Friday, September 27, 2013

Phage Therapy: An Old Idea Re-Emerging

Electron micrograph of phages attacking a bacterial cell
Bacteriophages, sometimes just called phages, are naturally-occurring viruses that can target and kill bacteria.  That's right, bacteria can get infected with viruses, too.

Phages were discovered in the early 20th century, and people immediately began to think that maybe they could use phages to treat bacterial infections.  However, this idea was more-or-less abandoned in the US and most of Europe after Alexander Flemming discovered penicillin in 1928 and the use of antibiotics grew more widespread.

At the the time, antibiotics were hailed as miracle drugs, which they were, and the thought of antibiotic resistance was not on any one's radar.  Phage research in the US continued and was critical to the development of many molecular biology techniques used today, but it mainly focused on intense research on a few strains of phage (including a famous one called lambda phage) that infect the bacteria E. coli.    

However, research into the use of bacteriophages to treat infections, often called "phage therapy," continued for many years in some countries, including the former Soviet Union and France.  Research into phage therapy continues in those countries today, and sometimes phages are even used to treat infections in people, but not yet in the US.

Because antibiotic resistance is becoming an increasingly dangerous global health threat (as we discussed previously), the "old" idea of phage therapy is making a comeback in the US.  Some people see phage therapy as an potential alternative to antibiotics that may "save" us in the era of antibiotic-resistant superbugs.  As some scientists and doctors are (quite alarmingly) saying that we are approaching the "post-antibiotic age," you might begin to hear more about bacteriophages or phage therapy.   Let's talk a little bit about the past, present, and future of phage therapy as well as some of its known pros and cons.

Bacteriophage Biology

The word bacteriophage comes from the word bacteria and the Greek word phagein, which means "to devour" or "to eat."  Bacteriophages are generally made of a shell of protein that contains DNA or RNA encoding a "genome" or genes that allow the bacteriophage to propagate once it has infected a bacterial cell.  When a bacteriophage attaches to a bacterial cell, it injects its genes into the cell, where it commandeers the cellular machinery of the bacterium to transcribe the phage genes and make proteins to form new bacteriophages.  At the same time, the phage genes are replicated so that copies can be packed into the newly forming bacteriophages.

B. anthracis plate showing spot of dead bacteria (a plaque) where phage was applied
Phages, like other viruses, cannot reproduce on their own.  They need to first infect a host cell and use components of that cell to make new copies of themselves.

Phages have two main types of "life cycles."  Some phages infect bacteria and immediately start the production of new phages, causing the bacteria to explode, or lyse, to release the new phages.  This kills the bacteria in the process.  The new phages (the progeny) can then go off and infect new hosts.  These kinds of phages are called lytic phages.

Other types of phages infect the cell and lie dormant for a while, integrating themselves into the host bacterial genome and replicating once each time the bacteria divide.  However, certain environmental triggers, such as nutrient depletion or other signals that relate to bacterial survival, can stimulate the dormant phages (called prophages) to begin to replicate in great numbers, producing progeny phages and causing host cell lysis and death.  These types of phages, with a delay between infection and replication, are called lysogenic phages or temperate phages.  In general, the best phages used in phage therapy are lytic phages, which can be used more quickly and more efficiently to eradicate bacterial populations.

Close-up of a plaque of lysed B. anthracis
Phages are everywhere.  There may be over 1,000,000,000,000,000,000,000,000,000,000 types of phage on the planet (that's 30 zeroes, or 10^30).  They can be found everywhere from soil and seawater to the skin and intestines of animals.  They are everywhere, but because they don't infect you, your body barely notices their presence.  They only infect bacteria.  Most of this is because they can only attach to and infect bacterial cells, but part of this is also because the phage genes cannot be replicated inside a human cell.  The machinery that transcribes genes to make proteins is different in bacterial cells and human cells.   The inability of phages to infect human cells is what makes antibacterial phage therapy both possible and attractive.               
Phages as a Human Therapy

The specific idea of using phages to kill infection-causing bacteria came very early in phage research.  The 1920s - 1930s were very enthusiastic periods for phage therapy; before antibiotics became dominant, people thought phages were the key to combating bacterial infections.  Without really understanding how phages worked or what the pros and cons of their use might be, many scientists thought phages would be the "magic bullet" to treating human infections. 

