Carl R. Merril, Dean Scholl, and Sankar Adhya




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Phage Therapy

Carl R. Merril, Dean Scholl, and Sankar Adhya


CARL R. MERRIL AND DEAN SCHOLL Section on Biochemical Genetics, NIMH, NIH, Bethesda, Maryland 20892. SANKAR ADHYA Section of Developmental Genetics, NCI, NIH, Bethesda, Maryland 20892


Introduction


The ability of bacteriophage (phage) to replicate exponentially and lyse pathogenic strains of bacteria suggests that they should play a vital role in our armamentarium for the treatment of infectious diseases. However, in spite of an initial enthusiasm, early clinical applications resulted in a negative shift of opinion concerning the therapeutic potential of phage. There are a number of factors that may have been responsible for this rejection of the use of phage as antibacterial therapeutic agents, particularly in countries that require certification based on the results of efficacy and pharmacokinetic studies in animals and humans. These factors include an initial lack of understanding of the narrow host range phage and an inability to purify phage preparations from bacteria products and debris. These contaminating materials often include bacterial exo- and endotoxins along with bacterial cellular components that tend to inactivate phage preparations when they are stored without further purification (60). Another major factor that affected the development of phage therapy was the successful introduction of antibiotics effective against a broad range of bacterial strains. With such antibiotics physicians could often successfully treat infections even before they determined the causative bacterial strain. The narrow host range of phage made such a practice questionable at best.

Despite the current wide spread use of antibiotics, an ever increasing prevalence of antibiotic resistant bacterial suggests that phage therapy merits reconsideration. In addition, knowledge gained since the initial phage therapeutic applications, concerning phage genetics, physiology and molecular biology, should provide beneficial information for current efforts to develop phage into a reliable therapeutic agent. However, there are still a number of gaps in our knowledge, including information on the interaction of phage with mammalian organisms including the reactions of innate and active mammalian immune systems to phage. We also need to develop methods to understand and improve on the pharmacokinetic behavior of phage. In addition, we need techniques to facilitate the rapidly determination of the best phage strain to use for each specific clinical infection before we can make full use of the therapeutic potential of phage.

As the discovery of antibiotics was one of the major events that overshadowed the development of phage as an antibacterial therapeutic agent we have divided the following historical introduction into pre-antibiotic and antibiotic eras.


Pre-antibiotic era phage therapy


Shortly after phage was discovered by Twort in 1915, Felix d’Herelle championed the concept of using them to treat bacterial infections (77; 19). d’Herelle’s first efforts were concentrated on the treatment of avian typhosis in chickens and shigella dysentery in rabbits. Following his reported success in these applications of phage as antibacterial therapeutic agents, he extended the use of phage to the treatment of bacillary dysentery in human infections. In pursuing his phage therapy studies he traveled around the world stimulating both basic and clinical phage research (75). While many of d’Herelle’s ideas concerning phage have proven correct, one idea that he proposed may have resulted in some of the clinical failures of phage therapy. d'Herelle suggested that there might be only one bacteriophage that could adapt to many bacterial hosts. He even named this perceived phage capacity, the “unicity of the bacteriophage” (80). This belief that there was but one phage that could adapt to all bacterial strains may have led clinicians at that time to use inappropriate phage strains. In 1928 d’Herelle was appointed to a professorship in the bacteriology department at Yale University School of Medicine (80). During his tenure at Yale he conducted a number of phage studies including efforts to “adapt” staphylococcus phage to resist inhibition by factors in human serum (20). Following his resignation from his position at Yale in 1934, d’Herelle played a major role in the establishment of an institute in Tbilisi, in Soviet Georgia. This Institute produced large quantities of phage for antibacterial therapy during and immediately following the Second World War and it is still actively pursuing phage therapy applications.

