“STRATEGY TO SLOW AND REVERSE ANTIBIOTIC RESISTANCE” : BACTERIOPHAGE THERAPY

1. PRESENTED BY-
RAJNANDINI
SINGHA
PHARM D
1
“STRATEGY TO SLOW AND
REVERSE ANTIBIOTIC
RESISTANCE”
PRESENTED BY-
RAJNANDINI SINGHA
PHARM D

2. OBJECTIVES
 What is antimicrobial resistance?
 Why antibacterial resistance is a concern?
 Factors of AMR
 Mechanism of antibiotic resistance
 Alternative approach to antibiotic resistance –
“BACTERIOPHAGE THERAPY”
 Bacteriophage classification and source.
 Lytic and lysogenic cycle.
 Preparation and Methods.
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3.  Bacteriophage receptors
 Mechanism of bacteriophage
 Clinical studies
 Complications
 Bacteriophage therapy over antibiotics
 Applications
 PHAGOBURN
 Conclusion
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5. INTRODUCTION
 Antimicrobial drug resistance is the ability to resist
the effects of the medication which was previously
used to treat them.
 This is responsible for millions of death world wide
and is consider as a major health care concern
nowadays.
 Antimicrobial resistance (AMR) threatens the
effective prevention and treatment of ever
increasing range of infections caused by the
microbes.
 The cost of health care for patient with resistance
infection is higher.
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7. ANTIMICROBIAL RESISTANCE
 Antimicrobial resistance is the ability of a
microorganism (like bacteria, viruses and some
parasites) to stop an antimicrobial such as
antibiotics, antiviral and antifungals) from working
against it.
 ANTIBIOTIC RESISTANCE=
MIC/MCC > TOXIC PLASMA CONCENTRATION
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9. WHY RESISTANCE IS A
CONCERN ?
Resistant
organism lead to
treatment failure.
Increased
mortality
Resistant bacteria
may spread in
community.
Low level
resistance can go
undetected.
Added burden on
health care cost.
Threatens to
return to pre-
antibiotic era.
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11. Environmental
factors
Increased
community acquired
infections.
Ineffective
Infection
control
program.
Widespread use of
antibiotic in animal
husbandry and
agriculture and as
medical cleansing
products.
High population
and overcrowding
Poor sanitation
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12. DRUG
RELATED
Irrational fixed
dose combination
of antimicrobials
Counterfeit
and
substandard
drug causing
sub optimal
blood
concentratio
n
Soaring use of
antibiotics
OTC
availability of
microbials
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13. Patient
related
Lack of sanitation
concept
Proverty
Lack of
education
Self medication
Misconception
Poor adhrence
to dosage
regimen
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14. Prescriber related
Inappropriate use of
available drugs.
Increased empiric poly
antimicrobial use.
Overuse of antimicrobial
Inappropriate dosing
Lack of current knowledge
and training.
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15. MECHANISM OF ANTIBIOTIC
RESISTANCE
INTRINSIC RESISTANCE
 1.LACK TARGET
 2. EFFLUX PUMPS INHIBITOR
 DRUG INACTIVATION
ACQUIRED RESISTANCE
 MUTATION
 PLASMIDS
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19. MECHANISM OF RESISTANCE GENE
TRANSFER
Transfer of r-gene from
one bacterium to another
 Conjugation
 Transduction
 Transformation
Transfer of r-genes between
plasmids within the
bacterium
 By transposons
 By integrons
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23. AN ALTERNATIVE APPROACH TO
ANTIBIOTIC RESISTANCE-
“BACTERIOPHAGE THERAPY”
A ONCE AND FUTURE SOLUTION

