Bacteriophage Therapy old idea new application Edited by Muslim Dhaher 2012
Introduction The emergence of pathogenic bacteria resistant to most, if not all, currently available antimicrobial agents has become critical problem in modern medicine, particularly because of the concomitant increase in immunosuppressed patients. The concern that humankind is reentering the ―preantibiotics‖ era has become very real, and the development of alternative antiinfection modalities has become one of the highest priorities of modern medicine and biotechnology
Bacteriophage definitionBacteriophages are viruses that parasitize bacteria. Bacteriophages were jointly discovered by Frederick Twort (1915) in England and by Felix dHerelle (1917) at the Pasteur Institute in France. Felix dHerelle coined the term ―Bacteriophage‖. Bacteriophage means to eat bacteria, and are called so because virulent bacteriophage can cause the compete lysis of a susceptible bacterial culture. They are commonly referred as ―phage‖. Phages are obligate intracellular parasites that multiply inside bacteria by makinguse of some or all of the host biosynthetic machinery. They occur widely in nature and can readily be isolated from feces and sewage. There are at least 12 distinct groups of bacteriophages, which are very diverse structurally and genetically .
Examples of phages T-even phages such as T2, T4 and T6 that infect E.coli Temperate phages such as lambda and mu Spherical phages with single stranded DNA such as PhiX174 Filamentous phages with single stranded DNA such as M13 RNA phages such as Q- beta
Phage life cycleThe following phases can be distinguished in the lytic bacteriophage developmental cycle:1. Adsorption of the phage on the bacterial cell by binding to a specific receptor.2. Injection of the nucleic acid into the bacterium.3. Expression of the phage early genes, synthesis ofearly proteins, most involved in the shutting down ofthe host bacterium systems and phage genomereplication.
4. Replication of the phage genome.5. Expression of the phage late proteins involved in the formation of new phage particles and lysis of the host bacterium.6. Assembly of the phage heads and tails and packaging of the genome.7. Lysis of the host bacterium and release of the new phage progeny
Schematic illustration of phage-induced bacteriolysis
The Problem Multidrug resistant bacteria are a serious health problem these days. Persistent use of broad spectrum antibiotics leads to further drug resistance in these organism. Rigorous research activities are ongoing to develop alternate methods of treatment of infections caused by these microorganisms. Phage therapy seems to be a good option for this problem.
Resistance is on the rise. An increasing number of pathogensare resistant to one or more drugs used to treat the diseases they cause!. Indeed, many diseases common in developing countries— including malaria, pneumonia, cholera, and dysentery—are increasingly caused by strains that are resistant to multiple drugs. This is true for diseases such as tuberculosis (TB) and infections such as Staphylococcus aureus (S. aureus) that afflict rich countries as well as poor ones.The problem is global: Drug-resistant TB is spreading rapidly to countries where it has not been seen before
The ability of the phages to kill the bacterialcells at the end of the infectious cycle is thecornerstone of the idea of using phages astherapeutic agents.
Electron micrographs of tailed phages. KVP20, KVP40, andKVP241 are Vibrio phages belonging to family Myoviridae;fMR11 is a Staphylococccus phage belonging to familySiphoviridae.
First Era of Bacteriophage Ernest Hankin, a British bacteriologist, reported in 1896 on the presence of marked antibacterial activity (against Vibrio cholerae) which he observed in the watersof the Ganges and Jumna rivers in India, and he suggestedthat an unidentified substance (which passed through fine porcelain filters and was heat labile) was responsible for this phenomenon Russian bacteriologist Gamaleya observed a similar phenomenon while working with Bacillus subtilis and the observations of several other investigators are also thought to have been related to the bacteriophage phenomenon However, none of these investigators further explored their findings
Frederick Twort, a medically trained bacteriologist for various reasons—including financial difficulties Twort did not pursue this finding from England, reintroduced the subject. The discovery or rediscovery of bacteriophages by d‘Herelle is frequently associated with an outbreak of severe hemorrhagic dysentery among French troops stationed at Maisons-Laffitte (on the outskirts of Paris) in July-August 1915, althoug d‘Herelle apparently first observed the bacteriophag phenomenon in 1910 while studying microbiologic means of controlling an epizootic of locusts in Mexico. Several soldiers were hospitalized.