Phage were also used as antibacterial therapeutic agents in Poland, France and they were distributed for clinical practice in the U.S. by a number of major pharmaceutical companies. In 1932 one European laboratory was reported to be distributing 50 liters of phage a day (39). Phage preparations were marketed by three major U.S. pharmaceutical companies. Eli Lilly & Co sold “Staphylo jel”, and other phage “jel” labeled products for streptococcus and colon bacillus. Phage products marketed by E. R. Squibb & Sons and the Swan-Myers division of Abbott Laboratories included a bacteriophage filtrate preparation for staphylococcus and a combined bacteriophage filtrate preparation for staphylococcus and colon bacillus, respectively (74). Problems with some of these commercial phage preparations were found to be caused by the use of organomercury compounds as preservatives. Such preservatives often resulted in loss of phage activity on storage. Variations in the phage strains from one lot to another but marketed under the same label served to further undermine the confidence of clinicians who might otherwise have used these preparations (74). Additional problems in early therapeutic applications of phage may have occurred because of the lack of refrigeration or adequate phage purification. Most phage preparations intended for therapeutic applications were only purified by passing the lysate through filters fine enough to remove the host bacteria. While such purification reduces the risk of bacterial infections it does not remove bacterial debris that can include bacterial endo- and exotoxins, these contaminants can result patient morbidity and mortality. Phage interactions with this material during storage of phage preparations can also result in loss of active phage. These were some of the issues that resulted in the establishment of a Council on Pharmacy and Chemistry by the American Medical Association. This Council concluded that the phage therapy was plagued by a lack of basic understanding and standards for purity or effectiveness (23). A recent review by Ho (32) presents additional information pertaining to this period.

Researchers interested in phage therapy were also concerned that even when phage with demonstrated in vitro antibacterial effects were used for clinical infections, factors present in the serum, tissue debris, cellular components, etc. would inhibit bacterial lysis by phage. They suggested that given these issues any positive effect of phage therapy on the course of an infection was probably due to stimulation of specific antibacterial immunity, and or non-specific phagocytic activity (24; 22).

By 1937 the state of affairs had deteriorated to the point that researchers such as Asheshov and his colleagues stated “no satisfactory evidence has yet been obtained that a phage exerts any significant effect on the course of an experimental infection” (5). Despite their skepticism, Asheshov and his colleagues recognized that part of the problem was associated with the difficulty of repeating experimental results due to failures of experimental design. By using a specific strain of bacteria and phage strains that were shown to be active in vitro against the selected bacterial strain, these researchers demonstrated the ability of the phage to rescue mice injected intraperitoneally with lethal concentrations of “Bacter. Typhosum.” They were able to rescue a significant number of mice even when the phage injections were given as late as 4 hours after the bacterial injection. Without phage treatment most of the animals died within 24 hours. Furthermore, they demonstrated that heat killed phage and non-specific phage strains were not able to rescue the animals. These experiments clearly showed that phage that display antibacterial activity against a particular bacterial strain in vivo may serve as an antibacterial therapeutic agent for the treatment of an animal with a systemic infection with that bacterial strain. They also demonstrated that the antibacterial effect of the phage is due to the physiological functions of the phage since heat killed and non-specific phage could affect no such rescue of infected animals.

Dubos et al. (22), at Yale University, addressed the concerns that factors in blood, tissue and bile might interfere with the lytic activity of phage and that such interference would render phage impotent as antibacterial therapeutic agents. They demonstrated that such interference effects, if present, were minimal as they were able to rescue mice infected intracerebrally with Shigella dysenteriae by injecting anti-Shiga phage into the general circulation. In these experiments they also observed a correlation between an increase in phage titer observed in the blood of infected animals and their rescue, suggesting that the rescue of the animals was due to phage functions including replication and that interfering factors, if present, were insufficient to inhibit the beneficial effects of phage as a potential antibacterial therapeutic agents.

These positive developments came too late to generate much enthusiasm for phage therapy in the Western World. By this time antibiotics were proving superior because of their activity against a broad range of bacterial hosts and in most cases their robust storage characteristics. While the use of phage therapy waned in Western medical practices it continued to be employed in Eastern Europe and parts of Asia. This was due in part to the restriction of information concerning the development of antibiotics, particularly penicillin, by the British and American Governments in the early phase of the Second World War. The Soviet and Polish phage literature concerning the therapeutic use of phage has been extensively reviewed (1). They note that the Soviet and Polish medical researchers studied the efficacy of phage therapy almost exclusively by qualitative clinical assessments of patients. Details of phage dosages and clinical criteria were reported in a “sketchy” manner. For these reasons most of the studies from Eastern Europe will not meet current standards in countries that require certification based on the results of efficacy and pharmacokinetic studies in animals and humans.