However, after World War II and the development of antibiotics, the idea of phage therapy began to dwindle in the US and most of Europe.  Antibiotics were much easier to produce in mass quantities and were very effective.  However, phage therapy remained an active area or research in the Soviet Union, continuing after the fall of the USSR in the countries of Russia and Georgia.  Lots of phage research also took place in France and Poland.  After the 1940s, research on phages in the US and most of western Europe focused almost solely on bacteriophages as curiosities that could be used to understand the general mechanisms of how viruses infect cells, without a lot of thought into their use in a clinical setting.  While this research has been important for the development of new techniques in molecular biology, US and European scientists and physicians are starting to come back around to the idea of using phages to treat human infection.  The driving force behind this is antibiotic resistance.  

While bacteria can gain resistance to phages through genetic mutations just like they can gain resistance to antibiotics, the key difference is that the phages can likewise mutate along with the bacteria.  It is easier to grow and isolate new phages to kill a resistant strain of bacteria than it is to develop new antibiotic chemicals in the lab.  This advantage is one reason why phage therapy is viewed today as a potentially important alternative to antibiotics.  A drawback to phages is that they can be much more difficult to isolate and purify, and in some cases, phages might need to be generated for the specific bacterial strains from the infection of an individual patient.  It is much easier to produce and store mass quantities of broad-spectrum antibiotics, but in the face of bacterial resistance to these antibiotics, the more individualized approach of phage therapy is attractive for hard-to-treat infections.               
The History of Phage Therapy
Frederick Twort
Bacteriophages were initially discovered by Frederick Twort in 1915, but the real start of hardcore phage research came from Felix d'Herelle in 1917.  His work   He pioneered the idea of human phage therapy and  influenced phage therapy research from France all the way to Russia, as we'll see below.

The early days of phage therapy were the medical-equivalent of the "wild west."  Patients often had adverse reactions to phage preparations, mostly due to certain products left over from culturing methods.  Propagating the phage in the lab means infecting bacteria with it to let it multiply; then, the bacteria need to be removed from the preparation or lysed (broken open to kill them), which can leave behind a lot of contaminants and bacterial components that your body can have immune reactions to.  At first, the methods had to be worked out by trial and error, but once the details were worked out, the phages themselves seemed to be very safe for human cells.

Early phage researchers developed techniques to make both phage "cocktails," which had activity against a variety of bacteria, as well as to generate bacteriophages specifically against for an individual infection.  Phages were injected intravenously, intrathecally (into the spinal fluid), subcutaneously, and topically.  It was a combination of science and art, and it was risky, but at that time, some infections meant almost certain death.  Physicians were willing to try phages as a last ditch effort.  And, once they began to refine the art of phage therapy, it appears to have been remarkably effective.   

Felix d'Herelle
Phages were first intentionally used to treat infections as early as 1919 in France; Felix d'Herelle, a French-Canadian microbiologist who worked at the Pasteur Institute, used phages to treat severe dysentery in children at the Hospital des Enfants Malades in Paris.  In Belgium, two physicians-scientists named Bruynoghe and Maisin wrote about treating Staphylococcus infections with phages in 1921.  Another physician-scientist named Asheshov used intravenously administered phage to treat cholera in India.

These early studies of d'Herelle and others led d'Herelle and his son-in-law, Theodore Mazure, to established a Laboratoire du Bacteriophage.  They began to produce therapeutic phage cocktails to treat infections.  The company that descended from this laboratoire produced commercially available phage cocktails until 1978, but even after this, phage therapy continued in France.  In the 1980s and 90s, the chief of clinical microbiology at the Pasteur institute, Henri de Montclos, ran a research team that that produced therapeutic phages, and he wrote extensively about the phage production techniques that his laboratory had perfected down to an art form.  However, after the start of the AIDS epidemic led to increases healthcare and pharmaceuticcal regulations, it became more and more difficult to produce individualized batches of phage, though a few French doctors have continued to use phages to treat particularly difficult infections.

