1. OUR ENEMY’S ENEMY CAN BE OUR BEST FRIEND
P R E P A R E D B Y
K A M A L S H R E S T H A
B S C M L T
Bacteriophage therapy for
antimicrobial resistant and
biofilm forming bacteria

2. Contents
 Antibiotic resistance
 Common types of resistances
 Mechanism of resistance
 Some recent and specific resistant type
 Biofilm
 Formation of biofilm
 Mechanism of resistance and Quorum sensing
 Bacteriophages
 History of bacteriophages therapy
 Current scenario in bacteriophages therapy
 Advantages of bacteriophages therapy
 Disadvantages of bacteriophages therapy
 Recent advances in bacteriophages therapy
 Summary and discussion

3. Antibiotic resistance
 Microorganism resistance to an antimicrobial drug
that was once able to treat an infection caused by
that organism.
 “this serious threat is no longer a prediction for the
future, it is happening right now in every region of
the world and has the potential to affect anyone , of
any age , in any places” –WHO

4. Classification of antimicrobial resistances
 Multidrug resistance(MDR)
 ≥1 agent of ≥3 antimicrobial agents
 Extensively resistance (XDR)
 ≥ 1 agent in al but ≤2 catergories
 Pan drug resistance(PDR)
 non- susceptible to all
Antimicrobial agent assign to
that bacteria

5. Mechanism of drug resistance
 Drug inactivation/ enzyme modification
 Alteration of target site
 Alteration metabolic pathway
 Reduce drug accumulation
 Biofilm formation

6. Biofilm
 Biofilm formation is a process whereby microorganisms
irreversibly attach to and grow on a surface and produce
extracellular polymers that facilitate attachment and
matrix formation, resulting in an alteration in the
phenotype of the organisms with respect to growth rate
and gene transcription.
 Biofilms are resistant to killing by antibiotics at
concentrations that are 10-1000 times greater than
concentrations needed to kill free-living or “planktonic”
 often lead to life-threatening systemic infections and
device failure

7. Steps of biofilm formation
Attachment of cell to any surface
Cell attach to surface irreversibly (secretion of
extracellular polymeric substance)
Cells adsorbed on surfaces replicatean grow on micro
colonies
Community grows into a three dimensional and
formation of mature biofilm
Some cell detach from the region of biofilm

9. Mechanism of resistance
1. Restricted penetration of antibiotics
2. Nutrient limitation, altered microenvironment
3. Adaptive response
4. Quorum sensing
5. Genetic alteration to persister cell

10. Bacteriophages
virus that infects and replicates with in bacterium
They do so by inserting their genetic material inside a
bacteria
composed of protein capsule containing either DNA or
RNA as their genome
They are the most common and diverse entities in
biosphere and thrives where there is a high bacterial
population
The phages are terraforming the planet.
Every second day the phages destroy approximately 50
percent of the Earth's bacterial population.

11. Different types of phages

12. History and taxonomy
 Ernest Hankin - 1896
 Frederick Towrt - 1915
 Felix d’Herelle – 1917
Taxonomy(ICTV)
 Order
Caudovirales Ligamenvirales not assign
Myoviridae lipoyhrixviridae Ampullaviridae
T4 phage Mu Acidious Bicaudaviridae,
etc.
filamentous virus
Siphoviridae Rudiviridae
λ phage, T5 sufolobus islandicus
virus
Podoviridae
T7 T3 phages

13. Life cycle

14. From where we can get phages
Obtain a fresh culture of bacteria and make a an overnight
suspension with suitable broth
Add certain volume of sample(5ml) + 0.5ml of overnight
suspensions + 0.5ml of 10x brothand incubate
Centrifuge the incubated suspension for 10 minsat 2500rpm
and take the supernatant ins separate tube and filter by using
<0.4micron filter
Take small volume from filtrate(10µl) and add to the lawn
culture of bacteria
Harvest the phage by using loop and add into another
suspension of same bacteria and store in refrigerator

15. Bacteriophage therapy
 Use of bacteriophages for the therapeutic purpose in
bacterial infection
 This method is still not approved in all countries
except Georgia
 This method is still being testing for the treatment of
antimicrobial resistance and biofilm forming
bacteria

