PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, INDIA 2009 VOL EXXIX SECTION-B __ PART IV Prospects of bacteriophage therapy in modern medicine MAYANK RAWAT, S. SURESHKANNAN! and RISHENDRA VERMA Division of Biological Standardization, Indian Veterinary Research Institute, lzatnagar - 243122, India. 1. Central University Laboratory, TANUVAS, Chennai, India. Reccived February 21, 2008, Accepted December 24, 2008 Abstract Bacteriophages (phages) were used for the therapy of infectious diseases soon after their discovery during the earlier part of twentieth century. Once antibiotics were discovered, interest in phage therapy waned in the Western and Asian countries. But countries of Eastern Europe and the former USSR. continued not only to practice this modality of treatment against infectious diseases but also developed it through consistent research efforts. The renewed worldwide approach to develop phage and Phage-based antibacterial products (popularly known iis “Enzybiotics’) into safe and reliable drugs because the virulent organisms are quickly developing resistancetoarange oF antibiotics. ‘The understanding of the physiology and genetics of phages has also considerably increased during last sixty years, Phages hhave now become a subject of esearch in terms of determination of safety, immunogenicity, efficacy and (pharmaco) kinetics so that they can be employed as therapeutic agents in the coutries that enforce regulations for commercialized pharmaceuticals. ‘Keywords: Bacteriophage, endolysin, multidrug resistant bacteria, Introduction Resistance of pathogenic bacteria to antibioties is fast becoming a grave medical and veterinary health problem all over the world. Bacteria are not only developing multiple resistance to @ range of antibi- otics, but also acquiring virulence genes thereby posing a double-fold threat to the human beings. The situ- ation has further been complicated by the fact that aie ed ooh 8 ufo ore eee ad} yea ae cha (etn) wr ete saree ae F ove A aT TY aM et Sata A ator are oft fiz Pree Bet A Reh Re A of mea wah AE eg ET ART he Ged B yorTOURCSTTE BA diame Oot & aa Gee aH TET HT tea oT sen after RRR argetarT sea aT A Ar oA rer Fst te Roh ener wore ore ar cea Tee ar ore ‘coterie wear #1 2 gee ate fears erat tt ot 2 Ewe 2 a i SARA a PR sateen Prieta we 8 1 Ro eT a Rt A) he orga err teen cg en Bk be PH, em B vor, weer, mmetearcren Ae (seta) cere 2 stot segeiare @ fea at 2 8 fore era mem gS et A ‘ob rat attr arodhoret ob Pram ory ae we ciate eee: sheeptoh, sherafirs, aetna wet ar since 1980 no new class of antibiotic has been discovered by any pharmaceutical industry or research organi- zation throughout the world in spite of huge research efforts and inputs! 2. This has led scientists to re- consider the approaches and compounds, many of which were not thought to yield desirable results in the past and were eventually abandoned especially, in the western countries. It is now accepted that those discontinued approaches or compounds may prove320 invaluable for our on-going fight against multi-drug resistant bacterial infections. Lytic bacteriophages have therefore, become the most acceptable and potent option for research during the last 30 years and Bacteriophage! Phage Therapy (PT) is rapidly re-emerging asa possible modality for treatment of bacterial infections’. The reasons for re-considering phage and phage-based prepa- rations as ‘anti-infective modalities with therapeutic potential’ are many and the principles of this approach are very simple. Each bacterium supports growth of bacteriophage/phages after receptor-mediated (0 the cell. The growing phage causes lysis of the host bacterial cell through enzyme-mediated ‘mechanisms with release of a number of progeny phages that attack specifically more bacterial cells and eventually eliminate the whole population. Both, lytic phage particles, and their gene coded products-the phage lysins, open up new opportunities of searching novel antibacterial agents with therapeutic potentials. Some of the recent publications on experiments demonstrating the anti- bacterial activity of lytic phages ©'6 © and the peptidoglycan hydrolyzing enzymes-the endolysins!™” ® have attracted a lot of attention as the targets of research for their therapeutic applications against many human and animal bacterial infections. ‘The general approach for direct use of lytic phage for therapy includes inoculation of bacteria of interest with their specific phages for production of homog- ‘enous therapeutic stocks *3. Selection of a phage for a particular bacterium is done by “Phage sen- sitivity test", They are then tested for their safety and can be used as therapeutic agents, Phages can be administrated by local, oral, intramuscular or intravenous routes. Therapeutic effect is more when directly applied on wound. Cocktail of different phages can be given in multiple injections**:®, Phage therapy has been used for single or multiple