Soviet physician-scientists also began to use phages to treat infections as early as 1920s-30s, and the Soviets (now Russians and Georgians) have had a long and seemingly successful track record of phage therapy.  Treatment of dysentery was a major concern in the early days of the USSR.  Infections resulting from battle became an even more important concern in the days of World War II.

The Soviets developed the first effective tablet for delivery of phage, though they developed technology to administer phages in just about every way imaginable.  However, compared with France, much less phage research was published out of Russia, as its potential military applications to cure battlefield infections made the Soviets consider phage therapy as a state secret.  Many of the publications that the Soviets did put out were in Russian and thus inaccessible to many non-Russian-speaking western scientist and physicians.  Phage production still remains an important therapy in Russia, with therapeutic phages now being produced by one of the major Russian pharmaceutical companies, Microgen.             

George Eliava
Much of the Soviet phage work occurred in the Soviet republic of Georgia.  In 1923, the microbiologist George Eliava collaborated with Felix d'Herelle to start the Bacteriological Institute in Tbilisi, the capital of Georgia.  After Eliava died, the Institute was renamed the Eliava Institute.   Bacteria from all over the Soviet Union were sent to the institute, where researchers developed and isolated phages against them.  Over 1200 people worked at the institute at its peak, with a production output capacity of 2 tons of phage preparations per week.  A large portion of this went to the Soviet army, which used the phage preparations to treat diarrhea and treat or prevent wound infections.

Georgia declared independence as the Soviet Union collapsed, but Georgian soldiers continued to use phage preparations produced by the Eliava institute.  The medical school in Tbilisi, Georgia, continues to run a "Surgical Infections and Phage Therapy" program to train surgeons in methods of treating infections using phage cocktails.  Several anti-infection phage cocktail products are publicly available in Georgia and Russia, even without prescription, made by both the Eliava institute and another Georgian company, Biochimpharm.  These include Intestiphage, which targets 20 varieties of bacteria which affect the gastrointestinal system, and Pyophage, which targets various bacteria for treatment of skin or wound infections.  These companies periodically update their phage products to make sure they target the most prominent strains bacteria present in the environment at any time.    
Another major force in phage therapy was Poland.  In 1954, the Hirszfeld Institute of Immunology and Experimental Therapy was founded in Wroclaw, Poland.  The institute has treated thousands of patients with phages and also made a made a significant contribution to phage research by thoroughly documenting and publishing about their experiences with phage therapy.  Because of the Hirszfeld institute, phage use in Poland became less of an experimental "shoot from the hip" last ditch effort for hard-to-treat patients.  It became a standard alternative when antibiotics were deemed ineffective or for otherwise chronic bacterial infections.  The Hirszfeld Institute still makes therapeutic phages today, and, since 2005, it has a center dedicated to treating antibiotic-resistant infections.    
Now, I don't want you to think that the US was completely out of phage therapy research.  Some human phage therapy research did go on in the US in the 1920s and 30s.  The pharmaceutical companies Eli Lilly and E.R. Squibb and Sons (part of Abbott Labs) commercially produced phage preparations, but they had difficulty with quality control as well as obtaining a consistent level of phage activity.  Like others around the world, US scientists and physicians had a lot of trouble developing effective phages at first, and as the US became able to mass produce antibiotics, the drive to study phage therapy dwindled. 

Two important review articles on phage therapy were published in the Journal of the American Medical Society, one in 1934 and one in 1941.  These papers suggested that it was difficult to conclude much from the published studies on phage therapy and suggested that the only convincingly positive data for phage therapy was from its use to treat localized Staphylococcus infections.  This led a lot of US physicians to focus their attention on antibiotics, which at the time seemed much more promising.  

Nonetheless, patients with typhoid fever were very successfully treated with phages in Los Angeles and Quebec in the 1930s.  Antibiotics at the time were not very effective against typhoid until the antibiotic chloramphenicol became available in 1949.  After chloramphenicol came into use, phage therapy for typhoid appears to have been largely abandoned in North America, because, as we've mentioned, it was was much easier to produce large quantities of chloramphenicol than it was to produce therapeutic phage.      

