Phage Therapy

Thomas Kalinoski

Abstract

Phage therapy is the application of bacteriophages to therapeutic effect in the body to control pathogenic bacteria. The use of phages has many potential applications for treating bacterial infections, especially infections that do not respond to conventional antibiotic treatment. Although the use of phages to control bacteria has been proposed decades ago, phage therapy use for human medicine has not yet been approved in most of the world and continues to undergo extensive research.  This review examines various research literatures involving the use of phages in human medicine. Included background information features some of the basic information already known about the bacteriophages and their potential applications. The study’s results reveal the findings on the efficacy of phage therapy as well as the present concerns about its safety and conclusions about its potential future in western medicine.

 

Introduction

Even from the start of widespread antibiotic use in the 1940’s it has been known that resistance strains of bacteria would become a problem in treatment of infections. Today, there are a diminishing number of antibiotics that can be used in many cases, and highly resistant strains are becoming more and more of a problem to health care professionals. Discovery of completely novel antibiotics is rare, and the development of more effective antibiotics cannot keep up with resistance. Therefore, use of bacteriophages to treat infections stands out as a promising alternative. Research on bacteriophages to treat specific diseases is widespread, and this review was conducted in an effort to amass this research and analyze its significance. The study brings together a variety of different research literature in order to review the critical points of current knowledge on phage therapy. In this review, the efficacy of phage therapy will first be examined, then the present concerns regarding safety of its use in humans. Finally, this review will address the issues and obstacles that need to be overcome for this technology to take hold in western medicine.

 

Background

Bacteriophages have been studied for many decades, resulting in a wealth of information on the topic. Bacteriophages, or simply phages, are viruses that exclusively infect bacterial cells. Bacteriophages were discovered in the early 20^th century, and not long after their discovery, they were already being tested therapeutically to fight against infections. The first study was conducted by Felix d'Hérelle at the Hôpital des Enfants-Malades in Paris in 1919 on a 12 year old boy with dysentery (10). According to the report, the boy’s symptoms ceased after a single administration. In 1923, George Eliava founded the Eliava Institute in Georgia, devoted to the development of phage therapy. Research and commercialization of phages soon began in Russia and the United States in the 1940’s, at the same time that antibiotics were taking off. The fact that early use of phage therapy was largely unreliable, and that the newly discovered antibiotics were seen as “wonder drugs”, ensured a loss of interest from most of the scientific community. Research into phage therapy continued in the Soviet Union, where it was isolated due to the cold war. Phage therapy continues to this day exclusively in Georgia at the Eliava Institute (8). A renewed interest to find alternative methods of treating bacteria due to increasing antibiotic resistance has increased the amount of recent research on the subject in the west. The fact that bacteriophages are much more specific agents than antibiotics, have few side effects, do not stress the liver, and are self-replicating, make phage therapy an attractive option (1).

 

Effectiveness of Phage Therapy

The efficacy of phage therapy has been documented in numerous studies over the past century, but many of them were poorly conducted and don’t hold up to the stringent standards of the modern scientific community. Recent credible research on the efficacy of phage therapy in a number of different studies can be found, however, some of which is documented below.

     Lytic Activity

Carmody and colleagues found that phage therapy was effective in the treatment of pulmonary infections due to species of Burkholderia cepacia in a study on mice (3). This study used the B. cenocepacia strains AU0728 and K56-2 isolated from sputum of patients with cystic fibrosis (CP), which were introduced to mice via tracheotomy. The mice were treated with B. cepacia specific phage twenty-four hours after infection by either intranasal inhalation or intraperitoneal injection. Bacterial densities in the lungs were determined seventy-two hours after infection. The treatment of infected mice with phage via intraperitoneal injection resulted in significant reduction in lung bacterial density after forty-eight hours relative to mock treated mice.

In studying the treatment of Klebsiella pneumonia mediated lobar pneumonia in mice, Chhibber and colleagues found that phage therapy can be effective, but the results vary significantly depending on treatment time (4), suggesting that there is a limited bacteria population size where use of phages can effectively check its growth. The study used phage SS, a phage specific for K. pneumonia. The phage preparation was injected by intraperitoneal route to mice immediately after infection in one group, and at 6 hours and 24 hours post-infection in two additional groups. The results showed that phage therapy has the potential to check the growth of K. pneumonia in the respiratory tract if initiated at an appropriate time, but a delay of even six hours rendered the treatment ineffective.

