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News Phage therapy

mr peabody

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Genetically modified viral cocktail treats deadly bacteria in teen

by Ashley Yeager | The Scientist | 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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mr peabody

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The business of antibiotic resistance: An unprofitable venture

by Louis Ngai | IMPRESS | Aug 19 2019

By the year 2050, it is predicted that antibiotic microbial resistance (AMR) will kill 10 million people every year. A major driver being the unhindered use of broad-spectrum antibiotics in produce, animals and their byproducts, and in clinical settings. Soon, a future where AMR is the norm, rather than the exception, is not a question of if but when. Like the rise of climate change, alarms have been raised long ago about the dangers should rampant antibiotic use be left unchecked. Critical medical procedures such as organ transplantations, caesarian sections, cancer chemotherapy and other invasive surgeries that rely on effective antibiotics may soon become more infrequent due to fear of secondary infections. While organizations like the Canadian Antimicrobial Resistance Surveillance System (CARSS) are in place to monitor AMR support policy and programs, on a global scale, the actions being taken are but a drop in the ocean.

AMR is by no means a new phenomenon, though only recently have we started to encounter on such a widescale. In fact, microbes isolated from 30,000-year old permafrost were identified to already harbor antibacterial resistant genes. This shouldn’t come as a surprise given that these organisms have had to co-exist with fungi and plants, which naturally produce antibiotics to avoid the toxic by-products produced by bacteria. The widespread and uncontrolled use of these drugs that came with the golden age of antibiotic discovery has accelerated the rate at which microbes develop resistance, selecting for populations that are in turn, able to further propagate these resistance genes across various communities.

Governments and organizations have begun taking action through the implementation of legislation, research funds, and improvements in regulations in order to develop additional antibiotics while curbing current antibiotic use. However, despite all these efforts to improve our current drug discovery landscape, antibiotics are not a profitable venture compared to the ten top-selling drugs in 2018. For example, Stelara, indicated for Crohn’s disease and psoriasis, is on the low end, earning a profit of $5.7 billion USD in 2018. Humira, indicated for arthritis and a myriad of other inflammatory conditions, topped the chart with a net profit of $19.9 billion USD, rounding out the top ten at an average of $8.24 billion USD in profits. By comparison, recent antibiotics sales averaged less than $50 million within a two-year period. From the standpoint of a company that answers to their shareholders, clearly antibiotics aren’t profitable. It should be no surprise then that since the 1980s, the number of drug companies with antibiotic discovery programs has fallen from 25 to just three, leaving only: Pfizer, Merck Sharp & Dohme (MSD), and GlaxoSmithKline (GSK).

Instead, small biotech companies have begun to take over development of antibiotics. Despite their efforts, the commercialization of new antibiotics has been met with difficulty. Take for example Achaogen, which launched the antibiotic plazomicin in 2018 and within 8 months, filed for bankruptcy on April 15, 2019. This situation is not unique to Achaogen. From a large pharmaceutical company’s standpoint, compared to long-term drugs that are used to treat diabetes or asthma, the short-term prescription of antibiotics leaves much to be desired in terms of financial return. On top of that, the ability of bacteria to quickly adapt to the drug means companies must spend more on R&D in order to keep up with the evolutionary arms race.

While small companies are able to receive support from government agencies like BARDA (the Biomedical Advanced Research and Development Authority) and CARB-X (Combating Antibiotic resistant Bacteria Biopharmaceutical Accelerator), they still lack the financial resources to get drug candidates through late-stage clinical development – a necessary step towards market release. In total, a small company will need at least $1 billion USD and approximately 10 years to push their drug for commercial use, a task that is challenging if not impossible without support from larger organizations and funding sources.

Like climate change, the business of AMR is the business of the world. Though efforts in some countries like Canada and the UK are pushing towards better antibiotic practices, in other nations like China, India, and the US, the rampant use of antibiotics is done in the name of short-term profit. In the coming years, more people could die from microbial infections than from cancer. In the coming years, large scale reforms will be needed in order to revitalize the antibiotic market and the drug market in general — not an easy task for a world obsessed with financial profits.

 
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Thomas Patterson and his wife with pictures of the superbug that nearly killed him, and the phage that saved his life.

Phages have evolved to become perfect predators of bacteria*

by Zoe Corbyn | The Guardian | 15 Jun 2019

In 2015, the scientist’s husband was almost killed by an antibiotic-resistant superbug, until she found a cure that is now saving others.

Infectious disease epidemiologist Steffanie Strathdee’s husband survived a deadly antibiotic-resistant bacterial infection thanks to her suggestion of using an unconventional cure popular in the former Soviet Union: fighting the bug with a virus. Now the global health expert at the University of California, San Diego, she has, along with her husband, Tom Patterson, who is also a scientist at the institution, written an account of their nine month ordeal – The Perfect Predator: A Scientists’s Race to Save Her Husband from a Deadly Superbug.

What was the superbug your husband got, and how did he contract it?

It is called Acinetobacter baumannii but nicknamed Iraqibacter because many wounded service members who have returned from the Middle East were infected with it. It was once a wimpy bacteria – mundane and harmless – but its superpower is its ability to acquire antibiotic-resistant genes. It now tops the World Health Organization’s list of the 12 mostly deadly superbugs to human health. It has infiltrated clinics and hospitals all around the world and sticks to hospital linens. Tom likely picked it up in Egypt while we were vacationing there in late 2015 – sequencing later showed it was an Egyptian strain. He came down in Egypt with what turned out to be acute pancreatitis, caused by a gallstone blocking his bile duct. He could have got [the superbug] from the Egyptian clinic we got him to. It could also have come from the swirling red dust we were exposed to while exploring pyramids – the pathogen is also found in soil. We really don’t know. But the football-sized cyst that formed in his abdomen as a complication of the gallstone provided a nice little apartment for the superbug to settle in and fester.

What is phage therapy and how does it work?

Phage therapy is the use of bacteriophages – viruses that attack bacteria – to treat infections. There are trillions and trillions of phages on the planet and they have evolved over millennia to become the prefect predators to bacteria. The phage latches on to and enters the bacterial cell where it takes over its machinery and turns it into a phage manufacturing plant. The newly minted phages then burst out and the bacterial cell dies. To work, phages have to be matched to the bacterial infection. But just how close the match needs to be depends on the bacterial species.

Phage therapy has been neglected in the west. Why?

Phages were first discovered in 1917 by a French-Canadian, Félix d’Herelle, but he had a hard time getting his work accepted – he wasn’t formally trained and was considered a vagabond scholar. Then, while phage preparations were being manufactured in the west up until the late 1930s, the scientific method hadn’t really been worked out. They were, for example, not being matched to specific bacteria and they were sold without being purified, so some actually could cause harm. After penicillin came to market in the early 1940s, phage therapy fell out of favour and political reasons kept it there. Former Soviet countries – where penicillin wasn’t so consistently available – had taken up phage therapy very vigorously. Western researchers and companies feared being labelled “pinko commie” sympathisers.

You got Tom out of Egypt and eventually into intensive care at your institution, UCSD. What turned you on to phage therapy and where did you find Tom’s phages?

We were running out of options to save Tom, whose infection had spread throughout his body, so I started exploring unconventional cures. I found a paper that mentioned phage therapy. Although I’d learned of phages in college, this was the first I had heard of using them as a treatment. My colleague Chip Schooley, chief of infectious diseases at the UCSD school of medicine, agreed it was an interesting idea and said if I could find phages that matched Tom’s bacterial infection he would contact the Food and Drug Administration and get approval to use them for compassionate use. I knew what that meant: officially, Tom was dying.

I began cold-emailing phage researchers asking for help. A phage researcher at Texas A&M University turned his lab into a kind of command centre, looking for phages active against Tom’s specific infection in his centre’s phage library and in sewage and barnyard waste – wherever you find a lot of bacteria is where you find the phages that kill them! He also wrote to researchers all over the world asking them to send any phages they had that might work, which they did. But phages for Acinetobacter baumannii are very picky. Tom’s life depended on finding a match in time. In three weeks, which included a week spent on purification, a cocktail of four phages was ready to give to Tom. The navy – who our FDA contact also put us in touch with – produced a second four-phage cocktail from their phage library.

Did it work at once?

We began the intravenous phage therapy, but Tom’s bacteria quickly became resistant to all the phages except one in the navy’s cocktail. We didn’t realise that a lot of the phages across both cocktails were very similar to one another and were all trying to enter the bacteria by the same receptor. The bacteria’s successful attempt at evading one phage therefore offered resistance to the rest. Luckily, looking again for more phages that would match, the navy found another in the murky waters of a sewage treatment plant in Maryland. From bog to bedside, so we say! It was powerful because it hit a different receptor. Coincidentally, we found there was synergy with one of the antibiotics Tom was getting. Tom woke up three days after we began the treatment and he fully cleared his infection within three months.

How did Tom’s case break new medical ground?

He was the first in the US to get intravenous phage therapy for a systemic superbug infection. In the past when phage therapy has been used, it has usually been topically – sprinkled on somebody’s skin – or inhaled with a nebuliser. Going intravenous was really risky. Even in the former Soviet countries, where phage therapy has been used for decades, they don’t often treat intravenously because they don’t have the hi-tech capability to purify their phage of the bacterial debris that accumulates when you prepare them in large quantities, so there’s the risk of septic shock. We think of what Tom received as 21st-century phage therapy.

Since Tom’s case, you have assisted other patients to get phage therapy and you now, in addition to your day job, co-direct a new phage therapy centre at UCSD – the Centre for Innovative Phage Applications and Therapeutics – the first in North America. How many people have you helped to date?

After Tom’s case was publicised, people contacted me from all over the world wanting phage therapy. Strangers had helped us, and I felt I had an obligation to help them. So far, we’ve treated six other patients here at UCSD, and advised on a couple of dozen cases elsewhere across the US and internationally. We haven’t always been successful, because sometimes we have been contacted too late. But the FDA is now making it easier for people to get phage therapy earlier in the course of their infections. What’s needed now are clinical trials to see if it works on a broader scale.

I understand your centre helped in the case reported last month of the British teenager Isabelle Holdaway. She became the first person to be treated successfully with a genetically modified phage for a superbug infection following a double lung transplant.

Her mother had heard about Tom’s case and asked her UK doctor whether he could consider phage therapy. He contacted Schooley, the doctor who ended up treated Tom and now co-directs the centre with me. Isabelle’s infection – Mycobacterium abscessus – is in the same genus as tuberculosis and was fully resistant to antibiotics. She was receiving hospice care. The team asked a researcher with a mycobacterium phage library at the University of Pittsburgh to help, but most of the phages he found that matched were not predatory enough. So they tweaked one genetically – clipping out a gene – to ensure it killed the bacteria rather than going dormant. The phage was administered intravenously, like with Tom, and Isabelle left the hospital in a week. I wept for joy. Isabelle is now finishing her A-levels and learning to drive.

Even if phage therapy is proven to work, is widespread use ever going to be achievable when it is essentially personalised therapy? How does it scale up?

What is needed are readily available phages to match whatever organisms we face in the bacterial world. Instead of having to resort to environmental sources every time – like we did in Tom’s case with the sewage – imagine a large, ever-expanding, open-source phage library that researchers and students contribute to from around the world, which could be accessible globally as a resource. Genetic tweaking is also clearly a way of expanding the range of bacteria that picky phages like Tom’s or Isabelle’s are able to attack.

What about the problem of the bacteria becoming phage-resistant?

Bacterial resistance to phage can be expected, but how quickly it occurs depends on a number of factors. In Tom’s case, it emerged fast. Isabelle has been treated for about a year and resistance hasn’t emerged yet, since her bacteria is slow-growing. And there are a few ways that issue can be overcome, like using a phage cocktail where different phages “hit” different bacterial receptors. In the future, genetically modified or synthetic phages could be developed that are harder for bacterial resistance to overcome.

What’s your advice to people who do find their loved ones hospitalised with superbug infections? Shout for phage therapy?

For any serious illness, you need to be an advocate. Get educated about what’s going on and be actively involved in your loved one’s care. In the case of a superbug, understand what options are left in terms of antibiotics and also what kinds of side effects exist, because some last-resort antibiotics, like colistin, can be very hard on the body. Because phage therapy is experimental, you can’t necessarily get it if there are still antibiotic options available. If there are not, phage therapy may be possible.

*From the article here:

 
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mr peabody

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Phage therapy could beat drug resistant illnesses

BioSpectrum | 1 April 2020

Dr. Ramdas Kambale, Sr.VP, Vetphage Pharmaceuticals shares talks about phage therapy.

In 2008, a superbug caught from a New Delhi hospital claimed the life of a Swedish patient. British scientists who found this "superbug" in New Delhi's public water supply, named it New Delhi Metallo-beta-lactamase-1 after the Indian capital, causing much hue and cry in India. The drug resistant gene including its new variants has since been found in over 100 countries including in one of the last "pristine" places on Earth -- a Norwegian archipelago close to the North Pole! The spread of this superbug that was found to be resistant to all available antibiotics on Earth is a distressing sign.

One of the leading healthcare challenges of our times is the emergence of “superbugs” which are nearly impossible to treat. These ‘superbugs’ or drug-resistant micro-organisms are claiming an increasing number of lives every year. If the superbug threat is not dealt with, the deaths from drug-resistant infections could increase to a whopping 10 million every year by 2050 from around 700,000 currently. In fact, medical researchers have pointed out that the burden of such resistant infections is comparable to that of tuberculosis, HIV/AIDS and influenza put together. This has prompted researchers and medical experts to look for viable alternative treatments and has elicited fresh interest in an age-old intelligence, called phage therapy.

Is Phage Therapy the answer?

Bacteriophages also called phages are bacteria-attacking microorganisms that devour selected bacteria without causing any harm to the host. Phages are all around us on our hands, our eyelids, animal intestines as well as in the soil but they don’t hurt us. They are natural organism made up on only genetic material namely DNA and RNA plus protein. Microbiologist Félix Hubert d’Herelle identified and explained the role bacteriophages can play in treating bacterial infections way back in 1917. He identified phages as virus-like organisms that could kill bacteria without any harmful effects and also coined the term “phage therapy”. However, the discovery of antibiotics put to rest any research or interest in phages. As bacteria evolve and develop resistance to existing antibiotics, the superbug concern has once again ignited research and experiment in phages. In fact, apart from treating bacterial infections, phages can also make our food supply safer.

Phage researchers today also have the technological tools needed to rapidly analyze the genomes of bacteria and phages, and find effective treatment pathways. In 2019, a 62 year old man in Minnesota was told by his doctors that he would have to have his leg amputated after over 10 years of failed treatments including multiple antibiotics and 17 surgeries to cure a stubborn infection. However, in his quest to find potential alternative treatment, led him to an organization that specialized in treating with phages. The man became the 14th person worldwide to be treated with phage therapy and ended up getting rid of his chronic infection.

Phage Therapy in rearing healthy poultry

Poultry is one of the fastest growing segments of the agricultural sector in India today. While the production of agricultural crops has been rising at a rate of 1.5 to 2 percent per annum that of eggs and broilers has been rising at a rate of 8 to 10 percent per annum. As a result, India is now the world's fifth largest egg producer and the eighteenth largest producer of broilers. Nonetheless the bigger question is whether or not they are fit for consumption.

In a recently concluded study it was established that most of the antibiotics being used in the poultry and aquaculture industry for farmed animals are increasingly losing their activity against pathogenic microorganisms. Moreover, the use of antimicrobial agents in animal husbandry has been linked to the development of resistant bacteria. If not kept in check use of antibiotics during poultry production can threaten the safety of such products through microbial residues as well as help spread microbial resistance. This has prompted many countries to withdraw antibiotics from being used in animal production as well as set up regulatory authorities for selected antibiotics as well encourage the use of bacteriophages. This is largely because phages are safe as they are only able to infect bacterial cells not human or animal cells. Without the presence of their bacterial host they become inactive within 48 hours.

Phages when consumed as part of animal feed keep the animals safe from bacterial infections. Moreover, they also do not damage the beneficial micro biome balance in animals. Phage therapy is now emerging as a useful tool in controlling bacterial infections among poultry while also encouraging growth of healthy poultry.

How we identify and use phages

Proteon Pharmaceuticals, one of the pioneering organizations working to introduce phage therapy in animal husbandry, has the most advanced Artificial Intelligence-supported technology to determine whether phages are lytic or not. When dealing with phages it is important that only lytic phages are used in animal health. This is because lysogenic phages are dormant and embed themselves in the bacterial cell wall to live off it without destroying it. On the other hand Lytic phages cause lysis which is destruction of the bacteria.

Protean produces phage-based feed additives for destruction and prevention of bacterial infection in farmed animals. These feed additives when administered prophylactically help prevent infection in poultry and can also therapeutically reduce preexisting infections such as Salmonella. Furthermore, given to poultry mixed with water, it is easy to apply and use. Its results are scientifically verified and based upon well-understood mechanisms of action, meaning that it works reliably across diverse farm environments.

 
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Bacterial predator could help reduce COVID-19 deaths

by Mary Ann Liebert | University of Birmingham | 26 June 2020

A type of virus that preys on bacteria could be harnessed to combat bacterial infections in patients whose immune systems have been weakened by COVID-19, according to an expert at the University of Birmingham and the Cancer Registry of Norway.

Called bacteriophages, the viruses are harmless to humans and can be used to target and eliminate specific bacteria. They are of interest to scientists as a potential alternative to antibiotic treatments.

In a new systematic review, published in the journal Phage: Therapy, Applications and Research, two strategies are proposed, where bacteriophages could be used to treat bacterial infections in some patients with COVID-19.

In the first approach, bacteriophages would be used to target secondary bacterial infections in patients’ respiratory systems. These secondary infections are a possible cause of the high mortality rate, particularly among elderly patients. The aim is to use the bacteriophages to reduce the number of bacteria and limit their spread, giving the patients’ immune systems more time to produce antibodies against COVID-19.

