This document discusses techniques for engineering bacteriophages (phages) to enhance their potential as antimicrobial agents. It describes various methods for genetically modifying phage genomes, including homologous recombination, recombineering, and rebuilding genomes in vitro or in yeast. Synthetic phages have been engineered with broader host ranges or the ability to deliver genes conferring antibiotic sensitivity. Phage lysins have also been developed as antimicrobials targeting pathogens like MRSA. Overall, the document outlines how phage engineering is an area of active research with applications for treating antibiotic-resistant bacteria.

2. Phage Engineering for
Antimicrobials
Dr. J Sai Prasad
Assistant Professor
Agricultural Microbiology
Professor Jayashankar Telangana State Agricultural
University, Hyderabad. T.S.

3. Contents
• Introduction
• Techniques for Engineering Phages
• Synthetic Phages for pathogen control
• Phage derived antimicrobials
• Drug delivery system by engineered
Phages
• Conclusion
• Future perspectives

4. Introduction
• Bacteriophages (phages) the most abundant biological particles on earth.
Highly versatile and potential for various applications.
• Phages are viruses that infect bacteria; their self-replication depends on access
to a bacterial host having additional advantages for their use as antimicrobials.
• Discovered independently by Frederick Twort in 1915 and by Félix d’Hérelle in
1917 and used early on as antimicrobial agents.
• The rising tide of antibiotic resistance has revived interest in phages as
antibacterial agents.
• Unlike most antibiotics, phages are typically highly specific for a particular
bacterial species or strains and thus expected to target effects on commensal
microflora
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

5. • Phages are used not only to treat and prevent human bacterial infections but also
to control plant diseases, detect pathogens and assess food safety
• Like certain antibiotics, phages can cause rapid and massive bacterial lysis via
release of lipopolysaccharides [LPS], which induce adverse immune responses in
the human host
• Bacteria frequently live in biofilm communities surrounded by extracellular
polymeric substances (EPS), which act as a barrier to phage penetration.
• By genetically engineering phages, it may be possible to overcome many of
these limitations.

6. TECHNIQUES FOR ENGINEERING
PHAGES

7. • Homologous
recombination is a
natural phenomenon.
• A reporter gene, usually
encoding luciferase or a
fluorescent protein, is
commonly cloned along
with the gene of interest
to facilitate the
identification of mutant
phages by detecting the
reporter.
Homologous Recombination
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

8. Bacteriophage Recombineering of Electroporated DNA
• Co-electroporating the
recombineering
substrates, i.e., phage
DNA and dsDNA, into
electrocompetent
bacterial cells carrying a
plasmid that encodes
proteins promoting high
levels of homologous
recombination, such as
the RecE/RecT-like
proteins.
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

9. In Vivo Recombineering
• Bacterial cells carrying a
defective prophage and the
pL operon under the control
of a temperature-sensitive
repressor (A) are infected
with the phage to be
manipulated (B) and
subsequently transformed
with dsDNA or ssDNA (C).
• Following phage infection, the
recombination functions are
induced by heating the mid-
log-phase bacterial culture to
42°C. Recombination then
occurs (D), after which
recombinant phage particles
are recovered (E).
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

10. Rebuilding/Refactoring Phage Genomes In Vitro
• Phage genomes can be manipulated and edited in vitro before they are
introduced into their bacterial hosts.
• Once the phage DNA has been purified (A), it is digested using native
restriction sites (B), and independent pieces (C) can be subcloned and further
manipulated (D). Once released from the vector, the recombinant section is
ligated to the rest of the phage genome (E) and electroporated into the phage
host for recovery of engineered phage particles (F).
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

11. Yeast-Based Assembly of Phage Genomes
• Propagating phage genomes in a bacterial host can be toxic for the host, thus
limiting the efficiency of phage genome engineering. It can overcome by using
Saccharomyces cerevisiae
• Purified phage DNA (A) is electroporated into S. cerevisiae together with linear
YAC molecules (B). Recombination in the yeast cell enables genomic
subcloning (YAC backbone in green) (C), which upon YAC purification and
electroporation (D) allows the recovery of functional phage particles in
bacteria(E).
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