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25. BACTERIOPHAGE
 Bacteriophages are viruses
capable of infecting and killing
bacteria.
 They have been referred to as
bacterial parasites ,with each
phage type depending on a single
strain of bacteria act as host.
 Bacteriophage carry only the
genetic information needed for
their protein coats. They require
precursor, energy generation and
require ribosome supplied by their
bacterial host cell.
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27. BACTERIOPHAGE CLASSSIFICATION
 At present ,over
5000
bacteriophage
have been studied
by electron
microscopy and
have been divided
into 13 virus
family
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29. SOURCES OF BACTERIOPHAGE
 In human and animal intestine.
 In running water.
 In the soil.
 sewages
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30. Administration
 Phages can usually be freeze-dried and turned into pills without
materially reducing efficiency.
 Temperature stability up to 55 °C and shelf lives of 14 months
have been shown for some types of phages in pill form.
 Application in liquid form is possible, stored preferably in
refrigerated vials.
 Oral administration works better when an antacid is included, as
this increases the number of phages surviving passage through
the stomach.
 Topical administration often involves application to gauzes that
are laid on the area to be treated.
 IV phage drip therapy was successfully used to treat a patient
with MDR Acinetobacter baumannii in Thornton Hospital at UC
San Diego
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32. LYTIC AND LYSOGENIC CYCLE
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35. EFFLUX PUMPS INHIBITOR
 Cytoplasmic membrane transport proteins.
 Major mechanism for resistance in antibiotics like
tetracycline.
 The system pump solutes out of the cell.
 Efflux pumps allows the microorganism to regulate their
internal environment by removing toxic substance , including
antimicrobial agents .
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40. PREPARATION
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41. METHODS
 1. Phage were mixed with bacterial cells for 10 mins( long
enough for adsorption but too short for further infection).
 2. The mixture is diluted by 10,000.( only those cells thst are
bound phage in the initial incubation will contribute to the
infected population ; progeny phage produced from those
infections will not find host cells to infect).
 3. Incubate the dilution. At intervals, a sample can be remoced
from the mixture and the number of free phage counted using
a plaque assay.
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42. PHAGES CROSSES THE
COMPLEMENTATION TEST
 MIXED INFECTION:
A single cell is infected with two
phage particles at once.
MIXED INFECTION (CO-
INFECTION)
1. It allows one to perform
phage crosses.
If two different
mutants of the same phage co-
infect a cell, recombination can
occur between the genomes.
The frequency of the genetic
exchange can be used to order
genes on the genome.
 2.It allows one to assign
mutation to complementation
group.
If two different
mutant phage co- infect the
same cell as a result each
provides the function that the
other was lacking, the two
mutants must be in different
genes(complementation
group). If not, the two mutation
are likely in the same gene.
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43. TRANSDUCTION AND RECOMBINANT
DNA
 During infection, a phage might pick up a piece of bacterial
DNA ( Mostly happens when a prophage excises from the
bacterial chromosome ).
 The resulting recombinant phage can transfer the bacterial
DNA from one host to another, known as specialized
transduction.
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49. BACTERIOPHAGE THERAPY
OVER ANTIBIOTICS
 Bacteriophage treatment offers a possible alternative to
conventional antibiotic treatments for bacterial infection.
 It is conceivable that, although bacteria can develop resistance to phage,
the resistance might be easier to overcome than resistance to antibiotics.
 Bacteriophages are very specific, targeting only one or a few strains of
bacteria.
 Traditional antibiotics have more wide-ranging effect, killing both harmful
bacteria and useful bacteria such as those facilitating food digestion. The
species and strain specificity of bacteriophages makes it unlikely that
harmless or useful bacteria will be killed when fighting an infection.
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A few research groups in the West are engineering a broader spectrum phage,
and also a variety of forms of MRSA treatments, including impregnated wound
dressings, preventative treatment for burn victims, phage-impregnated
sutures.
Enzybiotics are a new development at Rockefeller University that create
enzymes from phage.
Purified recombinant phage enzymes can be used as separate antibacterial
agents in their own right.
Phage Therapy also has the potential of preventing or treating
infectious diseases of corals. This could assist with decline of coral around
the world.

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52. APPLICATION
 1. IN FOOD INDUSTRY:
 Bacteriophage bioprocessing
is 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.
 Phage application can be also
use to control the presence
of biofilm in the food
processing environment.
 2.IN AGRICULTURE AND
FISHERIES-
 Bacteriophage have been
considered 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 fruit and vegetable or
the pathogenic E.Coli.
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54. EXAMPLES NO:1
 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 beneficent micro flora was only partially effective.
 A preventive group was infected with C. jejuni at day 4 of a 10 day phage
treatment.
 A therapeutic 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 pfeventive and therapeutic
group were observed.
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55. EXAMPLE NO :2
 KILLING OF MYCOBACTERIUM AVIUM AND MYCOBACTERIM TUBERCULOSIS
BY A MYCOBACTERIOPHAGE DELIVERED BY A NON – VIRILENT
MYCOBACTERIUM.
 Mycobacterium smegmatis, an a virulent mycobacterium , is used to
delivered the lytic phage TM4 where both M. avium and M. Tuberculosis
reside within macrophages
 This 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 the number of viable intracellular
bacilli.
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58. SCIENTIFIC REASON BEHIND THE GANGA WATER
PURITY!!
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59. PROBLEMS
 PROBLEM NO 1: Host
range
 Solution: cocktails of
bacteriophage
 PROBLEM NO 2: Bacterial
debris present in phage
preparation.
 Solution : Modern
technology allows density
centrifugation and other
methods of purification.
 PROBLEM NO 3: Lysogeny
 Solution: Use only lytic
phage that are strong
candidates for clinical trails
 PROBLEM NO 4: Anti- phage
antibodies.
 Solution: In treating chronic
infection, it may be
possible to administer
higher dose of phage, to
compensate for those that
are cleared by interaction
with neutralizing antibodies.59

60.  Phagoburn was a European Research &
Development (R&D) project funded by the
European Commission under the 7th
Framework Programme for Research and
Development. The project was launched in
June 2013 and ended in February 2017.
 Phagoburn aimed at evaluating phage
therapy for the treatment of burn wounds
infected with bacteria Escherichia
coli and Pseudomonas aeruginosa.
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61. CONCLUSION?
 Multi drug resistant bacteria have opened a second window
for phage therapy.
 Phage therapy can then serve as a stand – alone therapy for
infection that are fully resistant.
 It will also then be able to serve as co-therapeutic agent for
infection that are still susceptible to antibiotics, by helping to
prevent the emergencies of bacterial mutants against either
agent.
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62. REFERENCES
 1.Lima-Mendez, G., Toussaint, A. & Leplae, R. A modular view
of the bacteriophage genomic space: identification of host and
lifestyle marker modules. Res. Microbial. 162, 737–746 (2011).
 Zinder, N. D. & Lederberg, J. Genetic exchange
in Salmonella. J. Bacteriol. 64, 679–699 (1952).
 Phage therapy: An alternative to antibiotics in the age of multi-
drug resistance
Derek M Lin, Britt Koskella, and Henry C Lin
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