Therapeutic Uses of Bacteriophages - Research Paper Example

THERAPEUTIC USES OF BACTERIOPHAGES Introduction Bacteriophages or phages are the most abundant and ubiquitous eubacterial and archaeal viruses, suitably described as ‘viruses of the prokaryotes’. Ever since their almost simultaneous discoveries by Twort in 1915 and D’Herelle in 1917, the use of phages in treatment of bacterial infections has been explored (Ackermann, 2010). However, with the advent of antibiotics in the year 1928 this field of research was largely ignored. The 1960s saw the revival of interest in bacteriophages and their significance in molecular biology research was explored. In modern world, bacteria are developing resistance to a wide range of antibiotics, the problem being graver in developing countries where low doses and discontinued treatment course fails to eliminate bacteria that are resistant to low dose of antibiotics such as tuberculosis, pneumonia and dysentery causing bacteria. Moreover since in more than half of the world antibiotic production is used for animals mainly as growth promoters in industrialized animal husbandry, these antibiotics easily reach human body and further cause cross resistance to the antibiotics used in human medicine (Lorch, 1999). Thus the urgent need to find alternatives to antibiotic therapy has led us to the field of phage therapy which has not only been well researched, but also applied successfully in many European countries and the erstwhile Soviet Union. This paper aims to study the relevance and applicability aspects of phage therapy by exploring the history of progress, mechanism of action and current researches in the field of phage therapy. History The earliest reports of the affectivity of phages as antibacterial agents can be traced back to 1896 when Hankin reported presence of an antimicrobial agent effective against Vibrio cholera, in waters of rivers Ganges in India. The discovery remained unexplored till D’Herelle’s rediscovery of bacteriophages in 1917. D’Herelle recognized the significance of his discovery and widely practiced and promoted phage therapy in several countries like Vietnam, Holland, India, USA, France and Soviet Union, using the viruses for treatment of human dysentery, cholera, chicken cholera, bovine hemorrhagic fever, Bubonic plague and many other staphylococcal and streptococcal infections (Ackermann, 2010). The interest in phages peaked in 1930s with a total of 560 publications exclusively on the subject in the period spanning 1920-1930. However, with the discovery of antibiotics and inconsistent results for phages due to euphoria dominating rationale in conduct of clinic trials lead to loss of interest in phage therapy till its revival in the last few decades (Ackermann, 2010). Thus history of phage and phage therapy can be demarcated in to four phases: “early enthusiasm, critical skepticism, abandonment, recent interest and appraisal” (Summers, 2001). Meanwhile the research on phage therapy flourished in Soviet Union since the country was dealing with a grave situation of antibiotic resistance even in 1950s (table 1). The Institute of Bacteriophage Microbiology and Virology founded in Georgia in 1923 collected specimens of antibiotic resistant bacteria from all over the country and developed corresponding phage effective in controlling them. Besides Soviet Union, Poland was another major site of relevant researches, however lack of publications in English, along with political barriers limited the use of these publications to Russian and Polish scientists till the end of cold war and the advent of internet (Lorch, 1999). The modern phase of phage studies began with D. E. Bradley’s publication of phage morphology and classification, and was followed by explosive and still expanding interest in phages (Ackermann, 2010). Table 1: Major Human Phage Therapies Performed In Soviet Union and Poland (Sulakvelidze et al., 2001) Principle Host cell lysis is a consequence of two different mechanisms observed in different types of bacteriophages. In double stranded DNA phages of gram positive as well as gram negative bacteria a holin-endolysin system is responsible for host cell lysis (figure 1). Endolysins are a group of enzymes including lysozymes, endopeptidases, transglycosylases, amylases that are contribute to the muralytic activity and are dependent on holing for their activation. Holins on the other hand are small proteins capable of forming lesions of host cytoplasmic membranes thus providing signals to the endolysins and enable access of endolysins to cell wall peptidoglycan. In the ssDNA phages and SSRNA phages, no endolysin or holin are produced, but a single gene is responsible for host cell lysis. Phage encoded proteins interact with host enzymes responsible for peptidoglycan biosynthesis, causing an imbalance or inhibition of its synthesis, thus causing host cell lysis (Takac et al., 2005). It must also be remembered that of the two types of bacteriophages; lytic and