Reinventing phage therapy

ABSTRACT
Each year, thousands of people die from bacteria resistant to antibiotics. Alternatives drugs are urgently needed. A surprising ray of hope is actually a blast from the past and they are phages, viruses that kill bacteria, but not us. Unfortunately, the public thinks only of viruse as being harmful , when in fact they can also be our best friends, just as there are bacteria in the body, without which we would not function properly. Phages were discovered independentally by Felix d'Herelle and Fredrick Twort in around 1915, when infections were still a major cause of illness and death. They noted that a 'component' from the faeces of dysentry sufferers, when fed back to patients, produced a remarkale recovery. Chicken farmers have also discovered that if birds which survive a bacterial illness are introduced back into the main shed, other birds will pick up the phages in their droppings and the whole flock will then recover. Phage therapy became popular from the 1920s, until the introduction of penicillin 20 years later. Only in the countries of the Eastern block did the therapy survive and thrive. Now western researchers and companies are working on its comeback.
INTRODUCTION
BACTERIOPHAGE
A bacteriophage is any one of a number of viruses that infect bacteria . The term is commonly used in its shortened form , as phage . Bacteriophages consists of an outer protein hull enclosing genetic material can be ssRNA , dsRNA , ssDNA or dsDNA between five and five hundered kilo base pairs long with either circular or linear
arrangement. Bacteriophages are much smaller than the bacteria they destroy usual between twenty and two hundered nm in size . Phages are estimated to be the most widely distributed and diverse entities in the biosphere . Phages are found in all reservoirs populated by bacterial host , such as soil or the intestine of animals . One of the densest natural sources for phages and other viruses is sea water .


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PHAGE THERAPY
Phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections . Although extensively used and developed mainly in former Soviet Union countries for about ninty years , this method of therapy is still being tested elsewhere for treatment of a variety of bacterial and poly – microbial biofilms infections and has not yet been approved in countries other than Georgia . Phage therapy has mainly potential applications in human medicines , dentistry , veterinary science and agricujture . An important benefit of phage therapy is that bacteriophage can be much more specific than more common drugs so can be chosen to be harmless to not only host organism but also other beneficial bacteria . A smaller effective dose can be used in phage therapy . On the other hand specificity also a disadvantage , a phage will only kill a bacterium if it is a match to the specific strain . Thus mixture are often applied to improve the chances of success or samples can be taken and an appropriate phage identified and grown . Phages tend to be more successful than antibiotics where there is a biofilm covered by a polysaccharide layer , which antibiotics typically cannot penetrate .
Following the discovery of bacteriophages by Frederick Twort and Felix d’Herelle in 1915 and 1917 , phage therapy was immediately recognized by many to be a key way forward for the eradication of bacterial infections . A Georgian , George Eliava was making similar discoveries .
But when antibiotics were discovered in 1941 and marketed widely in the USA and Europe, Western scientists mostly lost interest in further use and study of phage therapy for some time . At the same time Russian scientists continued to develop already successful phage therapy to treat the wounds of soldiers in field hospitals . During world war second the Soviet Union used bacteriophages to treat many soldiers infected with various bacterial diseases e.g. dysentery and gangrene. The success rate was as good as, if not better than any antibiotic . However, due to the scientific barriers of the cold war this knowledge was not translated and did not proliferate across the world .

MODE OF ACTION OF BACTERIOPHAGE
Bacteriophages may have a lytic cycle or a lysogenic cycle . To enter a host cell, bacteriophages attach to specific receptors on the surface of bacteria, including lypopolysaccharides , teichoic acid , proteins or even flagella . This specificity means that a bacteriophage can only infect certain bacteria bearing receptors that they can bind to, which in turn determines the phage's host range. As phage virions do not move independently, they must rely on random encounters with the right receptors when in solution . Bacteriophages use a syringe-like motion to inject their genetic material into the cell. After making contact with the appropriate receptor, the tail fibers bring the base plate closer to the surface of the cell. Once attached completely, the tail contracts, possibly with the help of ATP present in the tail injecting genetic material through the bacterial membrane.
With in minutes , bacterial ribosomes start translating viral mRNA into protein. For RNA-based phages, RNA replicase is synthesized early in the process. Proteins modify the bacterial RNA polymerase so that it preferentially transcribes viral mRNA. The host’s normal synthesis of proteins and nucleic acids is disrupted, and it is forced to manufacture viral products instead. These products go on to become part of new virions within the cell, helper proteins which help assemble the new virions, or proteins involved in cell lysis. Phages released via cell lysis. Released virions are described as free and unless defective are capable of infecting a new bacterium.













