. For example, a 6-month-old girl with a congenital laryngeal abnormality was completely cured of severe tracheobronchitis, which had developed after tracheotomy, by inhalations of the Pseudomonas aureginosa bacteriophage. The pathogen that had caused her disease was identified as the Pseudomonas aureginosa strain, resistant to almost all antibiotics approved for infants. The girl received the bacteriophage twice a day for a week; after the pathogen and infection disappeared, the tracheotomy tube was removed; now the girl is quite well.

As for treating chronic drug-resistant urinary infections, it has been found that the bacteriophage should be administered directly onto the bladder. This cured a patient with post-surgery chronic cystitis caused by a “bouquet” of antibiotic-resistant enterobacteria. A set of specific bacteriophages had been administered for 10 days on a daily basis; the urine tests after the treatment showed no pathogenic flora

The experience of the Soviet Union, Georgia, and Poland has shown that a successful use of bacteriophages requires not only a clinical facility, but also a laboratory production site with a collection of phages and the staff skilled in identifying bacteria as well as in selecting and isolating bacteriophages for individual patients.

The question here is whether large-scale production of bacteriophage preparations is reasonable. The answer is yes, because the problem of narrow specialization of bacteriophages is solved in part by making phage cocktails containing several (sometimes, several tens of) different phages that can hit different target pathogen strains. Evidently, it is easier and faster to select the necessary phage cocktail for an individual rather than to test many individual phages from a large collection.

For all that, bacteriophages are not likely to replace antibiotics completely: these preparations are complementary and applicable to different situations. When a patient is seriously ill, with a good reason to suspect a bacterial infection, there is no time for experiments in selecting the proper phage preparation. The only satisfactory solution then is a broad-spectrum antibiotic.

The Russian Federation has currently the largest-scale production of bacteriophages. Mikrogen, a research and production facility, and the world leader in this area, manufactures a wide range of phage preparations. Therapeutic bacteriophages are also produced in Georgia, at the Eliava International Phagotherapy Center, which comprises both production facilities and a clinic with a vast collection of bacteriophages. European and the United States clinics, where phage therapy has not been officially approved yet, cooperate with this center under a medical tourism program. The Phage Therapy Unit with the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences produces bacteriophage preparations for experimental clinical application when treating patients who are non-sensitive to antibiotics

However, bacteriophage therapy is preferable when you deal with a chronic infection or with a disease caused by multi-drug resistant bacteria. In the case of chronic illnesses, such as otitis, the physician has enough time to administer a phage cocktail or to select a specific phage. Another example is a post-surgery infection with an antibiotic-resistant bacterial strain, which causes rapid deterioration of the patient’s state; here phage therapy can be the only option.

Wide experience in the clinical use of bacteriophages acquired over the last 100 years demonstrates the promising future of phage medical technologies. Further efforts of the experts working in this area, in combination with synthetic biology tools, will certainly create preparations with incomparably higher efficiency than that of the currently available phage cocktails.

However, several factors unrelated to science hinder the advances in designing and producing “medicinal” bacteriophages. The fact is that bacterial viruses are very easily reproduced, which offers exciting possibilities for their counterfeiting, thereby infringing on the rights of bonafide manufacturers. The requirements for phages as therapeutics have not been established yet either. It is only clear that they should be different from the requirements for synthetic drugs. Bacteriophage genomes are diverse; so, if a personalized approach is used, they should be selected individually.

Still, biotechnologists as well as researchers and physicians hope that these safe and efficient preparations will take their rightful place in the toolkit of therapies for infectious diseases.

 
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Phage therapy saves patient with drug-resistant bacterial infection*

Science Daily | May 8, 2019

The patient, a 15-year-old girl, had come to London's Great Ormond Street Hospital for a double lung transplant.

It was the summer of 2017, and her lungs were struggling to reach even a third of their normal function. She had cystic fibrosis, a genetic disease that clogs lungs with mucus, and plagues patients with persistent infections. For eight years, she'd been taking antibiotics to control two stubborn bacterial strains.

Weeks after the transplant, doctors noticed redness at the site of her surgical wound and signs of infection in her liver. Then, they saw nodules -- pockets of bacteria pushing up through the skin -- on her arms, legs, and buttocks. The girl's infection had spread, and traditional antibiotics were no longer working.

