Phage therapy

PROCEDURE OF THE DAY

Phage therapy

Phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Although extensively used and developed mainly in former Soviet Union countries for about 90 years, this method of therapy is still being tested elsewhere for treatment of a variety of bacterial and poly-microbial biofilm infections, and has not yet been approved in countries other than Georgia. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture.[1] If the target host of a phage therapy treatment is not an animal, however, then the term "biocontrol" (as in phage-mediated biocontrol of bacteria) is sometimes employed rather than "phage therapy".

A hypothetical benefit of phage therapy is that bacteriophages can be much more specific than more common drugs, so they can be chosen to be indirectly harmless not only to the host organism (human, animal, or plant), but also to other beneficial bacteria, such as gut flora, reducing the chances of opportunistic infections. They also have a high therapeutic index, that is, phage therapy gives rise to few if any side effects, as opposed to drugs, and does not stress the liver. Because phages replicate in vivo, a smaller effective dose can be used. On the other hand, this specificity is also a disadvantage: A phage will only kill a bacterium if it is a match to the specific strain. Thus, phage mixtures are often applied to improve the chances of success, or samples can be taken and an appropriate phage identified and grown.

Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics, particularly in the country of Georgia.[2][3][4] They tend to be more successful than antibiotics where there is a biofilm covered by a polysaccharide layer, which antibiotics typically cannot penetrate. [5] In the West, no therapies are currently authorized for use on humans, although phages for killing food poisoning bacteria (Listeria) are now in use.[6]

History

Following the discovery of bacteriophages by Frederick Twort and Felix d'Hérelle[7] in 1915 and 1917, phage therapy was immediately recognized by many to be a key way forward for the eradication of bacterial infections. A Georgian, George Eliava, was making similar discoveries. He travelled to the Pasteur Institute in Paris where he met d'Hérelle, and in 1926 he founded the Eliava Institute in Tbilisi, Georgia, devoted to the development of phage therapy.

In neighbouring countries including Russia, extensive research and development soon began in this field. In the USA during the 1940s, commercialization of phage therapy was undertaken by the large pharmaceutical company, Eli Lilly.

Whilst knowledge was being accumulated regarding the biology of phages and how to use phage cocktails correctly, early uses of phage therapy were often unreliable. When antibiotics were discovered in 1941 and marketed widely in the USA and Europe, Western scientists mostly lost interest in further use and study of phage therapy for some time.[8]

Isolated from Western advances in antibiotic production in the 1940s, Russian scientists continued to develop already successful phage therapy to treat the wounds of soldiers in field hospitals. During World War II, the Soviet Union used bacteriophages to treat many soldiers infected with various bacterial diseases e.g. dysentery and gangrene. The success rate was as good as, if not better than any antibiotic.[citation needed] Russian researchers continued to develop and to refine their treatments and to publish their research and results. However, due to the scientific barriers of the Cold War, this knowledge was not translated and did not proliferate across the world.[9][10]

There is an extensive library and research center at the Eliava Institute in Tbilisi, Georgia. Phage therapy is today a widespread form of treatment in that region. For 80 years Georgian doctors have been treating local people, including babies and newborns, with phages.

As a result of the development of antibiotic resistance since the 1950s and an advancement of scientific knowledge, there has been renewed interest worldwide in the ability of phage therapy to eradicate bacterial infections and chronic polymicrobial biofilm, along with other strategies.

Phages have been investigated as a potential means to eliminate pathogens like Campylobacter in raw food[11] and Listeria in fresh food or to reduce food spoilage bacteria.[12] In agricultural practice phages were used to fight pathogens like Campylobacter, Escherichia and Salmonella in farm animals, Lactococcus and Vibrio pathogens in fish from aquaculture and Erwinia and Xanthomonas in plants of agricultural importance. The oldest use was, however, in human medicine. Phages have been used against diarrheal diseases caused by E. coli, Shigella or Vibrio and against wound infections caused by facultative pathogens of the skin like staphylococci and streptococci. Recently the phage therapy approach has been applied to systemic and even intracellular infections and the addition of non-replicating phage and isolated phage enzymes like lysins to the antimicrobial arsenal. However, actual proof for the efficiency of these phage approaches in the field or the hospital is not available.[12]

Some of the interest in the West can be traced back to 1994, when Soothill demonstrated (in an animal model) that the use of phages could improve the success of skin grafts by reducing the underlying Pseudomonas aeruginosa infection.[13] Recent studies have provided additional support for these findings in the model system.[14]

Although not phage therapy in the original sense, the use of phages as delivery mechanisms for traditional antibiotics has been proposed.[15][16] The use of phages to deliver antitumor agents has also been described, in preliminary in vitro experiments for cells in tissue culture.[17].