Another thought came to me also.If this is true, the same thing willhave probably occurred in the sickman. In his intestine, as in mytest-tube, the dysentery bacilli willhave dissolved away under theaction of their parasite. He shouldnow be cured
d‘Herelle was assigned to conduct an investigation of the outbreak. During these studies, he made bacterium-free filtrates of the patients‘ fecal samples and mixed and incubated them with Shigella strains isolated from the patients. A portion of the mixtures was inoculated into experimental animals (as part of d‘Herelle‘s studies on developing a vaccine against bacterial dysentery), and a portion was spread on agar medium in order to observe the growth of the bacteria. It was on these agar cultures that d‘Herelle observed the appearance of small, clear areas, which he initially called taches, then taches vierges, and, later, plaques (68). D’Herelle’s findings were presented during the September 1917 meeting of the Academy of Sciences, and they were subsequently published in the meeting‘s proceedings. In contrast to Hankin and Twort, d‘Herelle had little doubt about the nature of the phenomenon, and he proposed that it was caused by a virus capable of parasitizing bacteria.
What is bacteriophage thearapyPhage therapy is the use of Bacteriophage viruses to combat bacteria instead of using antibiotics. Bacteriophages only attack bacteria nothing else which makes it useful in medicine as it does not affect the patient only the bacteria, it also has the bonus of being very specific to with bacteria it will attack. 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 .
Bacteriophage ignorationIt is interesting to note that phage therapy ceased to be used inthe West with the advent of the antibiotic era but has beenrediscovered because of the rise in antimicrobial-resistant bacteria. While the study and exploitation of phages flourished in the West, particularly in the development of molecular tools, its use in the former Soviet Union as a therapeutic tool has remained steady for over 80 years. Phage therapy has been the subject of numerous recent review articles (4-16). (Regrettably, the present article largely ignores the literature from the former Soviet Union because of language problems and lack of detail provided in the published studies.
Importance of phageEpidemiological fingerprinting of bacterial isolates (phage typing)In epidemiological work, it is necessary to track individual bacterialisolates from clinical specimens to their source (47). This is accomplished by fingerprinting the strains using a wide varietyof phenotype- or genotype-based typing methods. One of the classical procedures is phage typing, which is still used in Canada at the Laboratory for Foodborne Zoonoses (Guelph, Ontario) and the National Microbiology Laboratory (Winnipeg, Manitoba), for Salmonella and E coli strains. This procedure involves exposing the bacterial isolate to a battery of ‗typing‘ phages, and recording the pattern and degree of lysis. This approach offers import advantages, including the incredible specificity of phage, and a high degree of typability and reproducibility. Furthermore, in contrast to serotypingand pulsed field gel electrophoresis analysis, phage typing isrelatively inexpensive.
Use of bacteriophages to express peptides and proteins(phage display) Several systems have been developed to create peptide or protein fusions on capsid proteins of bacteriophages of coliphages lambda (59,60), M13 (61), T7 (62-64) and T4 (65). The M13 and T7 systems have been commercialized. These extremely elegant molecular tools have been used to identify antibody binding epitopes (66-68), amino acid residues involved in protein-protein interactions (69-71), peptides that mimic nonpeptide ligands (72), enzyme substrates and inhibitors, and have even been used to express proteins. One major advantage of this system over standard protein chemistry is that the sequence of the peptide insert can be rapidly aninexpensivel determined by DNA sequencing.
