Bacteriophages were used for the therapy of infectious disease soon after the early discovery of the twentieth century. Once antibiotic were discovered , interest in phage therapy were waned away from the Asian countries. But, developed a consistent research regarding the treatment and control of human and animal diseases using phage products. The world wide approach to develop phage and phage based antibacterial products into safe and reliable drugs because of multiple drug resistance organisms is growing very fast in our counties. Phage has a subject of research in term of determination of safety, immunogenicity and efficacy so that can be employed as therapeutic and vaccine components as well.
Persistence to pathogenic bacteria to antibiotics is fast becoming a grave medical and veterinary health problem. Bacteria not only developing multiple resistance to range of antibiotics which threaten to make new achievements of the modern medicine fatile, but also acquiring virulence genes (Fernandes, 2006), there by posing double fold threat to the human kind.
Bacteriophage therapy/PT is rapidly re-emerging as possible modality for treatment of bacterial infection. The growing phase of phage causes lysis of bacterial cell through enzyme mediated mechanism with release of a number of progeny phages that attack specifically more bacterial cells and eventually eliminate the whole bacterial population.
Phage therapy has been used for single/multiple infections caused by Strepto, Staphlyococcus, Enterobacter, Mycobacterium, E. coli, Salmonella and deadly Psuedomonas (Bunting, 1997; Alisky.1998). PT has been successful in treatment of secondary bacterial infection, septicemic wounds, and GIT infections in human patients and in veterinary practices and opened a new possibility of treating mastitis, metritis and pneumonia (Barrow and soothill, 1997). The phages can be administered by local, intramuscular, intravenous and spray. Therapeutic effect is more when directly applied on wound (Barrow et al., 2008).
In spite of some limitation like narrow host range, inadequate purity of phage preparation, poor stability and viability , failure to differentiate lytic and lysogenic phages and development of neutralizing antibodies, phages definitely have a role to play in the treatment and control of infectious organism, both independently and combination with peptidoglycon-hydrolyzing enzymes induced by them and antibiotics. Efforts towards exploiting the treatment of multi-drug resistant organism especially of Veterinary health importance are very meagre in our country until now, though countries in western and Eastern Europe world are engaged in extensive phage therapy and accumulated no of products and literature. The present review will analyze the prospects of phage and phage products as the anti-infective modalities having potential for safe, effective and biologically acceptable therapy.
Bacteriophage Biology and Interactions with Host Bacteria
Bacteriophages (phages) are bacterial viruses that play profound role in the evolution of their host. Whole genome sequencing of bacteria has revealed that phage elements contribute significantly to sequence diversity and can potentially influence pathogenicity.
It is certain that the variety of phages that have been isolated and characterized represent only a tiny fraction of the total. Bacteriophages cannot infect mammalian cells but specifically target bacteria. This specificity is highly refined and each phage will only attack one species or in some cases a single strain of bacterium. There are a variety of different morphological types of bacteriophage, the head (capsid) is a protein shell often in the shape of an icosahedron; this contains the viral genome that usually comprises double-strand (ds) DNA.
The tail may or may not be a contractile structure and to this are connected usually six tail fibres containing receptors at their tips that recognize attachment sites on the bacterial cell surface. Not all phages possess tails or tail fibres and here other attachment mechanisms are in place. Most of the bacteriophages of relevance to this review belong to three families the Myoviridae and the Podoviridae comprising 15 genera. The remaining phages occupy 10 families each with a small number of members.
Despite the large number of publications on phage therapy, there are very few reports in which the pharmacokinetics of therapeutic phage preparations is delineated. The few publications available on the subject suggest that phages get into the bloodstream of laboratory animals (after a single oral dose) within 2 to 4 h and that they are found in the internal organs (liver, spleen, kidney, etc.) in approximately10 h. Also, data concerning the persistence of administered phages indicate that phages can remain in the human body for relatively prolonged periods of time, i.e., up to several days (Alexander et al., 2001). As for their bactericidal activity, therapeutic phages were assumed to kill their target bacteria by replicating inside and lysing the host cell (i.e., via a lytic cycle). However, subsequent studies revealed that not all phages replicate similarly and that there are important differences in the replication cycles of lytic and lysogenic phages.
Recent Application of Phage Therapy
S.aureus is the most important organism associated with mastitis of ruminants. Though a lytic phage was successful used to treat mastitis induced by homologus S.aureus in mice model (Rawat et al., 2007), phage therapy for mastitis only partially successful. Because of reduced lytic efficacy of phage in presence of raw milk. In Veterinary practices, phage has been used to treat animals infected with Acinetobacter baumanii or Psuedomonas aeruginosa and mice with systemic and local infections associated with Vibrio vulnificeus.
Hauff and collegues have conducted series of interesting experiments to explore possibilities of phage therapy against Colibacillosis in broiler chicken. They administered phage through different routes either alone or contamination with enrofloxacin to treat severe E.coli respiratory infection. Similar experiments on phage therapy against Campylobacter jejuni colonization in broilers have given very emerging results.
Soothill and co-workers who explored the effectiveness of phage in controlling infections caused by P. aeruginosa and A. baumannii in burn wounds .These pathogens are particularly problematic in these cases since both species exhibit multiple resistance to antibiotics, can spread to cause generalised infections and can lead to rejection of skin grafts. Initially using guinea pigs as a model system, Soothill showed that the destruction of skin grafts by P. aeruginosa could be prevented by prophylactic administration of phage (Geoffrey, 2007).