Potential benefits

A hypothetical benefit of phage therapy is freedom from the adverse effects of antibiotics. Additionally, it is conceivable that, although bacteria rapidly develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics.

Bacteriophages are very specific, targeting only one or a few strains of bacteria.[18] Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The specificity of bacteriophages might reduce the chance that useful bacteria are killed when fighting an infection.

Increasing evidence shows the ability of phages to travel to a required site — including the brain, where the blood brain barrier can be crossed — and multiply in the presence of an appropriate bacterial host, to combat infections such as meningitis. However the patient's immune system can, in some cases mount an immune response to the phage (2 out of 44 patients in a Polish trial[19]).

Development and production is faster than antibiotics, on condition that the required recognition molecules are known.[citation needed]

Research groups in the West are engineering a broader spectrum phage and also target MRSA treatments in a variety of forms - including impregnated wound dressings, preventative treatment for burn victims, phage-impregnated sutures.[20] Enzobiotics are a new development at Rockefeller University that create enzymes from phage. These show potential for preventing secondary bacterial infections e.g. pneumonia developing with patients suffering from flu, otitis etc..[citation needed]

Some bacteria such as multiply resistant Klebsiella pneumoniae have no non toxic antibiotics available, and yet killing of the bacteria via intraperitoneal, intravenous or intranasal of phages in vivo has been shown to work in laboratory tests.[21]

Risks of Phage Therapy

Phase therapy also has disadvantages:

Unlike antibiotics, phages must be refrigerated until used,[22] and a physician wishing to prescribe them needs special training in order to correctly prescribe and use phages.

Phages come in a great variety. That diversity becomes a disadvantage when the exact species of an infecting bacteria is unknown or if there is a multiple infection. For best results, the phages should be tested prior to application in the lab. For this reason, phages are less suitable for acute cases. Mixtures consisting of several phages can fight mixed infections.

Another con is that like viruses, bacteria can become resistant to treatments, and in this case they can mutate to survive the phage onslaught. Mutant bacteria can be destroyed by other types of phages, however. And phages are found throughout nature. This means that it is easy to find new phages when bacteria become resistant to them. Evolution drives the rapid emergence of new phages that can destroy bacteria that have become resistant. This means that there should be an ‘inexhaustible’ supply.

Phages that are injected into the bloodstream are recognized by the human immune system. Some of them are quickly excreted and, after a certain period, antibodies against the phages are produced by the body. For this reason, it appears that one type of phage can only be used once for intravenous treatment.[23]

Application

Collection

In its simplest form, phage treatment works by collecting local samples of water likely to contain high quantities of bacteria and bacteriophages, for example effluent outlets, sewage and other sources.[2] They can also be extracted from corpses. The samples are taken and applied to the bacteria that are to be destroyed which have been cultured on growth medium.

The bacteria usually die, and the mixture is centrifuged. The phages collect on the top of the mixture and can be drawn off.

The phage solutions are then tested to see which ones show growth suppression effects (lysogeny) and/or destruction (lysis) of the target bacteria. The phage showing lysis are then amplified on cultures of the target bacteria, passed through a filter to remove all but the phages, then distributed.

Treatment

Phages are "bacterium specific" and it is therefore necessary in many cases to take a swab from the patient and culture it prior to treatment. Occasionally, isolation of therapeutic phages can typically require a few months to complete, but clinics generally keep supplies of phage cocktails for the most common bacterial strains in a geographical area.

Phages in practice are applied orally, topically on infected wounds or spread onto surfaces, or used during surgical procedures. Injection is rarely used, avoiding any risks of trace chemical contaminants that may be present from the bacteria amplification stage,and recognizing that the immune system naturally fights against viruses introduced into the bloodstream or lymphatic system.

The direct human use of phage might possibly be safe; suggestively, in August 2006, the United States Food and Drug Administration approved spraying meat with phages. Although this initially raised concerns since without mandatory labeling consumers won't be aware that meat and poultry products have been treated with the spray,[24] it confirms to the public that, for example, phages against Listeria are generally recognized as safe (GRAS status) within the worldwide scientific community and opens the way for other phages to also be recognized as having GRAS status.

Phage therapy has been attempted for the treatment of a variety of bacterial infections including: laryngitis, skin infections, dysentery, conjunctivitis, periodontitis, gingivitis, sinusitis, urinary tract infections and intestinal infections, burns, boils, etc.[2] - also poly-microbial biofilms on chronic wounds, ulcers and infected surgical sites.[citation needed]

In 2007, Phase 2a clinical trials have been reported at the Royal National Throat, Nose and Ear Hospital, London for Pseudomonas aeruginosa infections (otitis).[25].[26][27] Documentation of the Phase-1 and Phase-2a study is not available as of 2009[update].