Dr. Marcin Wojewodzic, a Marie Sklodowska-Curie Research Fellow in the School of Biosciences at the University of Birmingham and now researcher at the Cancer Registry of Norway, is the author of the study. He says: “By introducing bacteriophages, it may be possible to buy precious time for the patients’ immune systems and it also offers a different, or complementary strategy to the standard antibiotic therapies.”

Professor Martha R.J. Clokie, a Professor of Microbiology at the University of Leicester and Editor-in-Chief of PHAGE journal explains why this work is important: “In the same way that we are used to the concept of ‘friendly bacteria’ we can harness ‘friendly viruses’ or ‘phages’ to help us target and kill secondary bacterial infections caused by a weakened immune system following viral attack from viruses such as COVID-19.”

Dr. Antal Martinecz, an expert in computational pharmacology at the Arctic University of Norway who advised on the manuscript says: “This is not only a different strategy to the standard antibiotic therapies but, more importantly, it is exciting news relating to the problem of bacterial resistance itself.”

In the second treatment strategy, the researcher suggests that synthetically altered bacteriophages could be used to manufacture antibodies against the SARS-CoV-2 virus which could then be administered to patients via a nasal or oral spray. These bacteriophage-generated antibodies could be produced rapidly and inexpensively using existing technology.

“If this strategy works, it will hopefully buy time to enable a patient to produce their own specific antibodies against the SARS-CoV-2 virus and thus reduce the damage caused by an excessive immunological reaction,” says Dr. Wojewodzic.

Professor Martha R.J. Clokie’s research focuses on the identification and development of bacteriophages that kill pathogens in an effort to develop new antimicrobials: “We could also exploit our knowledge of phages to engineer them to generate novel and inexpensive antibodies to target COVID-19. This clearly written article covers both aspects of phage biology and outlines how we might use these friendly viruses for good purpose.”

Dr. Wojewodzic is calling for clinical trials to test these two approaches.

“This pandemic has shown us the power viruses have to cause harm. However, by using beneficial viruses as an indirect weapon against the SARS-CoV-2 virus and other pathogens, we can harness that power for a positive purpose and use it to save lives. The beauty of nature is that while it can kill us, it can also come to our rescue.” adds Dr Wojewodzic.

“It’s clear that no single intervention will eliminate COVID-19. In order to make progress we need to approach the problem from as many different angles and disciplines as possible.” concludes Dr. Wojewodzic.

Reference: Bacteriophages Could Be a Potential Game Changer in the Trajectory of Coronavirus Disease (COVID-19) by Marcin W. Wojewodzic, 23 June 2020,PHAGE: Therapy, Applications, and Research.

 
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Coronavirus demise could lie with phage nanoparticles

Genetic & Biotechnology News

Given the current global viral pandemic, it wouldn’t be difficult for most to think of viruses in only a negative context. However, over the past several years, a slew of researchers and data has come to light on the benefits of using phage viruses as therapeutic options.

Now, a team of investigators led by scientists at the Leibniz-Forschungsinstitut for Molecular Pharmacology (FMP) and Humboldt University (HU) has found a new use for phage in the fight against seasonal influenza and avian flu. The researchers developed a chemically modified phage capsid that “stifles” influenza viruses. Perfectly fitting binding sites cause influenza viruses to be enveloped by the phage capsids in such a way that it is practically impossible for them to infect lung cells any longer. This phenomenon has been proven in preclinical trials using human lung tissue and is being used for the immediate investigation of coronavirus infections.

Findings from the new study were published recently in Nature Nanotechnology through an article entitled “Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.”

Current antiviral drugs are only partially effective because they attack viruses like influenza and coronavirus after lung cells have been infected. It would be desirable—and much more effective—to prevent infection in the first place. This is exactly what the new approach from the current study promises. The phage capsid, developed by a multidisciplinary team of researchers, envelops flu viruses so perfectly that they can no longer infect cells.

“Preclinical trials show that we are able to render harmless both seasonal influenza viruses and avian flu viruses with our chemically modified phage shell,” explained senior study investigator Christian Hackenberger PhD, head of the department chemical biology at FMP and a professor for chemical biology at HU. “It is a major success that offers entirely new perspectives for the development of innovative antiviral drugs.”

The new inhibitor makes use of a feature that all influenza viruses have: There are trivalent receptors on the surface of the virus, referred to as hemagglutinin protein, that attaches to sugar molecules (sialic acids) on the cell surface of lung tissue. In the case of infection, viruses hook into their victim—in this case, lung cells—like a hook-and-loop fastener. The core principle is that these interactions occur due to multiple bonds, rather than single bonds.

It was the surface structure of flu viruses that inspired the researchers to ask the following initial question more than six years ago: Would it not be possible to develop an inhibitor that binds to trivalent receptors with a perfect fit, simulating the surface of lung tissue cells? The answer to the question lies with Q-beta phage, which has the ideal surface properties and is excellently suited to equip it with ligands—sugar molecules—as “bait.” An empty phage shell does the job perfectly.

“We present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus,” the authors wrote. “Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo, and in vivo.”

Lead study investigator Daniel Lauster, PhD, a former graduate student in the Group of Molecular Biophysics (HU) and now a postdoc at Freie Universität Berlin added that “Our multivalent scaffold molecule is not infectious, and comprises 180 identical proteins that are spaced out exactly as the trivalent receptors of the hemagglutinin on the surface of the virus. It, therefore, has the ideal starting conditions to deceive the influenza virus—or, to be more precise, to attach to it with a perfect spatial fit. In other words, we use a phage virus to disable the influenza virus!”

To enable the Q-beta scaffold to fulfill the desired function, it must first be chemically modified. Produced from E. coli bacteria, the researchers used synthetic chemistry to attach sugar molecules to the defined positions of the virus shell.

Several studies using animal models and cell cultures have proven that the suitably modified spherical structure possesses considerable bond strength and inhibiting potential. The study also enabled the research team to examine the antiviral potential of phage capsids against many current influenza virus strains, and even against avian flu viruses. Its therapeutic potential has even been proven on human lung tissue: when tissue infected with flu viruses was treated with the phage capsid, the influenza viruses were practically no longer able to reproduce.

Additionally, high-resolution cryo-electron microscopy and cryo-electron microscopy show directly and, above all, spatially, that the inhibitor completely encapsulates the virus. Moreover, mathematical-physical models were used to simulate the interaction between influenza viruses and the phage capsid on the computer.

“Our computer-assisted calculations show that the rationally designed inhibitor does indeed attach to the hemagglutinin, and completely envelops the influenza virus,” confirmed study co-author Susanne Liese, PhD, professor at Freie Universität Berlin. “It was therefore also possible to describe and explain the high bond strength mathematically.”

These findings must now be followed up by more preclinical studies. It is not yet known, for example, whether the phage capsid provokes an immune response in mammals. Ideally, this response could even enhance the effect of the inhibitor. However, it could also be the case that an immune response reduces the efficacy of phage capsids in the case of repeated-dose exposure, or that flu viruses develop resistances. And, of course, it has yet to be proven that the inhibitor is also effective in human

“Our rationally developed, three-dimensional, multivalent inhibitor points to a new direction in the development of structurally adaptable influenza virus binders. This is the first achievement of its kind in multivalency research,” Hackenberger concluded.

 
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Bacteriophages could be a game changer in the trajectory of COVID-19

by Marcin Wojewodzic | Genetic Engineering & Biotechnology News | 10 Jul 2020

The pandemic of the coronavirus disease (COVID-19) has caused the death of at least 270,000 people as of the 8th of May 2020. This work stresses the potential role of bacteriophages to decrease the mortality rate of patients infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The indirect cause of mortality in COVID-19 is miscommunication between the innate and adaptive immune systems, resulting in a failure to produce effective antibodies against the virus on time. Although further research is urgently needed, secondary bacterial infections in the respiratory system could potentially contribute to the high mortality rate observed among the elderly due to COVID-19. If bacterial growth, together with delayed production of antibodies, is a significant contributing factor to COVID-19’s mortality rate, then the additional time needed for the human body’s adaptive immune system to produce specific antibodies could be gained by reducing the bacterial growth rate in the respiratory system of a patient. Independently of that, the administration of synthetic antibodies against SARS-CoV-2 viruses could potentially decrease the viral load. The decrease of bacterial growth and the covalent binding of synthetic antibodies to viruses should further diminish the production of inflammatory fluids in the lungs of patients (the indirect cause of death). Although the first goal could potentially be achieved by antibiotics, I argue that other methods may be more effective or could be used together with antibiotics to decrease the growth rate of bacteria, and that respective clinical trials should be launched.

Both goals can be achieved by bacteriophages. The bacterial growth rate could potentially be reduced by the aerosol application of natural bacteriophages that prey on the main species of bacteria known to cause respiratory failure and should be harmless to a patient. Independently of that, synthetically changed bacteriophages could be used to quickly manufacture specific antibodies against SARS-CoV-2. This can be done via a Nobel Prize awarded technique called “phage display.” If it works, the patient is given extra time to produce their own specific antibodies against the SARS-CoV-2 virus and stop the damage caused by an excessive immunological reaction.

The virus that caused the pandemic

The coronavirus pandemic has caused the death of more than 270,000 people, as reported by 8th May 2020 by the World Health Organization (WHO). The crisis we observe is the joint effect of globalization and the properties of the new virus (SARS-CoV-2), which causes the disease, COVID-19. SARS-CoV-2 stands for “Severe Acute Respiratory Syndrome COronaVirus 2” describing one of the most dangerous symptoms in COVID-19. Although there have been past warnings of the threat that respiratory targeting viruses pose,1 the SARS-CoV-2 virus has spread at an unprecedented rate and it is devastating our health and economy globally. We urgently need multiple approaches to tackle this crisis.

This short communication attempts to highlight the potential for the use of natural bacteriophages to decrease the mortality rate among patients infected by the SARS-CoV-2 virus. COVID-19 patients can develop SARS, leading to atypical pneumonia2 that is mediated by cytokine storms.

Possible significance of bacteria in symptoms for COVID-19

The most probable entrance road of the SARS-CoV-2 to humans is the respiratory system, where the virus can disrupt its equilibrium.

The indirect cause of death in COVID-19 patients could be miscommunication between the innate and adaptive immunological systems.4 The adaptive immune response takes much longer than the innate immune response to begin effectively attacking a new pathogen. This means there is a period when only the innate immune system is fighting the infection and, in this period, the innate immune system’s response can become too aggressive when faced with a high virus load, causing it to damage other systems. The growth of the virus causes the innate immune system to secrete inflammatory material (fluid and inflammatory cells) into the lungs. As a result, the lungs become filled with fluid reducing the body’s ability to exchange gases.

The debris of dying and virally infected human respiratory cells can become a substrate for bacteria growth, a side effect of the virus infection. This growth of bacteria then causes the innate immune system to secrete additional inflammatory material in nearby alveoli. Bacterial infections seem to provoke a further reaction of the innate immune system, and they may interact with virus infections. This process accelerates as the virus continues to attack lung cells, and it thus creates more cell debris substrate for the bacteria to feed on. This can result in the innate immune system adding too much inflammatory fluid to the lungs, inhibiting gas exchange and resulting in an urgent need for ventilation, and it can cause sepsis and death.

The delay (or failure) of the production of antibodies specific to the virus could explain why SARS-CoV-2 is so dangerous for the elderly. A recent detailed review on immunity in COVID-19 summarizes state-of-the-art knowledge of the host’s immunological response to the virus, and it points out clear differences in disease progression between younger and older patients.

Immunosenescence (impairment of immune functions) can delay the production of antibodies and is usually expected in elderly patients (Figure 1B) which might be a part of the cause for the high age-dependent mortality observed in COVID-19 patients (Figures 1A, B). Although data for COVID-19 are still scarce, there is evidence that having previously contracted influenza predisposes the host to acquiring pneumococcal colonization and therefore there is a known mechanism for viral infections to cause bacterial colonization in the human respiratory system. Further, the co-occurrence of viruses and bacteria is well documented for other viruses.


Figure 1. Theoretical time courses of the SARS-CoV-2 virus growth (red curves), bacterial growth (purple curves), and host antibody production (blue curves) for four scenarios. (A) A young healthy individual who has no problems developing antibodies to the virus infection. (B) An old individual who experiences delayed antibody production, resulting in bacterial growth as well as increased virus growth. (C) An old individual for whom a bacteriophage cocktail against bacterial growth was introduced as a part of standard therapy. Increase of bacteriophages is marked (green curve) with the time of treatment (green arrow). The relationship between bacteriophages and bacteria can be described by the Lotkka-Voltera population model. The viral load does not decrease until the body’s natural antiviral antibodies are produced but more time is bought for this to happen. (D) An old individual for whom synthetic antibodies were introduced (brown curve), creating an immediate reduction in the viral load and once again buying time for the natural antibodies to be produced. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Although ecologists call this process a “succession,” medical doctors use the term “secondary infections.” For instance Staphylococcus aureus, Staphylococcus pneumoniae (pneumococcus), Aerococcus viridans, Haemophilius influenza, and Moraxella catarrhalis are bacteria typically found in influenza patients, as well as other respiratory commensals, which occasionally turn into pathogens causing infection.

A recent review suggests that bacterial infections, including Acinetobacter baumanii and Klebsiella pneumoniae, have been documented in COVID-19 patients, especially in the intensive care unit setting. Non-survivors were more likely to have sepsis and secondary infection, although detailed bacteriology results were not reported. Secondary infections were also positively correlated with steroid administration.

At least part of the high mortality rate attributed to COVID-19 could be due to bacterial infection of the respiratory system, although we still do not have an accurate estimate for the numbers. There might also be problems in producing reliable estimates for these numbers due to the overwhelming number of patients seen in clinics and the criteria for which patients are admitted to bacteriology tests, and at what point in the process. A recent report from Wuhan shows that at least 50% of patients dying developed secondary infections. The median time given for these secondary infections to develop is 17 days, although the range in time is quite large. It is plausible that bacterial infections begin to colonize before acute respiratory distress syndrome is developed.

In viral scenarios such as influenza, bacteria such as Pseudomonas aeruginosa are known to spread rapidly. In addition, the rapid and enormous response of the first-line, innate immunity system causes general inflammation that can change pulmonary structures (causing fibrosis), further reducing oxygen uptake and causing permanent damage to the respiratory tissue. This reaction can lead to the innate immunity system itself being the actual cause of death; however, the extent to which this reaction is caused by the body’s response to the SARS-CoV-2 virus or to which it is caused by its response to infection by bacteria (such as P. aeruginosa) is not yet known and I postulate may differ over the course of the infection.

The interplay between the time taken for the human body to develop antiviral antibodies and the role of bacteria in the death of older individuals is also not yet well known for COVID-19.

Integrative approach proposal

If bacterial growth, together with the delayed production of antibodies, is a significant contributing factor to COVID-19’s mortality rate, then the additional time needed for the human body’s adaptive immunity system to produce antibodies could be gained by reducing the bacterial growth rate in the respiratory system of the patient. If the growth of bacteria in lungs can be stopped, then the rate of liquid increase within the lungs should also decrease. However, as the growth of the virus is exponential, it might be necessary to decrease the viral load at the same time as the bacterial load to slow down the immunological response.

Natural bacteriophages’ potential — a direct weapon against bacteria

Bacteriophages are viruses that selectively attack specific species of bacteria and are otherwise harmless to animal cells, including humans. They were discovered 100 years ago by Frederick Twort and Félix d’Hérelle and are distributed throughout Earth’s ecosystems and over a broad bacterial host range, including bacteria naturally found in humans.

It has been shown that the attack of bacteriophages is specific, meaning that one species of bacteriophage targets only a single species of bacteria (or even a specific strain of one species). This specificity also points toward the “Red Queen” co-evolutionary process between these two players. The scenario of the attack is as follows: (1) The bacteriophage attaches itself to a susceptible bacterium, exclusively infects the host bacterial cell and (2) hijacks the bacterium’s biochemical machinery to produce multiple copies of itself. (3) The bacterium then undergoes destruction (lysis) and new copies of the bacteriophage are released and infect, exclusively, other bacteria of the same species in the neighboring areas.

Despite this known interplay between bacteriophages and bacteria, research into bacteriophages and their potential medical applications was largely abandoned for many years due to “The Antibiotics Revolution.” Antibiotics were adopted as the main way of treating bacterial infections due primarily to the fact that they are general purpose, as opposed to bacteriophages that specifically target a single species of bacteria. Other advantages include the fact that antibiotics are usually fast acting, efficient, and relatively cheap to manufacture. However, there are several drawbacks as well to the use of antibiotics. One of these is that, unlike bacteriophages, antibiotics can destroy beneficial bacteria in addition to harmful ones. More importantly, the overuse of antibiotics can cause bacteria to evolve resistances to them, resulting in antibiotic-immune “superbugs.”

In the current COVID-19 pandemic, around 70% of hospitalized COVID-19 patients worldwide receive antibiotics as part of their treatment. This raises the danger of the emergence of antibiotic-resistant strains of bacteria even higher and creates an even greater need for the development of alternative strategies to fight bacterial infections. Unlike antibiotics, bacteriophage treatments would be far less susceptible to the development of resistances, as the bacteriophage itself can also adapt to overcome any resistance that the bacteria develop.

It has also been suggested that the presence of bacteriophages can have positive effects on human health and patient recovery, suggesting that bacteriophages are to some extent responsible for homeostasis of the microbiota. For instance, a group investigating alternative treatments for Clostridium difficile, a bacteria that can infect the bowel and cause diarrhea, has identified a large set of bacteriophages that are effective at killing this pathogen. This method is now being transformed into a therapeutic treatment. We can find more examples of how bacteriophages are being used for human or animal models, in addition to different bioengineering methods using bacteriophages that are currently being developed.



Bacteriophages used for accelerated therapeutic antibody production against the virus

Despite the fact that bacteriophages’ potential to fight bacterial infections has only recently been rediscovered, they were successfully used as tools at the molecular level, leading to Nobel Prize awards.

