Final Aliaa-PHD Thesis

Zagazig University Faculty of Pharmacy
Isolation and characterization of bacteriophages infecting

Pseudomonas aeruginosa
A thesis submitted for partial fulfillment of the requirements for Doctoral
Degree of Philosophy in pharmaceutical sciences
(Microbiology and Immunology)
By
Aliaa Atef Taha Abdelghafar
Assistant lecturer of Microbiology & Immunology
Faculty of Pharmacy, Zagazig University
Under Supervision of
Prof. Dr. Ghada Hamed Shaker

Professor of Microbiology & Immunology
Dean of the faculty of Pharmacy
Sinai University, Kantara branch
Prof. Dr. Amira Mohamed El-Ganiny

Professor of Microbiology & Immunology
Ass. Prof. Dr. Momen Mahmoud Ez Elarab Askoura

Assistant Professor of Microbiology & Immunology
Department of Microbiology and Immunology

Faculty of Pharmacy
Zagazig University
(2023)
Acknowledgement
First and foremost, I thank ALLAH for helping me to complete this

work.
I would like to express my sincere gratitude from my heart to Prof. Dr.

Ghada Hamed Shaker (Professor of Microbiology and Immunology, Dean of the
faculty of Pharmacy, Sinai University, Kantara branch) for her kind supervision,
continuous support of my work and for her patience, motivation and valuable
advice. Her guidance helped me in all time of this work. I am sure it would not
have been possible without her help.
I would like to express my sincere gratitude to Prof. Dr. Amira Mohamed El-
Ganiny (Professor of Microbiology and Immunology, Faculty of Pharmacy,
Zagazig University) for her support and guidance to accomplish my
work.
I owe my deepest thanks to my adviser Ass. Prof. Dr. Momen Mahmoud Ez

Elarab Askoura (Assistant Professor of Microbiology and Immunology, Faculty
of Pharmacy, Zagazig University) for his valuable guidance, scientific and
technical support throughout the work, useful discussion in every step of my work,
patience, motivation, enthusiasm and constructive criticism.
I would also like to express my deepest appreciation to the staff members at

Microbiology and Immunology Department, Faculty of Pharmacy, Zagazig
University
Very special thanks to my friends who made the hard times always pass by with a
smile.
Aliaa Atef Taha Abdelghafar

Dedication
This work is lovely dedicated to my dear father
and my beloved mother who supported me
through work and always there whenever I
needed help.
To my lovely husband Abdelfattah and my
sweetheart Asser.
Special thanks for my lovely sisters Alshimaa,
Doaa, Lamiaa and Mariam.

List of contents
Page
Content
No.
List of abbreviations I
List of tables III
List of figures IV
Introduction 1
Literature Review 5
1. Structure and growth conditions 5
2. Pathogenesis and diseases caused by P. aeruginosa 6
2.1. Burn and wound infections (soft tissue infections) 7
2.2. Bacteremia 7
2.3. Lung infections 7
2.4. Urinary tract infections (UTIs) 8
2.5. Eye and ear infections 8
3. Virulence factors 9
3.1. Adhesins 10
3.1.1. Type IV pili-mediated adhesion 10
3.1.2. Flagella-mediated mucin binding 10
3.2. Secreted toxins and exoenzymes 11
3.2.1. Exotoxin A 11
3.2.2. Proteases 12
3.2.3. Phospholipase 12
3.2.4. Rhamnolipids 13
3.2.5. Other toxins 13
3.3. Alginate production 14
3.4. Pyocyanin 14
3.5. Biofilm formation 14
4. P. aeruginosa resistance to antimicrobial agents 17
4.1. Intrinsic antibiotic resistance 17
4.2. Acquired antibiotic resistance 18
4.3. Adaptive antibiotic resistance 20
5. Using bacteriophages (phages) as an alternative strategy for bacterial
infections control
21
6. Structure and classification of bacteriophages 22
7. Bacteriophage life cycle 25
7.1. Lytic cycle 26
7.2. Lysogenic cycle 26
8. Potential advantage of using bacteriophage in treatment of bacterial infections 27
9. Potential limitations or drawbacks of phage therapy 29
10. Phage application in biofilm degradation 31
10.1. Diffusion through biofilm water-channels 31
10.2. Enzymatic degradation 31
10.3. Adherence to motile bacteria 34
10.4. Tackling persister cells 34
11. Examples of therapeutic applications of bacteriophage 34
Materials and Methods
Materials 36
1. Experimental samples 36
1.1. Clinical specimens for bacterial isolation 36
1.2. Sewage samples 37
2. Culture media 37
3. Chemicals 40
4. Reagents and buffers 40
5. Materials used for genome sequencing 43
6. Antimicrobial discs used for antimicrobial susceptibility testing 43
7. Animal study 44
Methods 45
Results 58
Discussion 128
Conclusions and Recommendation 146
Summary 147
References 152
Arabic summary 1
List of abbreviations
Item Meaning
WHO World Health Organization
MDR Multidrug resistance
CF Cystic fibrosis
UTIs Urinary tract infections
T3SS Type III secretion pathway
LPS Lipopolysaccharides
eEF-2 Elongation factor 2
IFN-γ Human gamma interferon
PLC Phospholipase C
QS Quorum sensing
EPS Extracellular polymeric substances
c-di-GMP Cyclic di-GMP
RND Resistance-nodulation-division
ESBLs Extended-spectrum-β-lactamases
APH Aminoglycoside phosphotransferase
AAC Aminoglycoside acetyltransferase
ANT Aminoglycoside nucleotidyl transferase
ICTV International Committee on the Taxonomy of Viruses
BAVS Bacterial and Archaeal Subcommittee
dsDNA Double-stranded DNA
ssDNA Single-stranded DNA
ssRNA Single-stranded RNA
dsRNA Double-stranded RNA
OmpA Outer-membrane protein A
UV Ultraviolet
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
VAPGHs Virion-associated peptidoglycan hydrolases
PG Peptidoglycan
CFU Colony forming unit
MH Mueller-Hinton
TS Tryptone soya
TSI Triple sugar iron
TTC Triphenyl tetrazolium chloride
PBS Phosphate-buffered saline
IRB Institutional Review Board
I
CLSI Clinical Laboratory Standards Institute
OD Optical density
ODC Cut-off optical density
PFU Plaque forming unit
SM Saline-Magnesium buffer
TEM Transmission electron microscope
EOP Efficiency of plating
MOI Multiplicity of infection
ORF Open reading frame
Rpm Revolution per minute
MEGA Molecular Evolutionary Genetics Analysis
QUAST Quality Assessment Tool for Genome Assemblies
NCBI National Center for Biotechnology Information
IP Intraperitonially
ZU-IACUC Zagazig University Institutional Animal and Use Committee
XDR Extensively drug-resistant
NJ Neighbor-joining
II
List of tables
Table Title Page
No. No.
1 Bacterial species involved in assay of the host range 36
2 Source and number of P. aeruginosa clinical isolates 58
3 Biochemical identification of P. aeruginosa isolates 59
4 Antibiotic susceptibility of P. aeruginosa isolates 60
5 Classification of P. aeruginosa according to biofilm forming
63
capacity
6 Host range of isolated phages 73
7 Efficiency of plating (EOP) of vB_PaeP_PS28 and
79
vB_PaeM_PS3 phages
8 Predicted ORFs identified in the phage vB_PaeP_PS28
89
9 Homology of vB_PaeP_PS28 to other phages 96
10 Predicted ORFs found in vB_PaeM_PS3 104
11 tRNAs identified in the vB_PaeM_PS3 genome 120
12 Homology of phage vB_PaeM_PS3 to other phages genomes
122
III
List of figures
Figure Title Page
No. No.
1 Typical bacteriophage structure 23
2 Families of bacteriophages 25
3 Bacteriophage life cycle 27
4 Percentage of P. aeruginosa isolates resistance against tested
62
antibiotics
5 Quantitative evaluation of P. aeruginosa biofilm formation using
65
the crystal violet (CV) assay
6 Detection of phages by the spot assay 66
7 Plaque morphology of isolated phages on different P. aeruginosa
67
lawns by double agar overlay technique
8 Transmission electron microscope (TEM) images of a)
vB_PaeP_PS28, b) vB_PaeP_PS49, c) vB_PaeP_PS14, d) 68
vB_PaeM_PS3 and e) vB_PaeM_PS38
9 Thermal stability of isolated phages. a) vB_PaeP_PS28; b)
vB_PaeP_PS49; c) vB_PaeP_PS14; d) vB_PaeM_PS3 and e) 70
vB_PaeM_PS38
10 pH stability of isolated phages. a) vB_PaeP_PS28; b)
vB_PaeP_PS49; c) vB_PaeP_PS14; d) vB_PaeM_PS3 and e) 71
vB_PaeM_PS38
11 The antibiofilm activity of a) vB_PaeP_PS28, b) vB_PaeP_PS49, c)
vB_PaeP_PS14, d) vB_PaeM_PS3 and e) vB_PaeM_PS38 against 75
various P. aeruginosa isolates
12 One-step growth curve of a) vB_PaeP_PS28 and b) vB_PaeM_PS3 81
13 Bacteriolytic activity of phage vB_PaeP_PS28 against host strain
82
(a) and PAO1 (b)
14 In vitro planktonic cell lysis assay of vB_PaeM_PS3 at different
83
MOIs against host bacteria (a) and PS49 (b)
15 Genomic characterization of vB_PaeP_PS28 86
16 Comparative genomic analysis between phage vB_PaeP_PS28 and
97
related sequences
IV
17 Dot Plot comparisons of the genomic nucleotide sequences of
98
vB_PaeP_PS28 and related bacteriophages infecting P. aeruginosa
18 General features of vB_PaeM_PS3 genome
100
19 Dot Plot comparisons of the genomic nucleotide sequences of
102
vB_PaeM_PS3 and related bacteriophages infecting P. aeruginosa
20 Phylogenetic trees of vB_PaeM_PS3 based on the amino acid
sequences of major capsid protein (a) and terminase large subunit 121
(b)
21 In vivo characterization of the influence of phage vB_PaeP_PS28
124
on P. aeruginosa pathogenesis in mice infection model
22 Impact of phage vB_PaeM_PS3 on P. aeruginosa pathogenesis in
126
vivo
V
Introduction
Introduction
Pseudomonas aeruginosa (P. aeruginosa) is a widely distributed
opportunistic pathogen that could survive in water, soil, animals and humans. P.
aeruginosa is the major cause of nosocomial infections due to the flexibility and
adaptability encoded in its genome. Furthermore, P. aeruginosa is the causative
agent of wide variety of infections ranging from soft tissue infections to life-
threatening ones including bacteremia and pneumonia. P. aeruginosa could grow on
different medical equipment due to the presence of its essential binding factors such
as flagella, pili and the ability to form biofilms. Unfortunately, infections caused by
this bacterium are characterized by higher morbidity and mortality rates especially
in immunocompromised patients and those suffering from severe burns or cystic
fibrosis.
Numerous virulence factors contribute to P. aeruginosa pathogenesis including
hemolysins, rhamnolipids, proteases and biofilms. P. aeruginosa is capable of
forming biofilms which protect bacteria from environmental stresses and
phagocytosis. Additionally, it has been suggested that biofilm formation markedly
contributes to bacterial resistance to various antibiotics, including quinolones,
aminoglycosides and β-lactams leading to long term persistence. P. aeruginosa is
able to acquire resistance through mutation and horizontal gene transfer added to its
1
Introduction
intrinsic resistance to various antibiotics including beta-lactams. Therefore,
treatment of P. aeruginosa infections by conventional antibiotics has become a
major global challenge. Recently, due to increased antimicrobial resistance, there is
an urgent necessity for the development of alternative antimicrobial approaches to
efficiently control bacterial infections.
Latterly, researchers have indicated that phage therapy could be considered as an
effective approach to control P. aeruginosa infections and biofilms. Bacteriophages
also known informally as phages are viruses that selectively infect and replicate
within bacterial cell. They are the most predominant biological entities residing on
the biosphere. Phage therapy refers to the use of bacteriophages in treatment of
bacterial infections as potential alternatives to overcome limitations of antibiotics.
Phages possess remarkable advantages over antibiotics and are extremely specific to
their host bacteria as well as they do not cause harmful side effects to normal
microbiota. In addition, phages are easily isolated and highly effective at destroying
bacteria in biofilms.
Unlike chemical antibiotics, phages have different strategies for eliminating resistant
bacteria and have low environmental impact due to their natural origin. Furthermore,
they could target both Gram-positive and Gram-negative bacteria, and many reports
have shown that phages are highly effective against various multidrug resistance
(MDR( pathogens in humans and animals. Endotoxin released upon lysis of bacterial
2
Introduction
cells following treatment with phages is relatively lower compared to that released
by antibiotics Hence, phage therapy possesses a great potential to replace antibiotic
treatment in treatment of various bacterial infections.
The aim of work:
The current study aims to isolate and characterize virulent phages targeting P.
aeruginosa isolated from various clinical sources. The physical properties,
antibiofilm activity as well as whole genome sequencing of isolated phages will be
determined herein. Moreover, the influence of isolated phages on P. aeruginosa
pathogenesis in host will be assessed in vivo using mice infection model. The
findings of present study would be of great importance and helpful in treatment of
P. aeruginosa related infections.
For fulfillment of this purpose, the following aspects will be investigated:
1- Isolation and identification of P. aeruginosa from different clinical sources.
2- Determination of susceptibility of P. aeruginosa isolates to antimicrobial agents.
3- Isolation and propagation of bacteriophages targeting P. aeruginosa isolates.
4- Using electron microscopy for characterization of the morphology of isolated
bacteriophages.
5- Determination of pH and thermal stability of isolated bacteriophages.