In their study on the application of phage therapy to chronic bacterial prostatitis (CBP), Letkiewicz and colleagues found that phage therapy can be used to successfully treat resistant infections in tissues that are not readily accessible by antibiotics (7). This article is significant because it is one of few recent studies on phage therapy in humans. Conducted at the Institute of Immunology and Experimental Therapy in Poland, experimental phage therapy was tested on drug-resistant bacterial infections. After E. faecalis was cultured from infected prostate fluid, three patients suffering from CBP were treated rectally with a phage preparation active against the bacteria two times daily for 28-33 days. Treatment caused bacterial eradication, as well as reduction in prostate size and pain and significant increases in the maximum urinary flow in all cases.

     Immunomodulary Activity

There is also evidence in many studies that use of phages for the therapeutic control of bacterial infections influences the response by the immune system to fighting the infection. In addition to their role in the direct clearance of bacteria, many studies also document changes in cytokine levels, leukocyte recruitment, and antibody production.

In an article documenting the efficacy of phage therapy in the immunocompromised host, Borysowski and colleagues describe two major mechanisms of therapeutic effect due to phage therapy (2). The first relies on direct killing of bacterial cells by the lytic cycle, while the other depends on inducing an antibacterial immune response by either the phage particles themselves or constituents of dead bacterial cells, such as lipopolysaccaride (LPS). LPS, also called endotoxin, is a component of some bacterial cell walls and a powerful stimulator of the immune system.

                     

Figure 1. Endotoxin Release upon Cell Lysis. Retrieved from <http://www2.raritanval.edu>.

This study pointed out several experiments performed on immunocompetent mice that suggest direct killing of bacteria as the major mechanism of therapeutic effect. These researchers concluded that the only significant mechanism mediating the therapeutic effects of phage preparations is direct killing of bacterial cells by phage viruses.

Zimecki and colleagues found significant evidence of enhanced immune activity due to phages in their study on the prophylactic administration of bacteriophages in the treatment of Staphylococcus aureus infection in mice (11). Mice were injected with the immunosuppressant cyclophosphamide (CP) intraperitoneally and then administered S. aureaus intravenously four days later. S. aureus specific phages were administered intraperitoneally 30 minutes before infection. The results indicated that the highly elevated numbers of bacteria in CP treated mice were lowered by application of phages to values observed in mice not subject to CP treatment (with healthy immune systems). The results indicated efficient removal of bacteria by prophylactic administration of specific phages, but also revealed positive effects on the activity of the immune system.

In their study on the application of phage therapy to chronic bacterial prostatitis, Letkiewicz and colleagues found strong experimental evidence that purified phages may have an anti-inflammatory effect. They have been found to alter phagocyte function, decrease activation of the inflammatory cytokine nuclear factor-κB, and reduce platelet and T-cell adhesion. Their experiments also showed that phages can inhibit the formation of reactive oxygen species by neutrophils, which reduces tissue damage that accompanies the chronic inflammatory process. During their study, the researchers also observed a significant decrease in the level of C-reactive protein (an inflammatory marker). All of these anti-inflammatory properties contribute to reduction of symptoms.

Carmody and colleagues also describe anti-inflammatory effects of phages in their research on Burkholderia cenocepacia infection. In order to determine whether phage treatment could attenuate infection-associated lung inflammation, levels of proinflammatory cytokines TNF-α and MIP-2 were measured in the lungs forty-eight hours after treatment. In the AU0728 infected mice, it was observed that treatment with phage resulted in significantly reduced TNF-α level, whether administered via inhalation or intraperitoneal injection.  It was also found that mice treated via intraperitoneal injection had significantly reduced levels of MIP-2 in the lungs. To access the proinflammatory potential of phage alone, cytokine levels in mock-infected mice were measured after phage treatment twenty-four hours later. These mice showed no appreciable levels of either TNF-α or MIP-2 in the lungs forty-eight hours after treatment.

     Alternative Phage Treatments

In addition to the study of the efficacy of phages in the treatment of bacterial infections, notable research has been conducted in the study of the effectiveness of combinations of phage and antimicrobial therapy, as well as the use of lysins produced by phages to directly combat pathogenic bacteria.