infections caused by Streptococcus, Staphylococcus, Enterobacter, Mycobacterium, E.coli, Salmonella and deadly Pseudomonas*”: 8.8, Phage therapy has been suc- cessful in the treatment of secondary bacterial in- fections, septicemia wounds and GIT infections in human patients and in veterinary practice and opens up new possibility of treating mastitis, metritis, pneumonia and other economically important animal diseases. During last two decades, data have been accumulated to show that PT has become both an important alternative and an adjunct to antibiotics in the treatment of bacterial infections®: °°, Theoretical MAYANK RAWAT ef al and practical advantages that PT possesses over the conventional antibiotics are numerous and search for a specific phage against multi-drug resistant pathogen is much easier than the search for a new antibiotic. In spite of some limitations®! (Table: 1), phages definitely havea role to play inthe treatment of infections, both independently and in combination with the peptidoglycan-hydrolyzing enzymes induced by them and antibioties. Efforts towards exploiting this strategy of combating multi-drug resistant bacteria especially of veterinary health importance are very meager in ‘our country until now, though countries in western and eastern European world are engaged in extensive phage therapy research and an enormous amount of literature has been accumulated™®, Several patents on phage and phage-based preparations have also been awarded. 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. Bacter phage Physiology : New Opportunities Bacteriophages / phages are parasites for which bacteria are the natural hosts. Most of the bacteria that are pathogenic to humans or animals are hosts to specific phages. Phages invade bacterial cells, in case of lytic phages, disrupt bacterial metabolism and finally cause bacteriolysis™. The lytic phages express surface legends that are specific for receptors on the surface of host bacterial cells they are targeted to infect. Phages are the simplest and most abundant entities in the biosphere. It is anticipated that more than 10*” phages are present on the earth and roughly 5100 have been identified and reported®. Phages are classified into 13 different families based on their morphological features, type of nucleic acid, and presence or absence of lipid envelope. Among these, 4950 are ‘tailed’ phages, which are composed of an icosahedral head containing their genetic material and a syringe-shaped tail with several fibrils that selec- tively attach to specific receptors on host bacterial cell wall (Figure: 1). Tailed phages are classified into 3 families according to their morphological features of the tail: Myoviridae (contractile tail), Siphoviridae (long, non-contractile tail), and Podoviridae (very small tail). Rest of the phages representing only 4% of the total are classified into 10 families. They are cubic, filamentous or pleomorphic with dsDNA, ssDNA, dsRNA, or ssRNA genome™, Following injection ofBACTERIOPHAGE THERAPY IN MODERN MEDICINE, Fig. | - Electronmicrograph of a lytic phage against ‘Staphylococeus aureus. phage nucleic acid (DNA or RNA) in the cells metabolic machinery starts exclusive production of progeny phage particles, the number of which may range between 50 and 200. ‘There are two types of bacteriophages; yt virulent and temperate or lysogenic. Virulent phages induce infection of bacteria resulting cell and producing clear ‘Plaques’ (Figure: 2) in the awns of susceptible bacteria, This ability of the lytic phages makes them a potent candidate for anti-infective ‘therapy. Temperate phages, on the other hand, integrate theit DNA within the genome of host bacteria resulting inlysogenic infection and phage-genome is subsequently passed to all daughter cells of the host after division, Lysogenic phages are not therefore, considered as ‘candidates for therapeutic application. ‘The progeny of lytic phages is released from their bacteria by degrading the cell-wall peptidoglycan through production of two enzymes called lysins and holins (dsDNA phages) %, or by interfering with a cyto- plasmic enzyme UDP-N-acetyl glucosamine enolpyruvy! ‘ransferase (RNA phages Qb) involved in peptidoglycan biosynthesis. The phage-induced lysins have been found to be very important anti-bacterial agents with enormous potential for therapeutic use!” 35. It can ‘be emphasized therefore, that both lytic phages as such and their gene coded products open up new opportunities of searching novel antibacterial agents potentials. 