Using a technique called phage display, bacteriophages have the potential to quickly produce recombinant antibodies. This technique of producing antibodies was developed for MERS-CoV and successfully applied. In phage display, techniques blocking ACE2 interaction could be engineered from the serum of immune patients. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display. Before this, artificial antibody production was primarily done by using animals; however, this is both slower and less cost effective than using bacteriophage display techniques. Another benefit of this method is that monoclonal antibodies produced by bacteriophage display techniques can be humanized.

The use of antibody therapy for the control of viral diseases has already been reviewed and some therapies have been approved for human testing. As an example, the company ProteoGenix launched accelerated therapeutic antibody discovery by screening a naive antibody human library or an immune human antibody library (obtained from the plasma of COVID-19 survivors) by using the phage display technique. This demonstrates that accelerated therapeutic antibody discovery is highly feasible.

Therefore, there are two main ways that bacteriophages could be used to decrease the mortality rate of the COVID-19 pandemic. They can be used to decrease the population of bacteria in a patient’s respiratory system and/or bacteriophage display techniques can be used to efficiently manufacture synthetic antibodies against SARS-CoV-2 (Fig. 1D).

I propose a series of clinical trials for the use of cocktails of bacteriophages (that target the main species of bacteria known to cause respiratory problems) in treating COVID-19 patients and/or the use of phage display techniques to create synthetic antibodies that target SARS-CoV-2 in the early stages of infection.

Further considerations for bacteriophage therapy — bacteriophages as killers

The bacterial growth rate could potentially be reduced by the aerosol application of bacteriophages that prey on the main species of bacteria known to cause respiratory failures (Figure 1C). This can occur in a self-regulatory manner, similar to ecological prey–predator regulation. The exponential growth of the bacteriophage population (limited primarily by the population of the bacteria it preys on) should allow for a fast clearance, especially in cases where the bacterial population has already grown significantly. The relationship can be described by Lotka-Volterra or Kill-the-Winner population model.

In fact, we can already find evidence in literature that pneumonia could be cured by nebulized bacteriophages.41 Prophylactically administered bacteriophages reduced lung bacterial burdens and improved survival of antibiotic-resistant S. aureus infected animals in the context of ventilator-associated pneumonia. If needed, a selection of bacteriophages and optimal target bacteria could be quickly identified by a group of experts as the species of bacteria that commonly cause respiratory problems are well known and a bacteriophage that preys on a specific species can be quickly identified by screening methods. If needed, quantitative microbiome sequencing could potentially be used.

There are assumptions that need to be met during the clinical trials for the approach to work. (1) The cohort has to be chosen to have a high probability of developing bacterial infections. (2) It should be ensured to have the correct choice of bacteriophages that both target the optimal bacteria candidates and are most effective at reducing that bacteria’s population growth. (3) The bacteriophages should not interfere with the patient’s innate or adaptive immune system. (4) The patient does not have antibodies toward bacteriophages used, nor develops any antibodies toward bacteriophages to clear off the bacteriophage earlier than to SARS-CoV-2. We know from bacteriophage therapy in the pneumonia system that the rapid lysis of bacteria by bacteriophages in vivo does not increase the innate inflammatory response compared with antibiotic treatment. This is a promising finding and there seemed to be positive effects on the patient’s immune system. (5) Another obstacle could be a risk of a species of bacteria developing resistance to the bacteriophage, according to the co-evolutionary process mentioned. However, this would be much less serious than the antibiotic resistance problem as it would only reduce the effectiveness of that one bacteriophage and there is the possibility of the bacteriophage also adapting to overcome any resistance to it. (6) Finally, bacteriophages are so specific to one species of bacteria, and there is very little chance of the bacteriophage damaging any beneficial bacteria, but this should still be verified in clinical trials. It has to be noted that the point here is to decrease bacterial growth in critical time and therefore allow the patient more time to recover from the COVID-19 infection.

Decreasing the population growth rate of bacteria

The response to antibiotics may be slower or smaller than expected. This may be due to both antibiotic-resistant strains and slow diffusion rate of the antibiotics in that area due to bacterial biofilm formation.46 Also, in some cases, the penetration of antibiotics into target tissues is also dependent on the tissue type that was shown for lungs in tuberculosis scenarios. It has been shown that the sites of mycobacterial infection in the lungs of patients have complex structures and poor vascularization, which obstructs drug distribution to these hard-to-reach and hard-to-treat disease sites, further leading to suboptimal drug concentrations. Because of this, there is the potential for the use of bacteriophages (entering patients’ respiratory systems in a different way and acting differently to antibiotics) to decrease the mortality rate of patients infected by the SARS-CoV-2 virus.

Intensive use of antibiotics targeting COVID-19 in clinics can further lead to bacterial resistance spreading in the hospitals. Using bacteriophages could take pressure off this problem. This could also shed light on the use of bacteriophages to decrease this problem in post–COVID-19 scenarios.

Decrease the viral load by using Synthetic Antiviral Antibodies

There are also assumptions that need to be met during the clinical trials for the second approach to work. (1) The cohort has to be chosen to have a bad prognosis (age >80) and high viral load; (2) ensuring the correct choice of antibody that targets the virus epitope and nothing else in the human body; (3) the antibody should not cause failure of the immune system (anaphylactic shock); (4) the dose and frequency should be mathematically modeled; and (5) the delivery system should be efficient.

Gaps in knowledge

Before choosing the candidate bacteriophages, careful literature studies will need to be done to check for potential known interactions. For example, it has been shown that some bacteria can produce a biofilm when exposed to their relevant bacteriophages, which could be an obstacle for the development of these methods as a treatment for COVID-19 patients. Although most bacteriophages kill their bacterial hosts, others can live inside the microbes without killing them. Also, lessons from recent studies need to be carefully followed. For instance, complex immune dysregulation in COVID-19 patients with severe respiratory failure has been observed.

During the writing of this communication, the first immunological reviews were published, in which the authors identified major gaps in knowledge that need to be addressed by the scientific community. It is unknown how this may complicate any treatment and further investigation is needed.

High gain approach


However, if a treatment using bacteriophages therapy can be developed it is likely to prove practical as they can be produced both quickly and cheaply. Production of antibodies from the phage display techniques will have some costs of production but, owing to recent progress, the development should be simple. Bacteriophages can also be stored and transported easily. I believe that bacteriophages have the potential to be a practical tool in mitigating the SARS-CoV-2 pandemic, especially in patients with secondary bacterial infection and high viral load. I believe that it is unlikely to have any significant side effects, and that it has the potential to save a great number of lives. The beauty of nature is that although it can kill us, it can also come to our rescue.

Marcin Wojewodzic is a systems biologist at the Cancer Registry of Norway, Institute of Population-Based Cancer Research, Etiology Group.

 
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Lysins unlimited: Phages’ secret weapon

by Julianna LeMieux, PhD | Genetic Engineering & Biotechnology News | 3 Aug 2020

Instead of recruiting whole phages into phage therapy armies, antibacterial campaigns may simply requisition the organisms’ battle-tested cell-wall-breaching enzymes

It was 1917 when Felix d’Herelle, at the Institut Pasteur in Paris, first proposed using bacteriophages (or phages)—viruses that infect bacteria—as a therapy for human bacterial infections. Although used for decades in parts of Europe, notably Russia, Poland, and the Republic of Georgia, phage therapy is only permitted in the United States under the “compassionate use” umbrella—when there is nothing else available.

The rise of multidrug-resistant bacteria that defy traditional antibiotics has forced clinicians to seek alternative measures to curb deadly infections. Two cases made headlines in recent years. In 2016, the life of Thomas Patterson, PhD, a professor of psychiatry at the University of California, San Diego, was saved by phage therapy after he developed a deadly Acinetobacter baumannii infection. (The story is recounted in The Perfect Predator, the book that Patterson co-authored with his wife, epidemiologist Steffanie A. Strathdee, PhD.) Last year, the life of an English teenager was saved after she developed an infection following a lung transplant for cystic fibrosis.

Although phage therapy offers a promising way forward, other investigators want to take a more direct approach using only the active ingredient of the phage—the lysins—responsible for killing bacteria.

Roger Pomerantz, MD, president and CEO of Contrafect, compares lysins and phages to quinine and the fever tree. For many years, natural products were used in their entirety because the active agent had not been described. Upon isolating the active agent of the fever tree, or the foxglove plant (the source of digitalis), tree bark and flowers are no longer used. Understanding and isolating the active agent of phages removes the need to use the entire phage, or the “tree bark.”

Phage lysins work by degrading the bacterial cell wall, which is composed of the bacteria-specific molecule peptidoglycan. Different bacteria have different components and organizations of peptidoglycan in their cell walls; different lysins are specific to these structures. Pathogens can have a slightly different composition than similar bacteria that are part of the normal flora. For example, the lysin for the gut pathogen Clostridium difficile will selectively kill C. difficile without harming other Clostridium present in the microbiota.

Around 2010, as awareness of the antibiotic resistance crisis was reaching a fever pitch, alternatives to antibiotics were being sought with a newfound urgency. One of the most promising set of candidates was under investigation in a laboratory on the Upper East Side of Manhattan, where researchers were discovering that lysins could be used clinically.

Night and day

Vincent Fischetti, PhD, the primary developer of the lysin technology, has been on the faculty at the Rockefeller University since 1973. He purified a phage lysin during his thesis work, using it to extract proteins from group A streptococci. Fast forward to the year 2000, Fischetti was, he recalls, “the right person at the right time.” He added lysin to the throats of mice that had been colonized with streptococcal bacteria. The bacteria died, and the idea to use lysins as a therapeutic was born. Fischetti obtained a broad patent, received two grants from the Defense Advanced Research Projects Agency (DARPA), and published a string of papers.

The differences between treating infections with phages and lysins, Fischetti explains, are “night and day.” Lysins are direct and kill instantly, and no resistance has been observed to date. Also, off-target effects are unlikely because peptidoglycan does not exist in mammalian tissue. Lysins are also very stable proteins—they can be frozen and lyophilized, and they are heat stable up to about 50°C.


When treated with lysins, a Gram-positive bacillus will externalize its cytoplasmic
membrane before it ruptures and dies, as shown by this electron micrograph from
the Rockefeller University lab of Vincent Fischetti, PhD. The bacillus, which maintains
a high internal pressure, succumbs after lysins cut a few peptidoglycan bonds. “Boom,
it’s going to explode!” exclaims Fischetti.


Moreover, lysins can infiltrate a biofilm, a bacterial community that normally offers bacteria extra protection from antibiotics. When biofilms are treated with antibiotics, only the organisms on the surface of the matrix are killed. In contrast, lysins dissolve biofilms from the top down. The bacteria burst open, revealing the next layer of the biofilm, making it vulnerable to further lysin exposure.

Graham Hatfull, PhD, a professor at the University of Pittsburgh specializing in phage biology, says there are vast numbers of different lysins, each with specific cell wall targets, creating a huge space for discovery and development. The lysins are relatively cheap and easy to produce. For several Gram-positive bacteria—which have only a single membrane located interior to the cell wall, giving lysins direct access to the peptidoglycan targets—good antibacterial activity has been shown in vitro. In short, Fischetti says, “They work.”

Getting lysins into patients

With the success of phage therapy in a couple of high-profile cases in recent years and the numerous advantages on paper of lysins as antibacterial drugs, one might ask: Why haven’t lysins received the same attention from the biopharma industry?


In addition to lysins, Contrafect develops amurins, phage-encoded lytic antimicrobial peptides. Still in the
discovery phase, these peptides have been shown to have in vitro activity against Gram-negative bacteria.
The electron micrograph shows lysis of the Gram-negative bacterium Pseudomonas aeruginosa. Treatments
to counter Pseudomonas pathogens are much sought after because the pathogens cause multidrug-resistant
nosocomial infections.


One reason is that the research did not leave Rockefeller University until about 10 years ago. That’s when Yonkers, NY-based biotech Contrafect approached Fischetti about licensing the technology to develop lysins as a therapeutic. Raymond Schuch, PhD, a research assistant professor in Fischetti’s lab at the time, joined Contrafect, where he is now vice president of research.

Schuch tells GEN that he made the move to Contrafect because it opened a whole new area of translational research that “we usually don’t think about in research laboratories.” Schuch continues: “We spent years identifying these lysins, defining their characteristics, showing that they confer therapeutic benefits in animals.” He wanted “to continue along the pathway of the development.”

Over the past decade, the small company of 25 employees has taken the Staphylococcus aureus lysin Exebacase into the clinic, completing Phase I and Phase II of a trial. Contrafect is currently enrolling patients in Phase III. This is the first and only lysin to enter human clinical trials in the United States. The data showed that Exebacase, given in combination with antibiotics, improved clinical outcomes in patients with Staphylococcus aureus bacteremia, including endocarditis, when compared to antibiotics alone.

Although Exebacase is further down the clinical trial pipeline than any phage therapy in the United States, phage therapy has attracted all the public attention. Few have heard of lysins. “I don’t really know why,” remarks Fischetti. “That’s the problem.” One reason may be that lysins have not yet been approved for use in the compassionate care cases that tend to garner attention. When asked if there are any known disadvantages to using lysin over phage, Fischetti is adamant: “There are none.”

Path of least resistance

One of the major problems with phage therapy is the ability of bacteria to develop resistance, similar to the resistance that is rampant with antibiotics. In the recent cases where phages were used for compassionate care, the medical teams opted to use a cocktail approach, believing that one or even two phages could lose efficacy at some point due to the onset of bacterial resistance.

For example, in the case last year of the 15-year-old cystic fibrosis patient Isabelle Carnell-Holdaway, who was treated for a disseminated Mycobacterium abscessus infection, three phages were used. Hatfull, who supplied the phages, says, “Three was a number that gave some confidence that we wouldn’t see resistance. The more phages," he notes, “the more effectively you can counter that concern.”


Left: A phage infects a bacterial cell to initiate a progeny-producing process that culminates in the release of newly assembled phages. Key players in this process are phage lysins, which travel through holin—a small membrane protein also made by the phage—to reach the bacterial cell’s peptidoglycan.
Right: Lysins can kill antibiotic-resistant bacteria. Here, they attack a Gram-positive bacterial cell’s peptidoglycan from the outside. Regardless of the direction the lysins’ attack, peptidoglycan is cleaved.


Hatfull observes that "lysins, unlike phages, are associated with very low or even undetectable levels of bacterial resistance, and this is a major advantage."

Why is resistance not a problem for lysins? Fischetti says that “it’s keyed into the way that the phages have evolved.” Lysins are essential for the survival and release of the phage. Their target is a critical component of the bacterial cell wall that is essential and cannot be changed easily. For this reason, lysins tend to retain their potency against bacteria, which find it far more difficult to acquire resistance to phage lysins than to phages or antibiotics.

Lysin smoothies

Fischetti has recently teamed up with Lumen Bioscience, a Seattle-based company founded in 2017 with technologies for bioengineering spirulina, a photosynthetic microbe consumed worldwide as a nutritional supplement and food source. The blue-green algae can allow lysins to be produced for a fraction of the cost associated with conventional techniques. In fact, Fischetti asserts that algae can take the cost of lysins “down to pennies a dose.”

Producing lysins traditionally requires a biomanufacturing system and complicated purification processes. Algae are extremely easy to grow in enormous quantities (they grow in tap water), and they are the only microbes that can be commercially farmed at these scales.

Brian Finrow, the CEO of Lumen, tells GEN that the Fischetti laboratory will generate the lysins and that Lumen will carry out the protein engineering and other activities to create therapeutic strains of spirulina and carry those forward to FDA-supervised clinical trials.

Fischetti says that "the spirulina could be used as a 'lysin factory,' producing large quantities of lysin to be purified. But it is not implausible that someone could eat spirulina that is expressing lysin to have the enzymes coat their intestinal tract." He adds that "this technology and collaboration opens interesting possibilities for lysin, including moving into the veterinary field." (As they are currently made, lysins are too expensive to be used for veterinary purposes.)

Time will tell

Hatfull is a proponent of phage therapy, but he says “the proof-of-principle studies [using lysins] are encouraging.” As with every young field, there are many unresolved questions. Will lysins be able to access niches of bacterial infection in vivo, as these enzymes are larger than typical antibiotic molecules? And although the early data in Contrafect’s clinical trial regarding the host’s immune response look promising, might immune responses to the lysin limit their action in patients for extended or repeated periods?

In addition, the utility of lysins for Gram-negative pathogens remains unclear because of the need to get the lysins past the outer bacterial membrane. Gram-negative bacteria, in contrast to Gram-positive bacteria, have a second membrane located exterior to the cell wall, making access to the peptidogly can layer more challenging. Fishcetti’s laboratory is working on this problem: the team has lysins that, when modified, can get past the outer membrane. But development of the Gram-negative lysins is behind the development of the Gram-positive lysins.

Phage and bacteria have been evolving together for a billion years, Fischetti explains. They have been battling back and forth, building systems so that nobody loses—and nobody wins—because “whoever wins, loses.”

"Phage therapy is trying to take a billion-year-old, established system, where nobody is supposed to win and force the phages to win," notes Fischetti. He adds, “That is not going to happen easily.” One challenge is that we lack a full understanding of the mechanisms of bacterial resistance. “We know about CRISPR,” he says, “but we don’t know about all of the other bacterial defense systems against phage.”

Fortunately, resistance to lysins has not been seen in the 20 years of working with them. Bacteria have built up resistance to both phages and antibiotics, but “they don’t know how to handle a lysin,” notes Fischetti. The lysins, he says, “take the bacteria by surprise.” Fischetti notes that his laboratory tries to force resistance. Doing so is proving to be difficult. If resistance is difficult for scientists to achieve in the laboratory, it may also be difficult for bacteria to achieve in nature.