Zimecki and colleagues describe the benefits of combined action of lactoferrin and phages in the treatment of infection of Escherichia coli and S. aureus in mice (12). Lactoferrin (LF) is a protein found in the secretory fluids of mammals and the secondary granules of neutrophils, and exhibits both antibacterial and anti-inflammatory actions. It has also been shown to enhance neutrophil production. In the experiment, mice were injected with E. coli and S. aureus, while the E. coli and S. aureus specific bacteriophages were administered intraperitoneally or orally one hour after bacteria injection. Lactoferrin was injected intravenously 24 hours before infection. The results showed that both oral and intraperritoneal phage administration proved effective in reducing numbers of bacteria in the liver, but the combined action of bacteriophages and LF produced a significant additive effect.

Fischetti and colleagues studied the antibacterial effect of bacteriophage lysins in various types of infections (5). Lysins, the phage protein that cause bacteria to rupture, or “lyse”, by breaking down their pepdidoglycan cell wall, can kill gram-positive bacteria seconds after contact. These researchers studied the efficacy of lysins in the treatment of different diseases as well as their potential to be used synergistically with antibiotics. In contrast to studies done with phages, this study found that one dose of lysin administered one hour after infection with S. pneumonia was not enough to fully eliminate the bacteria after 48 hours. However, when treatment was initiated at 24 hours and every 12 hours thereafter, 100% of mice recovered from otherwise fatal pneumonia. Since lysins cannot reproduce like phages, multiple enzyme doses or a constant infusion of enzyme is required to completely eliminate infection.

 

Safety of Phage Therapy

Although bacteriophages are generally regarded as safe, there are a few concerns that are addressed by the literature.  Some concerns address some of the same problems associated with antibiotics, such as acquired resistance, while other concerns are directed exclusively at the safety of phages for use as a therapeutic agent.

     Concerns with Lytic Activity

In their introduction to the potential of phage therapy in the treatment of infections, Kropinski and colleagues introduced the concern over some phages, called temperate phages, being able to potentially transfer virulent genes between strains of bacteria, a process called “lysogenic conversion” (6). Temperate phages undergo the lysogenic cycle to integrate their DNA into the bacterial DNA, which is then replicated in each bacterial daughter cell.  Virus DNA can sometimes carry genes that increase bacterial pathogenicity. Also, infection by temperate phages does not always lead to propagation and cell lysis so this type of phage does not kill 100% of bacteria. Therefore, only lytic phages, where infection leads exclusively to cell death and lysis by the lytic cycle, have the potential to be used as a therapeutic agent.

                         

            Figure 2. Bacteriophage Lysogenic and Lytic Cycle. Retrieved from <http://www.mining.ubc.ca/cerm3/>.

The direct killing and lysis of bacteria by lytic phages, however, brings up new concerns in the safety of this therapeutic agent. Matsuda and colleagues addressed the role that phage therapy can have in the increased expression of inflammatory mediators which can lead to dangerous complications in patients (9). Lysis of some species of bacteria can lead to endotoxin release, which causes a tremendous increase in pro-inflammatory cytokine production, possibly resulting in toxic shock. Endotoxin release is also a potential problem with antibiotics that cause lysis. These researchers studied the efficacy of mutant A3 T4 (LyD) phages, which do not express holin, a protein needed to lyse bacterial cell walls. Since these are lytic phages, infection will always lead to cell death, but since they lack holin, should not lead to cell lysis. A study was conducted on mice with peritonitis caused by E. coli. Mice injected with LyD phages showed a significant survival advantage, with 81% survival in 48 hours. These findings were compared to control mice (0%) and wild type phages (52%). LyD phage therapy decreased endotoxin levels as well as cytokine release.

     Immune Response

In contrast to the positive effects on the immune system that phages showed in some studies, there has been a concern over a possible negative immune response to phages, especially after repeated treatment. An acquired immune response to phages or their derivatives through antibodies can severely limit the treatment potential of these viruses. 