321 Fig. 2- Plaques produced by a lytic phage in the lawa of a ‘Staphylococcus aureus isolate. Bacteriophage Therapy Early phase History of bacteriophage discovery is very lengty, debatable and controversial subject, Ernest Hankin’®, ‘British bacteriologist reported in 1896 that the waters of rivers Ganges and Yamuna possessed marked antibacterial activity. He particularly suggested that aan unidentified substance that was heat- liable and passed through fine porcelain filters was responsible for this phenomenon and for keeping a natural check ‘on cholera epidemics in India, Two years later a Russian bacteriologist observed similar phenomenon against Bacillus subtilis. Similar observations were subsequently reported by several other investigators and are now thought to have been related to bacteriophage phe- nomenon. Edward Twort’? a medical bacteriologist from England and Felix d'Herelle™ a French-Canadian microbiologist atthe Pasteur institute, Paris, independently reported isolation of filterable entities capable of estroying bacteria and producing clear lytic zones in the bacterial lawns. d'Herelle initially called these lytic zones ‘taches’, then ‘taches vigeas"-and later “plaque’. Both Twort and d'Herelle were jointly given credit for discovery of bacteriophages", a phenom- enon, which was probably observed by Hankin and other microbiologists earlier, with the waters of Ganges and Yamuna but did not explore their findings further. TThe term bacteriophage was derived from the fusion of two words that are thought to be of Gréek origin “Baxeenpov’ meaning “small sticks” (denoting bac-322 ‘Table 1 - Enperienced/Perceived Problems with Phage Therapy. MAYANK RAWAT ef al. PROBLEM Nutrow host-range/lytic spec rum of phages Inadequate purity ofpaage prepar rations Poor stability andlor viability of phage preparations Failure to differentiate lytic and Iysogenie phages Development of neutralizing an- tibodies Lytic efficacy: in vitro verses in OBSERVATIONS: (One o” the possible causes for PT failures. Phage preparations used in earlier times were crude lysates of host bacteria containing (uni- is much faster than de ‘velopment of antibodies by the host, Body fluids such as serum, pus, milk, ascites ‘uid CSF may inhibit infectivity of phages i POSSIBLE SOLUTIONS ‘© Develop a large collection of well-charac- terized phages for a broad range of patho- ons ofan epidemiological rca. © Determine phage susceptibility ofthe tar ‘net pathogen before using the phage prepa- ration for threapy. © Selection broad host-range phage/phages for therapeutic applications and/or use poly ‘lent phage cockiails having lytic activity against majority of strains of the target pathogen. © Develop methods for rapi phage’phages for aspecific © Molecular techniques can be used to enhance hhostrange of certain phages. Use improved techniques for obtaining phage preparations with high purity ‘@ Use advanced purification techniques to purity phages. ‘© Ensure bacterial sterility of the prepara tions. ‘@ Determine viability and ttre of phage be~ fore clinical application Carefully select lytic phages for therapeutic pur- poses. This is also eritical for avoiding horizon- tal transfer of a bacterial toxin, antibioti resist~ ance and virulence genes by lysogenic phages. Develop therapeutic phages and endolysins that are less antigenic. Rescarch on phage-mediated endolysins.BACTERIOPHAGE THERAPY IN MODERN MEDICINE ‘Table 2 - Antibiotics vs, Phage Therapy : Theoretical Considerations* 323, ANTIBIOTICS Usually wide. Both the targeted pathogen and the normal flora ‘may be susceptible to the action. ‘May affect the microbial balance sxdversely leading tothe second- ary bacterial infections and Aysbiosis. Bacterial L-forms not susceptible to antibiotics. ‘Well documented forall antibiot- igs, Antibiotics are metabolized and eliminated from the body and ‘do not necessarily coaventrate at the site of infection ‘© Frequent reports of intesti- nal disorders, secondary ‘bacterial and mycotic infec- tons, hypersensitivity reac- tions? In veterinary practice antibiotic residues in milk and meat, Resistance to antibiotis is not limited to target pathogen only. Time consuming (may require several years). Expensive. No new antibiotic group has been dis- covered since last 30 years, ‘Adapted from Sulakvelidze etal (2001) BACTERIPHAGES Spectrum of activitiy ‘Narrow. Only the targeted pathogen is suscep- tible . Pharmacokinetics Replicate atthe infection site. Available where ever bacteria multiply? Selfreplicating and self limiting. Safety © No adverse side-effects (except a few mi nor) documented, specifically in human in- feetions. (© Horizontal transfer of bacterial toxin, anti- bioti resistance and virulence genes. Pos- le with lysogenic phages. Resistance Even ifany pathogen becomes resistant to one phage, it remains susceptible to a plethora of other phages having similar host range. ‘Technology Every lytic phage isolated against any pathogen ‘ean have some therapeutic potential. Phage encoded endolysins have opened-up pos- sibilities of using entirely new group of antibacterials the “enzybioties’ as potent thera- peutic agents. COMMENTS ‘Narrow spectrum and high specifiy of activ- ity of phages has both advantages and disadvan- ‘tages. Though the normal flora remains unaf= fected, the procedute for selection of candidate phage with