3
Introduction
6- Determination of host range of isolated bacteriophages.
7- Investigating the role of isolated bacteriophages in controlling biofilm formation
by P. aeruginosa.
8- Genomic characterization of isolated bacteriophages.
9- Characterization the influence of isolated bacteriophages on P. aeruginosa
pathogenesis in vivo using mice infection model.
4
Literature Review
Literature Review
Pseudomonas aeruginosa is a widespread Gram-negative, aerobic, rod-

shaped pathogen characterized by high intrinsic antibiotic resistance (Lyczak et al.,
2002; Schweizer, 2003). P. aeruginosa is a ubiquitous organism that can colonize
many diverse environmental settings and could be isolated from animals, plants and
humans. Due to minimal nutritional requirements and bacterial tolerance to variety
of physical conditions, P. aeruginosa is able to survive and persist in both
community reservoirs and hospital settings such as swimming pools, whirlpools,
contact lens solution, humidifiers and soil (Lister et al., 2009).
P. aeruginosa is an opportunistic pathogen and a major cause of nosocomial

infections with significant mortality rates. It causes a wide variety of infections
ranging from self-limiting skin infections to life-threatening pneumonia and
septicemia (Goncalves-de-Albuquerque et al., 2016). It has been recently identified
as critical pathogen by the World Health Organization (WHO) and represents one of
the biggest threats to public health due to increasing incidence of multidrug
resistance (MDR) P. aeruginosa infections (Daikos et al., 2021). This resistance
could be related to bacterial inherited antibiotic resistance or to the acquisition of
resistance genes through mobile genetic elements (Breidenstein et al., 2011).
Treatment of infections caused by MDR P. aeruginosa strains is a great challenge
with the remaining limited options of antibiotics and higher cost (Bassetti et al.,
2018; Matos et al., 2018).
1. Structure and growth conditions
P. aeruginosa is a heterotrophic, motile, rod-shaped bacterium about 1–5 µm

long and 0.5–1.0 µm wide and arranged as singly, or pairs or in short chains (Diggle
and Whiteley, 2020). Being a Gram-negative pathogen, P. aeruginosa has a
5
Literature Review
cytoplasmic membrane with a phospholipid bilayer and outer membrane with a

phospholipid inner face and a lipopolysaccharide outer layer. The outer membrane
of P. aeruginosa contains numerous proteins including lipoproteins and channels
(Remans et al., 2010). P. aeruginosa derives its energy from oxidation rather than
fermentation of carbohydrates and does not require specific organic growth factors.
It can grow on media supplemented with only acetate for carbon and ammonium
sulfate for nitrogen. Furthermore, it can grow under anaerobic conditions using
nitrate as an alternative electron acceptor (Arai, 2011). The optimum growth
temperature at 25°C to 37°C, indeed, it can survive in broad temperatures ranging
from 4-42°C that distinguishes it from other Pseudomonas species. P. aeruginosa is
an important soil bacterium that is capable of breaking down polycyclic aromatic
hydrocarbons. Moreover, it is often detected in water-reservoirs polluted by animals
and humans, such as sewage and sinks inside and outside of hospitals (Stover et al.,
2000). In addition to its nutritional versatility, P aeruginosa resists high
concentrations of salt, weak antiseptics and many commonly used antibiotics.
Hence, these properties explain its ubiquitous nature and contribute to its
preeminence as a cause of nosocomial infections (Fazzeli et al., 2012). Most strains
of P. aeruginosa produce one or more pigments, including pyocyanin (blue-green),
pyoverdine (fluorescent yellow-green), pyorubin (red-brown) and pyomelanin
(brown to black, not common) (Orlandi et al., 2015).
2. Pathogenesis and diseases caused by P. aeruginosa
P. aeruginosa is a causative agent of a diverse array of infections including

both community, hospital-acquired infections, pneumonia, cystic fibrosis, urinary
tract infections and burn infections (Al-Dahmoshi et al., 2020). These infections
characterized by high mortality and morbidity especially in immunocompromised
6
Literature Review
patients and those suffering from severe burns or cystic fibrosis due to increased
prevalence of antibiotic resistant strains (English and Gaur, 2010).
2.1. Burn and wound infections (soft tissue infections)
Impairment of host immunity, loss of skin physical barrier and P. aeruginosa

virulence factors contribute to success of P. aeruginosa as a pathogen in the burn
patients. P. aeruginosa was found to be responsible for invasive infections in burn
patients, hence about of 75% of all deaths in patients with severe burn infections are
related to sepsis from invasive burn wound infection (Barrow et al., 2004). In
addition to wounded skin injury, inhalation injury is common in burn patients. This
results in edema and impairment of the normal mucociliary clearance mechanism,
thus making these patients more susceptible to upper respiratory tract infections and
pneumonia (Church et al., 2006).
2.2. Bacteremia
Bacteremia is the most serious nosocomial infection caused by P. aeruginosa

and is generally associated with higher mortality and persistence than other infecting
pathogens particularly related to device-related bacteremia (Rello et al., 1994).
When P. aeruginosa spread from a local site infection, it enters the bloodstream by
disruption of epithelial and endothelial tissue barriers (Kurahashi et al., 1999).
Ecthyma gangrenous and infarcted skin lesions are considered as primary indicators
of P. aeruginosa sepsis especially in neutropenic patients (Pier and Ramphal, 2005).
2.3. Lung infections
The human respiratory tract is the most favorable environment in which P.

