Phages – An Introduction Where we can find Taxonomy Phages? Phage characteristics Culture-commercial production Fates of Phages Administration Antibiotic Resistance- Growing Problem Examples Phage Therapy Enzybiotics History Phages in other industries Why phage therapy????? Future Initial problems Challenges Prerequisites for Phage Conclusion therapy
A bacteriophage is any one of a number of viruses that infect bacteria They do this by injecting genetic material Bacteriophages are among the most common and diverse entities in the biosphere Phages are widely distributed in locations populated by bacterial hosts It has been estimated that there are more than 100 different phage species and at least 10 phages for each bacterium
This group established the International Committee for the Taxonomy of Viruses whose objective, since 1971, is to continually update taxonomic guidelines. The ICTV placed the tailed bacteriophages under the order of the Caudovirales Within this order there are three families: the Myoviridae, with long, contractile tails, the Siphoviridae with long, non-contractile tails, and finally, the Podoviridae with short, stubbed tails and a striking lack of features. Each of these three families may be further broken down into genera Using this grouping there is 1 order, 13 families and 31 genera.
The genome sizes of phages vary enormously, from a few thousand base pairs up to 498 kilobases in phage G A bacteriophage particle or virion consists of a single or double stranded (ss of ds) DNA or RNA molecule, encapsulated inside a protein or lipoprotein coat Tail morphologies- long, flexible tails; double- layered, contractile tails ; short, stubby tails The size of the phage heads is correlated to the genome size being packaged, and varies in diameter between 45 and 100 nm.
Main reason is abusive use over the past twenty years The resistance phenomenon represents not only a important healthcare issue but also an economic problem Penicillin fails to completely eradicate Streptococci in up to 35 % of patients Infections caused by Streptomyces agalactiae in pregnant women cannot be treated with antibiotics because they increase the risk of abortion
Phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections This method of therapy is still being tested for treatment of a variety of bacterial and poly-microbial biofilm infections Has not yet been approved in countries other than Georgia Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture.
Bacteriophages are much more specific than antibiotics Have a high therapeutic index In the West, no therapies are currently authorized for use on humans, although phages for killing food poisoning bacteria (Listeria) are now in use.
From 1920 to 1940, phage therapy was extensively used to treat various infectious diseases In 1915, Félix dHerelle observed "clear spots" on bacteria culture. In 1917, dHerelle presented a note to the "Académie des sciences de Paris‖ In 1919, there was an epidemic of "fowl typhoid" in France First administration of phages was given in 1921 at the Hôpital des Enfants-Malades (Paris).
In Paris, "le laboratoire du bactériophage" produced many phages directed against common infectious diseases In 1945, a new era appeared in Western countries with the golden age of antibiotics Phage therapy was abandoned in the Western world, but maintained (it seems) on a large scale in Poland and the USSR where infections continued being successfully treated.
Problem 1- host range Problem 2- bacterial debris present in phage preparations Problem 3- attempts to remove host bacteria from therapeutic preparations Problem 4- rapid clearance of phages Problem 5- lysogeny
Levin and Bull 2004 suggest that phage therapy only needs to decrease the numbers of infecting bacteria to a level where the host defenses can take care of the remaining bacteria. Various prerequisites that should be met:1. Phage therapy should not be attempted before the biology of the therapeutic phage is well understood.2. Phage preparations should meet all the safety requirements3. Phage preparations should contain infective phage particles
…4. The phage receptor should be known.5. The efficacy of phage therapy should be tested in an animal model.
In humans and animal intestines In running water In the soil Effluent outlets Sewage from corpses .
D‘Herelle‘s commercial laboratory in Paris produced at least five phage preparations against various bacterial infections. The preparations were called Bacte-coli-phage, Bacte-rhinophage, Bacte-intesti-phage, Bacte-pyo- phage, and Bacte-staphy-phage Therapeutic phages were also produced in the United States. In the 1940s, the Eli Lilly Company (Indianapolis, Ind.) produced seven phage products for human use
Lyophilized phages were shown to be superior to liquid preparations. Repeated cycles of freezing and thawing were not linked to activity loss acidity below pH 3.5 decreased the phage activity substantially only egg yolk had some protective properties on the phage preparation Under dry conditions the phage preparation resisted temperatures at least up to 55°C
Orally Topically on infected wounds Application in liquid form is possible, stored preferably in refrigerated vials Injection is rarely used.
Phage therapy reduces Campylobacter jejuni colonization in broilers Attempts to prevent Campylobacter colonization of chickens by biosecurity measures have proven extremely difficult Probiotic treatment with lactic acid bacteria and competitive exclusion with beneficient microflora was only partially effective Aprevention group was infected with C. jejuni at day 4 of a 10-day phage treatment Atherapeutic group was phage treated for 6 days, starting 5 days after C. jejuni colonization of the broilers had been established
Treatment was monitored by enumerating Campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content A clear log decline in C. jejuni counts in both preventive and theraputic groups were observed
Killing of Mycobacterium avium and Mycobacterium tuberculosis by a Mycobacteriophage Delivered by a Nonvirulent Mycobacterium Tuberculosis is a serious public health problem that results in millions of deaths around the world each year Mycobacterium smegmatis, an avirulent mycobacterium, is used to deliver the lytic phage TM4 where both M. avium and M. tuberculosis reside within macrophages
These results showed that treatment of M. avium– infected, as well as M. tuberculosis–infected, RAW 264.7 macrophages, with M. smegmatis transiently infected with TM4, resulted in a significant time- and titer-dependent reduction in the number of viable intracellular bacilli.