16. Why phage therapy

17. History of phages therapy
 1919- 111940 , golden age for phage therapy
 1919 Felix d’Herelle extensively studies about the phenomenon of
bacteriophage and also used in human suffering from dysentery at
Hôpital des Enfants-Malades in Paris.
 1921 – 1st reported case of bacteriophage used to treatment of bacterial
infection in human by Richard Brugnoghe and Joseph Maisin
 D'Herelle's commercial laboratory in Paris produced at least five phage
preparations against various bacterial infections.
 1940 discovery of antibiotic halted the research on bacteriophage except
in east Europe
 Eliava Institute of Bacteriophage, Microbiology, and Virology
(EIBMV) of the Georgian Academy of Sciences, Tbilisi, Georgia, and the
Hirszfeld Institute of Immunology and Experimental Therapy
(HIIET) of the Polish Academy of Sciences, Wroclaw, Poland were
among the institute which done most of its work in bacteriophage therapy

18. Bacteriophage in animal trials
 Smith et al – 1982, successful use of phage to experimental
E. coli infection in mice. Phages treatment reduce the no. of
bacteria by many fold in different animals. Rekindled the
concept of bacteriophage therapy in West
 Soothil et al- (1988-1994), successfully treated the
experimental disease caused by Pseudomonas and
Acinetobacter in mice and guinea pig and suggested that it
might be efficacious in preventing infection of skin graft and
burn patient.
 Bogovazova et al- 1191, reported that phages are
efficacious and non-toxic (no gross and histological
changes) even after the use of 3,500 fold higher than
that use in humans , in mice and guinea pigs.

19. Bacteriophage in human trials
 Polish paper:-1983-1985
 Slopek et al published the SIX paper on effectiveness of the bacteriophage against
infection caused by MDR and found that bacteriophages are 75-100% effective(>94%)
 Another study also reported the effectiveness of bacteriophages therapy in meningitis
 Soviet paper:- 1963-1964
 In Georgia the effectiveness of bacteriophage to treat dysentery was determined and
found that overall 3.8 fold lower incidence of dysentery in children given anti-shigella
bacteriophage orally.
 In many other study yielded the similar result but main drawback of soviet studies
was it lacks the information required for the determination efficacy of bacteriophage
therapy
 Other studies
 Zhukov-Verezhnikov et al -1978, compared the effectiveness of
specially adapted bacteriophage to commercially available bacteriophage and
found that adapted bacteriophages are 5-6 fold more effective.
 Meladze et al- 1982, compared the effective ness of bacteriophage to
antibiotic and found to superior to antibiotic with lesser side effect

20. Advantages of phage therapy
 highly specific
 Very effective
 Harmless/very low side effect
 Phage are intelligent drug
 Bacteria fully resistant to phage hasn’t yet discovered
 Easy availability
 Low cost for the preparation
 Effective against most resistant bacteria
 Effective even to mature form of biofilm
 Phage can be genetically modified
 Individual component can be used to treat patient
 Phages mutate at a higher rate than bacteria and are able to
respond fast to possible phage- resistant bacteria.

21. Disadvantages/problems of phage
therapy
 Efficacy of phages in human hasn’t been full determined
internationally
 High specificity has hindered its effectiveness to many
bacteria
 Purity of bacteriophages suspension
 Bacteria resistant to phages
 Large size of phages
 Intracellular pathogen
 Clearance of phages by reticulo-endothelial system
 Formation of antibodies against bacteriophages
 Release of cellular toxins during cell lysis
 Can carry harmful gene to bacteria(lysogeny)
 Difficulty in administration

23. Prerequisites of bacteriophage therapy
 Phage therapy should not be attempted before the
biology of the therapeutic phage is well understood.
 Phage preparations should meet all the safety
requirements
 Phage preparations should contain infective phage
particles,
 The phage receptor should be known.
 The efficacy of phage therapy should be tested in an
animal model.

24. Recent advances to tackle the problems
 Cocktail of bacteriophages:-
 Broader phages Ellen et al , 1998 has succeed to isolates a
different bacteriopages with broader host range such as SN-1,
SN-2, SN-T, and SN-X, AB1157, BHR3, BHR4, and BHR5 which
can infected staphylococcus natans Pseudomonas aeruginosa
and escherichia coli.
 Merril et al- 1996 succeed to produce a mutant bacteriophage
by serially passaging phages through animal which can stay to
circulation more longer period of time
 Drug- delivery technologies
Kim et al- 2008, conjugated the bacteriophage to polyethylene
glycol(PEG) and found that this conjugation has the increase the
sustainability of bacteriophages in circulation and also decrease
the production of Th1 and interleukin factor showing decrease
immune response