REINVENTING PHAGE THERAPY
Phage enzymes may offer powerful novel method to wipe out anthrax bacteria in seconds.This experiment done by scientist in recent years but this is a old stratigy which is called reinvention of phage therapy.
Although phage therapy is used from hundred years ago but still its reinvention is necessary as the people not aware so much about this phage therapy . Many harmful diseases increase the need of reinvention of phage therapy ‘as described below;
Multidrug-Resistant (MDR) Bacteria Have Created a Need for Phage Therapy;
Several species of bacteria have become resistant to most antibiotics, with some strains being resistant to all antibiotics. One example is vancomycin-resistant Enterococcus faecium , a low-virulence pathogen that now frequently causes fatal bacteremias due to
complete resistance . Another example is vancomycin intermediate-resistant Staphylococcus aureus strains of which have recently emerged in three nations and are known to have killed 4 patients to date. Such strains spread throughout Japanese hospitals within a year of their first appearance. Unfortunately, it has been demonstrated that some
hospital strains of methicillin-resistant S. aureus (MRSA) that are widespread have become vancomycin resistant upon exposure of the patients to vancomycin. Experts predict that S aureus will progress to become completely resistant to vancomycin , that
when this occurs, millions of people will die each year to control. Based on such developments and impending developments with pathogens such as MRSA and VRE,
opinion leaders have been warning that we are entering the “Post-Antibiotic Era”. While pharmaceutical companies are developing new antibiotics to counter the trend, it has been shown that half a century of global antibiotic abuse has equipped the surviving bacteria with “supergenes” that enable them to quickly resist new classes of antibiotics, even those to which from infections that had until recently been fairly easy they have never been exposed. Examples of the “supergenes” are mutations that enable bacteria to pump out several classes of antibiotics alter the antibiotic binding sites on ribosomal subunits, so that several different classes of antibiotics can no longer inhibit those subunits. As a consequence, in recent years, by the time newer antibiotics have gone through clinical trials and have reached the market, 20% or more of clinical isolates in the hospitals are already resistant to them at the time of regulatory approval, and within a few more years the majority of strains are resistant.
Animal Models of Phage Therapy
From the 1950s to the 1980s there was little published on the subject of phage therapy. Then papers began to appear demonstrating the utility of phage therapy in animal models. For example, phages were shown to be effective in rescuing rats from fatal systemic infections (induced with E. coli) in rescuing calves and lambs from fatal diarrhea (induced with E coli) in rescuing chicks from fatal diarrhea (induced with S.typhimurium) and in preventing destruction of skin grafts in burned rabbits by Pseudomonas aeruginosa . MERRIL et al. demonstrated in 1996 that mice with fulminant E. coli bacteremia could be rescued by phages, and that long-circulating phage variants were superior to the wild-types .In one of those studies cited, Smith and Huggins demonstrated that, in rats inoculated with a lethal intramuscular dose of E coli, a single injection of a phage preparation was more effective than multiple injections of antibiotics (chloramphenicol, tetracycline,etc.). This work was replicated in 1997 by LEVIN and BULL , who used mathematical modeling in a population dynamics approach to study the titers of phages and bacteria in the animals. The investigators concluded that the reason a single injection of phage was superior to multiple injections of antibiotics was that the phages grew exponentially in number, overwhelming the bacteria present.
Current Status of Human Phage Therapy Efforts
Poland. Phage therapy is practiced in Poland, albeit on a small scale. In the mid-1980s a series of papers was published by a group led by the late Prof. S. S´ lopek and his colleagues, including Dr. M. Mulczyk and Dr.B Weber-Da²browska, working at the L. Hirszfeld Institute of Immunology and Experimental Therapy . These papers reported on 550 cases of suppurative bacterial infections in humans. Most of the cases were chronic; most were resistant to all available antibiotics; and most had not been referred for this form of therapy until all else had failed, meaning that it was often quite late in the disease progression.
The bacterial pathogens targeted included Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. The phages used by these investigators are reported to have cured approximately 90% of the cases.The criteria of cure were cessation of suppuration and, where applicable, complete closure of wounds/fistulae. These investigators administer phages orally, because they are aware of the hazards of administering them parenterally (not all of the bacterial debris has been removed). They pre-treat the patients with antacids and gelatin in order to protect the phages from destruction by gastric acidity. These same investigators have published evidence that phages administered orally to humans in this manner do in fact reach the bloodstream. The Polish investigators have been rigorous in matching the phages to the bacterial strain infecting the given patients. Their practice, as stated in the published reports, is to culture the bacteria during the course of treatment, so that the occurrence of a mutant resisting the phage can be countered by switching to a different phage strain. The group also has panels of multivalent phages available, for use in fulminant infections (such as septicemia with acute respiratory distress syndrome) where time is insufficient to classify the offending bacteria or to match phages to bacteria. The group now has statistics on the treatment of approximately 1 300 cases. The overall cure rate across the spectrum of pathogens and sites of infection is approximately 86% . A criticism of the work by S´ lopek’s group is that the absence of placebo controls means the power of suggestion cannot be definitively ruled-out. It is clear that the difficulties of that nation’s economy over recent decades has denied the investigators the financial resources needed to enroll matched cohorts in a placebo arm of a clinical trial. While the criticism is valid, and absolute proof of principle can be obtained only through placebo-controlled trials, nevertheless the usefulness of the data is improved by the detailed statistical accounting of the percentages of complete, partial and nil response.
The Republic of Georgia. The work started in Tblisi in the 1930s by d’Hérelle and his Georgian colleague, Eliava, continues to this day. In the 1970s, under the direction of Dr. Teimuraz Chanishvili, the Eliava-d’Hérelle Institute had a large staff manufacturing considerable quantities of phage preparations per year, primarily for the control of dysentery in the troops of the Soviet Army. This group has anecdotal evidence of the efficacy of phage therapy. They report, for example, that in certain adult and pediatric hospitals it is routine for their phage preparations to be administered topically on surgical incisions. Given the lack of statistical analysis,there is little to be said other than the anecdotal reports are encouraging that phage therapy can be useful.

