PHAGE THERAPYPast history, Present scenario and Future prospects

Presented By:-

Aprna Sabharwal

L-2010-BS-67-D

Contents Phages – An Introduction Taxonomy Phage characteristics Fates of Phages Antibiotic Resistance-

Growing Problem Phage Therapy History Why phage

therapy????? Initial problems Prerequisites for Phage

therapy

Where we can find Phages?

Culture-commercial production

Administration Examples Enzybiotics Phages in other

industries Future Challenges Conclusion

Phages

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

Taxonomy

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.

Phage characterstics

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.

Contd…

Fates of Phages

Antibiotic resistance- Growing problem

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

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.

Contd…

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.

History of phage therapy

From 1920 to 1940, phage therapy was extensively used to treat various infectious diseases

In 1915, Félix d'Herelle observed "clear spots" on bacteria culture.

In 1917, d'Herelle 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).

Contd…

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.

Contd…

Why phage therapy?

Initial problems

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

Prerequisites for phage therapy

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

Contd…

4. The phage receptor should be known.5. The efficacy of phage therapy should be

tested in an animal model.

Where we can find phages?

In humans and animal intestines In running water In the soil Effluent outlets Sewage from corpses .

Culture-Commercial production

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

Contd…

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

How phages can be administrated

Orally Topically on infected wounds Application in liquid form is possible, stored

preferably in refrigerated vials Injection is rarely used.

Examples

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

Contd…

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

Contd…

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

Contd…

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.

Efficacy trials in humans

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

Enzybiotics

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

Contd…

Contd…

Lysins…

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’.

Mode of action:-

Contd…

The majority of lysins described to date exhibit only one kind of muralytic activity, whereas relatively few possess two separate enzymatic domains

Contd…

Use of phages in other Industries 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

Contd…

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.”

Contd…

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

Future implications- Phage display

Phages as vehicles for vaccines delivery

Phage typing

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

Challenges

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

References 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:

Contd… 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