25. Genomic modification of bacteriophage:-
 Non-lytic/non-replicative
 Hagens et al– 2003, genetically modified filamentous bacteriophages by
replacing the export protein gene with restriction endonuclease but not holins
which are lethal to bacteria but do not induce lysis of bacteria.
 Lu and colins-
 In 2007, show that phage can be genetically modified to
disrupt the barrier like biofilm, they inserted dispersin-B(
glycoside hydrolase known to degrade biofilm in Escherichia
bacteriophage T7.
 In 2009 ,they genetically modified M13mp18 phages that
overexpress the lecA3 gene which decrease the SOS
response(DNA repair) on the presence of quinolones and
found to be effective in combating resistance bacteria.

27. Bacteriophage product
 Enzybiotic – Nelson et al
 Lysin:- endolysins or murein hydrolases are the hydrolytic
enzymes produced by bacteriophages in order to cleave the
host’s cell wall during the final stage of life cycle.
 Recombinant enzymes acting on cell wall can be uses for
therapeutic purpose rather than a whole bacteriophage
 Lysin effectiveness to eliminate the infection has been
shown by many study
 e.g. Nelson et al-2001 use C 1 bacteriophage lysin to treat a
experimental infection with streptococci of upper
respiratory tract in mice. And shows the high rate of activity

28. List showing specific bacteriophage ant their
lysin for different bacteria

29. continue

30. Protein antibiotics
 Some small phages do not have the genes for holin or
lysin proteins.
 Instead, they produce a protein that inhibits a step in
murein monomer synthesis. Their inhibitory gene
products are known as “protein antibiotics”
 the E protein of the single-stranded (ss)-DNA
bacteriophage φX174 (Microviridae), (ii) the L protein
of the ss-RNA bacteriophage MS2 (Leviviridae), and
(iii) the A2 protein of the ss-RNA bacteriophage Qβ
(Alloleviviridae) are some of protein that can induce
cell lysis in similar manner to that of penicillin.

31. A Trojan Horse Approach
 Killing of Mycobacterium avium and Mycobacterium
tuberculosis by a Mycobacteriophages delivered by a
Non-virulent Mycobaterium(M smegmatis)
 Infected M smegmatis with a TM4 phages(a broader
ranges phage that infect from fast growing
mycobacteria to slow growing Mycobacteria)
 Result shows that its able to kill tha micobacteria in
free living state as well as that are inside the
macrophage

32. Bacteriophages vs antibiotics

33. Use of phages in other industries
 In food industry
 Bacteriophage bioprocessing , a means of reducing
bacteria from food product by using bacteriophages
 This non-thermal intervention has been
demonstrated to control the growth of many bacteria
e.g. campylobacter and salmonella on chicken
listeria in meat etc.
 In agriculture and fisheries

34. Phage typing
 Is also known as the use of sensitivity pattern to
specific phages to precisely identifying the microbial
strains
 It implies the use of a set of bacteriophages provided
by international agency for the typing of certain
species of bacteria for epidemiological purpose

35. Phage for the detection of bacteria
 Wild phage detection system:-

36. reporter bacteriophages :-
 modified phages used
as a reporting gene
carrier, introducing a
gene of interest into
the host bacteria upon
infection
 Eg luciferase
expressing gene(lux
and luc)
galactosidase(lacZ),
bacterial ice
nucleation(inaW) ,
green fluorescent
protein(gfp)
expressing gene

37. Phages receptor binding proteins
 This are unique protein located on the tail fibers , which
binding to the host receptors induce the translocation of
genetic materials
 Bacteria can be detected by using these protein like a
antibody which binds to the specific bacterial.
 Offers better stability against many environmental
factors such as pH temperature and different
enzymes(proteses)
 Binding affinity can be tailored to the requirement
 Singh et al. demonstrated the use of cysteine-tagged P22
phage RBPs on gold surface for capture
 and detection of Salmonella enterica serovar
Typhimurium.

39. Conclusion
 Multidrug resistant bacteria have opened a second
window for phages therapy
 Modern innovation combined with careful scientific
methodology, can enhance mankind’s ability to
make it work this time around
 Phage therapy can stand alone therapy for infectious
that are fully resistant
 It will also then be able to serve as a co-therapeutic
agents for infections that are still susceptible to
antibiotic by helping to prevent the emergence of
bacterial mutant against either agent

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41. Continue
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