Department of Microbiology, Islamic Azad University, Falavarjan Branch

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IN THE NAME OF GOD

• Department of Microbiology, Islamic Azad
  University, Falavarjan Branch
• Microbial Biotechnology
• By
• Keivan Beheshti Maal

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Bacteriophage Applications and Biotechnology
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Bacteriophage
Definition Bacteriophage (phage) are obligate
intracellular parasites that multiply inside
bacteria by making use of some or all of the host
biosynthetic machinery (i.e., viruses that infect
bacteria.)
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What is a Bacteriophage ?

• Viruses that attack bacteria
• Non-self replicating
• Made up of mostly proteins and DNA
• Bacterial specific
• Able to infect most group of bacteria
• Isolated from soil, water, sewage and most
  bacterial living zones
• Number of progenies in a cell 50-200
• Inject their genome into host cell
• Lytic cycle (virulent)
• Lysogenic cycle (temperate)

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Bacteriophage properties

• Phages are ubiquitous and can be found in all
  reservoirs
• populated by bacterial hosts, e.g., soil or
  animal intestine.
• One of the densest natural sources for phages
  other
• viruses is sea water, where up to
  109 virions/ml
• found at the surface, and up to 70 of marine
  bacteria may be infected
• The dsDNA tailed phages, or Caudovirales, account
  for 95
• of all the phages reported in the scientific
  literature

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What phages do to Host Cell
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Lytic Life Cycle
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As lytic phage propagate, bacteria are destroyed
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Discovery of Bacteria Infecting Viruses

• Frederick W. Twort given first credit for phages
  1915
• Found by studying
• micrococcus colonies

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Naming of the Viruses

• Felix D Herelle
• Born in Montreal1873
• Medical bacteriologist
• Rediscovery of
• Bacteriophages 1917

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First Electron Micrograph

• Luria and Anderson 1942 first electron
  micrograph picture
• of a T2 phage
• Anderson also discovered the phages adsorbed by
  the tail by
• critical point technique

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Bacteriophage history in a glance

• 1915-1917 discovery
• 1920 bacteriophage base therapy
• 1940 pioneering studies of physiology
• and phage-host relationships
• 1950 molecular biology techniques for studing
• structure and genetics of
  bacteriophages
• 1970 use of many phage enzymes in cloning
• 1990 phage displayas powerful technique in
• identification of biomolecules
• 2000 transfer of toxin genes in invironment by
• phages (concern)
• Nowadays bacteriophage applications in medical
• biotechnology and industrial-food
  microbiology

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Bacteriophage Classification

• Based on two major criteria
• phage morphology and shape of the phage (electron
  microscopy)
• nucleic acid properties

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How many kinds of Bacteriophage?

• Over 5000 bacteriophages have been studied by
  electron microscopy which can be divided into 13
  virus families

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Electron micrographs of different phages

• B. caldotenax
• aJS025
• bJS017
• cJS027
• B. stearothermophilus
• dJS017
• B. anthracis
• e8724/25
• St. camosus
• fSt.c

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13 Bacteriophage families
Double stranded DNA, Non-enveloped
Double stranded DNA, Enveloped
Single stranded RNA
Double stranded RNA
Single-stranded DNA
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13 Bacteriophage families
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Bacteriophage Applications

• Bacteriophage therapy
• Bacteriophage mediated microbial control
• Bacteriophage enzymes
• Bacteriophage display
• Baceriophage typing
• Bacteriophage as biological tracer
• Monitoring and validation tool
• Bacteriophage based diagnostics
• Bacteriophage as cloning vector
• Bacteriophage for biodegradation

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Phage can be used biologically-based
antimicrobial system

• Phage produce products that disrupt the bacterial
  systems (antimicrobial proteins)
• Enzymatic
• Lysozymes
• B-glucosidases
• Nucleases
• Proteases
• Non-enzymatic
• Very effective on microbes (bacteria, viruses,
  fungi, etc.)
• Some evidence effective on spores
• Probably not useful for toxins
• Bacteriocins- produced by bacteria
• Antimicrobial peptides (AMPs)- produced by higher
  organisms

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Bacteriophage therapy

• Reducing of bacterial load by lytic phages or
  engineered phages
• Administration ways
• Orally topically systematically
• Use of free phages or phage infected bacteria
  (very much experimental)
• Usage during first step infection
• Catch infection on time before harden of
  infection eradication

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Bacteriophage therapy

• Key aspects
• 1. proper phage choice
• 2. quantity of delivery
• 3. Timing of treatment
• Advantages
• 1. unable to modify degrade animal metabolism,
  highly specific
• 2. self replicating -gt self amplifying -gt
  efficacy enhancement
• 3. ubiquity and diversity of bacteriophages
• 4. active against antibiotic resistant
  organisms
• 5. used as an alternative in antibiotic-allergic
  persons

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Bacteriophage therapy

• In eastern Europe spraying of E.coli phages at
  room surfaces, objects, toilets in hospitals
  (very effective)
• Tretment and prophylaxis of systemic E.coli
  infections of human, mice and diarrhoeal disease
  in calves
• Control and treatment of Ps. Aeroginosa and
  Acintobacter baumanii in burn states

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Bacteriophage therapy

• Exponential Biotherapies (Rockville, MD)
• Vancomycin resistant Enterococcus facium and
• Streptococcus pneumoniae
• Phage Therapeutics (Bothell, WA)
• Staphylococcus aureus and Staphylococcus
  epidermidis
• Intralytix, Inc. (Baltimore, MD)
• Salmonella in meat and poultry
• Biopharm Ltd. (Tblisi, Georgia)
• Infections associated with burns
• University of Idaho
• Escherichia coli O157H7 in cattle