With the increasing promise of antibiotics and the increasing ability to make large amounts of synthetic antibiotics, US and UK physicians and scientists became less enchanted with the magical phage, particularly with the increasing promise of antibiotics.  However, in 1941, editorials published in The Lancet and the British Medical Journal reported on the use of phages to treat diarrhea (dysentery) and gangrene in Soviet soldiers.  This was an important glimpse into the "secret" and "exotic" Soviet phage therapy program.  The US National Research Council then sponsored a series of quite successful and elegant animal research studies testing phages against Shigella dysenteriae, a important cause of dysentery.  These studies showed that phages were extremely effective in treating S. dysenteriae infection in mice and that phages could even cross the blood-brain barrier and attack infections in the brain.  However, these studies, while promising, weren't enough to spur a renaissance in phage therapy research in the US.    

Phages as One of the First Forms of  "Personalized Medicine"

In today's age of genome sequencing and genomics, physician-scientists love to use the term "personalized medicine."  The early phage pioneers were practicing one of the first forms of true personalized medicine long before the idea was in vogue.  They often developed therapies specifically directed at the strains of bacteria each patient was infected with.  And they were doing it all on the fly, learning about phages as they went.  It's kind of amazing to think about, and even more amazing that they were very often quite successful.  
Unfortunately, a lot of the past phage therapy research is difficult for modern English-speaking scientists to properly interpret and draw conclusions from.  Two major problems with a lot of the old data in phage therapy is that (1) often it is written in languages that they don't speak (French, Russian, Polish, etc.) in obscure or hard-to-find discontinued journals (ie, you can't just copy and paste it into Google Translate) and (2) because often each case was treated individually, there's no placebo or control group to compare with as their would be in a modern clinical trial.  In the Soviet Union, for example, phage therapy came to be considered as a standard of care, and thus many trials in this country compared various phage cocktails against each other, with no group receiving placebo or antibiotics.  Without a real control group, it is hard to determine what effects the phages had in these trials as well as how this would compare with antibiotics or other treatments. 

Present and Future of Phage Therapy in the US

Beginning in the 1980s and 90s, scientists in the US and Canada have become more interested in the prospects of phage therapy as an alternative to antibiotics.  Because antibiotics and phages kill bacteria differently, antibiotic-resistant bacteria can still be targeted by phages and bacterial resistance to phages can be overcome by mutations in the phages themselves.  While many questions about the safety and efficacy (clinical effectiveness) of phages still remain, there is a small but growing movement calling for closer examination of phage therapy in the US as an alternative to antibiotics.  Because phage use will not induce the same resistance mechanisms as antibiotics, it has even been suggested that phages would not only be effective against antibiotic-resistant bacterial strains but also help to extend the useful lifetime of conventional antibiotics.

One area where US phage use is moving forward is the use of phages to prevent food poisoning.  In 2006-7, the US Food and Drug Administration (FDA) approved the use of a phage preparation against Listeria monocytogenes for application to meat.  Listeria monocytogenes is a species of bacteria that can cause the disease listeriosis in some people after ingestion of contaminated food.  This product, called ListShield, was developed by the biotech company Intralytix, which  also developed another phage product, EcoShield, that protects against pathogenic E. coli.  EcoShield was approved by the FDA in 2011.  These products appear to be safe and effective, and open the door up to other phage-based food additives to control bacterial contamination.      

Pros and Cons of Phage Therapy

Phages are unique among antibacterial therapies in their ability to increase their numbers in the presence of bacteria.  While antibiotics need to reach a certain concentration (a "therapeutic dose") in order to inhibit key enzymes or kill bacteria by other means, relatively few phages can successfully take down a large bacteria population because of their intrinsic ability to replicate.  This has been termed "auto-dosing" and may even lead to some infections being treated with a single dose of phage.

One other advantage of phage therapy that has been touted is the lack of significant side effects seen with application of purified preparations of phages.  Many studies on phage immunology in animals and humans have suggested that the immune system only mildly reacts to the presence of phages.  This may be due to the fact that humans and other animals have been "conditioned" over the course of their evolution to the presence of phages, which are everywhere in the environment.  Adverse reactions in the early days of phage therapy have been attributed to residual bacterial antigens in the preparations or other components of the bacteria/phage culture medium rather than the phages themselves.