Antibiotic era phage therapy


It was thirty years after the Dubos' work before animal studies were again performed to investigate the efficacy of phage to treat bacterial infections. This hiatus was due in large part to the success of antibacterial chemotherapeutics such as the sulfonamides, discovered in 1935 followed by the antibiotics during and following the Second World War. However, some of the resistance may have been due in part to effect of theoretical explanations that arose to explain the perceived failure of phage therapy. These are best captured by the following statement from Stent’s 1963 book, Molecular Biology of Bacterial Viruses.


Just why bacteriophages, so virulent in their antibacterial action in vitro, proved to be so impotent in vivo has never been adequately explained. Possibly the immediate antibody response of the patient against the phage protein upon hypodermic injection, the sensitivity of the phage to inactivation by gastric juices upon oral administration, and the facility with which (as we shall see presently) bacteria acquire immunity or sport resistance against phages, all militated against the success of phage therapy”.


As suggested by Stent, antibodies, produced by the adaptive immune system, may be of importance for the inactivation of phage, particularly in individuals repeatedly exposed to a specific phage strain. This ability of phage to provoke an antibody response in normal individual has been used over the past three decades by Ochs and his colleagues, who employ the phage phi X174, to study normal individuals and patients with immune deficiencies (56,57). They demonstrated that the adaptive immune system of normal individuals, who are naive to a particular phage strain, requires a few days to develop a detectable antibody level and about 2 weeks for a maximal antibody response. However, the innate immune system has been show to be able to rapidly eliminate phage administered systemically. This was investigated in an experiment, published in 1973, that demonstrated that phage injected systemically in germ-free mice were removed rapidly, by the liver and spleen (reticuloendothelial system, RES of the innate immune system), from the circulatory system even though these mice displayed no antibody activity against the phage (30). The authors suggested that this rapid elimination of phage in intact animals “may explain the limited success reported for the phage treatment of infectious diseases.” They also suggested that the rapid rate of phage elimination could be slowed by overwhelming the RES with colloidal particles. Recently a less intrusive method was discovered to circumvent this rapid systemic elimination of phage. This method employs genetic selection to find mutant phage strains with reduced rates of clearance by the RES by employing serial passage techniques for the selection of such variants (50). Infected mice treated with these long-circulating phage variants recovered more rapidly and their symptoms were less severe than those mice treated with wild type phage. In these experiments long circulating lytic mutant Lambda and P22 phage were developed to treat mice infected intraperitoneally with E. coli and S. typhimurium respectively. In the case of Lambda phage the mutants displayed a single base change in the capsid E gene that resulted in the substitution of a lysine for the normal glutamatic acid residue in this capsid protein. Given the large number copies of this protein in the phage capsid the resulting alkaline shift associated with the mutant phage may have been associated with the capacity of this phage to remain in the circulatory system for an extended period of time.

Another concern that: “bacteria acquire immunity or sport resistance against phage” expressed by Stent, was addressed experimentally by Smith and Huggins (67). As predicted by Stent, Smith and Huggins found phage resistant bacterial mutants following the use of a single dose of an E. coli K1 strain specific phage to treat mice that had been infected either intramuscularly or intracerebrally, with a K1 strain of E. coli. Interestingly, the phage treatment of these infected mice still proved to be more efficacious than treatment with antibiotics. In fact, phage resistant bacterial mutations occurred with less frequency following phage therapy then the antibiotic resistant mutants that appeared after antibiotic therapy. Furthermore, the phage resistant bacteria mutants that were observed were found to be lacking the K1 capsule (the receptor for phage attachment) which is associated with a loss of pathogenicity.