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Bacteriophage mediated microbial control

• Control of bacterial contamination in food
  industries e.g. Pseudomonas fragi in milk and
  Pseudomonas sp in beef and steaks
• Control of bacterial contamination for water born
  pathogens such as Vibrio cholera
• Control of bacterial contamination for air born
  pathogens in the hospital and environmental
  Mycobacteria
• Control of bacterial contamination in poultry
  industries pathogens such as Campylobacter
• Control of plaque forming bacteria such as
  Streptococcus mutans, St.
• sunguis and St. sobrinus and Lactobacillus
  acidophilus by addition of
• bacteriophages to toothpaste, chewing gum
  and sweets
• Control of biofilm forming bacteria like
  listeria, Escherichia and
• Pseudomonas sp. in different industries
  (compete with undiffusible
• chemicals and antibiotics

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Bacteriophage enzymes

• Use of enzymes and other products as tools for
  molecular biology techniques specially
  thermophylic products from thermophyl phages

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Construction of Genomic DNA and cDNA phage
libraries

• Making Genomic DNA library for
• - Sequencing
• - Knock out mice production
• Making ESTs library for
• - To fined full length cDNA
• - Bioinformatics analysis
• - Expression analysis
• - There are more than 106 expressed sequence tags
  (ESTs) in databases (http//www.ncbi.nlm.nih.gov/d
  bEST/index.html)
• - To focus on a known protein with interesting
  biological function (and, ideally, a known
  structure)
• - To search for family member and other species
  gene homologue

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Phage display technology

• Phage display is a powerful screening tool
• permitting the discovery and
• characterisation of proteins that interact
• with a desired target
• A protein is displayed on the surface of a
• phage as a fusion with one of the coat
• proteins of the virus and the DNA that
• encodes this protein is housed within the
• virion
• A process of biopanning is used to
• rescue phage that display a protein that
• specifically binds to a target of interest

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Bacteriophage display

• A polypeptide can be displayed on the phage
  surface by inserting the gene coding for the
  polypeptide in the phage genome
• capable of performing a function, typically the
  specific binding to a target of interest

phenotype (binding)
p?
tip of phage
genotype
Phage displaying a binding protein
(redrawn from Viti 1999)
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Biopanning
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Construction and application of phage
antibody libraries

• Display of antibody fragments on bacteriophage
• the favored format of antibody fragment is
  single-chain FV (scFV)

antigen binding site
VH
Fab (50 kD)
CH1
VL
CL
CH2
whole Ab (150 kD)
CH3
FV (25 kD)
scFV (27 kD)
Schematic representation of different antibody
formats (redrawn from Viti 1999)
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scFV Antibody Phage Display

• Antibodies have been exploited for therapeutics
  and targeting
• Traditionally relied on long process of
  generation and screening
• Antibody phage display library contains 107
  unique scFV molecules
• Affinity binding allows rapid selection of scFV
  which bind target of interest

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Bacteriophage typing

• First practical applications of bacteriophages
• Very spesific technique for identification of
• bacterial strains according to their phage
• sensitivity
• Has been stablished for detecting bacteria
• such as Staphylococccus, Salmonella,
• Escherichia, Mycobacterium, Listeria,
• Campylobacter

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Bacteriophage as biological tracer

• For tracing air born and water (ground waters)
  movement
• Coli phage T4 was successfully used to trace
  ground
• water flow for 1.6 km (Southern Missouri,
  U.S.A)
• Advantages
• Small size, negligible impact on water
  quality,
• detectable in low number, adaptable to
  filtration
• recovery method
• Use of T4 for detection of contamination of
  sewage in
• water wells (New Zeland)
• Other phages
• MS2, PRD1, f2

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Monitoring and validation tool

• Use of bacteriophage as a model for evaluating
  and testing of filtration systems in removing
  dangerous viral particles such as HIV and SARS,
  HBV
• Seratia marcescens active phage and coliphage MS2

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Bacteriophage based diagnostic

• Rapid and accurate detection tool for targeted
  bacteria
• Phages vs Abs
• 1.Simple and economical
• 2.Producible in large amounts at low cost
• 3. Use of luciferase gene (lux) in phage ?
  expression in bacterium ? light emission
• -have been used to detect enteric bacteria in
  food, L.monocytogenes in foods and environmental
  samples

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Lysogenic Bacteriophages Examples of Virulence
Factors Carried by Phage
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BacteriophageThe Flesh-Eating Bacteria

• Then it rapidly kills tissues causing gangrene
  conditions.
• If treat early with antibiotics and removal of
  infected tissue then amputation and death can be
  averted.
• There are between 500-1500 case in the U.S.A.
  each year
• Flesh-eating bacteria has a death rate of 20-50

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BacteriophageRelatives of Flesh-Eating Bacteria

• Other Group A Streptococci which have acquired
  virulence factors
• Scarlet Fever Toxin
• Streptococcal Toxic Shock Syndrome

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Bacteriophage Therapeutic Uses

• Bacteriophage has been used to fight many
  bacterial infections
• Some examples of diseases treated with phage
  therapy
• staphylococcal skin disease
• skin infections caused by Pseudomonas
• Klebsiella
• Proteus
• E. coli
• P. aeruginosa infections in cystic fibrosis
  patients
• neonatal sepsis
• surgical wound infections
• Likewise, bacteriophage has also been used to
  treat animal disease.

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• Thank you for your Attention