therapeutic potential becomes time dependent, Methods for selection of phages with therapeutic potential needs further research. (See text) Phages have peculiar pharmacokinetics. Since there is “exponential growah’ of phages at the site of infection, less frequent administrations ‘may be required to achieve optimal therapeutic effect, More studies required for understanding pharmacokineties of PT (Text). ‘© The minor side effects sonietimes ob- served for therapeutic phages may have been due to large scale bacteriolysis at the sime of infection Effects of post antibiotic release of endotoxins are also frequently e- ported. ‘© Careful selection of lytic phages is needed before therapeutic application, Antibiotic resistance isa serious problem with antibiotic therapy whereas, itis not @ problem for PT. Selection of new phages (c.g, against phage oF antibiotic resistant pathogen) is relatively arapid process which ean be accomplished within days tr weeks, Isolation of new phages should be- come an ever-ongoing process of natural sclee~ ‘ion and conservation324 teria) and, ‘phagien’ meaning “to eat”. But by a careful analysis, both words appear to be of “Sanskrit” origin. Especially, the word ‘phagien’, the etymology of which can be traced to the Sanskrit word Bhogen or Bhojie. ‘Not long after his discovery, d'Herelle began using phages to treat dysentery. That was probably the first attempt to use bacteriophages therapeutically”. Single administration of anti-dysentery phages resulted in complete recovery of a 12-year old boy with dys- entery at the Hospital des Enfant Maladies. The efficacy of phage preparation was further confirmed when three more patients could be successfully treated with one dose of the preparation”, The results of these studies were not published, Bruynoghe and Maisin in 1921* documented first successful use of bacteriophages for therapy of staphylococcal skin infections and published the first report of successful phage therapy. thas also been documented that 'Herelle later used -various phage preparations to treat thousands of people having cholera and bubonic plaque in India”, Soon after those initial reports, several companies began commercial production of phages against various bacterial pathogens. At least five bacteriophage prepa- rations were commercially produced in d'Herelle’s Laboratory in Paris. . The preparations were called- Bacte’-coliphage, Bacte'-rhinophage, Bacte’ pyophage, Bacte'-intensi-phage and Bacte-staphyphage"®. They were marketed by a Company which later was known as LOreal® (France). Phages for therapeutic use were also produced in USA. Seven phage products for human use were produced by Eli Hilly Company of Indianapolis during 1940s. These preparations were targeted against staphylococci, streptococci, E.coli and other com- mon bacterial agents. They were produced as phage- lysed, bacteriologically sterile broth cultures of tar- geted pathogen and were named as colo- lysate, ento- lysate, neiso-lysate and staphylo-lysate Discovery and widespread use of antibiotics in Europe, America and Asian countries after 1940 eclipsed the idea of d'Herelle that bacteriophages might be effectively employed to control bacterial diseases. Though, the western world explored the option of “bacteriophage therapy" ina cursory fashion and eventually abandoned it®, interest in this anti-infec~ tion modality was consistently maintained in the countries ofthe former U.S.S.R and Poland, where bacteriophage therapy was used extensively since d'Herelle’s times. In these countries, phage therapy was used with MAYANK RAWAT ef al. a wide application in the areas of otolaryngology, stomatology, ophthalmology, dermatology, pediatrics gynecology, post-surgery wound infections, urology and pulmonology“*+5 though the effectiveness of phages remained @ matter of controversy in the Western world, Since, the results of those studies were exclusively published in non-English, Russian, Georgian and Polish journals, which were not readily accessible and un- derstandable to the scientists of other parts of the world, they almost remained entirely unnoticed until late 1970s. Two institutes, The Eliava Institute of Bacteriophage, Microbiology and Virology (EIBMV) Georgia (http2!