12. Cell-Free Transcription-Translation Systems
• Advantages of using in vitro
or yeast-based genome
modification is that phage
genomes can be engineered
without causing toxicity to
the host.
• Purified phage genome DNA
is combined with cell-free
expression systems (A) that
enable gene transcription
(B), translation (C), DNA
replication (D), and
assembly of whole phage
particles (E).
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

13. SYNTHETIC PHAGES FOR PATHOGEN
CONTROL
Natural Phage-Based Antimicrobials
Sl
No
Name Mechanism Antimicrobial / control
org.
1 PhagoBurn, a
clinical
trial by the Rose. T
et al., (2014)
Normal phages have
shown no adverse effects
related to its application
Evaluate phage cocktail for
the treatment of E. coli and
P. aeruginosa infections in
burn wound patients
2 Bruttin and Brüssow
(2005)
Treated E. coli T4 phage
orally to 5 human
volunteers.
Controls the E.
coli diarrheal infections.
No adverse effects
observed in a safety study.
Rose. T et al, (2014) & Bruttin and Brüssow (2005)

14. Engineered Phages for Enhanced Antibacterial Activity for
individual pathogen
Sl
no
Name Mechanism Antimicrobial / control
org.
1 Edgar et
al. (2012)
Engineered temperate phages
to deliver genes encoding rpsL
and gyrA, which confer
sensitivity to streptomycin and
nalidixic acid antibiotics into
bacteria respectively
E. coli K-12 resistant to
these antibiotics were then
lysogenized with the
engineered phages (carrying
rpsL or gyrA), and restores
antibiotic efficiency by
reversing pathogen
resistance.
Edgar et al. (2012), Appl Environ Microbiol 78:744–751.

15. Engineered Phages with Shifted or Broadened Host Ranges
Sl
n
Name Mechanism Antimicrobial / control org.
1 Lin T-Y
et al.,
A hybrid T3 and T7 phage (T3/7)
was devised, in which part
of the tail fiber gene of T3 (gp17)
was replaced with that of phage
T7.
The T3/7 recombinant phage
exhibited a broader host
range and a better
adsorption efficiency than
the wildtype phages, i.e., T3
and T7 individually
2 Marzari
et al.
Filamentous coliphage-fd by
adding a receptor-binding
domain from the filamentous
phage IKe.
Coliphage fd, which
normally infects E. coli, was
also engineered to recognize
Vibrio cholerae.
Lin T-Y et al., (2012) & Marzari et al. (1997)

16. PHAGE-DERIVED ANTIMICROBIALS
Lytic
enzyme
Model Target pathogens Result summary
Phage-
derived
lysins
ABgp46 In vitro MDR A. baumannii,
P. aeruginosa, and
S.typhimurium
Cross-inoculation significantly
reduced bacterial density
PlyCD In vitro Clostridium difficile Reduced C. difficile colonization
PlyG In vitro Bacillus anthracis Eliminated B. anthracis spores
and vegetative cells
PlyF307 Murine MDR A. baumannii i.p. treatment rescued mice from
lethal bacteremia
PlySs2 Murine Streptococcus
pyogenes and MRSA
i.p. treatment reduced mortality
from lethal bacteremia
Cpl-1 Murine Streptococcus
pneumoniae
i.p. treatment rescued mice from
lethal pneumonia
Cocktail
6
In vitro,
murine
MRSA Effective against biofilms in
vitro and protected mice from
lethal sepsis
Lin DM et al . World J Gastrointest Pharmacol Ther 2017; 8(3): 162-173