Now, a new personalized treatment is helping the girl heal. The treatment relies on genetically engineering bacteriophages, viruses that can infect and kill bacteria. Over the next six months, nearly all of the girl's skin nodules disappeared, her surgical wound began closing, and her liver function improved, scientists report May 8, 2019, in the journal Nature Medicine.

"The work is the first to demonstrate the safe and effective use of engineered bacteriophages in a human patient," says Graham Hatfull, a Howard Hughes Medical Institute (HHMI) Professor at the University of Pittsburgh. "Such a treatment could offer a personalized approach to countering drug-resistant bacteria. It could even potentially be used more broadly for controlling diseases like tuberculosis."

"The idea is to use bacteriophages as antibiotics -- as something we could use to kill bacteria that cause infection,"
Hatfull says.

Phage hunters

In October 2017, Hatfull received the email that set his team on a months-long bacteriophage-finding quest.

A colleague at the London hospital laid out the case: two patients, both teenagers. Both had cystic fibrosis and had received double lung transplants to help restore lung function. Both had been chronically infected with strains of Mycobacterium, relatives of the bacterium that causes tuberculosis.

The infections had settled in years ago and flared up after the transplant. "These bugs didn't respond to antibiotics," Hatfull says. "They're highly drug-resistant strains of bacteria."

But maybe something else could help. Hatfull, a molecular geneticist, had spent over three decades amassing a colossal collection of bacteriophages, or phages, from the environment. Hatfull's colleague asked whether any of these phages could target the patients' strains.

"It was a fanciful idea," Hatfull says, and he was intrigued. His phage collection ¬- the largest in the world -- resided in roughly 15,000 vials and filled the shelves of two six-foot-tall freezers in his lab. They'd been collected from thousands of different locations worldwide -- largely by students.

Hatfull leads an HHMI program called SEA-PHAGES that offers college freshmen and sophomores the opportunity to hunt for phages. In 2018, nearly 120 universities and colleges and 4,500 students nationwide participated in the program, which has involved more than 20,000 students in the past decade.

There are more than a nonillion (that's a quadrillion times a quadrillion) phages in the dirt, water, and air. After testing samples to find a phage, students study it. They'll see what it looks like under an electron microscope, sequence its genome, test how well it infects and kills bacteria, and figure out where it fits on the phage family tree.

"This program engages beginning students in real science," says David Asai, HHMI's senior director for science education and director of the SEA-PHAGES program. "Whatever they discover is new information." That basic biological info is valuable, he says. "Now the phage collection has actually contributed to helping a patient."

That wasn't the program's original intent, Asai and Hatfull say. "I had a sense that this collection was enormously powerful for addressing all sorts of questions in biology," Hatfull says. "But we didn't think we'd ever get to a point of using these phages therapeutically."

Experimental therapy

The idea of phage therapy has been around for nearly a century. But until recently, there wasn't much data about the treatment's safety and efficacy. In 2017, doctors in San Diego, California, successfully used phages to treat a patient with a multidrug-resistant bacterium. "That case, and the rise of antibiotic resistance, has fueled interest in phages," Hatfull says.

Less than a month after he heard about the two infected patients in London, he received samples of their bacterial strains. His team searched their collection for phages that could potentially target the bacteria.

They tested individual phages known to infect bacterial relatives of the patients' strains, and mixed thousands of other phages together and tested the lot. They were looking for something that could clear the whitish film of bacteria growing on plastic dishes in the lab. If a phage could do that, the team reasoned, it might able to fight the patients' infections.

In late January, the team found a winner -- a phage that could hit the strain that infected one of the teenagers. "But they were too late," Hatfull says. "The patient had died earlier that month. These really are severe, life-threating infections," he says.

His team had a few leads for the second patient, though: three phages, named Muddy, ZoeJ, and BPs. Muddy could infect and kill the girl's bacteria, but ZoeJ and BPs weren't quite so efficient. So Hatfull and his colleagues tweaked the two phages' genomes to turn them into bacteria killers. They removed a gene that lets the phages reproduce harmlessly within a bacterial cell. Without the gene, the phages reproduce and burst from the cell, destroying it. Then they combined the trio into a phage cocktail, purified it, and tested it for safety.