aeruginosa could colonize well and causes both chronic infections in the lung of
cystic fibrosis (CF) patients and acute nosocomial pneumonia. Up to 90% of
individuals suffering from CF become infected with P. aeruginosa resulting in
7
Literature Review
higher morbidity and mortality rates. In the majority of cases, P. aeruginosa

colonization of airway leads to chronic infections which are highly resistant to
therapy particularly in immunocompromised patients (MacEachran et al., 2007;
Döring et al., 2014).
2.4. Urinary tract infections (UTIs)
Urinary tract infections (UTIs) are the most common acquired healthcare
infections and account for over 30% of all nosocomial infections (Klevens et al.,
2007). UTIs can be classified as uncomplicated or complicated. In uncomplicated
UTI, Escherichia coli is the primary causative agent responsible for up to 80% of
cases with other Gram-negative microbes such as Klebsiella pneumoniae and P.
aeruginosa being less frequently detected (7%–15%). However, complicated UTIs
are more frequently caused by uropathogenic P. aeruginosa, which shows a higher
prevalence of antimicrobial resistance and great propensity to form biofilms on
medical devices than E. coli or K. pneumoniae. Initially, P. aeruginosa adheres to
the urinary tract using pili that facilitate invasion into host tissues and promoting
inter bacterial interactions (Kline et al., 2010). P. aeruginosa exhibited different
mechanisms to survive along with stress in the bladder such as starvation and
immune responses. It can persist and cause recurrent infections through biofilm
formation and morphological changes (Kostakioti et al., 2013).
2.5. Eye and ear infections
P. aeruginosa is one major causative agent of bacterial keratitis which is

responsible for 6% to 39% of bacterial keratitis cases (Varaprasathan et al., 2004).
P. aeruginosa is highly virulent and produces proteases and toxins which are the
most predominant virulent factors in keratitis (Fleiszig and Evans, 2002).
Additionally, P. aeruginosa is one of the major causative agents of severe corneal
8
Literature Review
ulcers. P. aeruginosa ulcers are generally thought to be more difficult to treat and
result in worse visual outcomes than other bacterial corneal ulcers (Green et al.,
2008; Sy et al., 2012).
P. aeruginosa frequently colonizes the external auditory canal and commonly

causes chronic suppurative otitis media (Afolabi et al., 2012). Chronic suppurative
otitis media is a chronic inflammation of the middle ear characterized by persistent
middle ear drainage through the perforated tympanic membrane for more than 6
weeks (Mittal et al., 2015). Chronic suppurative otitis media is one of the largest
public health burdens worldwide leading to hearing loss and life-threatening central
nervous system complications, including brain abscess and meningitis (Chew et al.,
2012).
3. Virulence factors
P. aeruginosa exhibited several mechanisms of virulence that contribute to

variety of human infections including motility (flagella and pili), immune evasion
(elastase and alkaline protease), antibiotic resistance (pump efflux and modifying
enzymes), cytotoxicity (exotoxin A, type III secretion pathway (T3SS) and
pyocyanin), iron scavenging (proteases and siderophores) and finally biofilm
structure (alginate and rhamnolipids) (Tam et al., 2010). P. aeruginosa has evolved
a complicated regulatory network to control expression of virulence factors for
bacterial survival (Pukatzki et al., 2002). These virulence factors beside intrinsic
drug resistance and efflux systems largely contribute to bacterial persistence and
pathogenicity (Stover et al., 2000).
3.1. Adhesins
Adhesion of P. aeruginosa to the host tissues is a crucial early step in infection

and pathogenesis. P. aeruginosa uses cell surface components or appendages to
9
Literature Review
promote the attachment to cells or to inanimate surfaces. There are different

adherence factors: type IV pili; mediating adhesion to epithelial host cells and
flagella; binding to mucin on epithelial cells.
3.1.1. Type IV pili-mediated adhesion
P. aeruginosa surfaces have retractable and flexible type IV pili that are hair-
like filamentous appendages. Pili filaments are homopolymers of pilin protein and
have multiple functions, such as surface motility, adhesion, biofilm formation,
immune evasion, transformation of DNA, cell signaling and phage attachment
(Berne et al., 2015). Twitching motility depends on type IV pili which play an
important role in mediating adherence to mucosal surfaces and subsequent
colonization (Zolfaghar et al., 2003). Together with flagella, pili also facilitate
swarming motility, a highly coordinated type of motility on semi-solid surfaces
(Yeung et al., 2009). Pili can also lead to aggregation causing the bacteria to form
microcolonies on target tissues, effectively concentrating the bacteria in one location
and potentially offering protection from the host immune system and from
antibiotics (Sriramulu et al., 2005).
3.1.2. Flagella-mediated mucin binding
P. aeruginosa has a single polar flagellum which is composed of a flagellin

and responsible for multiple functions, such as adhesion, motility and biofilm
formation. P. aeruginosa mutants lacking flagella or motility display reduced
invasion of epithelial host cells and pathogenicity (Dasgupta et al., 2003). Several
studies have shown that the receptors for flagella-mediated adhesion are mucins,
which lubricate and protect tissues from pathogens. Recent studies demonstrated that
flagella mediate adhesion to heparan sulphate chains on epithelial host cells, which
results in the activation of the epidermal growth factor receptor and subsequently
10
Literature Review
promote bacterial invasion (Bucior et al., 2012). Flagella are also responsible for P.
aeruginosa swimming motility in low-viscosity environments. This process occurs
through rotation, producing a force moving the bacterium forward (Sampedro et al.,
2015). Nevertheless, flagellar attachment is important in initial biofilm
establishment, whereas the motility mechanism is associated with dispersal in the
final biofilm steps (Guttenplan and Kearns, 2013).
3.2. Secreted toxins and exoenzymes
P. aeruginosa secretes various toxins and exoenzymes including exotoxin A,

rhamnolipid, elastases, alkaline protease and phospholipase C, all of which
contribute to bacterial virulence.
3.2.1. Exotoxin A
Exotoxin A is one of the most toxigenic virulent factors secreted by P.

aeruginosa. It is a member of a class of enzymes termed mono-ADP-
ribosyltransferases. ADP-ribosyltransferase activity of this protein modifies and
inactivates elongation factor 2 (eEF-2), resulting in the inhibition of protein
synthesis and cell death (Wedekind et al., 2001). In animal experiments, exotoxin
A-producing strains showed a 20-fold increase in virulence compared with exotoxin
A-deficient mutants that induced less severe infections (Michalska and Wolf, 2015).
Generally, exotoxin A belongs to the two-component AB toxin family, composed of
an A domain with enzymatic activity and a B domain as cell binding subunit
(Odumosu et al., 2010). Exotoxin A requires site-specific activation by furin-like
endoproteases in order to intoxication process upon uptake by mammalian cells
(Morlon-Guyot et al., 2009).
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Literature Review
3.2.2. Proteases
Elastases (LasA and LasB) are the most abundant proteases that are secreted
by P. aeruginosa and aid in the breakdown of host tissues which could facilitate
invasion and/or amino acid metabolism (Spencer et al., 2010). One of the substrates
of elastases is elastin which is the element of the connective tissue with high stability
against most proteases. LasA was shown to enhance the elastolytic activity of
Pseudomonas elastase and other proteases. LasB is a neutral zinc metalloprotease
that uses an autoproteolytic mechanism to remove the pro-peptide which is required
for its secretion. In addition, LasB plays role in biofilm formation and necessary for
LasA activation (Cathcart et al., 2011; Yu et al., 2014). Furthermore, alkaline
protease is a Zn+2 metalloprotease that increases iron availability via breakdown of
transferrin, improves amino acid metabolism and aids in immune evasion (Laarman
et al., 2012). Alkaline protease can also degrade host immune complements, human
gamma interferon (IFN-γ) and inhibit neutrophil oxygen consumption (Zhang et al.,
2012).
3.2.3. Phospholipase
P. aeruginosa can secrete different types of phospholipases such as

phospholipase A, C and D. Phospholipase A releases fatty acids from phospholipid
substrate as well as it could be implicated in apoptosis of host cells and possible
generation of reactive oxygen species (Tielen et al., 2011). Phospholipase C (PLC)
has been found to release phosphate esters. P. aeruginosa produces a haemolytic and
non-haemolytic version of phospholipase C PlcH and PlcN, respectively and found
commonly in UTI isolates (Wargo et al., 2009). Finally, phospholipase D plays a
role in bacterial persistence and chronic infections (Jiang et al., 2014).
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Literature Review
3.2.4. Rhamnolipids
Rhamnolipids are biosurfactants and surface-active amphipathic molecules.