/www.geocities.com/hotsprings/spa/5386) and Institute of Immunology and Experimental Therapy, Polish Academy of Seience, Wroclaw, Poland (http! surfer itd. pan. wroc.pl/index! htm) contributed sig- nificantly by using phages for therapy of clinical cases and for PT research. Though, these studies, mostly on human patients, did not formally meet ‘the current strict criteria of controlled clinical research, they still strongly suggest a very high efficacy of phage treatment and its safety with almost no adverse side effects, In the 1980s, increasing problem with the an- tibiotic resistant bacteria once again motivated the entists of the western world to re-try the forgotten ‘magic-bullets of Stalin, Smith and Huggins (1982- $7) 7 carried out their classical studies to prove that phages were superior to antibiotics when used to treat E. coli induced diarrhea in claves, piglets and lambs. Since then, a lot of interest has been generated among the scientific community of the world and several studies on various aspects of phage therapy have been conducted on terrestrial animal species ranging from chickens to human. Recent studies Some exhaustive reviews on the direct use of selected phages under experimental conditions and in clinical settings, especially in human beings, have been published during recent times’ * 19:42:48 59, Slopek® et al. have documented a 6 year account (1981-1986) of their phage therapy experiences in $50 human patients suffering from various bacterial infections in Poland. The infections were caused by pyogenic Staphylococcus spp. and Gram-negative bacteria (Klebsiella spp. E. coli, Proteus spp and Pseudomonas spp). Out of those S08 cases (94%)326 Poultry meat® disinfectants for surfaces, fomites, hospital walls, instruments and wounds. Bacteriophages can adapt to the environmental variations. Due to this flexibility, emergence of phage-resistant bacterial populations has not been reported. Moreover, ac- tively lytic phages can be selected with a relatively rapid process in comparison to developing a new antibiotic against every antibiotic resistant or even phage- resistant bacterium by ever-ongoing process of natural selection™, Practical use of Bacteriophage Therapy: Challenges: Challenges to implementation of BT in human beings and animals have been identified arid discussed in details in various reviews”. 71, Skurnik and Strauch’! have recommended that before attempting PT, 5 ‘pre- requisites’ with respect to identification of phage: bacteria system, safety and storage of phage preparations, understanding of phage-receptor and validation of efficacy by animal experimentation should be taken into consideration. Promising phage strain for therapy: New methods for early selection: Before direct use of a bacteriophage for therapy, the best phage strain is selected from a panel of phages, much in the way the sensitivity test is conducted for selection of antibiotic. Because of the narrow host range of most phages, selection of a ‘promising" therapeutic phage can only be done after determi nation of susceptibility of target pathogen against a set of phages for the bacterial species of concer. ‘This is achieved by screening the bacterial isolate against the set of phages. When the traditional methods are employed, this process may take days, limiting the use of PT only to chronic or slowly progressing infections. However, some methods that have been developed recently permit the identification of both the infectious agent and a suitable phage within a day or less. ‘One such approach is the use of phages that contain receptor genes such as luciferase”, Similar results can be achieved by using phage without marker genes and adding luciferin and luciferase in the phage and bacterial incubation mixture. ATP generated during lysis of the bacterial cells will induce light production from luciferase system. Here bacterial lysis will serve to identify phage susceptibility without the need to suse genetically engineered phages'®. In addition, mass MAYANK RAWAT et al spectrometry’, DNA micro-array technology also offer possibilities for rapid identification of bacterial agents and therapeutic phages”, Host-range of phages: Development of a large collection of well-char- acterized phages for isolates of a pathogenic species of an epidemiological area is one of the most im- portant approaches to tackle this otherwise advan- tageous characteristic of phages. Determination of phage susceptibility of the target pathogen before using the phage preparation for therapy is the other essential step before selection of therapeutically potent phages fora particularcase. Recent developments in the methods for rapid accomplishment of this process will help in early selection of therapeutic phage preparation for a clinical case, In addition, selection of broad host-range phage/ phages for therapeutic applications and/or use of polyvalent phage cocktails having lytic activity against majority of strains of the target pathogen can over- ‘come the limitation of narrow host range of the phages. Recently, molecular techniques have also been used to enhance host-range of certain phages”. Lysogeny Lysogenic phages are poor candidates for therapy. They do not grow rapidly to produce progeny phages and associated bacteriolysis that is required for their therapeutic efficacy. In order to be therapeutically effective, the selected phage must be lytic against the bacterial pathogen**. The guiding principle is that only lytic phages should be considered as candidates for the purpose of PT. The ‘preferred’ should be sequenced for homologies to known gene for lysogeny. Phage immunogenicity : ‘As phages are common