"Lysins could buy us time (maybe decades) by killing antibiotic-resistant bacteria until new methods are discovered," Fischetti speculates. "Using lysins," he adds, "is simply taking advantage of something that phages have figured out—of something that has helped them survive—and using it to our own benefit."

Broad- vs. narrow-spectrum lysins

Antibiotics can be classified as broad spectrum (affecting a wide swath of bacteria) or narrow spectrum. Which category makes a more desirable therapeutic is a matter of continuing debate.

Killing pathogenic bacteria without damaging the healthy bacterial components of the microbiome is a priority. This is one of the reasons why phages are so appealing, as they are targeted killers of their cognate bacteria.

Specificity, however, isn’t necessarily the best practice. According to some researchers, there are instances, for example, in treatments of polymicrobial diseases, where a more broad-spectrum approach is desired. Lysins seem to have both corners covered.

“You can identify broad-spectrum lysins if you need them and narrow-spectrum lysins for most applications,” says Vincent A. Fischetti, PhD, a researcher at Rockefeller University. "There are so many different lysins available, it is straightforward to find molecules that are narrow in scope." In fact, as Fischetti notes, this categorization was one of the early hurdles when trying to engage pharmaceutical companies, which wanted broad-spectrum compounds.

 
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Phage therapy: Turning the tables on bacteria

by Julianna LeMieux, PhD | GEN | 6 Mar 2019

When engineered to incorporate CRISPR components, phages may overwhelm bacterial defenses or transform bacterial functions.

Throughout the life and death (and nonlife) struggle between bacteria and bacteriophages, bacteria try to cut apart the genetic material that bacteriophages deploy when they try to commandeer bacterial resources. And now the bacterial weapon of choice, the immune system known as CRISPR, is starting to cut both ways. CRISPR components are being engineered into bacteriophages, arming them against pathogenic bacteria, including antimicrobial-resistant bacteria. In phage therapies, CRISPR-wielding bacteriophages may kill bacteria or compel them to carry out useful functions. For example, after suffering a few thrusts of the CRISPR sword, otherwise recalcitrant bacteria may have no choice but to express therapeutic proteins.

The swashbuckling ways of engineered phages are possible only though the patient work of highly disciplined researchers. For example, two research teams—one based at Rockefeller University, and one at the Massachusetts Institute of Technology (MIT)—began independent investigations of phage therapy that culminated in a close and productive collaboration.


Luciano Marraffini, PhD, and Timothy K. Lu, MD, PhD

Antibacterial teamwork

Five years ago, Luciano Marraffini, PhD, was leading work at Rockefeller to undercut the Gram-positive Staphylococcus aureus, while Timothy K. Lu, MD, PhD, was heading a group at MIT that was taking stabs at the Gram-negative Escherichia coli. In both labs, bacteria were being set upon by phages that had been weaponized with CRISPR-Cas technology.

The similarity in the projects was discovered when Xavier Duportet, PhD, an MIT graduate student familiar with Lu’s lab, talked to his friend David Bikard, PhD, a postdoctoral fellow in Marraffini’s lab. When Lu’s Boston area lab and Marraffini’s New York lab started sharing data, they formed a bond that withstood the usual interlab rivalries (as well as the divisions that must have emerged during sports season).

The labs produced a pair of papers showing, for the first time, that a Cas system targeting a bacterial chromosome could efficiently kill bacteria. Published back to back in Nature Biotechnology, the papers described work that not only delivered scientific value, but also held commercial potential. With Duportet and Bikard at the helm, the work led to the birth of the Eligo Biosciences, a Paris-based company that has assigned itself the task of developing targeted, CRISPR-based antimicrobials to kill antibiotic-resistant bacteria.

Using phages to kill bacteria is a century-old idea that has been slow to come to fruition. To date, no prospective phage therapy has become an FDA-approved drug. But phage therapy continues to inspire research because it could overcome antibiotic resistance, which is reaching dire proportions. It has been estimated that by 2050, 10 million people will die each year from antimicrobial-resistant infections.

Research into phage therapy is exciting, Marraffini tells GEN, because it could “generate a new antimicrobial that will prevent the worst-case scenario from happening.” In the past, people used phages without modifying or engineering them. At present, however, researchers are going farther. For example, scientists at Eligo are adding payloads into phages that directly target bacteria in an orthogonal way to rapidly kill the bacteria.

“Leveraging phage-based killing of microbes is more complicated than it may sound,” notes Lu. Natural phages can kill bacteria, but in many cases, bacteria have evolved resistance to phage activity. "One way to circumvent bacterial resistance is through the genetic modification of exterior phage components—an art in which Lu is a “master,” says Marraffini. There are also intracellular barriers such as phage restriction mechanisms and, as noted earlier, the bacterial immune system called CRISPR (clustered regularly interspaced short palindromic repeats).


Eligo Biosciences is working to leverage existing microbial populations in the gut, lungs, vagina, skin, etc.
The company’s technology, Eligobiotics, takes a targeted approach to functionalize the microbiome.


In the CRISPR realm, Marraffini’s expertise is invaluable. He helped pioneer CRISPR research by collaborating with Feng Zhang, PhD, to illustrate CRISPR’s capacity in human cells. A microbiologist by training, Marraffini notes that it is particularly exciting to see CRISPR advances being applied to antimicrobial-resistant bacteria.

Eligo has developed technology that avoids indiscriminate killing. Associated with current broad-spectrum antibiotics, this sort of killing can eliminate commensal bacteria and accelerate the evolution of drug resistance. The company’s targeted approach is emphasized in the company’s name, which includes “eligo,” a Latin word meaning to choose or select.

Duportet says that while a phage may be able to bind to all the bacteria in a species, by using a guide RNA that is specific for a genetic sequence present in one strain of bacteria and absent in another, it will kill only the bacteria that carry that specific genetic target. In addition, CRISPR-Cas9 can be programmed to cut in 10 different regions of the bacterial genome, which would challenge the bacteria to evolve resistance in 10 different places—a highly unlikely event.

Banking on Cas3

Another company that is engineering CRISPR-wielding delivery vehicles is Locus Biosciences, a 2015 spinout of North Carolina State University (NCSU). Locus is working to introduce Cas3, a nuclease that completely obliterates the cell’s DNA, to bacteria.


Rodolphe Barrangou, PhD

Locus’ technology emerged from the NCSU labs of Rodolphe Barrangou, PhD, and Chase Beisel, PhD. Barrangou, distinguished professor at NCSU, pioneered Cas3 research. Beisel, currently an assistant professor at the Helmholtz Centre for Infection Research in Germany, contributed to (and continues to work on) phage packaging.

Paul Garofolo, co-founder of Locus and currently the company’s CEO and Joe Nixon, senior vice president of business development, knew that Cas enzymes in addition to Cas9 represented untapped potential, and they were particularly interested in Cas3. Not only did it “seem to have the best mechanism of action,” Garofolo recalls, but it also avoided the sort of intellectual property drama surrounding Cas9. Barrangou adds that building the company on Cas3 was “obviously valuable, creative, different, inventive, and novel” while being “not obvious to others.”

Locus sources a panel of clinically relevant clinical isolates and then performs high-throughput screening (through their acquisition of EpiBiome last summer) to identify a block of phages that have infectivity to those clinical isolates. That cocktail of phages will be the starting point for engineering. The company also sequences the clinical isolates to identify conserved genes. These genes are used to build the CRISPR targets.

CRISPR allows selective and precise removal of the one genotype of interest by targeting one distinct locus in that strain. Barrangou explains that being precise at this point in the process is of paramount importance. In fact, it is the cornerstone of the company’s technology. It is no coincidence that the company name includes the word “locus.”

“You have to pick the right place,” Barrangou insists. “You can take two strains that are 99.9% identical, but the locus of interest—the locus that is different for each of them—enables you to segregate the two.” He adds that "CRISPR is like a sniper in its ability to target the locus of interest."

Another application that interests both Locus and Eligo is the microbiome. The companies hope to manipulate complex bacterial populations in a sequence-specific manner. With the growing understanding that certain resident bacteria cause disease, the ability to kill bacteria in a targeted way may lead to treatments for microbiome-specific alterations. This fits nicely with the overall goal that Marraffini described. That is, to create a smarter antimicrobial that does not wipe out all the bacteria in the body. Such an antimicrobial preserves the good bacteria while targeting the bad ones.

“This will help us develop new technologies to edit the gut microbiota to treat many gastrointestinal diseases,” asserts Casey Theriot, PhD, an assistant professor at NCSU College of Veterinary Medicine and a scientific advisory board member at Locus. “[It will also advance the] rational design of the gut microbiota, which will aid in precision medicine.”

Any advances in this realm, according to Duportet, “rely on the advance of the understanding of the microbiome and moving from a correlation between specific strains and a disease to a causative relationship.” To that end, Eligo is working on some targets through undisclosed partnerships with microbiome companies.

It’s not all about killing

In addition to killing bacteria, Eligo is focused on developing “microbiome gene therapy” which involves putting nonlethal payloads into phages to functionalize the microbiome. The goal is to harness the bacteria resident in the body to express proteins that may serve as biotherapeutics, degrading toxins or otherwise promoting health.

A similar approach has already achieved this by producing genetically modified probiotics. For example, the work being done at Synlogic to lower levels of phenylalanine in people with phenylketonuria. However, most of these probiotics do not colonize the gut for a very long time. So, after these probiotics stop being introduced, they disappear in a matter of weeks. Eligo would rather target resident microbes that are already adapted to the environment.

Beyond bacteria

Cas3, which shreds rather than snips DNA, is already being loaded into bacteria-killing phages. Adapting Cas3 (Type I CRISPR) technology to eukaryotic cells is a long-range goal for Locus. Charles Gersbach, PhD, a Locus scientific co-founder and an associate professor of biomedical engineering at Duke University, is helping the company reach this goal through the development of highly targeted technologies for editing eukaryotic genome sequences, altering epigenomic regulation, and rewiring cellular gene circuits.

“Type I CRISPR systems could potentially be used for all of the same applications that other DNA-targeting CRISPR technologies are used for, plus more,” notes Gersbach. "The Cascade complex that targets DNA serves as a scaffold to which one may add nuclease enzymes that cut DNA, or transcriptional modulators that control gene expression." The addition of Cas3 to Cascade could potentially serve as a means for the elimination of unwanted DNA such as viral infections, cancerous gene sequences, and chromosomal abnormalities. Gersbach adds that this is something that current CRISPR systems cannot do.

Alternative means of delivery are needed, however, if Cas3 is to target eukaryotic cells with any efficiency. Type I CRISPR-Cas systems can be delivered by any of the same delivery vehicles that other CRISPR systems and gene therapies use, including viral vectors, nanoparticles, and electroporation.

“The Cas9 systems that have dominated our attention make up only a small fraction of total CRISPR systems in nature,” Gersbach points out. “The type I CRISPR systems that we are now working with actually make up the vast majority, and it is incredibly exciting to open up that area of biology for the genome engineering community to explore all the different ways these additional diverse systems could be used in biotechnology and medicine.”

Phage therapies for a new century

Both Locus and Eligo issued Series A funding announcements in the fall of 2017. Since then, Eligo has been laying low, building the company and working with a focus on research. Locus, however, recently announced a collaboration and license agreement with Janssen Pharmaceuticals, part of Johnson & Johnson, to develop, manufacture, and commercialize Locus’ CRISPR-Cas3-enhanced bacteriophage (crPhage™). The partners intend to target two undisclosed bacterial pathogens. Locus will receive $20 million in initial payments and is eligible for up to a total of $798 million in potential future development and commercial milestones, as well as royalties on any product sales.

Offering a scientific prediction for 2019 to STAT, Steffanie Strathdee, PhD, associate dean of global health sciences, University of California, San Diego, and co-director of the Center for Innovative Phage Applications and Therapeutics, cited advances in CRISPR gene editing and phage therapy. “[These] will coalesce,” she said, “and we will witness the first genetically modified phage cocktails being used to cure patients with multidrug-resistant bacterial infections.” Strathdee added that “this will attract new players in the biotech and pharma space and will provide new momentum to bring phage therapy into clinical trials in the United States.” Indeed, both Eligo and Locus report that they are starting clinical trials soon.

However, Lu predicts that antibiotics will always be a key component in the fight against bacteria because, despite their limitations, antibiotics are cheap and easy. Even if the source of the infection is unknown, it can be effectively treated with antibiotics. Treatments that rely on phages are more complicated. Although phage therapies are more complicated, they must be pursued, Lu maintains, because they promise to counter antimicrobial-resistant infections, a task that may soon exhaust our current armamentarium.


Martha Clokie, PhD

Opening a new phase of phage discovery with microbiologist Martha Clokie

GEN caught up with Martha Clokie, PhD, professor of microbiology at the University of Leicester, UK, to ask her about the future of phage therapy. Just days earlier, Clokie had agreed to serve as the editor-in-chief of PHAGE: Therapy, Applications, and Research, a journal that Mary Ann Liebert, Inc. plans to launch later this year.

Clokie started her career studying the molecular evolution of plants, but she migrated to the world of cyanobacteria so that she could work on “something that evolved a bit faster.”

The world of cyanobacteria, she came to appreciate, is profoundly influenced by phages, which are vast in number.

Research into cyanobacteria/phage links deepened when Clokie discovered that marine phages contain photosynthesis genes. She showed that a phage can do more than exert selective pressure on a cyanobacterium’s infection-survival mechanisms. It can also acquire genes from bacterial prey, extending to its host a valuable characteristic—ensuring that energy (and therefore more phages) are produced. Phages, then, aren’t just parasites. They can be partners in physiology.

“Getting phages to the clinic has been a tough road,” notes Clokie. "A stable, high-titer phage therapy is both hard to produce and tricky to regulate." Encouragingly, Clokie adds "there is a new level of engagement from the regulatory agencies." She observes that more people from the FDA are attending phage meetings than ever before.

“When you look at it,” notes Clokie, you see that “there is very little money spent on phage research—dribs and drabs” compared to other research. However, the pressing need for novel ways to combat antimicrobial resistance may change research priorities." She points to the example of the funding being directed toward Locus Biosciences, surmising that their interesting approach coupled with having the CRISPR-Cas3 string in their bow likely proved useful when seeking funding.

When asked to choose this moment’s most exciting phage research, Clokie responds, “It is much too hard to narrow it down.” She adds that "even though people have been studying phages for a century, we are just now entering a new phase of phage discovery.”

 
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Turning the phage on infectious diseases

by Laura Masters | PHYS.ORG | 17 Sep 2020

Not all viruses infect humans and cause pandemics. In fact, some viruses can help us out—by infecting bacteria.

Viruses that infect bacteria are called bacteriophages, or simply phages. They are the most abundant organism on the planet.

Being natural predators, these viruses hijack bacteria and turn them into phage-building factories. The newly produced phages collect inside the bacterium until eventually the cell dies and millions of new phages come pouring out.

And it's this bacteria-killing ability that makes phages a promising treatment for bacterial infections in humans.

Release the phages

Dr. Lucy Furfaro is a microbiologist at The University of Western Australia who has been intrigued by phages from the moment she learned about them.

Currently, phages are used only where antibiotics have failed and there are no further treatment options left to try.

"At the moment, the use of phages to treat patients is restricted to compassionate use. You need to get special access from regulators," says Lucy.

Robust safety and efficacy information is needed before phages can become part of routine care.

The good, the bad and the bug-ly

Currently, the go-to treatment for bacterial infections is, of course, antibiotics. Phage therapy promises two distinct advantages over these drugs.

Firstly, phages are very specific. Phages infect one type of bacteria or sometimes a very small number of closely related bacteria. Antibiotics, on the other hand, work on a much broader scale.

A single antibiotic drug will kill many different bacteria—including the good ones. This can disrupt our microbiome—the ecosystem of microbes that live on and inside of us—which can have a substantial impact on our health and wellbeing.

Phage therapy may also overcome a second consequence of antibiotic use—superbugs.

Bacteria are constantly evolving to protect themselves from antibiotics. And they can share these new abilities with other bacteria too. The result is scary—infections with superbugs can be difficult, even impossible, to treat.

Dr. Furfaro: bacteriophage hunter

Lucy's research focuses on understanding our natural exposure to phages. The self-described bacteriophage hunter takes samples from all kinds of environments. Her focus is on places where both the bacteria and the phages are abundant—such as household garbage and human wastewater treatment facilities.

Once back at the lab, Lucy screens the newly isolated phages for their bacteria-killing ability. This involves adding them to agar plates where bacteria are growing and seeing which of the bacteria they can kill.

Lucy understands that the idea of using phages as treatment can seem pretty frightening for some people.

"It's just really scary to say, "I want you to take this virus. I want to make sure people understand that they don't infect human cells—they're only infectious against the bacteria."

By looking at all of the phages we safely encounter in our daily life, her research will help to reassure people that phages aren't dangerous. It will also help researchers get a better understanding of how our bodies might respond to phage therapies as a new treatment option.

If the research goes well, this is one treatment option that's sure to go viral.

 
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A micrograph of phage therapy in action against alcoholic liver disease.

Phages: the tiny viruses that could help beat superbugs

by Clément Girardot \ The Guardian | 23 Sep 2020

Bacteriophages were superseded by modern antibiotics, but scientists believe they could be key to conquering antimicrobial resistance.

It is, say enthusiasts, the cure that the world forgot. An old therapy that could take on the new superbugs.

Discovered in 1917 by French Canadian biologist Félix d’Hérelle, phages – or bacteriophages – are tiny viruses that are natural predators of bacteria. In many countries they were supplanted during the second world war by antibiotics but continued to be used for decades in eastern Europe.

They are now being seen by some scientists as a complement – and perhaps an alternative –to antibiotics, the overuse of which has led to increasing bacterial resistance and the advent of the superbug.

Tobi Nagel, a California-based biomedical engineer, launched Phages for Global Health (PGH) in 2014 to help developing countries fight antimicrobial resistance (AMR). She had become disillusioned with the inequality in the pharmaceutical industry, in which she worked for 15 years, and says: “We have 30 years until the worst of this crisis. Phages could be made into drugs in less than 10 years."