Rhamnolipid biosynthesis is carried out by gene products of rlhA, rlhB and rhlC
whose expression is regulated by a quorum sensing (QS) system. Rhamnolipids were
initially identified as heat-stable haemolysins which can affect the function of
macrophages and also inhibit ciliary function of human respiratory epithelia (Thakur
et al., 2021). Latterly, the rhamnolipids were also found to modulate swarming
motility and biofilm architecture in P. aeruginosa. Rhamnolipids expression is
upregulated under iron-limited environments and promotes twitching motility and
leads to detachment of P. aeruginosa biofilms (Glick et al., 2010). P. aeruginosa
secretes two hemolysins, rhamnolipids and phospholipase C which appear to act
synergistically to degrade lipids and lecithin (Wargo et al., 2011).
3.2.5. Other toxins
The four known exoenzymes (ExoS, ExoT, ExoU and ExoY) are secreted by
T3SS in P. aeruginosa. Among these exoenzymes, ExoU may be responsible for the
greatest virulence (Engel and Balachandran, 2009). ExoS and ExoT are closely
related bifunctional proteins that induce mitochondrial apoptosis in host cells and
immune system evasion. ExoU (also known as PepA) is extremely cytotoxic and
was found in most clinical isolates (Feltman et al., 2001). ExoU leads to rapid death
of variety of mammalian cell types in vitro including macrophages, epithelial cells
and fibroblasts. Furthermore, ExoU interacts with a host cell factor that activates its
phospholipase A2 activity. This activity then causes cell death by utilizing plasma
membrane phospholipids as substrates (Rabin et al., 2006).
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Literature Review
3.3. Alginate production
Alginate is a polysaccharide polymer and its overproduction enhances

bacterial persistence, poor prognosis and high rates of morbidity and mortality rates
of chronic infections, particularly in CF patients (Stapper et al., 2004). Alginates
protect P. aeruginosa from various environmental stresses including host immune
response by inhibiting complement activation and decreasing phagocytosis by
neutrophils and macrophages, as well as by sequestering the free radicals that are
released from these cells (Balasubramanian et al., 2011). The overproduction of
alginate is responsible for the development of excessive slimy or mucoid biofilms.
Mucoid biofilms are known to be more resistant to host immune system attack and
antibiotic treatment as compared with non-mucoid biofilms (Moradali and Rehm,
2019).
3.4. Pyocyanin
Pyocyanin gives P. aeruginosa colonies their distinct color and causes

oxidative stress to the host, disrupting host catalase and mitochondrial electron
transport (Lau et al., 2004). Purified pyocyanin has been shown to induce apoptosis
in neutrophils and inhibit phagocytosis by macrophages (Bianchi et al., 2008).
3.5. Biofilm formation
Biofilms effectively promote P. aeruginosa colonization, improve bacterial

resistance to antimicrobial agents and counter the host immune system (Tuon et al.,
2022). Biofilm is a complex aggregate of bacterial cells adhered to both biotic and
abiotic surfaces. This microbial community is encased within a matrix of
extracellular polymeric substances (EPS), extracellular DNA (eDNA), and matrix
proteins, which play an important role in the structural maintenance and drug
resistance of biofilms (Ghafoor et al., 2011). P. aeruginosa can synthesize at least
14
Literature Review
three types of exopolysaccharides: alginate, Pel polysaccharide, and Psl

polysaccharide (Mann and Wozniak, 2012).
Formation of bacterial biofilm is a continuous process that includes four main stages
(Saxena et al., 2019). Bacterial attachment to a living or non-living surface
represents the initial stage of biofilm formation. Adhesion occurs through reversible
or irreversible attachment processes (Abebe, 2020). Reversible adhesion is a
transient attachment of the free-living cells to the surface which causes a weak
adhesion mediated by non-specific van der Waal’s, electrostatic and Lewis’s acid-
based electronic forces (Tian et al., 2021). In contrast, irreversible adhesion is a
permanent adhesion which causes strong attachment of the bacteria to any surface
and mediated by bacteria specific adhesion pili and flagella. Interestingly,
hydrophobic and non-polar surfaces such as plastics and teflon provide a higher
adhesion than hydrophilic and polar surfaces such as metals and glasses (Jamal et
al., 2018).
In the second stage of biofilm development, attached bacteria gradually divide

and form aggregates called microcolonies and begin to synthesize EPSs. EPSs act as
a physical barrier between biofilm cells and surroundings to provide protection from
various stress conditions such as antimicrobial exposure or immune cell attack (Roy
et al., 2018).
The formation of microcolonies from layered cells and small clusters leads to the
generation of a thin biofilm to begin the maturation phase and the synthesis of EPS
matrix (polysaccharide, protein and eDNA). Clusters develop into macrocolonies
with the displacement of cells from the substratum to form channels and voids,
which facilitate the exchange of nutrients and waste products through biofilm
(Toyofuku et al., 2016). Polysaccharide forms the core of the matrix whereas eDNA
is involved in horizontal gene transfer. The maturation of the biofilm causes
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structural changes as well as many changes in the expression of genes coding for
different virulent factors. These changes include loss of cellular motility by
expressing flagella-free phenotypes, reduction in protease and phospholipase C
synthesis, decrease in the synthesis and release of toxins as well as production of
rough and sometimes mucus-like polysaccharide to better adapt to certain conditions
of the biofilm microenvironment (Guła et al., 2019). The morphological changes in
the biofilm enable metabolic adaptation under aerobic and anaerobic conditions that
results in metabolically distinct microcolonies, whose presence provide an important
metabolic state to resist antimicrobials surrounding the environment (Moormeier
and Bayles, 2017).
The final stage of biofilm development is bacterial dispersion in which sessile

cells converted to planktonic cells that attach to another surface or start a new cycle
of biofilm development (Kim and Lee, 2016). Dispersion is an active event triggered
by self-synthesized signaling molecules such as fatty acid signaling molecules and
environmental conditions such as oxygen depletion, nutrient availability and
starvation which ultimately result in the overall reduction in the levels of cyclic di-
GMP (c-di-GMP) (Rumbaugh and Sauer, 2020). Low levels of c-di-GMP lead to the
upregulation of genes involved in cell motility such as flagella biosynthesis and EPS
degradation enzymes such as endonucleases and glycoside hydrolases.
Concurrently, genes involved in bacterial attachment and synthesis of EPS are also
downregulated (Amankwah et al., 2021). Therefore, all these phenotypes related to
low c-di-GMP levels lead to increased bacterial motility, reduced matrix synthesis
and finally biofilm dispersion (Toyofuku et al., 2016).
4. P. aeruginosa resistance to antimicrobial agents
P. aeruginosa is highly resistant to various classes of antibiotics and

antibacterial agents that makes Pseudomonas infections difficult to control. P.
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aeruginosa has been recently listed by WHO as one of three bacterial species in
which there is a critical need for the development of new antibiotics to treat
infections (Tacconelli, 2017). Moreover, excessive use of antibiotics accelerates
development of multidrug resistant P. aeruginosa strains, leading to the
ineffectiveness of the empirical antibiotic therapy. P. aeruginosa displays resistance
to various antibiotics including aminoglycosides, quinolones and β-lactams (Hirsch
and Tam, 2010). Generally, the major antibiotic resistance mechanisms of P.
aeruginosa can be classified into intrinsic, acquired and adaptive resistance
(Hancock and Speert, 2000).
4.1. Intrinsic antibiotic resistance
The intrinsic antibiotic resistance refers to innate bacterial ability to diminish

the efficacy of a specific antibiotic through inherent structural or functional
characteristics (Blair et al., 2015). P. aeruginosa has been shown to possess a high
level of intrinsic resistance to most antibiotics through restricted outer membrane
permeability. Most antibiotics should penetrate the cell membrane to reach
intracellular targets to treat P. aeruginosa infections. The outer membrane of P.
aeruginosa acts as a selective barrier to prevent antibiotic penetration, is an
asymmetric bilayer of phospholipid and lipopolysaccharides (LPS), embedded with
porins that form β-barrel protein channels (Delcour, 2009). Moreover, efflux pumps
play an important role in expelling toxic compounds out of the bacterial cell.
Particularly, the proteins belonging to the resistance-nodulation-division (RND)
family of efflux pumps play a key role in antibiotic resistance in P. aeruginosa (Li
and Nikaido, 2009). P. aeruginosa strains have been found to overexpress multiple
efflux pumps which enhance bacterial antibiotic resistance and lead to the
development of multidrug resistance. Therefore, the use of efflux pump inhibitors
has emerged as a potential therapeutic strategy for treatment of P. aeruginosa
infections (Shigemura et al., 2015).
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In addition, production of enzymes that break down or modify antibiotics is

one of the major mechanisms of intrinsic resistance in P. aeruginosa. Many
antibiotics have chemical bonds such as amides and esters that are susceptible to
hydrolysis by enzymes such as β-lactamases and aminoglycoside-modifying
enzymes (Wolter and Lister, 2013). P. aeruginosa possesses an inducible ampC gene,
encoding the hydrolytic enzyme β-lactamase. This enzyme is able to inactivate β-
lactam antibiotics (Wright, 2005). Some P. aeruginosa isolates have been found to
produce extended-spectrum-β-lactamases (ESBLs) which confer a high degree of
resistance to the majority of β-lactam antibiotics including penicillins,
cephalosporins and aztreonam (Rawat and Nair, 2010). P. aeruginosa secretes
various enzymes that plays a predominant role in its resistance to aminoglycosides
including aminoglycoside phosphotransferase (APH), aminoglycoside
acetyltransferase (AAC) and aminoglycoside nucleotidyl transferase (ANT)
(Ramirez and Tolmasky, 2010).
4.2. Acquired antibiotic resistance
In addition to the high level of intrinsic antibiotic resistance of P. aeruginosa,