New phage diagnostic tools In addition to the classical uses of phages in molecular biology and diagnostic microbiology, new tools have been developed. The phage amplification assay, for example, is a simple yet elegant way to identify the presence of specific pathogens in food products. The intracellular replication of phage and concomitant lysis of the susceptible bacteria leads to an increase in free phage, which can be easily measured
SPECIAL ADVANTAGESPhages have specific properties which give themadvantages as therapeutic agents. They are self- replicating as well as self-limiting. They continue to multiply and penetrate deeper as long as local infection as present. This is in sharp contrast to antibiotics which decrease in concentration below the site of infection. Phages are lytic against specific bacteria so they can be targeted more specifically than antibiotics which are active against a group of bacteria. Phages do not harm normal intestinal microflora.6 Antibiotics have side effects which can be serious. But phages have been used in
millions of patients without any reported side effects. Phages can be used prophylactically as well as in established infections. The self-perpetuating nature of phages in the presence of susceptible bacteria, makes multiple administrations7,8 unnecessary. It also allows transfer of administered phages between animals in a farmyard.9,1
The advantages and disadvantages of phage therapy Advantage RemarkPhages are very specific and do not As a result, there are no side effectsharm the useful bacteria that live in like diarrhoea or secondary infectionsand on the body. such as those that occur in treatment with antibiotics.Due to their specificity, phages donot cause a selection of resistancesin the useful bacteria that live in andon the body.We are constantly ingesting Because they are harmless, phagesphages. In general, they are can be used for combating harmfulharmless to human beings. When bacteria in fattening animals andwell-purified phages are used, few food.side effects have been describedfor all types of administration
Some resistant Antibiotic-resistantbacteria that have been bacteria are generallyselected during not less virulent.treatment with phagesare less virulent andcan be fought by theimmune systemPhages are also activeagainst bacteria thathave become resistant toantibioticsPhages can begenetically modified inorder to make up forsome of theirdisadvantagesPhages are an Bacteria that have‘intelligent’ drug. They become resistant to amultiply at the site of the certain antibiotic ofteninfection until there are become resistant tono more bacteria. Then other drugs more easily.they are excreted
Phage disadvantage The great specificity of phages is a disadvantage when the exact species of infecting bacteria is unknown or if there is a multiple infection. Bacteria have a type of ‘immune system’ that destroys the hereditary material of some penetrating phages. Only suitable phages can conquer this ‘immune system’ Infections whose agents are hidden in the interior of human cells may be inaccessible to phages
Results of bacteriophagetreatment Septicemia S. aureus, E. coli, Klebsiella, Proteus, PseudomonasPurulent otitis S. aureus, media Klebsiella, Pseudomonas Varicose S. aureus, E. ulcers of coli, lower Klebsiella, extremities Proteus, PseudomonasMucopurulent S. aureus, E. chronic coli, bronchitis, Klebsiella, laryngitis, Proteus, rhinitis Pseudomonas
Bronchopneumoni S. aureus, E. coli, 57 47 a, Klebsiella, empyema Proteus, PseudomonasPleuritis with fistula S. aureus, E. coli, Klebsiella, Proteus, Pseudomonas Suppurative S. aureus, E. coli, peritonitis Klebsiella, Enterobacter, Proteus, Pseudomonas Urinary tract S. aureus, E. coli, infections Klebsiella, Proteus, Pseudomonas Pyogenic arthritis S. aureus, E. coli, and Klebsiella, Proteus, myositis PseudomonasSuppurative osteitis S. aureus, E. coli, after Klebsiella, Proteus, bone fractures Pseudomonas
CONCLUSIONIn medicine today phages find many applications. Theyare used for typing of clinical bacterial strains for in situbacterial detection through labelled phages, (TB, Listeria),phage display system for vaccines, control of food pathogensand for drug and gene delivery using defective phage withtargeted receptor. Phage therapy for eliminating multidrugresistant bacteria is gaining importance. However, there is aneed to carry out further studies on phages as therapeuticagents using specific phage strains against the correspondingbacterial hosts. Phages should be essentially free ofcontaminating bacterial toxin and also capable of evadingthe reticulendothelial system