“I was becoming increasingly frustrated that the drugs I was working on in the US, which typically cost $1bn to develop, were not accessible to most people living in developing countries.”


Unlike antibiotics, phages must be used in a highly targeted way, because each phage is effective against only a limited number of bacteria. They have, says Nagel, undergone important therapeutic and commercial development.

“In the near future, phages will be secondary to antibiotics as they can still work against most pathogens. Phages will be the last option when you have no choice,” says Sivachandran Parimannan, a researcher at the Centre of Excellenceat AIMST University in Kedah, Malaysia.

According to the World Health Organization (WHO), antimicrobial resistance is a rising threat to global health, jeopardising decades of medical progress and transforming common infections into deadly ones. A UN report published last year suggested yearly deaths from drug-resistant diseases could rise from the current 700,000 to 10 million in 30 years if no action is taken.

“In principle, phages are cheaper and quicker to develop than conventional drugs, can be designed to minimise future bacterial resistance and have no reported side-effects,” says Nagel. “They can be produced with relatively simple equipment that is readily available to scientists in developing countries, which are the most endangered by the rise of AMR.”

According to a 2014 study commissioned by British authorities, by 2050 approximately 90% of deaths attributable to AMR are expected to occur in Africa and Asia.

Research in phage therapy, relaunched over the past 10 years in Europe and the US, is still in its infancy in developing countries. Phage for Global Health hopes to promote a transfer of skills and knowledge.

There are downsides – phages are slower than antibiotics. Not readily available, they cannot be used in an emergency setting and time is usually needed to find the right phage to target the relevant bacteria. They have a narrow spectrum and are less stable than chemical drugs.

Phage therapy tends to be used in a personalised way which makes comparisons difficult and it is likely they are more efficient against certain bacteria, while antibiotics are more efficient against others, so new studies suggest it is better to combine both. Phage therapy centres such as the ones that exist in Poland and Georgia claim to have a success rate of 75-85%.

More research is needed to know if phage use has any negative effect on the human body, but so far few side-effects have been reported.

“There are specific needs in developing nations. Even common bacterial infections will have their own strains associated with specific countries,” says Martha Clokie, professor of microbiology at the University of Leicester and a trainer with PGH. “For example Salmonella food poisoning is a problem worldwide, but each African country will likely have different strains of the bacteria and therefore need specific phages.”

Since 2017, four two-week workshops have been organised in Africa, training about 100 scientists who have passed on what they have learned to more than 1,000 students.

A fifth workshop planned for Malaysia has been postponed to next year due to the pandemic. The global disruption has led Nagel to shift some activities online. Part of the learning material will be soon available on the website phage.directory thanks to a grant from the Mozilla Foundation.

“The training in Malaysia will sensitise more researchers from south-east Asian countries to the use of phages, which is not limited to human health but has applications for agriculture, livestock and food,” says Heraa Rajandas, a lecturer at AIMST, which will host the event.


An agar plate containing bacteriophage at a laboratory in Bengaluru, India.

At the end of the workshops, trainees know how to take phages from nature, isolate those corresponding to the target bacteria and characterise them, making use of DNA sequencing, to ensure that the phage does not have undesirable genetic properties.

Due in part to a lack of technological, financial and human resources, scientific teams working in the global south often focus more on animal and plant health, as well as food decontamination.

“The use of antibiotics by Ugandan farmers is massive, and this is a selection pressure for resistant organisms which can get to the human beings,” says Jesca Nakavuma, a microbiologist at Makerere University in Kampala who hosted the first PGH training in 2017. With funding from the African Union, she is working on phage cocktails that act against fish pathogens for aquaculture and now hopes to market them.

“There are also other ongoing projects from former participants on bovine mastitis, crop pathogens and on bacteria which are multidrug resistant such as E. coli, Klebsiella and Pseudomonas,” says Clokie.

PGH is also coordinating two transnational research programmes involving scientific institutions from Europe, North America and Africa. “In Kenya, two teams I collaborate with are working on phage cocktails against Campylobacter and Salmonella to decontaminate poultry meat, which is the cause of many food-borne infections,” says Nagel. The second project aims to test cholera phages in Bangladesh and then apply this treatment in the Democratic Republic of the Congo.

While producing phage-based drugs for food decontamination is already possible in some countries, the development of phage therapy for human health faces bigger hurdles. The lack of clinical trials meeting international standards means that – outside the former Soviet bloc – access to phages is either nonexistent or restricted to compassionate use. Many countries lack an appropriate regulatory framework.

In western countries, the manufacture of medicines must comply with strict criteria before they reach the market. GMP standards can increase the production cost of a drug by 10 times; this is a major obstacle for all players. There are other forms of controlled production which might enable developing countries to access drugs they desperately need at prices they can actually afford,” says Nagel, who, with several colleagues, has outlined the role the WHO could potentially play in overseeing the use of phage-based products in developing nations.

The WHO has not officially included phage therapy in its action plan against antibiotic resistance, but Nagel hopes this could change if new clinical trials prove positive.

 
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Lehigh University

Phage therapy gaining traction as a way to fight antibiotic-resistant bacterial infections*

by Emily Henderson, B.Sc.| News Medical Life Sciences | 28 Oct 2020

A phage is a virus that invades a bacterial cell. While harmless to human cells, phages are potentially deadly to bacteria, since many phages enter a cell in order to hijack its machinery in order to reproduce itself, thus destroying the cell.

While this is bad news for bacteria, it may be good news for humans. There is a growing need to develop new treatments that effectively attack deadly strains of bacteria that have become resistant to other medicines.

Already used with success in some parts of the world, phage therapy is gaining traction as a more widespread way to fight antibiotic-resistant bacterial infections and even, at some point, some viral infections including, according to a recent article possibly COVID-19.

Among the challenges: a virus type known as a prophage. A phage enters a bacterial cell and, instead of destroying it, takes up residence. Called a "prophage," it fights off other viruses' attempts to invade.

According to Vassie Ware , a professor in Lehigh University's Department of Biological Sciences, many bacterial strains contain prophages.

"These prophages," she says, "may provide defense systems that would make therapeutic uses of phages more challenging. In order to eradicate a pathogen, phages may need to overcome an already-in-residence prophage's defense systems."

Ware and her team recently conducted a study that focused on a phage called Butters that attacks a bacterial strain related to mycobacteria that cause tuberculosis or other human infections.

The group uncovered a two-component system of Butters prophage genes that encode proteins that "collaborate" to block entry and subsequent infection of some phages, but not others.

While the Butters prophage cannot protect the bacterial cell against all phage attacks, they discovered that more than one defense system is present in the Butters prophage defense repertoire. These weapons, they discovered, are specific for different types of phages.

These findings were published in an article earlier this month in mSystems, a journal of the American Society for Microbiology.​
Previous findings by several members of our research team working with other collaborators showed that prophages express genes that defend their bacterial host from infection by some specific groups of phages. For Butters, no genes involved in defense against specific phages had been previously identified.
With our experimental approach, we expected to identify genes involved in defense against infection by several phages, but were not expecting to uncover interactions between the two proteins that affected how one of the proteins functions in defense
."
Vassie Ware, Professor, Department of Biological Sciences, Lehigh University​
The Ware/Buceta team used a multidisciplinary approach to identify the genes and interactions. They utilized bioinformatics tools to predict structural features of proteins encoded by genes expressed by the Butters prophage and to probe databases for the presence of Butters genes within known bacterial strains.

Molecular biology techniques were used to engineer mycobacterial strains to express phage genes from the prophage.

Microbiology experiments included immunity plating efficiency assays for each engineered bacterial strain to determine if the gene in question would protect the engineered bacterial strain from infection by a particular phage type.

"This strategy," says Ware, "allowed identification of specific genes as part of the defense mechanism against specific viral attack."

They also conducted microscopy experiments for live-cell imaging to visualize the cellular location of phage proteins within engineered bacterial cells and to show a functional interaction between the phage proteins in question.

Biochemical experiments determined that the phage proteins likely interact physically as part of the defense mechanism.

"Collectively, these approaches provided data that allowed the team to construct a model for how the Butters prophage two-component system may function in defense against specific viral attack," says Ware.

Adds Ware: "The diversity of defense systems that exists demonstrates that efforts to establish generic sets of phage cocktails for phage therapy to kill pathogenic bacteria will likely be more challenging."

In addition to advancing phage therapy development, the team's discovery may also be important for engineering phage-resistant bacteria that could be used in the food industry and in some biotechnology applications.

*From the article here:
 
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When a virus is the cure

by Nicola Twilley | The New Yorker | 14 Dec 2020

As bacteria grow more resistant to antibiotics, bacteriophage therapy is making a comeback.

Some years before Joseph Bunevacz came to America, and decades before he got sick, he taught the Beatles how to ski. Or so he told me when I visited him at his home, on the arid northeastern slopes of the mountains that separate Los Angeles from the Mojave Desert, to learn more about an experimental medical treatment that he was hoping to receive for a strange and persistent infection in his blood. His wife, Filomena, took me through his medical history, consulting a stack of yellow legal pads in which, for the past five years, she has recorded countless tests and treatments. Yet Bunevacz, a bright-eyed seventy-nine-year-old with a shock of white hair, wearing an official Hungarian Olympic tracksuit, just wanted to tell wild, improbable stories about his younger years.

Born in Hungary in 1941, he trained as an athlete in his teens, as a way, he said, of escaping Communism. Short and not particularly muscular, he opted for dinghy sailing, reasoning that a lack of homegrown competition (Hungary has no coast, after all) might enable him to qualify for the national team, compete overseas, and then defect to the West. In 1960, after a respectable performance at a regatta on Lake Chiemsee, in Germany, his plan succeeded. He ended up in Munich, working in a department store and selling newspapers in the evenings. One night a year or two later, as he tells it, he heard music—catchy, melodious, altogether irresistible—drifting out of a club. The band was performing again the following night, so he came back early and struck up a conversation with the four young Liverpudlians. He took them to a ski resort nearby, he told me, and it quickly emerged that winter sports were not yet part of their repertoire. “You don’t believe me!” he exclaimed. (He wasn’t wrong.)

A love of music, Bunevacz said, brought him to America before the sixties were over. After hearing the gospel singer Mahalia Jackson perform in a Munich church, he moved to Detroit, and then travelled around the country working in hotels—in the kitchen, then behind the desk, and, eventually, as a manager at the Sheraton in Waikiki, at the time the world’s largest. There, he told me, he met the crooner Al Martino and the jazz pianist Oscar Peterson. He reminisced about later travels with the Hungarian National Olympic Committee, and lectured me on the best way to make strudel, smoked Hungarian sausage, and the fruit brandy pálinka.

Whenever Bunevacz paused for breath, Filomena, a retired nurse, filled me in on the dates of his various scans, his handful of colonoscopies, his gall-bladder operation, his bile-duct stent, the surgical removal of his upper colon, and his trips to urgent care. “Do you know how many blood cultures they have done on this man?” she said. “When I was a nurse, the patients who were this sick—they died.”

Despite his irrepressible good humor, Bunevacz is, indeed, very unwell. His case is also something of a medical mystery. His symptoms—fever, nausea, abdominal pain, and diarrhea—are easily explained: he is being poisoned by E. coli bacteria in his bloodstream. But it’s not clear what has been causing the infection to recur. When I saw him, Bunevacz had been going to his local emergency clinic every month, in order to receive huge doses of antibiotics, but after each treatment ended the infection would return. For years, doctors from across the country have scanned him, probed him, and sliced him open to inspect or remove the tissue in which they suspect the E. coli may lurk. Nothing has made the slightest difference.

“Honestly, I would have thought he would have died from this a year ago,” Emily Blodget, his infectious-disease consultant at the University of Southern California’s Keck Hospital, told me. Bunevacz is an optimist by nature, but the cost—financial as well as personal—of the procedures, along with the recurring fevers and pain, not to mention the side effects of the antibiotics, have begun to seem overwhelming. “I would try anything,” he said, in a rare moment of seriousness.

Late last year, the Bunevaczes’ daughter came up with a new suggestion: an emergency treatment, not yet approved by the F.D.A., that had saved the life of a man in San Diego. “She called and said, ‘Mom, you have to get Dad to do phage therapy,’ ” Filomena told me. “P-H-A-G-E,” Bunevacz clarified, nodding. So Filomena asked Blodget whether he might be a candidate for this mysterious new medicine.

Phages, or bacteriophages, are viruses that infect only bacteria. Each kingdom of life—plants, animals, bacteria, and so on—has its own distinct complement of viruses. Animal and plant viruses have always received most of our scientific attention, because they pose a direct threat to our health, and that of our livestock and crops. The well-being of bacteria has, understandably, been of less concern, yet the battle between viruses and bacteria is brutal: scientists estimate that phages cause a trillion trillion infections per second, destroying half the world’s bacteria every forty-eight hours. As we are now all too aware, animal-specific viruses can mutate enough to infect a different animal species. But they will not attack bacteria, and bacteriophage viruses are similarly harmless to animals, humans included. Phage therapy operates on the principle that the enemy of our enemy could be our friend. If Bunevacz’s doctors could find a virus that infected his particular strain of E. coli, it might succeed where antibiotics had failed.

“I’d heard of it,” Blodget said, when I asked her how she’d responded to Filomena’s question about phage therapy. “But in the past it was thought of as kind of fringe.” Recently, though, she’d seen reports describing patients whose long-standing, sometimes life-threatening bacterial infections had been eradicated by phage. Last year, a paper published in Nature Medicine documented the role of phages in saving the life of a teen-age cystic-fibrosis patient in the U.K., who was stricken with a bacterial infection after a double lung transplant. Another case study described how phages helped save a Minnesota man’s leg, which had become infected after knee surgery.



In the past five years, phage research has accelerated, with a proliferation of publications, conferences, and pharmaceutical-company investment. This enthusiasm reflects the ever-growing threat of antibiotic-resistant bacteria and a dearth of new antibiotics available to fight them. In 2016, the United Nations pronounced antibiotic resistance “the greatest and most urgent global risk.” Without reliable antibiotics, even relatively routine surgery—Cesarean sections, hernia repair, appendix or tonsil removal—could be deadly. One analysis published in a leading British medical journal estimated that, without antibiotics, one in seven people undergoing routine hip-replacement surgery might die from a drug-resistant infection. Already, some seven hundred thousand people die each year as a direct result of drug-resistant infections, a number that is predicted to rise to ten million by 2050.

The bacteria plaguing Bunevacz haven’t yet developed resistance to the full range of antibiotics, but Blodget told me that they inevitably would. Soon after Thanksgiving last year, he was identified as a viable candidate for the therapy, and Blodget told him that she thought it was worth a try. “I said, I don’t think it’s going to hurt, and it can possibly help,” she recalled. “I mean, at this point, there’s nothing else to do.”

The explanation for Blodget’s initial hesitance can be found in phage therapy’s complicated history. Although it is still considered an experimental treatment in the U.S., phages have been used to treat and prevent bacterial infections since their discovery, more than a century ago. For many American doctors, the obvious next question is: If they actually work, wouldn’t we know by now?

Part of the problem with phages is that they were discovered almost too early—far in advance of the technology and scientific understanding required to use them effectively. In 1915, a British bacteriologist named Frederick Twort reported the existence of an infectious agent capable of killing bacteria, but he didn’t pursue the finding. It was left to a French-Canadian scientist, Félix d’Hérelle, to name and describe phages, in 1917. Unfortunately, d’Hérelle was an autodidact working as a volunteer at the Institut Pasteur, in Paris. What’s more, he recklessly claimed that phages were the basis of the human immune response, in direct opposition to the Nobel Prize-winning research of the institute’s Brussels director, Jules Bordet, who had demonstrated that immunity was based on antibodies. D’Hérelle, with a lack of restraint that was apparently characteristic, described his superior’s work as laden with “monstrosities.” Bordet responded by championing Twort’s prior observation of phages; as a result, the credit for the discovery remains controversial.

D’Hérelle realized that bacteriophages congregated wherever bacteria did, and that a particularly fruitful source was effluvia from sick humans. He would mix fetid water with meat bouillon, wait until any bacteria had fed and multiplied, then pass the murky soup through a porcelain filter fine enough to remove the bacteria and leave the phages. He then evaluated the filtered dregs by pouring them into a test tube filled with the target bacterium. The results were promising. After “proving” the safety of phages by feeding them to himself, his young family, and some of his colleagues, d’Hérelle went on to inject them into the swollen lymph nodes of four people who had bubonic plague, effecting a seemingly miraculous cure. Phages were briefly all the rage: in 1925, Sinclair Lewis used them to tackle a fictional outbreak in his Pulitzer Prize-winning novel, “Arrowsmith.”

Still, Bordet and his admirers in the research establishment remained firmly opposed to the treatment, and many scientists considered the promise of phage therapy to be, at best, oversold—a perception that was not helped by d’Hérelle’s own rhetoric when he travelled to India at the behest of the British government, pouring phages into wells and promising an end to cholera. At this time, no one had seen a phage. An E. coli bacterium, two-thousandths of a millimetre long, is almost as small as the shortest wavelengths of light visible to the human eye under magnification, whereas the phages that attack it are a tenth of that size, or a hundred times smaller than the smallest thing we can see. Only with the invention of the scanning electron microscope, in 1937, did phages become visible, but because the first images were published in Nazi Germany it was years before British and American scientists saw them. Even today, most scientists “see” a phage only by the destruction it has wreaked on bacteria in a petri dish—clear, glassy zones of death scattered across a soupy, yellowish microbial lawn.



In the thirties, d’Hérelle, who was sympathetic to Communist ideals, was invited by Stalin to help establish a center for phage-therapy research in Tbilisi, in the Soviet republic of Georgia. During the Second World War, Soviet and German military medics carried vials of phages as part of their field kits, to prevent infection of wounds and burns. That connection with America’s adversaries made phages seem ideologically suspect to many in the West: as the medical historian William Summers has written, phage therapy acquired a “Soviet taint” in the postwar period, becoming “scientifically unsound because it was politically unsound.”