Another advantage of phage therapy may be its specificity.  Strong, broad-spectrum antibiotics can wipe out both harmful disease-causing (pathogenic) bacteria as well as the helpful bacteria that normally live in the human body (ie, those that live in the intestine), whereas phages engineered against specific pathogenic strains of bacteria may have the desired antibacterial effects without harming these "good" bacteria.  However, this specificity could also be a disadvantage in some cases.  If not all of the strains of pathogenic bacteria causing an infection are targeted by the phage cocktail that is administered to the patient, infection with the phage-resistant bacteria could continue.  However, it may be that phage killing of all of the bacteria isn't necessary if the immune system can pick up the slack and clean up the remaining bacteria.  The relative benefits of using specific phages vs broad-spectrum phage cocktails still remains to be studied thoroughly.

One potential con to the application of bacteriophages is that the phage-induced lysis of bacterial cells can result in the release of large amounts of toxins contained within certain types of bacteria.  These are called endotoxins, and they can cause significant immune reactions or otherwise damage tissue.  In medicine, the body's reaction to endotoxins released by bacteria is sometimes called the Jarisch-Herxheimer reaction.  This is a more important concern with infections inside the body (systemic infections), where large amounts of endotoxins can be released into the bloodstream and could cause fever or toxic shock.  In the treatment of skin infections like wounds or burns, endotoxin release is much less of a concern.  This is one of the reasons that phage preparations need to be purified extremely well when used intravenously, as residual endotoxins from the bacteria used to prepare the phage can result in adverse immune reactions.  Some, but not all antibiotics also cause bacterial lysis and cause endotoxin release, so endotoxin release is a concern both for antibiotics and phages.  Some phage scientists have suggested that, because some bacteriophages shut down the production of bacterial proteins while they are replicating, it may be that any phage-stimulated release of endotoxin will be balanced by a general reduction in the bacterial production of endotoxin.  Again, this is an aspect of phage therapy that is not yet fully understood.    

Some Final Thoughts About Phages

Despite the fact that phage therapy was largely overlooked by the English-speaking medical community for decades, there's a lot of evidence that suggests phages have the potential to be an effective form of antibacterial therapy that may overcome some cases of antibiotic resistance.  In light of the increasing danger brought about by our reliance on antibiotics, we need to seriously consider and study phage therapy as an potential weapon in our medical armament against the increasing numbers of drug-resistant "super bugs."   

Phage therapy is certainly not a "magic bullet."  There are real concerns that need to be addressed about the effects of putting phages into people, and the preparation of phages is far more intricate and complicated than the production of synthetic antibiotics.  There's still a lot of research to be done on phage therapy.  Many clinical trials will need to be carried out to determine both the safety and efficacy of therapeutic phages before they will be accepted into US medical practice.  Because of the uniqueness of phage therapy, scientists and doctors will need to come to some consensus as to what benchmarks need to be met for the medical community to accept phage therapy.  Determining the efficacy of phages may not be all that difficult, but proving the safety of a biological therapy that itself can replicate and evolve may end up being a monumental task.  Part of our acceptance of phage therapy may depend on just how fast the danger of antibiotic resistance increases in the coming years.

This will all take time and will cost a lot of money, but the potential public health benefits could be game-changing in the war against bacteria.  That's just one more reason why we need to keep funding scientific and medical research.  Our future likely depends developing new and more effective antibiotics as well as on exploring valid alternative avenues like phage therapy.         

Text ©2013, TheMadScienceBlog.   Images are from the public domain.  

Sources and Further Reading (All Freely Available)
  • Image of the B. anthracis bacteria plate is from the CDC Public Health Image Library Image # 11752.
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  1. I am confident that Phage Therapy is a potential strategy to overcome antibiotic resistance. We just need time and further study to prove it.

  2. I'll guess advances in gene sequencing will be able to more efficiently match a phage to the specific bacterium targeted for elimination. The economic benefit to the company that works smart this way are beyond huge.