the acquired resistance greatly contributes to the development of multidrug resistant
strains. The acquired resistance of P. aeruginosa can be achieved by either
mutational changes or horizontal transfer of resistance genes (Breidenstein et al.,
2011). Mutational changes are able to decrease antibiotic uptake through
modification of antibiotic target sites as well as overexpression of efflux pumps and
antibiotic-inactivating enzymes. Spontaneous mutations can affect the expression or
function of a specific porin, thereby reducing bacterial membrane permeability and
increasing antibiotic resistance (Fernández and Hancock, 2012). Poonsuk et al.
(2014) demonstrated that bacteria employ energy-dependent efflux systems to
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prevent intracellular accumulation of toxic compounds and pump the toxic

molecules out of the cells. Thus, P. aeruginosa clinical isolates with overexpressed
efflux pumps have decreased susceptibility to antibiotics. Similarly, mutation
causing overexpression of antibiotic-inactivating enzymes is another well-
characterized mechanism of acquired resistance (Munita and Arias, 2016). Some P.
aeruginosa clinical isolates have overproduction of β-lactamases caused by
mutations in a β-lactamase inducible gene ampC, which greatly increased bacterial
resistance to cephalosporins (Berrazeg et al., 2015).
In addition to mutational changes, P. aeruginosa can acquire resistance genes

carried on plasmids, transposons, integrons and prophages via horizontal gene
transfer from the same or different bacterial species. Integrons are genetic elements
that insert mobile gene cassettes into a specific genetic site via site-specific
recombination and was found to play a critical role in dissemination of antibiotic
resistance among P. aeruginosa strains (Odumosu et al., 2013). The main
mechanisms of horizontal gene transfer involve transformation, transduction and
conjugation (Arber, 2014).
4.3. Adaptive antibiotic resistance
Adaptive resistance increases the ability of a bacterium to survive antibiotic

attack due to transient alterations in gene and/or protein expression in response to an
environmental stimulus, and it is reversible when the stimulus is removed (Sandoval‐
Motta and Aldana, 2016). In P. aeruginosa, the best characterized mechanisms of
adaptive resistance are the formation of biofilm and the generation of persister cells
(Taylor et al., 2014).
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Even bacteria that are deficient in intrinsic resistance or lack protective mutations
can become less susceptible to antibiotics when they grow in a biofilm. The general
mechanisms of biofilm-mediated resistance protecting bacteria from antibiotic
attack involve prevention of antibiotic penetration, altered microenvironment
inducing slow growth of biofilm cells, induction of an adaptive stress response and
persister cell differentiation (Stewart and Costerton, 2001). Persister cells are able to
remain viable due to the existence of a dormant state that shuts down the synthesis
of the antibiotic targets. Persister cells do not proliferate in the presence of
antibiotics; however, they resume growth once the antibiotics are removed. Thus,
persister cells in biofilms are believed to be responsible for prolonged and
recalcitrance of chronic infections (Van den Bergh et al., 2017).
As a result of increased P. aeruginosa resistance to various antimicrobials, the

development of new antibiotics or alternative therapeutic strategies for treatment of
P. aeruginosa infections is urgently required. New antibiotics with novel modes of
action have been explored in recent years (Cigana et al., 2016). Recent studies have
reported several novel non-antibiotic therapeutic approaches that are highly effective
in killing antibiotic-resistant P. aeruginosa strains. These approaches include
inhibition of bacterial quorum sensing, use of iron chelation, vaccine strategy,
nanoparticles, antimicrobial peptides and phage therapy. These therapeutic
approaches can be used as either an alternative to or in combination with
conventional antibiotic treatments (Chatterjee et al., 2016). Of note that,
bacteriophages are considered to be potential bioagents for the prevention and
control of bacterial infections and biofilms due to their infection diversity and
specificity. It is supposed that they can be used alone or in combination with
antibiotics to treat bacterial infections (Melo et al., 2019).
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5. Using bacteriophages (phages) as an alternative strategy for bacterial

infections control
Bacteriophages (phages) were first discovered by Frederick Twort and Felix

d’Hérelle in the early 20th century (Wittebole et al., 2014). In 1917, d’Hérelle
isolated phages and successfully treated soldiers suffering from bacillary dysentery
in World War I. In 1921, the use of phages for clinical treatment continued when
Richard Bruynoghe and Joseph Maisin injected Staphylococcus specific phages into
and around opened lesions, successfully treating Staphylococcal skin disease in
human patients (Sulakvelidze et al., 2001). Bacteriophages can be defined as viruses
that infect and replicate within bacterial cells. They are the most ubiquitous and
diversified biological group residing on the biosphere. Due to their obligate
requirement of a bacterial host, phages are abundantly found distributed essentially
anywhere their host exists in various environments. They are generally found across
different habitats like soil, river, sea and desert. They are also associated with various
excretions and human body fluids (Divya Ganeshan and Hosseinidoust, 2019). It
was known that hospital sewage is a good reservoir for phage isolation. As reported
by many studies, phages that were isolated from wastewater of medical centers are
used in applications against resistant bacteria that cause diseases (Jeon et al., 2016).
Bacteriophages play an important role in maintaining community dynamics and
bacterial diversity via the "kill the winner" phenomenon. According to this theory,
bacteriophages contribute to population diversity by selectively eliminating
dominant bacterial species. More than 1023 phages are infecting their hosts on the
planet at any given time (Kirsch et al., 2021).
6. Structure and classification of bacteriophages
Virus taxonomy is currently the responsibility of the International Committee

on the Taxonomy of Viruses (ICTV), and the Bacterial and Archaeal Subcommittee
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(BAVS) within the ICTV that focuses on phages. The system is based on different
phage properties including the virus genome, the structure of virus capsid and
whether it is enveloped or not, the host range and sequence similarity (Chibani et al.,
2019). Phages are initially classified into families and each family is further
categorized according to capsid structure, genetic material and the mechanism of
their mRNA production (Divya Ganeshan and Hosseinidoust, 2019).
Most bacteriophages are composed of an icosahedral head associated with a helical

symmetry protein tail. The head is formed by the capsid that surrounds and protects
the nucleic acid as well as possessing proteins which confer specificity for certain
bacterial cells (Adriaenssens and Brister, 2017). The neck connects the head to the
tail that ensures genome release when the virion is bound to the host cell. The distal
portion of the tail has a basal plate to which tail fibers and spicules are attached
which have proteins capable of binding to membrane receptors of certain bacteria
(Figure 1). The tail may have different contractility, dimensions and may exhibit
accessory structures such as spicules, collar, lipid envelope or absence of envelope
(White and Orlova, 2019).
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Figure 1. Typical bacteriophage structure adopted from Mansour (2017).
Bacteriophages could be classified according to genetic materials into double-

stranded DNA (dsDNA, the major), single-stranded DNA (ssDNA), single-stranded
RNA (ssRNA), and double-stranded RNA (dsRNA; very rare). Phage particles may
be classified morphologically to tailed, cubic, filamentous and pleomorphic. The
majority of viruses are tailed and represent over 96% of phages. In contrast, cubic,
filamentous and pleomorphic phages are generally rare and possess narrow host
ranges (Ackermann, 2009).
Virions of tailed phages contain dsDNA and have icosahedral or elongated heads.
Tails are helical and generally provided with fixation structures (baseplates, spikes,
fibers). They have no envelope. Tailed phages are the most ubiquitous of all viruses
and extremely varied in size and structure, DNA content and composition, genome
structure, proteins, antigenic and biological properties. Tailed phages constitute the
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Literature Review
order Caudovirales with three families divided into Siphoviridae, Myoviridae and
Podoviridae (Figure 2). All three families have icosahedral head but differ in tail
morphology. Phages related to the Siphoviridae family characterized by simple,
noncontractile tail with flexible or rigid tubes. Siphoviruses are considered as the
most numerous of tailed phages (61%). Tails of Myoviridae virions consist of a neck,
a contractile sheath, and a central tube (25% of tailed phages). The third family is
Podoviridae which is characterized by short and noncontractile tails. Podoviruses
may be more related to siphoviruses than to myoviruses (Ackermann, 2009).
The term “cubic” denotes cubic symmetry and icosahedral shape. Some types
contain lipids in envelopes or internal constituents, which are invariably ether and
chloroform sensitive. Cubic phages include 5 families: Microviridae, corticoviridae,
Tectiviridae, Leviviridae and Cystoviridae. Moreover, filamentous phages include 3
families: Inoviridae, Lipothrixviridae and Rudiviridae, while pleomorphic phages
include 3 families: Plasaviridae, Fuselloviridae and Guttaviridae (Ackermann and
Prangishvili, 2012). Phages may also be classified according to their specific host
for instance the staphylococcal phage family, the Pseudomonas phage family, and
so on, the place where they live (marine virus vs. other habitats) and their life cycle
(Wittebole et al., 2014).
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Figure 2. Families of bacteriophages adopted from Orlova (2012).
7. Bacteriophage life cycle
Bacteriophages are routinely classified according to their biological cycle into