In 1963 a total of 30,769 children (6 months to 7 years old) were enrolled in Tbilisi, Georgia, in an oral phage prophylaxis trial against bacterial dysentery. The children were followed for 109 days. Phage administration was associated with a 3-8 fold decrease in dysentery incidence Phage exposure also decreased the incidence of any form of diarrhea
The term ― enzybiotic ‖ was used for the first time in a paper by Nelson et al 2001 Other names that are used with respect to enzybiotics are lytic enzymes and peptidoglycan hydrolases Most important characteristics of enzybiotics are a novel mode of antibacterial action The capacity to kill antibiotic - resistant bacteria Another feature is the low probability of developing bacterial resistance
Lysins or endolysins are double - stranded DNA bacteriophage – encoded enzymes that cleave covalent bonds in peptidoglycan. The term ― endolysin ‖ was introduced to the scientific literature by F. Jacob and C. R. Fuerst to stress that enzyme molecules act on peptidoglycan from within the bacterial cell in which they are synthesized Recombinant enzymes acting on the cell wall from outside the cell (e.g., those used for therapeutic purposes) were referred to as lysins rather than endolysins Another name proposed to designate a lysin is ‗virolysin‘.
The majority of lysins described to date exhibit only one kind of muralytic activity, whereas relatively few possess two separate enzymatic domains
In food industry:- Bacteriophage bioprocessing is bacteriophage control as practiced in the factory particularly as means of reducing food bacterial loads Phage-based control of pathogens is a non-thermal intervention, and has been demonstrated to control the growth of Campylobacter and Salmonella on chicken skin, Salmonella enteritidis in cheese Listeria monocytogenes on meat and on fresh-cut fruit Phage application has also been studied as a method to control the presence of biofilms in the food processing environment, Such a treatment is potentially useful in the control of L. monocytogenes
Food and drug administration concerning to use of phage in food industry:- ―……for the safe use of mixture of bacteriophages as an antimicrobial agents on foods, including meat and poultry products neither an environmental assessment nor an environmental impact is required.”
In agriculture and fisheries:- Bacteriophages have been considered, for example, to control Salmonella infestation of cut fruit The most successful use of phage therapy, already in practice, has been in the control of fish pathogens To avoid contamination of food products with Listeria monocytogenes, Salmonella on cut vegetables and fruits, or the pathogenic Escherichia coli O157 : H7, phage therapy is now being advocated for use in the food and livestock market
Phage typing is also known as the use of sensitivity patterns to specific phages for precisely identifying the microbial strains The sensitivity of the detection would be increased if the phages bound to bacteria are detected by specific antibodies The technique has most extensively been used for the detection of Mycobacterium tuberculosis, E.coli, Pseudomonas, Salmonella, Listeria, and Campylobacter species
Novelty Specificity of phages Efficacy and other technical challenges Regulatory approvals Patent protection. Market acceptance
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
Borysowski J and Gorski A (2010) Enzybiotics and their potential applications in medicine Enzybiotics: Antibiotic Enzymes as Drugs and Therapeutics Pp 1-22. L. Hirzfeld Institute of Immunology and experimental therapy, Wroclaw, Poland Broxmeyer L et al Killing of Mycobacterium avium and Mycobacterium tuberculosis by a Mycobacteriophage Delivered by a Nonvirulent Mycobacterium: A Model for Phage Therapy of Intracellular Bacterial Pathogens J Infect Dis 186: 1155-60 Brussow H (2005) Phage therapy: the Escherichia coli experience Microbiol 151:2133-40 Carlton R M (1999) Phage therapy: Past history and Future prospects Arch Immunol Ther Exp 47: 267-74 Haq I U et al (2012) Bacteriophages and their Implications on Future Biotechnology: A Review J Virol 9:
Hermoso J A et al (2007) Taking aim on bacterial pathogens: from phage therapy to enzybiotics Curr Opin Microbiol 10:1-12 Inal J M (2003) Phage Therapy: a Reappraisal of Bacteriophages as Antibiotics Arch Immunol Ther Exp 51:237-44 Skurnik M and Strauch E (2006) Phage therapy:Facts and Fiction Int J Med Microbiol 296 :5-14 Sulakvelidze A (2011) The Challenges of Bacteriophage Therapy Eur I Pharm 10:14-18 Thiel K (2004) Old dogma, new tricks—21st Century phage therapy Nat Biotechnol 22:31-36 Wagenaar J A (2005) Phage therapy reduces Campylobacter jejuni colonization in broilers Vet Microbiol 109:275-83