Still, as late as 1961, phage therapy had some American adherents, including Elizabeth Taylor, who received a dose of staph bacteriophage when she developed near-fatal pneumonia during the filming of “Cleopatra” and needed an emergency tracheotomy. By then, however, phage therapy had been superseded by penicillin, which had become widely available in the West after the war and quickly established itself as the preferred treatment for bacterial infections. Doctors in Eastern Europe continued to prescribe phages—delivered both topically and orally in powders, sprays, and syrups—but their counterparts on the other side of the Iron Curtain had, for the most part, barely even heard of them. Phages were still studied—Francis Crick and James Watson, two of the discoverers of the double-helix structure of DNA, both conducted phage research—but they were not part of modern medicine in Western Europe and the United States.

The rise of antibiotic-resistant bacteria was predicted by Alexander Fleming, the Scottish bacteriologist who discovered penicillin. In 1945, just seventeen years after his accidental breakthrough, he warned, “There is the danger that the ignorant man may easily under-dose himself, and by exposing his microbes to nonlethal quantities of the drug, make them resistant.” As early as 1947, penicillin-resistant staphylococcus bacteria were found in hospitals in England, but few heeded Fleming’s warning.

Antibiotics were systematically overused and abused (including as a growth aid in factory-farmed livestock), giving rise to a microbiological arms race, in which bacteria mutated new forms of resistance and scientists raced to develop powerful new classes of antibiotic. To make matters worse, fears of antibiotic resistance have, in recent decades, created a perverse incentive in medical research: new antibiotics, to remain effective, must be used sparingly, as so-called antibiotics of last resort. As a result, it is almost impossible to recoup the cost of developing them. No significant new antibiotics have been introduced since the nineteen-eighties, and, in 2001, the World Health Organization issued an urgent call to action to tackle antibiotic resistance. Phages were ready for their renaissance.

In November, 2015, Steffanie Strathdee, an infectious-disease epidemiologist at the U.C. San Diego School of Medicine, went on a vacation to Egypt with her husband, Tom Patterson, a professor of psychiatry. After visiting the pyramids, Patterson, sixty-eight at the time, became violently sick with what they at first assumed was food poisoning. But Egyptian doctors gave him a diagnosis of acute pancreatitis, and he was medevaced to Frankfurt, where tests revealed that he also had an abscess infected with a deadly, drug-resistant strain of Acinetobacter baumannii. Doctors tested his infection against fifteen powerful antibiotics, but only three had even a slight effect. Another air ambulance brought Patterson home to San Diego, where, within weeks, his infection evolved immunity to those three antibiotics, too. Patterson’s organs had begun to fail—first his heart and his lungs, and soon, it seemed, his kidneys—and he went into a coma. By the third week of February, 2016, his doctor, Robert Schooley, warned Strathdee that they were out of options.

Searching the biomedical literature for alternative treatments, Strathdee found a reference to phage therapy. She and Schooley, a human virologist by training, started contacting phage researchers around the world to see if any of them had a virus that might kill Patterson’s bug. They received phages originally isolated from sewage plants, Texas dirt, and lagoons of swine and cattle manure; colleagues then grew them in bulk and purified the resulting solution. Schooley received special approval from the F.D.A. to inject some phages into the plastic tubing draining fluid from Patterson’s abdominal cavity, near where the infection had originated, and to pump others directly into a vein. Three days later, Patterson emerged from his coma; after a few months, he was discharged, his infection entirely eradicated.

As Patterson underwent months of physical therapy and rehabilitation, Strathdee and Schooley began publicizing his case, describing it in a scientific paper, giving talks, and providing expert testimony to the National Institutes of Health. In July, 2018, they founded the first phage-therapy center in North America, the Center for Innovative Phage Applications and Therapeutics (IPATH), at U.C. San Diego, and began to build a library of phages. Patterson and Strathdee published a joint memoir about his miraculous recovery, and, as word started to spread, e-mails, calls, and Facebook messages began to flood in from people desperately hoping that phages could help their loved ones, too. It was Patterson’s case that Joseph Bunevacz’s daughter had heard about, and late last year Strathdee promised to take me along on her next phage-trapping expedition, as part of a national search to identify a phage that could kill Bunevacz’s pernicious E. coli.

Finding phages is not in itself particularly challenging: they are by far the most abundant biological entities on earth. According to one estimate, there are ten million trillion trillion phages, which is more than every other organism, including bacteria, combined. The average teaspoon of seawater holds five times more phages than there are people in Rio de Janeiro; for every grain of sand in the world, there are a trillion phages. But the best place to find phage that will kill drug-resistant bacteria is where people or animals have shed them—in other words, sewage.



The timing of a successful phage hunt in Southern California is thus strongly correlated with rainfall: during a severe storm, sewage-laced runoff pours straight into the ocean at a rate of millions of gallons a minute, leading health departments to close beaches and ban swimming and surfing for days. A year ago, after a brief downpour, I drove to Carlsbad, just north of San Diego, to meet Strathdee and Patterson for a day of phage hunting. First, though, we stopped for lunch at their favorite Mexican restaurant, a hole-in-the-wall called Juanita’s, a few blocks from the beach. “This taco was the first solid food I had back when I got out of the hospital in 2016,” Patterson said. “Didn’t stay down for long.” Patterson, now seventy-three, is lanky and youthful, all relaxed grin, Hawaiian shirt, and Southern California chill; Strathdee is Canadian by birth, and talks so fast that she frequently runs out of breath. Over carnitas, Patterson began describing the hallucinogenic experience of being in a coma. “I was a snake,” he explained. “And that’s not easy for people to grasp.” A man in the next booth leaned over and asked, “Are you Tom?” He’d seen Patterson and Strathdee speak at a local community college a few months before, and was curious whether phage therapy might one day help his daughter, who suffers from cystic fibrosis. “Tom’s the face of phage now,” Strathdee said. “Someone had to be.”

We drove ten minutes up the coast to a brackish wetland called Batiquitos Lagoon. Patterson parked just off I-5, which bisects the lagoon, and, with semis rumbling in the background, prepared to take a postprandial nap. Strathdee handed me a lunch cooler containing her phage-hunting kit and set off at a brisk pace toward the water. The path had turned to mud in the previous day’s rain, but above us the sky was bright blue, streaked with the wispiest of clouds, and the air smelled briny, with a strong sulfuric tang. As the freeway’s roar softened in the distance, I heard a frog croak, and we passed a large Leucadia Wastewater District truck, equipped with a cylindrical holding tank and a complicated set of pipes and pumps. Strathdee was delighted. “That’s the hydro-cleaning truck,” she said. “The sewage outflow must be blocked.”

For the next hour, I followed Strathdee as she dove into bulrushes and squelched through puddles, her acid-washed jeggings and swirly-patterned hoodie providing the opposite of camouflage. We filled vials with dubious brown liquid from the end of a rusted pipe, from water that had a coyote turd floating in it, and from the rotting, shrimp-scented swampy edges of the slough. We labelled each sample with a date and a number and dropped them in ziplock bags in the cooler. Then she and Patterson drove home, and I took our spoils to U.C. San Diego, to meet Hedieh Attai, a postdoctoral researcher. Attai joined the IPATH team to work on a new clinical trial of phage therapy that Robert Schooley is preparing to launch, and she spends most of her time refining a technique for measuring bacteria levels in sputum samples coughed up by patients with chest infections. “But we’re always looking for phages to build up our library,” she said cheerfully, as I handed her the cooler.

Attai keeps a freezer of E. coli, Enterococcus, and Pseudomonas—three of the six pathogens that together cause most hospital-acquired infections. To see if Strathdee and I had found anything useful, she would pit the unknown phages in our sludge samples against these heavyweights of the bacterial world. Wearing a lab coat, goggles, and gloves, she put a dish of nutrient-rich jelly on a turntable and then, in a process that resembled coating a frying pan with oil, swirled it to distribute a layer of pathogenic E. coli. Elsewhere, our samples were sucked through a filter with pores small enough to remove any bacteria, leaving only the phages. The previously murky liquid came out crystal clear—it looked good enough to drink. “I can’t let you do that,” Attai said, with a nervous laugh. She did, however, let me draw the phage samples into a syringe and squirt a series of identical droplets onto the bacterial film.

If none of the phages we’d found were capable of attacking these particular bacteria, the pathogenic microbes would continue growing undisturbed. But, if the liquid contained a single phage that was a match for this particular host, that phage would bind to the bacterial cell membrane and insert its genome into the fluid-filled interior. Once inside an E. coli cell, the phage would take over, mimicking and exploiting the bacterium’s own signalling pathways in order to force the cell’s protein-manufacturing machinery to start printing out copy after copy of the phage genome instead. Eventually, the E. coli cell would become so stuffed with phage copies that it would burst, releasing a horde of phages ready to invade the next bacterial cell. We would know in a day or two if our phage had been successful by the appearance of a circle of dead microbes puncturing the thick layer of E. coli.

Across the U.C. San Diego campus from IPATH is the office of Saima Aslam, a transplant specialist who has probably become the leading phage-therapy physician in the United States, having treated ten patients, with more pending, and advised on a number of other cases around the country. She came to phages in a roundabout way: transplants require immunosuppression, leaving her patients vulnerable to hospital-acquired infections, which are, increasingly, antibiotic resistant.

In the waiting room the day I visited was a man in his early eighties named Napoleon Del Fierro, a retired electrician, originally from the Philippines, who had served in the U.S. Navy. He was there with his wife, Violeta, a former nurse, and their son, Dino, a pediatric dentist. While he rested his head in his hand to sleep, occasionally blinking his eyes slowly open, his family and Aslam told me about his case. A few years ago, after suffering from congestive heart failure for nearly a decade, he’d had a pump implanted just under his sternum to take over the work of circulating blood around his body. Almost immediately, the area had become infected with Pseudomonas. “The pump is so infected, it’s eroding the bone, and so he’s got a couple of holes where pus just constantly comes out,” Aslam said. “The infection is a slime layer on the device—we call it biofilm—and his immune system and antibiotics can’t get to it.” The pump couldn’t be replaced—Del Fierro would not survive the surgery required to remove something so deeply embedded—and so the infection just smoldered, with bacteria sloughing off into his bloodstream and occasionally sending him into septic shock.



Violeta had read about Tom Patterson’s case in People magazine; Napoleon’s daughter Divina wrote one of the hundreds of pleading e-mails that Strathdee routinely receives and forwards to Aslam. By the time I met Del Fierro, it was four months since he had undergone his first round of phage therapy: a surgeon had opened him up, removed pus and dead tissue, and applied phages directly to the device; then he was given further doses of phage, in combination with antibiotics, intravenously for six weeks. “He looked great—everything was great,” Aslam said. “I really thought we had eradicated his infection.” But, as soon as she stopped his antibiotic dose, the infection came back. Aslam admitted that she was “very, very disappointed.” Still, she told the family that she’d just heard that researchers had found a couple of phages that were highly active against his Pseudomonas, and she was preparing the paperwork to secure F.D.A. approval for another round.

Later, after the family had left, Aslam told me that she was trying to keep their and her own expectations low. “You know, he’s eighty-three, he’s got a device in his heart, he’s got this very drug-resistant infection, he’s failed a course of therapy already,” she said. “But I hope it cures him. I want to cure him.”

The excitement created by success stories like Patterson’s is itself infectious. But Aslam explained that phage therapy is still a long way from being a standard treatment. Because phage cocktails are classed as experimental drugs, each patient requires a waiver from the F.D.A. and approval from the review board of whatever medical facility is involved, and health insurance doesn’t cover any of the costs. Despite an abundance of inspiring case studies, there haven’t been good clinical trials of phage, the next step before it can become part of standard medical care. “There’s amazing promise, and we’ve had some wonderful outcomes,” Aslam said. “But each time I do this I feel like I have ten other questions—maybe I should do it this way or that way?”

She worried that the dose initially applied to Del Fierro’s heart pump hadn’t been high enough, but the research to determine the right dose hasn’t yet been done. It’s also possible that biofilms like the one on his device are not suitable for phage treatment. They are anaerobic and made of polysaccharides, and some scientists believe that environments with lots of sugars and no oxygen can cause phages to lose their killing ability and become more “temperate,” coexisting in harmony with their bacterial hosts. On the other hand, lab studies seem to show that some phages release enzymes that could help them penetrate biofilms.

One of Strathdee and Schooley’s goals with IPATH has been to conduct the first clinical trial of intravenous phage therapy, with cystic-fibrosis patients. They are hoping to establish basic therapeutic principles: the best dose, and the best way of administering it; how the phages interact with a bacterial host in the human body; what side effects there might be. Schooley’s major challenge has been securing a phage supply. “We could have started it two and a half years ago if we had a phage source,” he said. The pandemic has delayed the trial yet further. In the meantime, a handful of labs and small startups volunteer their time and their phage libraries to help Aslam and others treat sick patients; finding an institution or a company that is willing and able to invest in the basic clinical trials needed to learn how phages work has been all but impossible.

Forest Rohwer, a microbial ecologist at San Diego State University, pointed to a more fundamental problem. In a dynamic ecosystem, whether a coral reef or our bodies, enemies and friends are situational rather than static. Indeed, phage viruses are responsible for creating the majority of pathogenic bacteria in the first place, thanks to their ability to move genes around. An E. coli bacterium is usually harmless until it acquires virulence genes from an invading temperate phage. A cholera outbreak is both triggered by phages and halted by them: one kind of phage donates a virulence gene to cholera bacteria, causing it to expand its range, only for another kind to hijack those newly vulnerable pathogenic bacteria to make copies of itself. Sick or healthy humans are just a side effect. Although Rohwer is excited about phage’s therapeutic possibilities—his lab purified part of Tom Patterson’s phage cocktail—he worries that our ambitions to manipulate an entire ecosystem within the human body might overstep our abilities, and that the unintended consequences might be as unwelcome as the pathogenic bacteria itself. “They can kill you, no problem,” he said. “You get the wrong phage and the right bacteria and you’re dead.”

Phage therapy thus continues to be a boutique affair—just a few patients, each treated with a personalized phage cocktail scavenged from moldy eggplants, cesspools, and pig farms. It’s also hit-and-miss: the phages that Strathdee and I collected at Batiquitos Lagoon turned out, unfortunately, not to be a good match for Joseph Bunevacz’s infection.



In mid-January, Napoleon Del Fierro began receiving a phage injection, twice daily, through a port in his arm. There were four phages in his dose, all isolated from wastewater-treatment facilities near Walter Reed Army Institute of Research, which prepared the treatment. When I visited him, at the end of the month, he was asleep after a big morning: he’d finished a breakfast of oatmeal and managed to get out of bed for the first time in two weeks. “He was sitting up,” Violeta said. “I hope that’s the start.” We sat together by his bed while Violeta told me how they met, back in Manila; his brother borrowed her sister’s textbooks after school. A nurse came in and, as she rearranged his blankets to tuck him in more comfortably, gave us the good news that Del Fierro’s latest sample results had just come back and showed significantly lower levels of Pseudomonas.

By February 10th, the medical team decided that Del Fierro was healthy enough to continue treatment from home. But, just as he was about to be discharged, he began vomiting dark-brown fluid, and his temperature soared. He had suffered a gastrointestinal bleed, and fluid from his abdomen had entered his lungs, causing aspiration pneumonia. Meanwhile, the Pseudomonas levels in his bloodstream had crept up again. Although he could no longer speak, it was clear that he was now in considerable pain. On the afternoon of February 22nd, his family gathered around his bedside, and his heart pump was switched off. He died a few minutes later.

When I spoke with Divina after the funeral, she told me that she still believed in the promise of phage therapy. “It just didn’t have a chance to perform,” she said. “It was up against such a big obstacle, in a vessel that was so compromised. I’m just eternally grateful they even gave it a shot.” Aslam, however, was discouraged. “That’s the second Pseudomonas biofilm infection I’ve treated where the outcome has been really difficult,” she said. “We try to help everyone, but we really need clinical trials to figure out why in some cases it just doesn’t work.” Scientists in the IPATH team had begun analyzing samples from Del Fierro, to try to understand why therapy failed, but this work is now on hold because of COVID-19.

There was better news from Baylor College of Medicine, where researchers had isolated phages that were active against Joseph Bunevacz’s E. coli infection. As Southern California emerged from late-spring rains into a dazzling superbloom, Filomena texted me a photo of the couple embracing on a hillside blanketed with poppies. As it turned out, the coronavirus outbreak was about to slow everything down, and it was late fall before his treatment received F.D.A. approval. This month, Bunevacz should finally be able to start his phage therapy. “It’s a beautiful life,” he said when I met him. “And I’d like to push it a little longer.”

 
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Scientists investigate phages that can kill the world's leading superbug*

by Monash University | PHYS | 12 Jan 2021

A major risk of being hospitalised is catching a bacterial infection.

Hospitals, especially areas including intensive care units and surgical wards, are teeming with bacteria, some of which are resistant to antibiotics—they are infamously known as 'superbugs.'

Superbug infections are difficult and expensive to treat, and can often lead to dire consequences for the patient.

Now, new research published today in the prestigious journal Nature Microbiology has discovered how to revert antibiotic-resistance in one of the most dangerous superbugs.

The strategy involves the use of bacteriophages (also known as 'phages').

"Phages are viruses, but they cannot harm humans," said lead study author Dr. Fernando Gordillo Altamirano, from the Monash University School of Biological Sciences.

"They only kill bacteria."

The research team investigated phages that can kill the world's leading superbug, Acinetobacter baumannii, which is responsible for up to 20 percent of infections in intensive care units.

"We have a large panel of phages that are able to kill antibiotic-resistant A. baumannii," said Dr. Jeremy Barr, senior author of the study and Group Leader at the School of Biological Sciences and part of the Centre to Impact AMR.