Phase 1 clinical trials are underway in the South West Regional Wound Care Center, Lubbock, Texas for an approved cocktail of phages against bacteria, including P. aeruginosa, Staphylococcus aureus and Escherichia coli (better known as E. coli).[citation needed]

Reviews of phage therapy indicate that more clinical and microbiological research is needed to meet current standards.[28]

Distribution

Phages can usually be freeze-dried and turned into pills without materially impacting efficiency.[2] In pill form temperature stability up to 55 C, and shelf lives of 14 months have been shown.[citation needed]

Application in liquid form is possible, stored preferably in refrigerated vials.[citation needed]

Oral administration works better when an antacid is included, as this increases the number of phages surviving passage through the stomach.[citation needed]

Topical administration often involves application to gauzes that are laid on the area to be treated.[citation needed]

Obstacles

General

The high bacterial strain specificity of phage therapy may make it necessary for clinics to make different cocktails for treatment of the same infection or disease because the bacterial components of such diseases may differ from region to region or even person to person.

In addition, due to the specificity of individual phages, for a high chance of success, a mixture of phages is often applied. This means that 'banks' containing many different phages are needed to be kept and regularly updated with new phages.

Further, bacteria can evolve different receptors either before or during treatment; this can prevent the phages from completely eradicating the bacteria.

The need for banks of phages makes regulatory testing for safety harder and more expensive. Such a process would make it difficult for large scale production of phage therapy. Additionally, patent issues (specifically on living organisms) may complicate distribution for pharmaceutical companies wishing to have exclusive rights over their "invention"; making it unlikely that a for-profit corporation will invest capital in the widespread application of this technology.

As it has been known for at least thirty years or more, mycobacterias such as Mycobacterium tuberculosis have specific bacteriophages.[29]. As for Clostridium difficile, which is responsible of many nosocomial diseases, no lytic phage has yet been discovered but some temperate phages (integrated in the genome) are nevertheless known for this species which opens encouraging avenues.

To work, the virus has to reach the site of the bacteria, and viruses do not necessarily reach the same places that antibiotics can reach.

Funding for phage therapy research and clinical trials is generally insufficient and difficult to obtain, since it is a lengthy and complex process to patent bacteriophage products. Scientists comment that 'the biggest hurdle is regulatory', whereas an official view is that individual phages would need proof individually because it would be too complicated to do as a combination, with many variables. Due to the specificity of phages, phage therapy would be most effective with a cocktail injection, which are generally rejected by the FDA. Researchers and observers predict that for phage therapy to be successful the FDA must change its regulatory stance on combination drug cocktails.[30] Public awareness and education about phage therapy are generally limited to scientific or independent research rather than mainstream media.[31]

The negative public perception of viruses may also play a role in the reluctance to embrace phage therapy.[32]

Safety

Phage therapy is generally considered safe. As with antibiotic therapy and other methods of countering bacterial infections, endotoxins are released by the bacteria as they are destroyed within the patient (Herxheimer reaction). This can cause symptoms of fever, or in extreme cases toxic shock (a problem also seen with antibiotics) is possible.[33] Janakiraman Ramachandran, a former president of AstraZeneca India who 2 years ago launched GangaGen Inc., a phage-therapy start-up in Bangalore,[34] argues that this complication can be avoided in those types of infection where this reaction is likely to occur by using genetically engineered bacteriophages; which have had their gene responsible for producing endolysin removed. Without this gene the host bacterium still dies but remains intact because the lysis is disabled. On the other hand this modification stops the exponential growth of phages, so one administered phage means one dead bacterial cell.[4] Eventually these dead cells are consumed by the normal house cleaning duties of the phagocytes, which utilise enzymes to break the whole bacterium and its contents down into its harmless sub-units of proteins, polysaccharides and lipids.[35]

Care has to be taken in manufacture that the phage medium is free of bacterial fragments and endotoxins from the production process.

Lysogenic bacteriophages are not generally used therapeutically. This group can act as a way for bacteria to exchange DNA, and this can help spread antibiotic resistance or even, theoretically, can make the bacteria pathogenic (see Cholera).

The lytic bacteriophages available for phage therapy are best kept refrigerated but discarded if the pale yellow clear liquid goes cloudy.

Effectiveness

In Russia, phage therapies produced by manufacturers have been shown to have approximately a 50% success rate at eradicating target bacteria.

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