17. Lytic
enzyme
Model Target
pathogens
Result summary
Bioeng.
chimlysins
CHAPK In vitro MRSA Eliminated MRSA and dispersed
biofilms
ClyH Murine MRSA Treatment rescued mice from
bacteremia
Cpl-711 Murine S.
pneumoniae
Treatment rescued mice from
bacteremia
Ply187 Murine Staphylococcu
s aureus
Prevented bacterial
endophthalmitis
Lysin &
antibiotic
therapy
CF-301 Murine MRSA Lysin treatment was most
effective when combined with
vancomycin or daptomycin
MR-10 Murine Burn wound
infection
Lysin treatment was most
effective when combined with
minocycline
Lin DM et al . World J Gastrointest Pharmacol Ther 2017; 8(3): 162-173

18. Sn Engineered phages Drug activi Antimicrobial / control org.
1 Yacoby I. et al., (2006) used
filamentous phages (fd and
M13) to target S. aureus by
target-specific peptides on
the major coat protein
Enhancing
Antibiotic
Activity
Chemically conjugated the phages
with chloramphenicol, bound to
the target cells and chloramphenicol
was released, retarding bacterial
growth
2 Bar H et al., (2008) used
genetically modified
fUSE5-ZZ phages to
display a ligand that leads to
the target cancer cells and
then loaded with cytotoxic
drugs.
Delivery of
Anticancer
Drugs
The drug-carrying phages targeted
ErbB2-overexpressing human
breast adenocarcinoma. Once
endocytosed, the phages releasing
the drug inside the cancer cells and
resulting in about 50% inhibition of
target cell, a 1,000-fold
improvement of hygromycin.
Drug Delivery Systems by Engineered Phages
Yacoby I. et al. (2006) & Bar H etal., (2008)

19. Published findings on phage therapy in humans
and in animal models
Causative
agent
Model Condition Oral Result summary
Shigella
dysenteriae
Human Dysentery Oral All four treated individuals
recovered after 24 h
Vibrio
cholerae
Human Cholera Oral 68 of 73 survived in treatment group
and only 44 of 118 in control group
MDR S.
aureus
Human Diabetic
foot ulcer
Topical All 6 treated patients recovered
P.
aeruginosa
Murine Sepsis Oral 66.7% reduced mortality
S. aureus Rabbit Wound
infection
S.C. Co-administration with S. aureus
prevented infection
Lin DM et al . World J Gastrointest Pharmacol Ther 2017; 8(3): 162-173

20. Phage products
Sl no. Phage products Control organisms
1 List shield, Ecoshield and
SalmoFresh from
Intralytix
Listeria monocytogenes, E. coli
O157:H7, and S. enterica in foods or
food-processing environments.
2 Salmonelex and Listex P100 Salmonella and L. monocytogenes,
reduce contamination during food
processing
3 AgriPhage from OmniLytics Xanthomonas campestris and
Pseudomonas syringae
on tomato and pepper plants
Pires DP et al., 2016, Microbiol Mol Biol Rev 80:523–543

21. Conclusion of the Seminar
• The prodigious diversity of phages has led to powerful applications for
therapeutics, diagnostics, materials science, drug delivery systems and
vaccines
• The new genetic engineering technologies has led to a more precise and
accelerated modification of phage genomes.
• The use of phages and derived proteins for combating bacterial infections,
specifically for multidrug-resistant bacteria, shows phage therapy as either
an alternative or a supplement to antibiotics.
• Furthermore, techniques to contain the use of genetically modified phages
for materials science applications and to inactivate the phages after use may
also help to mitigate these issues
• Phage engineering is an area of research that is attracting intense interest
and has great potential utility, but it has yet to be fully exploited.

22. Future Perspectives
• Engineered phage for single homogenous biofilm control technology could be
expanded to target the heterogeneous extracellular composition of biofilms
• Many strategies for engineering phages require the ability to genetically modify
the bacterial hosts, which is still a challenge for many bacterial species.
• Despite the potential benefits, the acceptance of genetically modified phages for
real-world applications may vary across different regions of the world.
• In the case of human use, the choice of compelling areas of tremendous medical
need and explicit demonstrations of safety will both be important.