"But this superbug is smart, and in the same way it becomes resistant to antibiotics, it also quickly becomes resistant to our phages," Dr. Barr said.


Acinetobacter baumannii

The study pinpoints how the superbug becomes resistant to attack from phages, and in doing so, the superbug loses its resistance to antibiotics.

"A. baumannii produces a capsule, a viscous and sticky outer layer that protects it and stops the entry of antibiotics," said Dr. Gordillo Altamirano.

"Our phages use that same capsule as their port of entry to infect the bacterial cell."

"In an effort to escape from the phages, A. baumannii stops producing its capsule; and that's when we can hit it with the antibiotics it used to resist."


The study showed resensitisation to at least seven different antibiotics.

"This greatly expands the resources to treat A. baumannii infections," Dr. Barr said.

"We're making this superbug a lot less scary."

Even though more research is needed before this therapeutic strategy can be applied in the clinic, the prospects are encouraging.

"The phages had excellent effects in experiments using mice, so we're excited to keep working on this approach," said Dr. Gordillo Altamirano.

"We're showing that phages and antibiotics can work great as a team."

*From the article here :
 
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Phage viruses can make superbugs susceptible to antibiotics again

by Michael Irving | New Scientist | 12 Jan 2021

Viruses firmly hold the world’s attention at the moment, but we shouldn’t ignore the rising health threat that bacteria pose, too. The crafty critters are fast evolving resistance to antibiotics, meaning our best drugs could soon stop working entirely. Now researchers in Australia have found a way to bypass drug resistance in these so-called superbugs – by distracting them with predatory viruses.

Antibiotics were one of the most important medical breakthroughs of the 20th century, saving countless lives by clearing out infections that previously may have been lethal. Unfortunately, we’ve been locked in a biological arms race ever since, as bacteria develop better and better defenses against the drugs.

And the tide is slowly turning in their favor. Our last line of defense is already beginning to fail, with some bacteria now impervious to anything we can throw at them. Studies have predicted that if this trend continues, superbugs could kill as many as 10 million people a year by 2050.

In an effort to find new treatments, scientists are beginning to circle back to old, discarded ideas. At the top of the list is phage therapy, which uses bacteriophages – tiny viruses that prey solely on bacteria – to hunt down the superbugs. Since antibiotics were discovered soon after phages were, there was never a dire need to develop phage therapy further. Until now.

For the new study, researchers from Monash University set out to find a phage that would target and kill a superbug called Acinetobacter baumannii. This opportunistic bacteria, often acquired in hospitals, is currently priority target number one on the World Health Organization’s hit list.

The team identified a phage from wastewater that almost completely wiped out A. baumannii in lab culture tests. Unfortunately, the effect was short-lived, and it only took a few hours before the bacteria developed resistance to the phages. But there’s an intriguing upside to the story: in developing resistance to phages, the bacteria became vulnerable to antibiotics again.

"A. baumannii produces a capsule, a viscous and sticky outer layer that protects it and stops the entry of antibiotics," says Gordillo Altamirano, lead author of the study.

"Our phages use that same capsule as their port of entry to infect the bacterial cell. In an effort to escape from the phages, A. baumannii stops producing its capsule; and that's when we can hit it with the antibiotics it used to resist."

In tests, the phage therapy was found to re-sensitize the bacteria to at least seven different antibiotics that it was once resistant to. Phage therapy proved effective in tests on mice, raising hopes that the two could work well as a team in the future.

The research was published in the journal Nature Microbiology.

 
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CRISPR Phage offers precision medicine tool to fight bacterial infections

by Samantha Black, PhD | The Science Advisory Board | 20 Nov 2020

November 20, 2020 -- In an attempt to combat bacterial infections, Locus Biosciences is developing bacteriophage therapies that deliver CRISPR-Cas3 machinery directly to specific, targeted pathogens to obliterate them. Locus CEO Paul Garofolo discussed the company's technology and recent updates with The ScienceBoard.net.

Locus Biosciences comes from humble beginnings. In fact, it was more of a project than a fully fleshed out business plan. In the spring of 2015, Garofolo participated in an executive-in-residence program at the Poole College of Management as part of the Technology, Entrepreneurship, and Commercialization (TEC) Program at North Carolina State University (NC State).

Paul Garofolo, CEO of Locus Biosciences.

Paul Garofolo, CEO of Locus Biosciences.

As part of an entrepreneurship class, Garofolo was assigned to work with a team of students to develop a new business idea.

One student, Ahmed Gomaa, a PhD student at the time, came to the group excited about the idea of CRISPR-Cas3 systems. Ahmed explained how CRISPR-Cas3 works more like PacMan than as a pair of scissors. The team decided to pursue Ahmed's plan and presented their work at the end of the semester.

Garofolo, along with two NC State professors and one student, worked out a startup arrangement with the university and the company was officially launched in May 2015 -- just three weeks after the entrepreneurship course finished. The company quickly moved into a lab on NC State's centennial campus and then to the main campus for a year before moving to a more permanent location in Research Triangle Park.

Developing a complete tool

From the beginning, the Locus team knew that they had a powerful tool. But they were missing an important component to the system -- the delivery vector. During the first few years, the team worked through several options, including peptides, nanoparticles, and phages.

"Phage pretty quickly filtered to the top - in safety profile and use in humans," Garofolo explained.

By the time that Locus was moving off campus, in late 2016, they knew they had a working product and began to amass preclinical data. Throughout 2017, they continued to build out their preclinical animal dataset and closed $19 million in series A financing. They settled on their first lead asset for recurrent urinary tract infections, and this is when Locus really began to take off, according to Garofolo.

With a plan in place, Locus was able to meet with the U.S. Food and Drug Administration (FDA) in early 2018 to review its plan to submit an investigational new drug application for urinary tract infection and advance the candidate towards clinical trials.

"Using phage as an antimicrobial agent is hugely powerful," explained Garofolo.

Generally, the company gets 100-1,000 times greater activity in preclinical testing compared to standard of care antibiotics. This is achieved by delivering CRISPR-Cas3 systems that directly target specific pathogens, via engineered bacteriophages as part of CRISPR Cas3-enhanced bacteriophage (crPhage).

High-throughput screening is used to select phages that naturally target clinically relevant pathogens followed by synthetic biology processes to insert CRISPR-Cas3 constructs into phage genomes.

This novel modality gives the company precise control to target individual bacterial species of interest. These powerful tools immediately gained the attention of the industry, spurring collaboration and partnerships.

In January 2019, the company landed its first strategic partnership with Johnson & Johnson for the development, manufacture, and commercialization of crPhage targeting infections of the lung and other organs caused by two common pathogens.

The company announced on September 30 that it has partnered with the U.S. Biomedical Advanced Research and Development Authority (BARDA) on its lead asset -- the one they decided on back in 2018 -- targeting recurrent urinary tract infections caused by Escherichia coli. The groups will co-fund the development of this product through phase II/III clinical trials.

More recently, on November 10, the company announced that it had joined forces with the Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) on a second crPhage product targeting recurrent urinary tract infections caused by Klebsiella pneumoniae. This partnership will fund preclinical and phase I clinical development of the product.

Why phage?

Working with bacteriophages is advantageous for Locus, according to Garofolo. This is because there are already many generally recognized as safe (GRAS) phage products in the U.S. for food processing and agricultural applications. Given that, the FDA was very familiar with phages when Locus began regulatory discussions with the agency.

Garofolo explained that the company's experience in working with the FDA's Office of Vaccine Research and Review in the Center for Biologics Evaluation and Research has been extremely collaborative.

Because of the agency's familiarity with phage therapy, quality measures for clinical trial material such as identity testing, purity testing, and primary item specifications were relatively straightforward, as they already had a track record for evaluation. The agency and the company were already on the same page about potential manufacturing impurities inherent to bacteriophages (e.g. endotoxins).

Another benefit of working with phages versus other biologics is that phages can't infect human cells. Garofolo describes this as a huge win for the company.

On the other hand, phages are notoriously difficult to manufacture. In fact, Garofolo explained how in his whole professional career, prior to Locus, he did everything he could to avoid "phage-out" (many pharmaceutical biologic manufacturing processes are particularly susceptible to damage by bacteriophages). Therefore, the primary challenge in manufacturing Locus' crPhage product was getting a mainstream manufacturing organization to allow its phage product in the facility.

To overcome this, the company made a $12 million investment to internalize good manufacturing practice (GMP) capabilities with a 10,000-sq-ft facility. Now, the company has the independence to manufacture at will without the timing problems that other startup or medium sized biotech companies experience in working with contract manufacturing organizations (CMOs).

On the horizon

Currently, Locus is focused on finishing its phase I clinical trial of lead asset No. 1 (recurrent urinary tract infection caused by E. coli.). Garofolo reported that Locus just finished enrolling patients in their first phase I clinical trial, and they expect to report topline results in early 2021.

The company is also exploring applications of crPhage beyond infectious diseases. They have some interest in pursuing immunology indications including Crohn's disease and colitis, and they also have several oncology targets in mind.

"Wherever you know the bug that you need to get out of the body to have a therapeutic effect, we've got the world's most powerful tool to remove that bug," Garofolo explained.

 
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Are phages overlooked mediators of health and disease?

by Catherine Offord | Ther Scientist | 1 Feb 2021

Bacteria-infecting viruses affect the composition and behavior of microbes in the mammalian gut—and perhaps influence human biology.

When microbiologist Breck Duerkop started his postdoc in 2009, he figured he’d be focusing on bacteria. After all, he’d joined the lab of microbiome researcher Lora Hooper at the University of Texas Southwestern Medical Center in Dallas to study host-pathogen interactions in the mammalian gut and was particularly interested in what causes some strains of normally harmless commensal bacteria, such as Enterococcus faecalis, to become dangerous, gut-dominating pathogens. He’d decided to explore the issue by giving germ-free mice a multidrug-resistant strain of E. faecalis that sometimes causes life-threatening infections in hospital patients, and analyzing how these bacteria express their genes in the mouse intestine.

Not long into the project, Duerkop noticed something else going on: some of the genes being expressed in E. faecalis weren’t from the regular bacterial genome. Rather, they were from bacteriophages, bacteria-infecting viruses that, if they don’t immediately hijack and kill the cells they infect, can sometimes incorporate their genetic material into the bacterial chromosome. These stowaway viruses, known as prophages while they’re in the bacterial chromosome, may lie dormant for multiple bacterial generations, until certain environmental or other factors trigger their reactivation, at which point they begin replicating and behaving like infectious agents once again. (See illustration below.) Duerkop’s data showed that the chromosome of the E. faecalis strain he was using contained seven of these prophages and that the bacteria were churning out virus particles with custom combinations of these prophage sequences during colonization of the mouse gut.

The presence of viruses in Duerkop’s E. faecalis strain wasn’t all that surprising. Natural predators of bacteria, bacteriophages are the most abundant biological entities on the planet, and in many fields, researchers take their presence for granted. “Nobody really was thinking about phages in the context of bacterial communities in animal hosts," Duerkop says. “It would have been very easy to just look at it and say, ‘Oh, there are some phage genes here. . . . Moving on.’” But he was curious about why E. faecalis would be copying and releasing them, rather than leaving the prophages asleep in its chromosome, while it was trying to establish itself in the mouse intestine.

Encouraged by Hooper, he put his original project on hold in order to dig deeper. To his surprise, he discovered that the E. faecalis strain, known as V583, seemed to be using its phages to gain a competitive advantage over related strains. Experiments with multiple E. faecalis strains in cell culture and in mice showed that the phage particles produced by the bacteria didn’t harm other V583 cells, but infected and killed competing strains. Duerkop and his colleagues realized that, far from being background actors in the bacterial community, the phages “are important for colonization behavior” for this opportunistic pathogen.

The idea that a phage could play such a significant role in the development of the gut bacterial community was relatively novel when the team published its results in 2012. "Since then, it’s been pretty well established that phages can shape the assembly of microbial communities in the intestine, and that can influence the outcome on the host—either beneficially or detrimentally,” says Duerkop, who now runs his own lab at the University of Colorado School of Medicine in Aurora. There’s evidence that phages help bacteria share genetic material with one another, too, and may even interact directly with the mammalian immune system, an idea that Duerkop says would have had you “laughed out of a room” of immunologists just a few years ago.​

Predation by phages influences bacterial communities

Around the time that Duerkop was working on E. faecalis in Dallas, University of Oxford postdoc Pauline Scanlan was studying Pseudomonas fluorescens, a bacterial species that is abundant in the natural environment and is generally harmless to humans, although it’s in the same genus as the important human pathogen Pseudomonas aeruginosa. "Bacteria in this genus sometimes evolve what’s known as a mucoid phenotype—that is, cells secrete large amounts of a compound called alginate, forming a protective goo around themselves. In P. aeruginosa, this goo can help the bacteria evade the mammalian immune system and antibiotics, and when it crops up, it’s not good news” for the patient," Scanlan says. She was curious about what causes a non-mucoid bacterial population to evolve into a mucoid one and had found previous research suggesting that the presence of bacteriophages could play a role. Other studies documented high densities of phages in mucus samples from the lungs of some cystic fibrosis patients with P. aeruginosa infections.

Working in the lab of evolutionary biologist Angus Buckling (now at the University of Exeter), Scanlan grew a strain of P. fluorescens with a phage called Phi2 that specifically infects and destroys this bacterium. Cells with the gummy mucoid coating, the researchers noted, were more resistant to phage infection than regular cells were. What’s more, over generations, bacterial populations were more likely to evolve the mucoid phenotypes in the presence of Phi2 than they were in its absence, indicating that the phenotype may arise in Pseudomonas as an adaptive response to phage attack. Scanlan, now at University College Cork (UCC) in Ireland, notes that more work is needed to extend the findings to a clinical setting, but the results hint that phages could in some cases be responsible for driving bacteria to adopt more virulent phenotypes.

Such a role for viruses in driving bacterial evolution fits well with phages’ reputation as “the ultimate predators,” says Colin Hill, a molecular microbiologist also at UCC who got his introduction to phages studying bacteria used in making fermented foods such as cheese. Hill notes an estimate commonly cited in the context of marine biology—a field that explored phage-bacteria interactions long before human biology did—that phages kill up to 50 percent of the bacteria in any environment every 48 hours. “The thing that any bacterium has on its mind most, if bacteria had minds, would be phage,” Hill says, “because it’s the thing most likely to kill them.”

Several in vivo animal studies lend support to the idea that predatory phages help shape bacterial evolution and community composition in the mammalian microbiome. In 2019, for example, researchers at Harvard Medical School reported that phages not only directly affect the bacteria they infect in the mouse gut, but also influence the rest of the microbiome community via cascading effects on the chemical and biological composition of the gut. Observational studies hint at similar processes at work in the human gut. A few years ago, researchers at Washington University Medical School in St. Louis observed patterns of phage and bacterial population dynamics that resembled predator-prey cycles in the guts of children younger than two years old: low bacterial densities at birth were followed by decreases in phages, after which the bacteria would rebound, and then the phages would follow suit. The team concluded that these cycles were likely a natural part of healthy microbiome development.

Although researchers are only just beginning to appreciate the importance of phages in microbiome dynamics, they’ve already begun to explore links to human disease. Authors of one 2015 study reported that Crohn’s disease and ulcerative colitis patients showed elevated levels of certain phages, particularly within the viral order Caudovirales. They proposed that an altered virome could contribute to pathogenesis through predator-prey interactions between phages and their bacterial hosts. Other studies have explored possible phage-driven changes in the bacterial community in human diseases such as diabetes and certain cancers that are known to be associated with a disrupted microbiome. But the observational nature of human microbiome studies prevents conclusions about what drives what—changes in virome composition could themselves be the result of disruptions to the bacterial community, for example.
Currently, researchers are exploring the possibility of using predatory phages as weapons against pathogenic bacteria, particularly those that present a serious threat to public health due to the evolution of resistance to multiple antibiotics. "It’s the principle that the enemy of my enemy is my friend,” says Yale University virologist and evolutionary biologist Paul Turner. “If we have a pathogen that is in your microbiome, can we go in and remove that bacterial pathogen by introducing a predatory phage, something that is cued to only destroy [that pathogen]? Although the strategy was first proposed more than a century ago, we and others are trying to update it,”he adds.

Bacteria-infecting viruses affect the composition and behavior of microbes in the mammalian gut—and perhaps influence human biology.

When microbiologist Breck Duerkop started his postdoc in 2009, he figured he’d be focusing on bacteria. After all, he’d joined the lab of microbiome researcher Lora Hooper at the University of Texas Southwestern Medical Center in Dallas to study host-pathogen interactions in the mammalian gut and was particularly interested in what causes some strains of normally harmless commensal bacteria, such as Enterococcus faecalis, to become dangerous, gut-dominating pathogens. He’d decided to explore the issue by giving germ-free mice a multidrug-resistant strain of E. faecalis that sometimes causes life-threatening infections in hospital patients, and analyzing how these bacteria express their genes in the mouse intestine.

Not long into the project, Duerkop noticed something else going on: some of the genes being expressed in E. faecalis weren’t from the regular bacterial genome. Rather, they were from bacteriophages, bacteria-infecting viruses that, if they don’t immediately hijack and kill the cells they infect, can sometimes incorporate their genetic material into the bacterial chromosome. These stowaway viruses, known as prophages while they’re in the bacterial chromosome, may lie dormant for multiple bacterial generations, until certain environmental or other factors trigger their reactivation, at which point they begin replicating and behaving like infectious agents once again. (See illustration below.) Duerkop’s data showed that the chromosome of the E. faecalis strain he was using contained seven of these prophages and that the bacteria were churning out virus particles with custom combinations of these prophage sequences during colonization of the mouse gut.