entities in the environ- ment and are regularly consumed with foods and feeds, the development of neutralizing antibodies should not be, in theory, a significant hurdle during initial treat- ment of acute infections specifically in animals. Kinetics of lytic action of phage or lytic enzyme is much faster than development of antibodies by the host. Though, extensive animal studies and clinical experience of Polish scientists have documented that phage preparations do not have any adverse side effects, hypersensitivity or intoxication®®, phage immunogenicity is an impor- tant consideration for two reasons: first, because thereBACTERIOPHAGE THERAPY IN MODERN MEDICINE {sa possibility of adverse reactions and second, because of its effects on pharmacokinetics of PT particularly after multiple injections of phage. In order to address such hypothetical problems, phage-displaying peptide libraries or affinity matrixes made of antibodies from animals and humans, have been used to reduce the immunogenicity of phages. This type of approach has also been used for reducing immunogenicity’ and! or reactivity of circulating antibodies” to therapeu- tically important enzymes. Safety consideration: Horizontal transfer of harmful genes. ‘This problem can be addressed by careful se- lection of lytic phages before therapeutic application. Screening of therapeutic phages for toxin genes, either by biochemical or by DNA sequencing methods can also be done. The presence of toxin, antibiotic re- sistance and virulence genes can be determined by searching phage genomes against GenBank on-line using the Basic Local Alignment Search Tool (BLAST)® or similar programs. Bacterial endotoxins in phage preparations: Presence of even a very small quantity of bac~ terial endotoxins can be unsafe specially when ad- ministered parentrally. By using simple physico-chemical techniques of density gradient centrifugation and different forms column chromatography techniques, endotoxins- free phage preparations can be made, Intracellular pathogens: antibacterials? Phages as future It was found that Mycobacteria, specifically M. avium and M. tuberculosis, being intracellular patho- gens that replicate and survive within the mononuclear phagocytes were difficult to treat with lytic phages For addressing this problem, a benign species of Mycobacterium was used as a ‘Trojan horse” to deliver lytic phage into the macrophages infected with M. avium and M. tuberculosis. TM4 is a lytic mycobacteriophage that kills both M. avium and M. tuberculosis. When TM4 is delivered by transiently infected M. smegmatis with the phage (a non-virulent bacterial species found generally in smegma secre- tions from human genitalia), there is a rapid killing of both M. avium and M. tuberculosis within RAW 264.7 macrophages. The in vitro experiments M. avium and with M tuberculosis-infected macrophages 327 and M, smegmatis transiently infected with TM4 showed an unexpectedly large time and titer-dependent reduc- tion in the number of viable intracellular organisms. In vivo trials in mice have shown correlation with in vitro studies and has opened up a potentially novel concept to kill intracellular pathogenic bacteria, which warrants future development®!: 8, Phage products: Endolysins as therapeutic agents: Currently there are two types of purified phage- based products that have been evaluated as anti-microbial agents. These include bacteriophage lysins and phage tail-like bacteriocins. Bacteriophages infect their host organism to produce their progeny. Release of the progeny phages from the host cell at the end of reproductive cycle (which may last up to one hour) involves enzymatic deg- radation of bacterial cell wall. These cell wall hydrolyzing enzymes are phage gene mediated and consist of a olin and at least one peptidoglycan hydrolase, or lysin capable of dissolving the bacterial cell wall, The enzymatic action of lysins is tightly regulated by holins Which are expressed during the late stages of phage infection and form pores in the cell membrane. This allows the preformed lysins to gain access to the peptidoglycan and release the progeny phages” Depending on the enzymatic specificity, endolysins can be divided into 5 major classes: (i) N- acetylmuramidases (lysozymes) (ii) endo-8-N- acetylglucosaminidases (ii) transglycosylases which cleave the sugar moiety of the peptidoglycan (iv) endopeptidases which cleave the peptide moiety (v) N-acetylmuramoyl-L-alanine (amidases) which cleave tetrapeptide likage™ ®. ‘The name endolysin was coined in 1958 to designate a probably proteinaceous lytic substance synthesized in bacterial cells during phage multiplication and acting ‘on the cell wall from inside the cell®®, While lysins were known for many years: *, it was not demonstrated that purified or recombinant endolysins may constitute highly effective topical and systemic antibacterial agents until 2001 when Nelson and colleagues!” presented their