Uses;
Phages have been explored as means to eliminate pathogens like Campylobacter in raw food and Listeria in fresh food or to reduce food spoilage bacteria. In agricultural practice phages were used to fight pathogens like Campylobacter , Escherichia and Salmonella in farm animals, Lactococcus and Vibrio pathogens in fish from aquaculture and Erwinia and Xanthomonas in plants of agricultural importance. The oldest use was, however, in human medicine. Phages were used against diarrheal diseases caused by E. coli, Shigella or Vibrio and against wound infections caused by facultative pathogens of the skin like staphylococci and streptococci. Phage therapy therefore looks like a platform technology. This impression is reinforced by recent extension of the phage therapy approach to systemic and even intracellular infections and the addition of non-replicating phage and isolated phage enzymes like lysins to the antimicrobial arsenal.

Future Prospects for Phage Therapy
Infectious disease experts have warned that there is now a compelling need to develop totally new classes of antibacterial agents, ones that cannot be resisted by the same genes that render bacteria resistant to antibiotics. Phage therapy represents such a “new” class. We believe that the impediments cited above (bacterial debris in the preparations, rapid clearance in the body,etc.) can be overcome, freeing up the phages so that their attributes (such as exponential growth, and the ability to mutate against resistant bacteria) can be used to great advantage.
There are 3 additional attributes of phages that should be noted:
Host specificity. While the host specificity is somewhat of a drawback (requiring a matchup of phage to bacterial target, and/or the development of highly multivalent phages), it also offers the great advantage that the phages will not kill other species of bacteria.
Conditions where phage efficacy is predicted to be reduced would include
1) hypoxic sites, where bacterial replication is slower and therefore phage replication is reduced; and 2) chronic obstructive pulmonary disease, where high acidity and proteases would be expected to inactivate some percentage of the phages. Thus, e.g., phage therapy is not likely to kill off the healthy flora of the intestines, lungs or urogenital tract,and it is therefore unlikely to provoke the illnesses and deaths seen when antibiotics cause overgrowth of pathogens (such as Clostridia difficile and Candida albicans)! .
Genetic engineering. It is possible to genetically engineer phages to express new traits of potential value.
In so doing, scientists will have to deal with the legitimate concerns of regulatory agencies concerning recombinant organisms. The regulatory obstacles may be well worth the price, given the powerful engineering tools that are currently available.
Ideal candidates for co-therapy with antibiotics. If a given bacterium acquires resistance to a phage (e.g. by a mutation in the receptor site or in the endonuclease enzymes), that mutation is not likely to “teach” the bacterium to resist the antibiotics (which do not target those structures). Similarly, if a given bacterium acquires resistance to an antibiotic (e. g. by a mutation in the reflux pump or in the ribosomal subunits), that mutation is not likely to “teach” the bacterium to resist the phage (which does not target those structures). Thus, if the bacterium is exposed to both agents, the odds are remote that any resistance genes it starts to express (or acquires anew) will enable it to survive. There are reports that bacteria tend to mutate against antibiotics once in every 106 divisions, while they tend to mutate against phages once in every 107 divisions. Therefore the odds of a given bacterium mutating against a phage and an antibiotic at the same time would be the product of 106*107’ meaning it would likely take 1013 bacterial divisions for such a double mutation to occur. Given that low probability, the co-administration of phages and antibiotics may help prevent the emergence of bacterial resistance to antibiotics, thereby greatly prolonging their clinical usefulness . Just as multiple classes of anti-HIV medications are administered to AIDS patients, to prevent the emergence of resistant strains of that virus, so it is that co-therapy with phages and antibiotics may also prove to be of sgreat clinical value.
Conclusions
Multidrug-resistant bacteria have opened a second window for phage therapy. Modern innovations, combined with careful scientific methodology, can enhance mankind’s ability to make it work this time around. Phage therapy can then serve as a stand-alone therapy for infections that are fully resistant. It will also then be able to serve as a co-therapeutic agent for infections that are still susceptible to antibiotics, by helping to prevent the emergence of bacterial mutants against either agent.

References
www.phage.org.mansfield.ohio-state.edu/sabedan/bgnew026.htm
http://surfer.pan.wrac.pl/journals/aitefulltext/47z501.pdf
http://www.nature.com/nrd/journal/v6/n1/pdf/nrd2223.pdf
www.rockefelleruniversity
www.bacteriophagetherapy.info