The presence of viruses in Duerkop’s E. faecalis strain wasn’t all that surprising. Natural predators of bacteria, bacteriophages are the most abundant biological entities on the planet, and in many fields, researchers take their presence for granted. “Nobody really was thinking about phages in the context of bacterial communities in animal hosts," Duerkop says. “It would [have been] very easy to just look at it and say, ‘Oh, there are some phage genes here. . . . Moving on.’” But he was curious about why E. faecalis would be copying and releasing them, rather than leaving the prophages asleep in its chromosome, while it was trying to establish itself in the mouse intestine.
Predation is just one type of phage-bacteria interaction taking place within the mammalian microbiome; many phages are capable of inserting their genomes into the bacterial chromosome.

Encouraged by Hooper, he put his original project on hold in order to dig deeper. To his surprise, he discovered that the E. faecalis strain, known as V583, seemed to be using its phages to gain a competitive advantage over related strains. Experiments with multiple E. faecalis strains in cell culture and in mice showed that the phage particles produced by the bacteria didn’t harm other V583 cells, but infected and killed competing strains. Duerkop and his colleagues realized that, far from being background actors in the bacterial community, the phages “are important for colonization behavior” for this opportunistic pathogen.

The idea that a phage could play such a significant role in the development of the gut bacterial community was relatively novel when the team published its results in 2012. "Since then, it’s been pretty well established that phages can shape the assembly of microbial communities in the intestine, and that can influence the outcome on the host—either beneficially or detrimentally,” says Duerkop, who now runs his own lab at the University of Colorado School of Medicine in Aurora. There’s evidence that phages help bacteria share genetic material with one another, too, and may even interact directly with the mammalian immune system, an idea that Duerkop says would have had you “laughed out of a room” of immunologists just a few years ago.​

Predation by phages influences bacterial communities

Around the time that Duerkop was working on E. faecalis in Dallas, University of Oxford postdoc Pauline Scanlan was studying Pseudomonas fluorescens, a bacterial species that is abundant in the natural environment and is generally harmless to humans, although it’s in the same genus as the important human pathogen Pseudomonas aeruginosa. Bacteria in this genus sometimes evolve what’s known as a mucoid phenotype—that is, cells secrete large amounts of a compound called alginate, forming a protective goo around themselves. "In P. aeruginosa, this goo can help the bacteria evade the mammalian immune system and antibiotics, and when it crops up, it’s not good news for the patient," Scanlan says. She was curious about what causes a non-mucoid bacterial population to evolve into a mucoid one and had found previous research suggesting that the presence of bacteriophages could play a role. Other studies documented high densities of phages in mucus samples from the lungs of some cystic fibrosis patients with P. aeruginosa infections.

Working in the lab of evolutionary biologist Angus Buckling (now at the University of Exeter), Scanlan grew a strain of P. fluorescens with a phage called Phi2 that specifically infects and destroys this bacterium. Cells with the gummy mucoid coating, the researchers noted, were more resistant to phage infection than regular cells were. What’s more, over generations, bacterial populations were more likely to evolve the mucoid phenotypes in the presence of Phi2 than they were in its absence, indicating that the phenotype may arise in Pseudomonas as an adaptive response to phage attack. Scanlan, now at University College Cork (UCC) in Ireland, notes that more work is needed to extend the findings to a clinical setting, but the results hint that phages could in some cases be responsible for driving bacteria to adopt more virulent phenotypes.

Such a role for viruses in driving bacterial evolution fits well with phages’ reputation as “the ultimate predators,” says Colin Hill, a molecular microbiologist also at UCC who got his introduction to phages studying bacteria used in making fermented foods such as cheese. Hill notes an estimate commonly cited in the context of marine biology—a field that explored phage-bacteria interactions long before human biology did—that phages kill up to 50 percent of the bacteria in any environment every 48 hours. “The thing that any bacterium has on its mind most, if bacteria had minds, would be phage,” Hill says, “because it’s the thing most likely to kill them.”

Several in vivo animal studies lend support to the idea that predatory phages help shape bacterial evolution and community composition in the mammalian microbiome. In 2019, for example, researchers at Harvard Medical School reported that phages not only directly affect the bacteria they infect in the mouse gut, but also influence the rest of the microbiome community via cascading effects on the chemical and biological composition of the gut. Observational studies hint at similar processes at work in the human gut. A few years ago, researchers at Washington University Medical School in St. Louis observed patterns of phage and bacterial population dynamics that resembled predator-prey cycles in the guts of children younger than two years old: low bacterial densities at birth were followed by decreases in phages, after which the bacteria would rebound, and then the phages would follow suit. The team concluded that these cycles were likely a natural part of healthy microbiome development.

Although researchers are only just beginning to appreciate the importance of phages in microbiome dynamics, they’ve already begun to explore links to human disease. Authors of one 2015 study reported that Crohn’s disease and ulcerative colitis patients showed elevated levels of certain phages, particularly within the viral order Caudovirales. They proposed that an altered virome could contribute to pathogenesis through predator-prey interactions between phages and their bacterial hosts. Other studies have explored possible phage-driven changes in the bacterial community in human diseases such as diabetes and certain cancers that are known to be associated with a disrupted microbiome. But the observational nature of human microbiome studies prevents conclusions about what drives what—changes in virome composition could themselves be the result of disruptions to the bacterial community, for example.

Currently, researchers are exploring the possibility of using predatory phages as weapons against pathogenic bacteria, particularly those that present a serious threat to public health due to the evolution of resistance to multiple antibiotics. It’s the principle that “the enemy of my enemy is my friend,” says Yale University virologist and evolutionary biologist Paul Turner. “If we have a pathogen that is in your microbiome, can we go in and remove that bacterial pathogen by introducing a predatory phage, something that is cued to only destroy [that pathogen]? Although the strategy was first proposed more than a century ago, we and others are trying to update it,” he adds. (See “My Enemy’s Enemy” below.)

Phage lifecycle​

Phages can interact with bacteria in two main ways. In the first, phages infect a bacterial cell and hijack that cell’s protein-making machinery to replicate themselves, after which the newly made virus particles lyse the bacterium and go on to infect more cells. In the second process, known as lysogeny, the viral genome is incorporated into the bacterial chromosome, becoming what’s known as a prophage, and lies dormant—potentially for many generations—until certain biotic or abiotic factors in the bacterium or the environment induce it to excise itself from the chromosome and resume the cycle of viral replication, lysis, and infection of new cells.​

Phages provide a gene-delivery service for bacteria

Predation is just one type of phage-bacteria interaction taking place within the mammalian microbiome. Many phages are capable of inserting their genomes into the bacterial chromosome, a trick beyond the bounds of traditional predator-prey relationships in other kingdoms of life that adds complexity to the relationship between phages and bacteria, and consequently, to phages’ potential influences on human health.

This role for phages has long been of interest to Imperial College London’s José Penadés. Over the last 15 years or so, he and colleagues have described various ways in which many phages help bacteria swap genetic material among cells. He likens phages to cars that bacteria use to transport cargo around and says that, in his opinion, it almost makes sense to view phages as an extension of bacteria rather than as independent entities. “This is part of the bacterium,” he says. “Without phages, bacteria cannot really evolve. They are absolutely required.”

In the simplest case, the genetic material being transported consists of viral genes in the genomes of so-called temperate phages, which spend at least part of their lifecycle stashed away in bacterial chromosomes as prophages. These phages are coming to be appreciated by microbiologists as an important driver of bacterial evolution in the human microbiome, notes Hill. The lack of practical and accurate virus detection methods makes it difficult to precisely characterize a lot of the phages resident in mammalian guts, but microbiologists estimate that up to 50 percent are temperate phages, and, more importantly for human health, that many of them may carry genes relevant to bacterial virulence. Researchers have long known, for example, that many toxins produced by bacteria—including Shiga toxin, made by some pathogenic E. coli strains, and cholera toxin, secreted by the cholera-causing bacterium Vibrio cholerae—are in fact encoded by viral genes contained in the bacterial chromosome, and that infection by temperate phages that carry these genes may be able to turn a harmless bacterial population into one that’s pathogenic.

Evidence from other studies points to phages as capable of transporting not just their own genomes, but bits of bacterial DNA as well. In the best-studied examples of this phenomenon, known as bacterial transduction, tiny chunks of the bacterial genome get packed up into viral particles instead of or alongside the phage genome, and are shuttled to other bacterial cells. In 2018, however, Penadés and colleagues presented results showing that very large pieces of bacterial DNA can also be exchanged this way, in a process the team named lateral transduction. Not only does the discovery have implications for how researchers understand viral replication in infected cells, it shines light on a novel way for bacteria to share their genes. “With lateral [transduction] you can move huge parts of the bacterial chromosome,” says Penadés. The team first observed the phenomenon in the important human pathogen Staphylococcus aureus, and is now looking for it in other taxa, he adds. “Right now, for us, it’s important to show that it’s a general mechanism, with many bugs involved.”

Although the research is still in the nascent stages, this mechanism could help explain findings from University of Barcelona microbiologist Maite Muniesa and others who have been studying whether phages transport antibiotic resistance genes between bacterial cells, and whether they can act as reservoirs for these genes in the natural environment. Early studies on this issue had proposed that, like many toxin genes, antibiotic resistance genes might be encoded in viral sequences and thus transported to bacteria with the rest of the viral genome. But the idea wasn’t without controversy—a 2016 analysis of more than 1,100 phage genomes from various environments concluded that phage genomes only rarely include antibiotic resistance genes. That study’s authors argued that prior reports of these genes in phage genomes were likely due to contamination, or to the difficulty of distinguishing viral sequences from bacterial ones.

Nevertheless, Muniesa’s team has published multiple reports of antibiotic resistance sequences in phage particles, including in samples of meat products from a Barcelonan fresh-food retailer, and more recently in seawater samples—not only from the Mediterranean coastline but even off the coast of Antarctica, far from human populations that use antibiotics. “We were pretty surprised that we found these particles in this area with low human influence,” Muniesa says. Although her team hasn’t determined whether the antibiotic resistance sequences are of phage or bacterial origin, she suspects they might be bacterial genes that ended up in phage particles during lateral transduction or some process like it. “Bacteria are using these phage particles in a natural way to move [genes] between their brothers and sisters, let’s say,” she says. “It’s happening everywhere.”

Duerkop cautions that it’s not yet clear how often phage-mediated transfer of antibiotic resistance genes occurs or how significant it is in the epidemiology of drug-resistant infections in people. “It’s not to say that antibiotic resistance can’t be mediated through phage,” he says. “I just don’t think it’s a major driver of antibiotic resistance.”

Whatever its natural role, temperate phages’ ability to insert themselves into bacterial genomes could have applications in new antibacterial therapies. Viruses that insert pathogenicity-reducing genes or disrupt the normal expression of the bacterial chromosome could be used to hobble dangerous bacteria, for example—an approach that proved successful last year in mouse experiments with Bordetella bronchiseptica, a bacterium that often causes respiratory diseases in livestock. Using a phage from the order Siphoviridae, researchers found that infected B. bronchiseptica cells were substantially less virulent in mice than control cells were, likely because the viral genome had inserted itself in the middle of a gene that the bacterium needs to infect its host. What’s more, injecting mice with the phage before exposing them to B. bronchiseptica seemed to completely protect them from infection by the microbe, hinting at the possibility of using temperate phages as vaccines against some bacteria.

GUT WARS

Bacteria-infecting viruses, or bacteriophages, may influence microbial communities in the mammalian gut in various ways, some of which are illustrated here. Through predation, phages can influence the abundance of specific bacterial taxa, with indirect effects on the rest of the community, and can drive the evolution of specific bacterial phenotypes. Phages can also incorporate their genomes into bacterial chromosomes, where the viral sequences lie dormant as prophages until reactivated. Researchers have found that phages interact directly with mammalian cells in the gut, too. These cross-kingdom interactions could affect the health of their eukaryotic hosts.

Direct interactions between bacteriophages and mammalian cells

Despite growing interest in phages’ role in shuttling material among bacteria, some of the biggest recent developments in research on phages in the human gut have turned out not to involve bacteria at all. One of the key pieces of this particular puzzle was fitted by University of Utah microbiologist June Round and her colleagues, who as part of a phage therapy study a few years ago fed several types of Caudovirales phages to mice that were genetically predisposed to certain types of cancer and had been infected with a strain of E. coli known to increase that risk. “The premise was pretty simplistic,” recalls Round. “It was just to identify a cocktail of phage that would target bacteria that we know drive chronic colorectal cancer.”

The team was surprised to see that the phages, despite being viewed by most researchers as exclusively bacteria-attacking entities, prompted a substantial response from the mice’s immune systems—mammalian defenses that should, according to conventional wisdom, be indifferent to the war between bacteria and phages in the gut. Intrigued, the researchers tried adding their phage cocktail to mice that had had their gut bacteria completely wiped out with antibiotics. Still, they saw an immune response. "It was then," Round says, "that we realized that [the phages] were likely interacting with the immune system.”

Exploring further, the team found that the phages were activating both innate and adaptive immune responses in mice. In rodents with colitis, the phages exacerbated inflammation. Turning their attention to people, the researchers isolated phages from ulcerative colitis patients with active disease, as well as from patients with disease in remission and from healthy controls, and showed that only viruses collected from patients with active disease stimulated immune cells in vitro. And when the team studied patients who received fecal microbiota transplantation—an experimental treatment for ulcerative colitis that involves giving beneficial gut bacteria to a patient to try to alleviate inflammation and improve symptoms—the researchers found that a lower abundance of Caudovirales in a recipient’s intestine at the time of transplant correlated with treatment success.

By the time the team published its results in 2019, a couple of other groups had also documented evidence of direct interactions between phages and host immune systems. Meanwhile, Duerkop, Hooper, and colleagues reported that mice with colitis tended to have specific bacteriophage communities, rich in Caudovirales, that developed in parallel with the disease. Many of the types of phage they identified in the intestines of those diseased mice also turned up in high abundance in samples taken from the guts of people with inflammatory bowel disease, the researchers noted in their paper, supporting a possible role for phages in the development of disease.

Round says that researchers are still unsure about exactly why these trans-kingdom interactions are happening—particularly when it comes to host adaptive immune responses, which tend to be specific to a particular pathogen. She speculates that mammalian hosts might derive a benefit from destroying certain phages if those phages are carrying genes that could aid a bacterium with the potential to cause disease. Exactly how immune cells would detect what genes a phage is carrying isn’t yet clear.

Meanwhile, hints of collaboration between eukaryotic cells and phages have cropped up in the work of several other labs. One recent study of a phage therapy against P. aeruginosa found that phages and immune cells seem to act in synergy to clear infections in mice. Other work has indicated that phages bind to glycoproteins presented by cells along the mucosal surfaces of the mammalian gut and may provide a protective barrier against bacterial pathogens—a relationship that some microbiologists have argued represents an example of phage-animal symbiosis. Duerkop adds that there’s evidence emerging to support the idea that phages in the mammalian intestine not only can be engulfed by certain eukaryotic cells, but also might slip out of the gut and into the bloodstream to make their way to other parts of the body, with as yet undiscovered consequences.

Whether these mechanisms can be exploited for therapeutic purposes remains to be seen, but Round notes that they do raise the possibility of unintended effects in some circumstances if researchers try to use phages to influence human health via the gut microbiome. At least in the type of chronic inflammatory diseases she and her team have been studying, “we might just be making it worse” by using phages to target disease-causing bacteria, she says, adding that all research groups studying such approaches should take into account potential knock-on effects. Considering phages’ multiple interactions, with both bacteria and animal cells, she says, “it’s a lot more complex than what we’d appreciated.”

My Enemy’s Enemy

Bacteriophages’ ability to selectively target and kill specific bacterial strains has long been recognized as a possible basis for antimicrobial therapies. Proposed by researchers in Europe as early as 1919, phage therapy went on to be widely promoted in Germany, the USSR, and elsewhere before being overtaken worldwide by the soaring popularity of antibiotics in the 1940s. But the strategy has come back into fashion among many microbiologists, thanks to the growing public health problem of antibiotic resistance in bacterial pathogens and to the rapidly improving scientific understanding of phage-bacteria interactions.

Some of the latest approaches aim not only to target specific bacteria with phages, but also to avoid (or exploit) the seemingly inevitable evolution of phage resistance in those bacteria. One way researchers try to do this is by taking advantage of an evolutionary trade-off: bacterial strains that evolve adaptations to one therapy will often suffer reduced fitness when confronted with a second therapy, perhaps one that targets the same or similar pathways in a different way.



Yale University virologist and evolutionary biologist Paul Turner, for example, has studied how phages in the Myoviridae (a family in the order Caudovirales) can promote antibiotic sensitivity in the important human pathogen Pseudomonas aeruginosa. Turner and colleagues showed a few years ago that one such phage binds to a protein called OprM in the bacterial cell membrane, and that bacterial populations under attack from these phages will often evolve reduced production of OprM proteins as a way of avoiding infection. However, OprM also happens to be important for pumping antibiotics out of the cell, such that abnormal OprM levels can reduce bacteria’s ability to survive antibiotic treatment in vitro.

A handful of groups have published case studies using this kind of approach, known as phage steering, in humans. A couple years ago, for example, Turner and colleagues reported that a post-surgery patient’s chronic P. aeruginosa infection cleared up after treatment with the OprM-binding phage and the antibiotic ceftazidime. Researchers at the University of California, San Diego, in partnership with California-based biotech AmpliPhi Biosciences (now Armata Pharmaceuticals), reported similar success in a cystic fibrosis patient with a P. aeruginosa infection who was treated with a mixture of phages and with antibiotics. A Phase 1/2 trial for that therapy was greenlighted by the US Food and Drug Administration last October.

The complexity of the relationship between phages and bacteria, not to mention recently discovered interactions between phages and eukaryotic cells, has many researchers tempering optimism about phage therapy with caution. “There might be off-target effects to this that we hadn’t really thought about,” says University of Colorado School of Medicine microbiologist Breck Duerkop. "That said, thanks to research in the last few years, the black veil on phage therapy is, I believe, being lifted,” he adds, “which I’m really excited about because I think they have a ton of potential to be used in biomedicine.”

 
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