findings on effective clearance of group C streptococci from upper res- piratory tract of mice by a phage lytic enzyme, The most likely reason for this delay was the fact that ‘earlier antibiotic resistance was not a problem serious enough to compel the development of alternative antibacterial agents"? Owing to their specificity and328 high activity, endolysins have now become a subject of study for various in vitro and in vivo aims, in food science, in microbial diagnosties, and for treat- ‘ment of infections, Clearly, phage endolysins repre~ sent great tools for use in molecular biology, biotechnology and in medicine. Endolysins have attracted scientific brains throughout the world as these phage «gene products have vast potential for combating bacterial infections in a better and biologically acceptable manner, than indiscriminate use of antibiotics®, Because of their unique ability to cleave peptidoglycan in a generally species-specific manner, endolysins represent a novel class of antibacterial agents now named as ‘Enzybiotics* and provide a means of selective and rapid killing of pathogenic bacteria with no effect on the normal microflora”, Lytic enzymes were originally developed with a view to killing Gram-positive bacteria colonizing mucous membranes. This colonization is of great importance to medicine, as it provides a potential starting point for infection and contributes to the horizontal spread of bacteria within the community. Hence, owing to their rapid killing of bacteria in a species-specific manner, lysins provide a unique means of selective prophylaxis of infections without disturbing the balance of the indigenous microflora‘. In fact, several studies have clearly shown a great capacity of lytic enzymes for Killing bacteria colonizing mucous membranes of mice following topical administration’?: !%.®°, The other potential application of lysins may be the treatment of bacterial infections, the results of the first relevant studies being very encouraging” ?", Interestingly, it has been shown that antibodies, contrary to expec- tations, do not neutralize, but rather slightly decrease the antibacterial activity of lytic enzymes in vivo? This finding is very important, as it provides an additional argument in favor of the possibility of using lysins for the treatment of systemic bacterial infections. Endolysins and bacteriophages have a few sig- nificant features in common. These include a novel (as compared with antibiotics) mode of action targeted, species specific, narrow antibacterial range® 18.18.83 or sometimes genus-specific™. killing and, the capability to kill bacteria regardless of their antibiotic- sensitivity. On the other hand, there are also some major differences between them, as endolysins, unlike bacteriophages, do not have the capacity for expo- nential growth and are less likely to be used for the treatment of Gram-negative bacterial infections®. MAYANK RAWAT ef al However, owing to the lack of reports directly comparing the antibacterial activity of lysins and phages, no general conclusions can be drawn regarding the superiority of either modality. Since their discovery, endolysin therapy has been extensively used with a wide application. Several basic applications have been reported for endolysins, including: (i) the elimination of bac- terial colonization of mucous membranes!? (ji) the ‘treatment of bacterial infections!*2. (ii) the biocontrol of bacteria in food and feed”: % (iv) the protection of plants against phytopathogenic bacteria® Conclusion Bacteriophages and phage-induced lytic enzymes are bound to become an indispensable part of our armory against bacterial infections with or without currently available antibiotics. 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 only recently these antibacterials have once again attracted attention throughout the world. Phages have been reported to be more effective than the currently available anti- biotics in treating certain infection of man and animals and have distinct advantages over them. Continuous research is needed to establish a phage-base thera- peutic system in our country. Acknowledgement ‘The first author is grateful to the Department of Biotechnology (DBT), New Delhi for awardinga research project ‘Isolation, characterization, preservation and therapeutic use of bacteriophages against Staphylococcus aureus associated with ruminant mastitis’. (BT/PR4194/ ‘AAQ/01/158/2003) which helped in initiating research on Bacteriophage therapy in India and made this review possible. References 1. Payne D, Tomasz A. The challenge of antibiotic resistant bacterial pathogens: the medical need, the market and prospects for new antimicrobial agents. Curr Opin Microbial 2004; 7: 435-438. 2. Femandes P, Antibacterial diseovery and development - the failure of suecess? Nat Biotechnol 2006; 24: 1497- 1503. 3. Loreh A. Bacteriophage: alternativeto antibiotic? Biteohnal ‘and Develop Monitor 1999; 39: 14-17,