lytic (virulent) and lysogenic (temperate) phages (Van Regenmortel, 2007). In the
first stage of the infection process, the phage recognizes the receptors present on the
bacterial surface. A variety of components present in the bacterium such as flagella,
pill, capsule, LPS and proteins are potential receptors for phages (Rakhuba et al.,
2010). The initial contact with bacteria occurs through brownian movements of the
viral particle then a reversible bond may occur mediated by the tail fibers (Chiaruttini
et al., 2010). Afterwards, the specific and irreversible binding of phage protein with
the target receptors of the bacterium, allowing release of its genome through the
capsid into the bacterial cytoplasm (Kropinski, 2006). After phage introduces its
nucleic acid into the bacterial cell, the life cycle may diverge to lytic cycle or
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lysogenic cycle, depending on the type of phage and the physiological state of host
bacterium (Rakhuba et al., 2010).
7.1. Lytic cycle
During lytic cycle, phages bind to specific receptors on the surface of the host
cell and inject its genome. Next, the bacterial synthetic machinery is redirected to
the production of viral genome and proteins as well as viral transcription begins
(Rakhuba et al., 2010). Subsequently, the synthesis of structural and catalytic
proteins occurs to form the structures of new phages. Genes are expressed to encode
the synthesis of holin and lysine enzymes that are responsible for promoting cell
lysis. Finally, assembly and packing of newly formed phage particles occurs and
release in the external environment to infect other bacterial cells (De Smet et al.,
2017) (Figure 3).
7.2. Lysogenic cycle
Tempered viruses integrate their genome into the host genome with the help
of phage-encoded integrases to become a prophage. In this state, the phage genome
replicates along with the host cell (lysogen). Lysogenic phage can switch to a lytic
life cycle, particularly in response to environmental stresses (Ptashne, 2004).
Prophage induction is thought to be a survival strategy to aid phage release from
host cell at risk of death (Refardt and Rainey, 2010). Potent inducers of DNA
damage and phage induction include physical and chemical mutagens such as
ultraviolet (UV), mitomycin C and reactive oxygen species (Aanaes et al., 2011).
Several antibiotics have also been shown to trigger the lytic cycle, particularly those
that target DNA replication (fluoroquinolones such as norfloxacin and
ciprofloxacin) (López et al., 2014). Temperate phages are natural vectors for gene
transmission among bacteria and affect the phenotype of the produced lysogen
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(Cuenca et al., 2016). Several virulence factors or toxin encoding genes of

pathogenic bacteria have been associated with prophages, indicating that temperate
phages may play a significant role in increasing the host pathogenicity, on the other
hand, a virulence-attenuating role is not often reported for temperate phages (Chen
et al., 2020).
Figure 3. Bacteriophage life cycle adopted from Pyzik et al. (2021). Lytic cycle: The phage
uses the host cell to synthesize its own proteins. The heads and sheaths are assembled separately,
the new genetic material packed into the head and new daughter phage particles constructed then
burst into the surrounding environment. Lysogenic cycle: Following the injection of the phage
DNA into the host cell, it integrates itself into the host genome. The prophage genome is then
replicated passively along with the host genome as the host cell divides.
8. Potential advantage of using bacteriophage in treatment of bacterial

infections
Antibiotic resistance is now recognized as a health care emergency which

requires the development of novel means to combat bacterial pathogens. The
implantation of phage therapy could become an alternative strategy against MDR
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bacterial pathogens. Phage therapy can be defined as the use of phages as

antibacterial agents which is based on the fact that phages recognize, bind and
replicate within certain bacterial host cells causing rapid cell lysis and resulted in
significant reduction in bacterial numbers (Abedon and Thomas-Abedon, 2010).
They are active against both Gram-positive and Gram-negative bacteria including
MDR pathogens (Matsuzaki et al., 2003). Indeed, phages retain its activity against
bacteria exhibiting multiple mechanisms of antibiotic resistance (Hanlon, 2007).
Furthermore, the need for phage applications certainly exceeds its use in
human infections. The use of bacteriophages has been described in various situations
including food safety, agriculture, veterinary applications, industry and clinical
diagnostic application such as detection and typing of bacteria in human infection
(Wittebole et al., 2014). Phage therapy is supposed to have several advantages. It is
thought that phages are significantly safe and best tolerated as they interact with
bacterial cells only and do not interfere with mammalian cells without significant
adverse effects (Kakasis and Panitsa, 2019). Moreover, administration is easier, as
bacteriophages do not need repeated administrations shortly after one another over
several days, as is commonly required for antibiotics. In general, very few doses are
needed because of the increase in phage titer (concentration) at the site of infection
after the initial administration (Pouillot et al., 2012). Because of phage specificity,
phage therapy has a narrow antibacterial spectrum with an effect limited to one
single species or single strain within a species. In contrast to antibiotics whose effect
is limited to the site of infection, there is a wide distribution of phages upon systemic
administration, including the ability to penetrate the blood brain barrier, allowing
these agents to be used in cases of central nervous system infections (Gorski et al.,
2009). Interestingly, phages also display the capacity to disrupt bacterial biofilms
(Azeredo and Sutherland, 2008). Finally, the economic aspects related to phage
therapy look promising as the cost of phage therapy was lower than conventional
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antibiotic treatment. Moreover, Międzybrodzki et al. (2007) found that use of

bacteriophages significantly reduced healthcare costs (Międzybrodzki et al., 2007).
9. Potential limitations or drawbacks of phage therapy
Despite the previous numerous advantages of phage therapy, challenges and

limitations exist and must be considered including the optimal dose, route of
administration, frequency and duration of treatment still deserve to be investigated
carefully before widespread clinical trials. The major disadvantage of phage therapy
is the need to rapidly determine the precise etiological microorganism causing
infection with accuracy. The exquisite specificity of phage therapy against specific
pathogens is a major advantage but also a liability as most types of infections are
polymicrobial so that it is necessary to use an effective phage cocktail with a greater
spectrum of action (Divya Ganeshan and Hosseinidoust, 2019). Another concern of
phage therapy is the possibility of bacteriophages to transfer the DNA from one
bacterium to another. Therefore, the choice of bacteriophage for therapy is limited
to lytic phages rather than lysogenic phages (Gorski et al., 2009). Furthermore,
virion stability against various external and physical factors could account for some
difficulties in preparing stable solutions (Jończyk et al., 2011).
Lytic phages induce the lysis of bacteria, liberating various bacterial substances such
as endotoxin (LPS) from gram-negative bacteria. This may account for several side
effects on the host such as the development of an inflammatory cascade leading to
multiple organ failure. However, this potential issue applies to currently available
rapidly bactericidal antibiotics (Goodridge, 2010). Furthermore, bacteriophages are
composed of protein and genomic material that may interact with the body's immune
system. Therefore, they may be rapidly cleared from the body or may be inactivated
by the adaptive immune mechanisms. This could lead to a decreased efficacy in case
of prolonged or repeated applications (Dabrowska et al., 2005).
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Finally, bacteria have evolved mechanisms to resist specific phage. For instance,
loss or lack of receptor, structural modification and, or masking of the receptor will
prevent phage adsorption to the bacteria and prevent further ability to generate new
phages. Loss of receptor may occur when cell surface composition is changed, as
observed for Bordetella spp. (Liu et al., 2002). as well as for E. coli which modifies
the conformation of the outer-membrane protein A (OmpA), the receptor for T-even-
like phages (Riede and Eschbach, 1986). Secretion of various molecules (such as
exopolysaccharide by Pseudomonas spp. or glycoconjugates by
Enterobacteriaceae) may mask the receptor, but phages may counteract this by the
selection of a new receptor or by secreting exopolysaccharide degrading enzyme.
Other mechanisms of resistance include the degradation of phage DNA by
restriction-modification defense system or by Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) and the blocking of phage replication, transcription,
translation, or virions assembly by abortive infection system (Drulis-Kawa et al.,
2012). For instance, bacterial cells protect themselves by degradation of foreign
DNA using CRISPR-Cas system which acts as an adaptive bacterial immune system.
CRISPR-Cas system is activated when the virus enters the bacterium, recognizes the
exogenous DNA and enzymes (Cas), cut pieces of this material and introduce them
into a specific genomic region of the bacterium, called the CRISPR locus. In the next
viral infections, bacteria that contain these pieces of virus DNA inserted into the
CRISPR locus generate RNA from this sequence. This RNA will associate with the
Cas enzyme and then make its way to the viral DNA, which is then cleaved and thus
inactivated (Bondy-Denomy et al., 2013). Phages have conceived ways to disable
CRISPR-Cas systems. In total, 10 different families of anti-CRISPR proteins (Acr)
were identified in Pseudomonas temperate phages. Phages appear able to escape
CRISPR interference through specific mutations (Le Rhun et al., 2019). Fortunately,
the frequency of phage therapy resistance is reportedly low, so that isolation of novel
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active phages from the environment could provide a new possibility for treatment
(Kutter et al., 2010).
10. Phage application in biofilm degradation
Phage therapy has gained great attention to be used in therapy of drug

resistant bacteria as well as treating biofilms and associated infections (Kutateladze
and Adamia, 2010). Phages have developed specific strategies to penetrate the three-
dimensional structure of biofilm and to cope with different cell physiologies.
10.1. Diffusion through biofilm water-channels
Biofilms are highly hydrated structures formed by water channels that help
the diffusion of nutrients throughout the biofilm. Phages can diffuse through these
channels and penetrate the inner biofilm layers. In contrast to antibiotics, where
diffusion limitations lead to a depletion of the antibiotic concentration at the inner
biofilm layers, phages increase in number due to active replication. This fact leads
to the lysis of the sessile population inhabiting the inner layers, contributing to the
disturbance of the biofilm 3D structure (Pires et al., 2017).
10.2. Enzymatic degradation
Phages can encode enzymes such as endolysins and depolymerases that

degrade polymers and inhibit P. aeruginosa biofilm formation by disrupting the
extracellular matrix and increasing the permeability allowing antibiotics to reach the
inner layer of biofilms (Harper et al., 2014). It is worth mentioning that the cell lysis
itself, caused by phage propagation, leads to the release of bacterial cell content
directly into the biofilm milieu, thus bacterial enzymes are also incorporated into the
EPS degradation action. Therefore, an increasing number of studies have used
bacteriophages to treat biofilm infections (Soothill, 2013).
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Phage lytic proteins, such as endolysins and virion-associated peptidoglycan