In June 2018, doctors administered the cocktail to the patient via an IV twice daily with a billion phage particles in every dose. After six weeks, a liver scan revealed that the infection had essentially disappeared. Today, only one or two of the girl's skin nodules remain. Hatfull has high hopes: "The bacteria haven't shown any signs of developing resistance to the phages, and his team has prepped a fourth phage to add to the mix."

"Finding the right phage(s) for each patient is a big challenge,"
Hatfull says. "One day, scientists may be able to concoct a phage cocktail that works more broadly to treat diseases, like the Pseudomonas infections, that threaten burn patients."

"We're sort of in uncharted territory,"
he says, "but the basics of the young woman's case are pretty simple," he adds. "We purified the phages, we gave them to the patient, and the patient got better."

*From the article here:

 
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Phages Therapy saves man’s infected leg from amputation

by Ran Nir-Paz | Healio | April 16, 2019

A hospital in Israel has used bacteriophages and antibiotics to successfully treat a multidrug-resistant left tibial infection in a man who was injured in an auto accident, saving his leg from amputation, researchers said.

The bone was infected with two bacterial species — extensively drug-resistant (XDR) Acinetobacter baumannii and multidrug-resistant (MDR) Klebsiella pneumoniae, according to findings published in Clinical Infectious Diseases. After treating the infection, researchers noted that they were able to develop a phage-resistant A. baumannii mutant in vitro, and then isolate a new phage to combat it, “showing the potential flexibility of phage treatments.”

Ran Nir-Paz, MD , infectious disease specialist at Hadassah-Hebrew Medical Center, and Ronen Hazan, head of the phage therapy lab at Hebrew University, said the case contributes to the expanding knowledge of phage therapy in several ways.

“First, we succeeded to treat an infection with two bacteria in which we could show in the lab that they probably protect each other from the antibiotics. Second, we showed by simulation in the lab that if a resistant mutant would emerge, we would be able to treat it. Third, this work is a rare example of voluntary collaboration between clinicians,” they told Infectious Disease News.

“This was the first treatment in Israel, which we hope will lead to the establishment of an advance center of personal, adaptive and dynamic research and treatments of infectious diseases medicine.”

The study focused on a 42-year-old man admitted to the trauma unit of Hadassah-Hebrew University Medical Center after a motor vehicle accident with a left bicondylar tibial plateau fracture with compartment syndrome and a right distal femoral fracture, the authors explained.

Six weeks after admission, physicians discovered the left tibia infection with XDR A. baumannii and MDR K. pneumoniae. According to the study, the wound was treated with serial irrigation, debridement, the placement of a cement spacer due to bone loss, and 6 weeks of piperacillin/tazobactam, followed by an 8-week course of meropenem and high-dose colistin, all of which failed to clear the infection.

After the patient refused an above-the-knee amputation 7 months into his hospital stay, doctors turned to a combination of bacteriophage therapy targeting the two bacterial strains, plus IV meropenem and colistin.

Within days, there were signs of wound recovery, graft healing and elimination of subtle chronic bone pain in the patient’s leg, the authors reported. During an 8-month post-treatment follow-up, no positive cultures for either A. baumannii or K. pneumoniae were found. The wound closed, the patient’s pain subsided and blood, stool, urine and saliva samples were all clear of bacterial isolates, they reported.

“The main take-home lesson is that with the right tailored phage therapy combined with antibiotics it is, most probably, possible to treat any pathogen or even infection of multiple pathogens,” Nir-Paz and Hazan said. “Moreover, dynamic monitoring and adaptation of the treatment will promise success, even in cases of emergence of resistant bacteria.” – Caitlyn Stulpin

 
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Emily Monosson, environmental toxicologist

Bacteriophages to the rescue

Emily Monosson | The Scientist | Jul 17, 2017

This year I gave a presentation to public-health students at a university about options for controlling pests and pathogens that didn’t depend on industrial-age chemicals such as antibiotics and pesticides. When I asked if they’d ever heard of phage therapy—the use of bacteria-attacking viruses to fight infection—I was met with blank stares. When I finished sharing stories of desperate patients miraculously cured of antibiotic-resistant infections within days, I sensed a bit of skepticism, as if the crowd’s politeness was keeping them from asking: “If it’s so effective, how come we’ve never heard of this?” In this age of alternate truths and quack cures, it’s an appropriate question.