lysogenic; lysogenic bacteria are poor candidates for phage therapy since they get incorporated in the bacterial cell machinery and can live there for generations causing host cell lysis only in the presence of certain factors that exert physiological stress on the host such as a carcinogen (Sulakvelidze et al., 2001). Figure 1: Mechanism of endolysin mediated phage therapy (Fischetti, 2006) Pharmacokinetics Phage therapy has been theoretically considered to hold many advantages compared to antibiotics (table 2) and phages have been used successfully to control bacterial infections in humans, as well as experimentally infected animals. Despite the alleged superiority of phages over antibiotics for therapeutic purposes, the major issues to be considered in context to phage therapy are the Biology of the phage itself. While some phages are highly specific, others have broad host range like antibiotics. Thus not all phages can be utilized without specific information regarding causal bacteria. It is important to identify host range for particular strains of phages prior to their utilization. Next the problem of pharmacokinetics of the phage preparation due to them being live and replicating forms a major issue. Storage problems can be overcome by keeping them in lyophilized form since they are more stable and active compared to liquid forms. Whole phage preparation also several advantages compared to extracted components. They are not only more resistant to environmental conditions, but are also self replicating once inside the body (Veiga-Crespo & Villa, 2010). It is also difficult to formulate the exact dosage and preparations with exact proportions of the various components in the extracted components of the phage. Mathematical models capable of predicting the behaviour of phages in vivo conditions also need to developed to understand the details of the therapeutic mechanism in vivo. Table 2: Comparison of Prophylactic and Therapeutic Uses of Phage and Antibiotics (Sulakvelidze et al., 2001) Other aspects of phage pharmacokinetics that are incompletely understood and form prerequisites for their utilization as antibacterial agents in vivo conditions are mechanism of approach of phages to target bacteria. It has been reported that phages can cross blood brain barrier and are more effective than antibiotics in parts of body with poor circulation (Veiga-Crespo & Villa, 2010). The potential routes of administration of phages have been identified as oral, rectal, topical and parenteral, with topical route being most favoured for control of wound infections (Doresinski, 2009). Current Status During the last few decades many attempts have been made to review the historical aspects of phage therapy and many researches have been conducted in model and natural system to assess the suitability of various phage strains against the various species of antibiotic resistant bacteria. Researches using mice, chicken, calf and fish model systems have been conducted in recent years and have led to remarkable progress in developing an understanding of phage therapy. One of the most publicized series of studies on phage therapy was conducted in 1980s on E. coli O18:K1:H7ColV+, a known pathogen of calves by Williams Smith and colleagues. The study utilized phages that require K1 antigen for infection. They explored the rationale behind rapid development of resistance against phage therapy and isolated 9 anti K1 coliphages which were effective against E. coli in experimental mice infection. Affectivity of single dose treatment was reported to be higher than multiple doses of tetracycline, ampicillin, chloramphenical, or trimethoprim and sulfafurazole combination. The study by Smith et al was used to conduct similar researches for affectivity of phage therapy against chicken, mouse, fish, calves and beef pathogens. Most of these studies reported the effectiveness of phage therapy; however, more than half of the beef pathogens were reported to be resistant to phage pool. Similar data was obtained for phage therapy trials conducted on rabbit diarrhea pathogen (Summers, 2001). One of the chief model studies was conducted by Chighladze and associates against Salmonella. Salmonella phages were isolated combined in to a cocktail which was found to be lytic to 232 of the 245 Salmonella isolates which represented 21 sero and 78 PFGE types. When this cocktail was applied to an artificially contaminated surface, the Salmonella were reduced to undetectable levels within 48hrs. For many of the pathogens potential therapeutic lysins are now identified namely Bacillus anthracis, Streptococcus pyogenes, S. agalactiae,S, pneumonia, Enteroccocus faecalis, E. faecium etc (Doresinski, 2009). A cocktail of phages has been approved by FDA to be used in form of ready to eat meat and poultry products to control Listeria. monocytogenes infection (Fischetti et al., 2006). Phages have also been utilized as vehicles to administer vaccines and other therapeutically relevant