Smith and Huggins’ experiments demonstrating that phage injected intramuscularly could be used to treat an intracerebral infection, corroborating the findings of Dubos et al. (22) obtained 39 years earlier, in which phage injected into the circulatory system were successfully used to successfully treat an intracerebral Shigella dysenteriae infection in mice. These observations may help to mitigate concerns that phage, due to their larger size, will not be as effective in treating tissue infections as the lower molecular weight antibiotics (42).

Experiments by Soothill (71) showed that the lytic development of the phage is a critical parameter in the ability of phage to control bacteria in vivo. In separate experiments animals were given either intraperitoneal injections of Acinetobacter baumanii or Pseudomonas aeruginosa and then treated successfully using phage specific for each. However, in a similar experiment with Staphylococcus aureus treatment with phage failed. Staphylococcus phage used significantly less active in in vitro than the phages for the other organisms.

Experimentally induced septicemia and meningitis in chickens and calves have also been successfully treated with phage (7). In these studies, E. coli K1 strains were used along with the K1 specific R phage (the same phage strain as that used by Smith and Huggins). The phage was able to rescue chickens even when the administration was delayed until the onset of symptoms. Like the work by Dubos et al., 55 years earlier, in vivo phage multiplication was found in the brains of infected animals, even when the phage were injected intramuscularly. It is not clear if the capacity of the phage to cross the blood brain barrier was due to effects from an inflammatory response to the bacterial infection or whether phage can normally cross this barrier. It was also found that phage taken up by the spleen persisted in significant numbers for several days after injection, corroborating the findings of Geier et al. (30).

Much of the interest in reviving phage therapy has been fuelled by the appearance of antibiotic resistant bacteria. In this regard, Biswas et al. (10) showed that phage specific for vancomycin resistant Enterococcus faecium could rescue mice that were infected intraperitonially with bacteria. If titers of phage equivalent to the titers of infecting bacteria were given 45 minutes after infection, 100% of the mice were rescued and even when treatment was delayed for 24 hours, when the mice were moribund, 50% could still be rescued. Phage may also be used to treat antibiotic resistant intracellular pathogens. Broxmeyer et al. (12) have demonstrated that it is possible to use Mycobacterium smegmatis, an avirulent mycobacterium, as a vector to deliver the lytic phage TM4 to treat intracellular mycobacterium infections (with either Mycobacterium avium or Mycobacterium tuberculosis) in macrophages. These examples of efforts to develop phage therapy represent cases where specific needs exist for the treatment of clinical infections that are not treatable by current methods. As the occurrence of drug resistant pathogens increases we expect to see an increase in efforts to develop phage into a viable alternative to antibiotics.

All of the work described above involved the treatment of systemic infections with phages injected either intramusculary or intraperitoneally. Phage have been demonstrated to be effective therapeutic agents for the treatment of non-systemic infections. Several studies have shown that gastrointestinal infections can be treated by oral administration of phage. Smith and Huggins (67) and Smith et al. (68,69) showed that various phages could protect calves, pigs and lambs from gastrointestinal infections of enteropathogenic strains of E. coli. In addition Ramesh et al. (59), found that oral administration of a bacteriophage specific for Clostridium difficile could prevent ileocecitis caused by this organism in hamsters. Since C. difficile colonization of the gut is a common consequence of extended antibiotic treatment due to destruction of the normal gut flora, phage used in such a manner may prove to be useful in conjunction with antibiotic therapy.

A study by Soothill (72) showed that bacteriophage could prevent destruction of skin grafts by Pseudomonas aeruginosa. This bacterium commonly colonizes burn wounds and is often difficult to treat with antibiotics. There is an immediate need for a treatment this problem and topical application of phages to burn wounds with Pseudomonas infections may be an attractive way to re-introduce phages to the modern clinical setting.

Terrestrial animals are not the only candidates for phage therapy. Recent studies have shown that phage can be used to treat bacterial diseases of fish in aquiculture (53). In addition, phages have recently been shown to be effective in the treatment of bacterial blight of geranium (26) and bacterial spot on tomatoes (25).   

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