But phage therapy is nothing new, nor is it some fringe remedy. It was first used to cure Shigella infections early in the 20th century, to miraculous effect (although at the time, scientists were unaware of the nature of viruses). Once treated with phages isolated from fecal samples of spontaneously recovering dysentery sufferers, patients’ Shigella-induced fevers and bloody stools subsided within 24 hours. Within a decade, pharmaceutical companies on both sides of the Atlantic began developing various phage therapies. But then came antibiotics. And poor production practices by some pharmaceutical companies led to a couple of damning reviews of phage therapy in the Journal of the American Medical Association. All of this helped to close the door on phage therapy in Western medicine. The Cold War kept that door closed for decades to come.

Meanwhile, Russia, France, and Poland continued refining the therapy. Noticing the bacterial propensity to evolve resistance under pressure from killer viruses, researchers understood that they could capitalize on the even greater capacity of viruses for rapid evolution, updating phage cocktails as newly resistant bacterial strains emerged. The Phage Therapy Center in Tbilisi, Georgia, currently offers phage treatments.

Despite encountering skepticism about the effectiveness of phage therapy, I have also been asked by students desperate to find a cure for themselves, or a loved one, to recommend phage-friendly doctors here in the U.S. Other than suggesting that they ask their physician to look into phage therapies or do a Google search, I have had little to offer. But there are glimmers of hope. Now, Western scientists and physicians are trying to introduce the therapy into the American pharmacopeia. In July 2015, the National Institutes of Health organized a meeting of international bacteriophage scientists, entrepreneurs, and regulators hailing from the United States, France, Georgia, China, and elsewhere. Another workshop will be held this July in Rockville, Maryland. There are now clinical trials of phage-therapy products underway in the U.S. and in Europe. If successful, developers will soon be knocking on the FDA’s door.

Phage therapy is just one example of a disease-control approach that is more in tune with nature, whether we are concerned about protecting our kids or a field of strawberries. In my new book Natural Defense: Enlisting Bugs and Germs to Protect Our Food and Health, I explore a range of strategies that can help us reduce our dependence on chemicals, from antibiotics to pesticides. The control of microbial infections in humans, in particular, shares many characteristics with agricultural strategies. Rapid, more-accurate diagnostics will help both in the hospital and on the farm: new technological solutions promise to enable rapid disease detection and identification. Prevention can help protect us and the crops we grow. And sometimes the solutions in field and body are the same—phages are useful allies against bacteria in the food industry and in human medicine. As I write in the preface, these are just a few strategies. Some may work, others may not, but such combined efforts can help to reduce our dependence on 20th-century chemical cures. For too long, we have considered ourselves separate from the environment. But the sooner we begin working with, rather than against, nature for our food and health, the better off we will be.

Emily Monosson is an environmental toxicologist. She is an independent scholar at the Ronin Institute and an adjunct professor at the University of Massachusetts, Amherst.

 
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What is Phage Therapy?

Phage Therapy Center

Bacteriophages or "phage" are viruses that invade bacterial cells and, in the case of lytic phages, disrupt bacterial metabolism and cause the bacterium to lyse [destruct]. Phage Therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections.

Bacterial host specificity

The bacterial host range of phage is generally narrower than that found in the antibiotics that have been selected for clinical applications. Most phage are specific for one species of bacteria and many are only able to lyse specific strains within a species. This limited host range can be advantageous, in principle, as phage therapy results in less harm to the normal body flora and ecology than commonly used antibiotics, which often disrupt the normal gastrointestinal flora and result in opportunistic secondary infections by organisms such as Clostridium difficile. The potential clinical disadvantages associated with the narrow host range of most phage strains is addressed through the development of a large collection of well-characterized phage for a broad range of pathogens, and methods to rapidly determine which of the phage strains in the collection will be effective for any given infection.