particles in form of peptides or as DNA vaccines which are not the inherent active ingredient of the phage (Veiga-Crespo & Villa, 2010). Current studies in phage therapy involve the use of endolysin component of phage instead of the entire bacteriophage to control antibiotic resistant bacteria in animal models. The cell wall binding domains of the endolysins direct the enzyme molecules to their respective positions on the bacterial cell wall, while the catalytic site is able to cleave the bonds in the peptidoglycan of the cell wall to bacterial cell lysis. Three dimensional structure and catalytic mechanism of the endolysins is extensively being studied and the molecule itself being considered a promising antimicrobial agent with potentially important applications (Hermoso et al., 2007). Studies have also been conducted to explore the synergistic effects of various lysins and of lysins in combination with antibiotic. Prominent synergistic effects of lysine and antibiotic have been demonstrated against S pneumonia (Veiga-Crespo & Villa, 2010) Mayer and colleagues have been able to subclone the endolysin gene from a Clostridium difficile bacteriophage and express the gene in E. coli. The endolysin has been reported to be effective against epidemic strain of C. difficile along with 29 other strains. An expression of the gene in Lactobacillus lactis shows prospects for the utilization of L. lactobacillus as a potential delivery system to gastrointestinal tract (Doresinski, 2009). For human uses phages have been explored for treating rectal, surgical and burn wounds. The efficacy of phages has in fact been reported to be higher in treatment of murine burn wound infection, in comparison to antimicrobial agents such as gentamycin and silver nitrate (Kumari, et al., 2011). In a recent study efficacy of E. faecalis phage Φ SH-56 for controlling enterococci obtained from diabetic foot wound was demonstrated (Vinodkumar et al., 2011). Future Prospects Present state of issue of antibiotic resistance renders the need to develop alternatives to antibiotic therapy urgent. Phage therapy provides such an alternative, already studied and even time tested. However, before it can replace antibiotics as control agents for bacterial infections, it is imperative that the problems associated with its use for the same are directly addressed and means are devised to overcome them. Some of the major issues that need to be resolved include host range identification of phages, presence of bacterial debris in phage preparations, presence of host bacteria in therapeutic preparations of phages, rapid removal of phages from circulation due to them being identified as foreign protein particles, effective and accurate means of identification of lysogenic bacteria (against therapeutically more significant lytic bacteria), and finally appearance of antiphage antibodies in animals and humans. Theoretical solutions for each of these problems have been suggested and some of them are also dealt with at experimental level. However, it is imperative that long term feasible solution to each of these problems is sought before large scale and popular utilization of phage therapy can be recommended. Besides these further researches in the field of phage therapy can explore the possibility of using the genetic engineering to develop new therapeutically applicable traits in phages. Moreover the potential of the combined use of antibiotics and phages for therapeutic purpose can be further explored for exploitation of both of these individually effective strategies (Carlton, 1999). Conclusion Conclusively, with growing number of pathogenic bacteria becoming resistant to most if not all antibiotics and increase in the number of immunosuppressed patients, world seems to be reverting to the ‘pre antibiotic era’ (Sulakvelidze et al., 2001). Alternatives to antibiotics are therefore being explored, one of the most promising of which is phage therapy. Phage therapy has been practiced ever since the discovery of bacteriophages by Twort and D’Herelle and is based on the lytic and lysogenic properties of bacteriophages. Recent interest in the concept of phage therapy has lead to a series researches exploring the historical aspects of phage therapy as well as conducting fresh experiments to explore the potentials, problems, safety and comparative efficacy of phage therapy. As a consequence remarkable progress has been reported in use of specific isolated phages for against specific antibiotic resistant bacteria. Attempts have also been made to introduce vaccines and other therapeutic agents in to circulation using phages as carriers. Contrarily phage proteins or endolysins have been isolated and used as independent antibacterial agents or enzybiotics. Finally the most important possibility being explored engineered phages designed to act against specific pathogen. The field is promising and a cautious approach can provide a strong weapon in the fight against diseases. References Read More