hydrolases (VAPGHs) have high antimicrobial activity due to their ability to
hydrolyse the peptidoglycan (PG) from the cell wall (Latka et al., 2017). Phages can
produce a wide range of PG-degrading enzymes including muramidase, amidase,
and endopeptidase. The two latter enzymes are also classified as proteases. The
application of endolysins against Gram-negative pathogens is impaired by the
presence of a protecting outer membrane layer, however, the combination with
membrane permeabilizers turned out to significantly improve lysin efficiency. More
recently, genetic engineering allowed design of lysin/cationic peptide combination
called artilysins (Briers and Lavigne, 2015).
Phage lytic proteins offer interesting antibiofilm properties, for example, they
easily penetrate the biofilms and are active against low metabolically persister cells
(Gutierrez et al., 2014). Besides these, other valuable antimicrobial characteristics
include the lack of bacterial resistance to phage lytic proteins (Schmelcher et al.,
2011). Phages can also destroy biofilm structures by synthesis of enzymes such as
polysaccharide depolymerases in P. aeruginosa (Chegini et al., 2020).
Polysaccharide depolymerase is a hydrolase that can specifically bind and degrade
macromolecular carbohydrates on the host bacterial envelope (Yan et al., 2014).
Additionally, it could effectively degrade exopolysaccharides, inhibiting the
formation of host bacterial biofilms and destroying established biofilms (Pires et al.,
2016). Effective removal of biofilms can be achieved by using a combination of
lysin and depolymerase. In a previous study reported that a combination of these
enzymes significantly reduced viable cell counts compared to individual enzyme
treatment (Olsen et al., 2018).
Hyaluronidase enzyme is encoded by streptococcal prophages to degrade

capsular hyaluronic acid and reduce biofilm matrix viscosity. Moreover, lysogenic
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streptococci utilize these prophage-encoded enzymes as a virulence factor, digesting

the main component of the tissue extracellular matrix (Smith et al., 2005). Similar
to endolysins, the antibiofilm activity of these proteins could be enhanced when
combined with antibiotics, which may represent a promising strategy to combat
infections caused by drug-resistant and biofilm-forming pathogens (Majkowska-
Skrobek et al., 2018).
In addition, lipases are among other useful enzymes to disperse biofilms by

disrupting the lipidic bounds involved in cell-cell or cell-surface interaction. There
is little information about the existence of lipid hydrolysis activity in phages, and in
fact, lipases represent rare domains found within phage structural components (Pires
et al., 2016).
Another way in which bacteriophages inhibit biofilms is through the production of

enzymes that inhibit biofilm production. It has been reported that phages can be
genetically modified to induce the synthesis of quorum quenching lactamase,
thereby inhibiting bacterial biofilm formation (Pei and Lamas-Samanamud, 2014).
An engineered T7 phage incorporating the acyl homoserine lactonase AiiA can
inhibit QS activities in P. aeruginosa, ultimately inhibiting biofilm production.
Unlike polysaccharide depolymerases, which can degrade one or several related
polysaccharides, the T7AiiA phage can affect multiple bacteria in mixed-strain
biofilms, rather than the host bacteria alone. In addition to being directly used as a
tool to destroy biofilms (Whiteley et al., 2017).
10.3. Adherence to motile bacteria
Phages can attach reversibly to the appendices of motile bacteria such as

flagella. Therefore, phages may develop an active way of penetrating inside biofilm
matrix (Guerrero-Ferreira et al., 2011).
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10.4. Tackling persister cells
Unlike antibiotics, phages can infect and kill dormant and persister cells.
Previous studies showed that phages can replicate in late stationary cultures known
to be mainly composed of growth arrested cells. The process of replication can
initiate immediately after phages enter the target cell or as soon as cells restore their
normal growth. Furthermore, the release of intracellular material and the dispersion
of the biofilm trigger the metabolism of the persister population further activating
phage replication (Bryan et al., 2016; Melo et al., 2018).
11. Examples of therapeutic applications of bacteriophage
The first controlled clinical trial of a therapeutic bacteriophage preparation

was carried out in 2009, which showed efficacy and safety in antibiotic-resistant P.
aeruginosa chronic otitis (Chan et al., 2018). Next, in the same year, another
randomized, double-blind controlled trial addressed the safety of a phage cocktail
targeting P. aeruginosa, S. aureus and E. coli for the treatment of venous leg ulcers
(Pires et al., 2020). Sextaphage is one of such commercial pharmaceutical phage
composition from the Russian company Microgen. This phage therapeutic contains
phages against six specific pathogens for treating UTIs in pregnant women (Divya
Ganeshan and Hosseinidoust, 2019). In 2016, Paul Turner and colleagues reported
the isolation of a phage that could restore antibiotic sensitivity in MDR P.
aeruginosa. This phage was later used to treat a patient with a longstanding aortic
graft infection that did not respond to multiple surgical interventions and aggressive
antibiotic therapy with a single application of phage (Kutter et al., 2010).
Recently, Wu et al. (2021) have successfully demonstrated the efficacy of

phage therapy against secondary infection of Acinetobacter baumannii in COVID-
19 patients. This observation firmly establishes the applicability of phage therapy
34
Literature Review
for treating secondary bacterial infection in COVID-19 patients. Therefore,

accelerated effort should be made to bring phage therapy as a mainstream treatment
alternative to treat secondary infection in COVID-19 patients (Khan et al., 2022).
While a lot of previous studies have isolated several phages infecting P.

aeruginosa, many of these studies suffer from serious gaps regarding detailed
characterization of isolated phages and in vivo efficacy of isolated phages. For
instance, the genomes of some previously isolated phages have not been fully
characterized (Kumari et al., 2009; Azizian et al., 2015; Didamony et al., 2015;
Barazandeh et al., 2021). Furthermore, neither the antibiofilm potential of isolated
phages nor their in vivo antibacterial efficacy has been investigated in these studies
(Miyata et al., 2014; Cao et al., 2015; Shigehisa et al., 2016; Tang et al., 2018; de
Melo et al., 2019; Alvi et al., 2020; Enwuru et al., 2021; Namonyo et al., 2022).
Therefore, complete genome analysis, the antibiofilm activity and the ability of
isolated phages to reduce P. aeruginosa pathogenesis in vivo will be covered in detail
herein. In the current study, virulent phages targeting P. aeruginosa from various
clinical sources were isolated and fully characterized. The physical properties,
antibiofilm activity as well as whole genome sequencing of isolated phages will be
investigated. Moreover, the influence of isolated phages on P. aeruginosa
pathogenesis in host will be assessed in vivo using mice infection model. The
findings of present study would be of great importance and helpful in treatment of
P. aeruginosa related infections.
35
Materials & Methods
Materials and methods
Materials
1. Experimental samples
1.1. Clinical specimens for bacterial isolation
A total of one hundred and twenty clinical specimens were collected from
patients admitted to Zagazig University Hospital and El-Ahrar Educational
Hospital in Zagazig, Egypt during the period from April 2021 to August 2021.
Specimens were collected aseptically and transported to the microbiological
laboratory, Department of Microbiology and Immunology, Faculty of Pharmacy,
Zagazig University, where they were immediately processed.
In this study, different bacterial species were involved in assay of host range
of isolated phages in addition to Pseudomonas aeruginosa (P. aeruginosa)
reference strains. These bacterial strains include Staphylococcus aureus,
Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Salmonella
Typhimurium and are listed in table 1.
Table 1. Bacterial species involved in assay of the host range
Bacterial strain Source
P. aeruginosa ATCC 27853 Stock culture collection of
P. aeruginosa ATCC 9027 Microbiology and Immunology
P. aeruginosa PAO1 Department, Faculty of pharmacy,
E. coli ATCC 10536 Zagazig University
36
Materials & Methods
E. coli ATCC O26
E. coli ATCC O78
E. coli ATCC O157
S. Typhimurium ATCC 14028
K. pneumoniae ATCC 700603
Serratia marcescens
S. aureus ATCC 6538 Microbiological Resources Center

(Cairo MIRCEN), Egypt.
S. aureus ATCC 9295
1.2. Sewage samples
Ten sewage samples were collected from different regions in Al Sharkia

Governorate, Egypt in addition to Minya El-Qamh Hospital and Zagazig
University Hospital for phage isolation. The sewage samples were collected into
sterile 100 mL falcon tubes. The samples were directly transferred to the
microbiological laboratory, Department of Microbiology and Immunology, Faculty
of Pharmacy, Zagazig University, Egypt for isolation of active phages.
2. Culture media
Unless otherwise specified, all the culture media were prepared and
sterilized by autoclaving at 121°C for 20 min after pH adjustment to 7.3 ± 0.2.
37
Materials & Methods
(a) Ready-made culture media
Mueller-Hinton (MH) broth, tryptone soya (TS) broth, cetrimide agar,