Biswas et al., (2002) performed experiments using a mouse model of vancomycin- resistant Enterococcus faecium (VRE) infection. They isolated VRE-specific phage from raw sewage and selected one that showed an antibacterial effect against 79% of the studied strains in vitro for animal experiments. They showed first that intraperitoneally (i.p.) 45 min post infection administered phage was able to rescue mice from VRE bacteremia and that the rescue was associated with a significant decrease in bacterial numbers in blood.
Matsuda et al (2005) have explored the potential of lysis-deficient bacteriophagesin the treatment of mouse peritonitis. They used a lysis-deficient E. coli bacteriophage (t amber A3 T4) with a mutation in the gene for the production of holin. This was a genetically engineered mutant of T4 phage only able to infect E. coli. Hence these phages were able to infect host cells and to replicate within but not to lyse. The bacterial cells were thus killed but remained largely intact following infection. When used in an experimental murine peritonitis model, treatment with lysis-deficient phages was shown to improve survival of the animals compared with other treatment modalities. In addition, these phages killed the infecting bacteria but in a manner that significantly minimised the release of endotoxin and inflammatory cytokines compared with treatment using lytic phage.
Intracellular Phages as Future Antibacterial?
It was found that Mycobacteria, specifically M.avium and M.tuberculosis,being intracellular pathogens that replicate and survive within the mononuclear phagocytes were difficult to treat with lytic phage . For addressing this problem, a benign species of Mycobacterium was used as Trojan horse to deliver lytic phage into the macrophage infected with M. avium and M. tuberculosis. TM4 is a lytic phage that kills both M.avium and M.tuberculosis. When TM4 is delivered by transiently infected M. smegmatis with phage there is a rapid killing of both M. avium and M. tuberculosis with in RAW 264.7 macrophages. The in vitro experiments with M.avium and with M.tuberculosis-infected macrophages and M.smegmatis transiently infected with TM4 showed an unexpectedly large time and titre dependent reduction in the number of viable intacellular organism. In vivo trial in mice shown correlation with in vitro studies and has opened potentially a novel concept to kill intracellular pathogenic bacteria, which warrants future development (Eaton and Bayne-Jones, 2004).
Phage Lysate Bacterin as a New Vaccine to Control Bacterial Diseases
Since the earliest days of bacteriophage research, it has been reported that bacteriophage lysates could be effective at eliciting protective immunity against a variety of bacterial strains (Matsuzaki et al, 2005 and O’Flaherty et al, 2005). Many workers of the period believed that phage lysates were superior to preparations of whole bacteria for vaccination to prevent bacterial diseases. Later, Compton (1930) compared efficacies of plain-formalinized and lysed plague vaccines, and found that the lysate vaccine was superior to the formalin inactivated vaccine. The only investigation conducted on use of bacteriophage as immunizing agent against barbone in an emergency situation of severe outbreak, resulted in control outbreak in buffaloes in India and China during 1920s. The observations made on the biphasic protective response of phage lysate, and inverse correlations of dose of injection with onset of protective immunity are extremely significant.
Despite numerous examples of efficacy, phage lysates were not found superior in every system examined. This was perhaps due to insufficient understanding of the immune system in those days. Due to variability of observed results, study of bacteriophage as an immunogen was abandoned. For some time but recently, the potential of phage lysates as vaccines has been re-investigated and a patent has been awarded for control of Salmonella infections in poultry (Pasternack et al., 2009).
A phage lysate bacterin is defined as a composition comprising bacteria and/or fragments of bacteria killed by lytic bacteriophage which will induce an immune response, either cellular or humoral or both. The bacterial components in such a composition are produced by infection of bacteria by lytic bacteriophage followed by production of new bacteriophage particles released in a subsequent lysis of the bacteria in what is termed a lytic burst. Substantially all of the bacteria in the suspension are killed by the phage infection, meaning that at least 90%, preferably 95%, more preferably 99% of the bacteria in the suspension are killed by the bacteriophage. Preferably, residual live bacteria are removed by means such as centrifugation or filtration so as to render the bacterin bacteriologically sterile, particularly for the bacterial host organism used to make the bacterin.
The immediate attention need on Phage lysins, or endolysins as antimicrobial agents against gram positive bacteria and have been applied to a variety of pathogens, such as E. coli, B. anthracis and P. multocida. Over expression of recombinant endolysin could be made availability of endolysin in the market as antimicrobial agents.
Bacteriophage, phage induced lytic enzymes and phage lysates are bound to become an indispensible part of our armoury against bacterial infections with or without currently available antibiotics and vaccines. Although, the concept of phage therapy is not new and phages have been employed to treat a wide range of bacterial infections of humans and animals, it is the only recently these antibacterial and vaccine have once again attracted attention throughout the world. Phage and phage lysate have been reported to have more efficacy than antibiotics and vaccines. Continuous research is needed to establish phage-based therapeutic and vaccine system in our country.
The authors are grateful to the DIRECTOR of IVRI for providing facilities which initiate research on bacteriophage in India and made this review possible.
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