In the study conducted by Fischetti on lysins, the effects that antibodies have on the therapeutic effects of phage particles was examined. Since lysins are proteins that can stimulate an immune response, activation by the immune system could show a significant interference in the therapeutic effect, something that is not a problem in traditional antibiotics. To investigate this problem, rabbit hyperimmune serum raised against the pneumonia-specific phage enzyme Cpl-1 was tested for its effect on lytic activity. The results of the study found that highly immune serum slows, but does not block the lytic activity of Cpl-1. A study was then conducted on mice that tested positive for antibodies against Cpl-1 from a previous inoculation with the phage. After intravenous inoculation of pneumococci, mice with the antibody against Cpl-1 showed the same degree of reduction of bacteria as naïve mice, indicating that lysin antibodies have little to no neutralizing effect.

In their research article on Burkholderia cenocepacia pulmonary infection, Carmody and colleagues suggest that the effect of phage therapy is rapid and likely faster than specific immunity can develop. However, the effect of long-term exposure to circulating phage is unknown. Repeated dosing using phages with different antigens to avoid immunologic reactivity is a possible means to circumvent this problem. Most scenarios in clinical settings would involve cocktails of multiple phages with different receptor specificities.

 

Conclusion

The literature reviewed in this article has contributed to the understanding of the potential, as well as the present obstacles in bringing phage therapy into a mainstream reality. These articles present impressive research on the efficacy of phage therapy, as well as issues that need to be addressed for future studies. The results of the study on the effectiveness of phage therapy look encouraging in both direct lytic activity and immune system enhancement. The safety of this method raises some issues, and more research must be done before this therapy can gain wide acceptance for use in humans.

Most research that has been conducted formally has been on mice, and human studies and treatment remain exclusive to Eastern Europe. It will be a major challenge for phage therapy to take hold in western medicine because it challenges the conventional view of a “drug.” Unless pharmaceutical companies can make a sure profit and regulatory approval, many will be unwilling to invest in this technology.

By taking small steps in that direction, however, this technology can someday take hold in western medicine. Regulatory approval might be difficult for “live” viruses, but the use of bacteriophage lysins in the control of bacterial infections could gain easier acceptance. Continued research on new alternative therapies such as this will help expand our treatment options beyond antibiotics.

References

1. Abhilash, M., Vidya, A.G., Jagadevi, T. (2009). Bacteriophage Therapy: A War Against Antibiotic Resistant Bacteria. The Internet Journal of Alternative Medicine. 7 (1).

2. Borysowski, J.. (2008, Sep). Is phage therapy acceptable in the immunocompromised host?. International Journal of Infectious Diseases. 12(5), 466-471. from Science Direct.

3. Carmody, L. A. (2010, Jan). Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection. The Journal of Infectious Diseases. 201(2), 264-271. from Medline.

4. Chhibber, S. (2008). Therapeutic potential of bacteriophage in treating Klebsiella pneumoniae B5055-mediated lobar pneumonia. Journal of Medical Microbiology, 57 pp. 1508-1513.

5. Fischetti, V. A. (2008, Oct). Bacteriophage lysins as effective antibacterials. Current Opinions in Microbiology. 11(5), 393-400. from PubMed.

6. Kropinski, A. M. (2006, Sep). Phage Therapy- Everything Old is New Again. Medical Microbiology. 17(5), 297-306. from PubMed.

7. Letkiewicz, S.. (2010, Nov). The perspectives of the application of phage therapy in chronic bacteria prostatitis. Fems Immunology and Medical Microbiology. 60(2), 99-112. from ISA Web of Knowledge.

8. Levin, B., Bull, J. (2004). Population and Evolutionary Dynamics of Phage Therapy. Nature Review: Microbiology. (2), 166-173.

9. Matsuda, T.. (2005, Jun). Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and improves survival in a murine peritonitis model. Surgury. 137(6), 639-546. from ScienceDirect.

10. Sulakvelidze, A.. (2005, Jun). Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections . Drug Discovery Today. 10(12), 807-809. from ScienceDirect.

11. Zimecki, M.. (2009, Aug). Effects of prophylactic administration of bacteriophages to immunosuppressed mice infected with Staphylococcus aureous. BMC Microbiology. 17(9), 169-. from PubMed.

12. Zimecki, M.. (2008, Feb). The concerted action of lactoferrin and bacteriophages in the clearance of bacteria in sublethally infected mice. Postepy Hig Med Dosw. 7(62), 42-46. from PubMed.