Advantages over antibiotics

Phage therapy can be very effective in certain conditions and has some unique advantages over antibiotics. Bacteria also develop resistance to phages, but it is incomparably easier to develop new phage than new antibiotic. A few weeks versus years are needed to obtain new phage for new strain of resistant bacteria. As bacteria evolve resistance, the relevant phages naturally evolve alongside. When super bacterium appears, the super phage already attacks it. We just need to derive it from the same environment. Phages have special advantage for localized use, because they penetrate deeper as long as the infection is present, rather than decrease rapidly in concentration below the surface like antibiotics. The phages stop reproducing once as the specific bacteria they target are destroyed. Phages do not develop secondary resistance, which is quite often in antibiotics. With the increasing incidence of antibiotic resistant bacteria and a deficit in the development of new classes of antibiotics to counteract them, there is a need to apply phages in a range of infections.

Lytic phages are similar to antibiotics in that they have remarkable antibacterial activity. However, therapeutic phages have some advantages over antibiotics, and phages have been reported to be more effective than antibiotics in treating certain infections in humans and experimentally infected animals. For example, in one study, Staphylococcus aureus phages were used to treat patients having purulent disease of the lungs and pleura. The patients were divided into two groups; the patients in group A (223 individuals) received phages, and the patients in group B (117 individuals) received antibiotics. Also, this clinical trial is one of the few studies using i.v. phage administration (48 patients in group A received phages by i.v. injection). The results were evaluated based on the following criteria: general condition of the patients, X-ray examination, reduction of purulence, and microbiological analysis of blood and sputum. No side effects were observed in any of the patients, including those who received phages intravenously. Overall, complete recovery was observed in 82 percent of the patients in the phage-treated group as opposed to 64 percent of the patients in the antibiotic-treated group. Interestingly, the percent recovery in the group receiving phages intravenously was even higher (95 percent) than the 82 percent recovery rate observed with all 223 phage-treated patients.

 
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Phage Therapy treats deadly bacteria

by Ashley Yeager | May 8, 2019

Tweaking the genomes of two phages and combining them with a third phage helped to clear a persistent Mycobacterium infection in the patient.

Genetically modified viruses have successfully treated an antibiotic-resistant infection in a teenage girl, saving her life, researchers report today. While this approach using engineered phages—viruses that infect bacteria—has only been tested in one person, the technique could be developed to battle other persistent, “superbug” infections.

“This is actually a historic moment,” Steffanie Strathdee, a professor of medicine at the University of California, San Diego, who was not involved in the study, tells NPR. “This is the first time that a genetically engineered phage has been used to successfully treat a superbug infection in a human being.” Strathdee says. “It’s terribly exciting.”

In the study, a teenager from England was suffering from an infection with a strain of Mycobacterium, a relative of the bacterium that causes tuberculosis. The girl had cystic fibrosis (CF) and had been on antibiotics to control bacterial infections that cause complications related to the genetic condition. She then endured a double lung transplant to treat the disease. Afterward, one of the infections spread throughout her body, and there weren’t any antibiotics that could treat it. At that point, Graham Hatfull, a professor of biotechnology at the University of Pittsburgh, got involved. He’d been working with phages, collecting them from the environment. He started to analyze his collection to see if any could kill the bacteria causing the patient’s infection.

“The idea is to use bacteriophages as antibiotics—as something we could use to kill bacteria that cause infection,” Hatfull says in a Howard Hughes Medical Institute press release. His team identified three phages that appeared promising. One could infect and kill the bacterial strain making the patient sick. The other two weren’t as effective, so Hatfull and his colleagues tweaked the phages’ genomes, removing a gene, so the phages would kill the bacteria.

The team concocted a phage cocktail with all three viruses and tested it for safety on the patient’s skin. The cocktail was then given to the teen via IV. Several weeks later, a scan of her liver showed a significant drop in signs of infection, and it didn’t seem that the bacterial strain causing the infection was developing a resistance to the phage cocktail. Hatfull and colleagues are now testing a fourth phage to add to the treatment and looking for ways to apply the technique to other pathogenic bacteria.

“I still have reservations about whether this kind of approach could be developed into something that could be usable on a large scale,” Marcia Goldberg, an infectious disease specialist at Massachusetts General Hospital, tells STAT. “The amount of science that needs to go into developing a therapeutic against any single strain is huge.”

 
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