MacConkey agar, Simmons citrate agar, nutrient agar and triple sugar iron (TSI)
agar were obtained in dehydrated form (Oxoid, Hampshire, England). These media
were reconstituted before use and pH adjusted following the manufacturer's
guidelines.
(b) Prepared media
The following culture media were prepared from their components as

illustrated by Winn and Koneman, (2006) and Atlas, (2010). All materials used
were of microbiological and laboratory grades. The components of culture media
were constituted and sterilized by autoclaving at 121°C for 20 minutes after pH
adjustment.
2.1. Nutrient agar
Beef extract 3.0 g
Peptone 5.0 g
Sodium chloride 5.0 g
Yeast Extract 2.0 g
Agar-agar 20.0 g
Distilled water to 1000 mL
The pH was adjusted to 7.4 ± 0.2 .
38
Materials & Methods
2.2. Blood agar
The medium was prepared by adding 5% sheep blood to sterile molten

nutrient agar media after cooling to 50°C followed by gentle mixing and poured
into sterile petri dishes (Cruikshank et al., 1975).
2.3. Oxidative fermentative (O/F) medium
Peptone 2.0 g
Bromothymol blue 0.03 g
Agar 3.0 g
Dipotassium hydrogen phosphate 0.3 g
The medium was prepared and then sterilized by autoclaving without sugar. 10 g of
glucose was dissolved in 100 mL distilled sterile water, sterilized separately by
filtration through 0.22 µm membrane filters and incorporated into the media after
cooling to 60°C to a final concentration of 1% (w/v), then mixed well and
dispensed into sterile tubes each contained 5 mL aliquot.
2.4. Motility test medium
The medium was prepared according to Atlas, (2010) and adjusted to final
pH of 7.3.
Beef extract 3.0 g
Tryptone 10.0 g
39
Materials & Methods
Agar-agar 5.0 g
Five mL of 1% triphenyl tetrazolium chloride solution (TTC) was added to 1 liter

for better visualization of motility. Then, dispense in 5 mL aliquots into tubes and
autoclaved at 121°C for 15 min.
3. Chemicals
Peptone, agar-agar, tryptone and beef extract were purchased from Oxoid,
UK. Analytical grades of ethanol 95%, glycerol, glacial acetic acid, liquid paraffin,
potassium dihydrogen phosphate, ammonium oxalate, potassium iodide, iodine,
methanol, hydrochloric acid, sodium chloride, potassium chloride, crystal violet,
safranine, glucose, Tetramethyl-p-phenylenediamine dihydrochloride, yeast,
gelatin, sulphuric acid, sodium hydroxide, bromothymol blue, disodium hydrogen
phosphate and magnesium sulfate were purchased from Al-Nasr Pharmaceutical
Chemicals Company, Abou Zabel, Egypt (ADWIC). Tris base was obtained from
Fluka (Sigma-Aldrich).
4. Reagents and buffers
4.1. Gram stain (Koneman et al., 2006)
4.1.1. Hucker’s Crystal violet solution
Solution A:
Crystal violet 2.0 g
Ethyl alcohol 95% 20 mL
40
Materials & Methods
Solution B:
Ammonium oxalate 0.8 g
Both solutions A and solution B were mixed to achieve a 2% crystal violet

solution, filtered through coarse filter paper prior to use and stored at room
temperature.
4.1.2. Gram’s iodine
Iodine crystals 10.0 g
Potassium iodide 20.0 g
Grind the iodine and potassium iodide in a mortar and add water slowly with
continuous grinding until the iodine is dissolved. Iodine solution stored at room
temperature in a foil covered bottle.
4.1.3. Safranin solution
Safranin 2.5 g
Ethyl alcohol (95%) to 100 mL
Add 10 mL safranin and ethyl alcohol solution to 90 mL distilled water, then store
at room temperature.
41
Materials & Methods
4.2. Oxidase reagent
Tetramethyl-p-phenylenediamine dihydrochloride 1.0 g
The solution was prepared according to MacFaddin (2000) and maintained in a

dark container. The solution should be freshly prepared.
4.3. Phosphate-buffered saline (10X PBS) solution
Potassium chloride 2.0 g
Disodium hydrogen phosphate 11.5 g
Potassium dihydrogen phosphate 2.0 g
The pH was adjusted to 7.2-7.3. Then, the solution was autoclaved,

and diluted by adding 100 mL of 10X PBS to 900 mL of distilled water to make
one liter of 1X (PBS).
4.5. 1 M Tris-HCl buffer
Tris base 121.1 g
42
Materials & Methods
Adjust pH to 7.5 with the appropriate volume of concentrated HCl. Then, bring the
final volume to 1 liter with distilled water. The solution was autoclaved and stored
at room temperature.
4.6. Saline-magnesium (SM) phage buffer (Bonilla et al., 2016)
Magnesium sulfate 1.2 g
1 M Tris-HCl (pH=7.5) 50 mL
Gelatin 0.1 g
5. Materials used for genome sequencing
The genomic DNA was extracted using the QIAamp1 DNA Mini kit
(QIAGEN, Germany) following the manufacturer’s instructions. The preparation
of the library was carried out utilizing the Nextera XT DNA library preparation kit
(Illumina, USA).
6. Antimicrobial discs used for antimicrobial susceptibility testing
The antimicrobial discs used in antimicrobial susceptibility testing of P.

aeruginosa were obtained from Oxoid, Hampshire, England. These discs include
piperacillin tazobactam (TPZ, 110 μg), aztreonam (ATM, 30 µg), piperacillin
43
Materials & Methods
(PRL, 100 µg), ceftazidime (CAZ, 30 µg), cefepime (FEP, 30 µg), cefoperazone
(CEP, 75 µg), ciprofloxacin (CIP, 5 µg), gatifloxacin (GAT, 5 µg), amikacin (AK,
30 µg), tobramycin (TOB, 10 µg), gentamicin (CN, 10 µg), colistin sulfate (CT, 10
µg) and meropenem (MEM, 10 µg).
7. Animal study
Three-week-old healthy albino mice (Mus musculus) of the same weight

obtained from the Medical Research Center, Ain Shams, University, Cairo, Egypt,
were used in the current study.
44
Materials & Methods
Methods
1. Clinical specimens collection and P. aeruginosa isolation
Clinical specimens were collected from patients admitted to Zagazig

University Hospitals and El-Ahrar Educational Hospital in Zagazig, Egypt.
Specimens were collected aseptically and immediately transported to the
microbiology laboratory. The specimens were obtained from different clinical
sources include; burns, surgical wounds, urine, ear infections and endotracheal
aspirates according to the Declaration of Helsinki, and informed consent has been
obtained from the subjects. The Institutional Review Board (IRB) provided the
ethical approval under the number (ZU-IRB #10219). The samples were processed
according to the approved microbiological procedures (Winn, 2006). Specimens
were plated onto nutrient agar as non-selective medium as well as onto cetrimide
and MacConkey agar plates as selective medium and blood agar as an enriched
medium and incubated at 37°C under aerobic conditions for 24 hr (Cheesbrough,
2005).
2. Identification of P. aeruginosa isolates
Suspected P. aeruginosa isolates were picked from agar plates and

presumptively identified by Gram stain, colony morphology and biochemical
characters on culture media. P. aeruginosa isolates were primarily identified as
Gram-negative rods. P. aeruginosa isolates were further identified biochemically
for complete identification as reported by MacFaddin, (2000) and Koneman et al.
(2006).
45
Materials & Methods
2.1. Production of pigment on cetrimide agar
The tested isolates were grown on cetrimide agar and incubated overnight at
37°C. P. aeruginosa colonies were characterized by their bluish green color due to
the production of pyocyanin and pyoverdine pigments.
2.2. Oxidative-fermentative test (O/F)
Bacterial isolates were stabbed into duplicate tubes containing (O/F)

medium. One tube was covered with 2-3 mL of sterile liquid paraffin for anaerobic
conditions. Afterwards, both tubes were incubated for 48 hr at 37°C. The organism
that ferments sugar, produces yellow color (acid production) in both tubes, whereas
that utilizes the sugar only oxidatively, produces acid in the aerobic culture tube
only.
2.3. Oxidase test
Pure bacterial colonies were added to a filter paper that had been wetted with
freshly made oxidase reagent. Development of purplish-blue color within 10 sec
indicated a positive result.
2.4. Citrate utilization test
The tested isolates were streaked on the surface of Simmons citrate agar
slants and incubated at 37ºC for 24 hr. Positive result was observed by
transformation of the green color of the medium to blue color and bacterial growth.
2.5. Growth on blood agar
The bacterial isolates were streaked on the surface of blood agar plates and
incubated at 37°C for 24 hr. β- hemolysis (complete hemolysis) was observed.
46
Materials & Methods
2.6. Motility test
The bacterial isolate was stabbed into a test tube containing motility test
medium with needle inoculated with the pure single well isolated colony then
incubated at 37°C for 24 hr. Motile bacteria gave a diffuse spreading growth which
mean that movement from initial inoculation along a single stab line.
2.7. Growth at 42°C
Three tubes of tryptone soya broth were inoculated with the test organism
and one of the three tubes incubated at 37°C, the second at 42°C, the third at 44°C
using a thermostatically controlled water bath. The tubes were examined for
growth.
2.8. Growth on TSI agar
The slant was streaked with the test organism on its surface and stabbed with
the same needle in the butt. The inoculated tubes were incubated at 37ºC for 24 hr.
The formation of acid and gas in the butt with or without blackening due to
hydrogen sulfide production and the type of reaction on the surface were
determined.
3. Maintenance of bacterial culture
A single colony of P. aeruginosa isolates was picked up, streaked on nutrient

agar slants, incubated at 37°C for 24 hr and stored in refrigerator at 4°C for short
term preservation. The purity of each strain was checked monthly by streaking and
subculture. For long term preservation of isolates, glycerol (20%) was added to
overnight culture of MH broth and kept at -80°C (Vandepitte et al., 2003).
47
Materials & Methods
4. Antimicrobial susceptibility testing of P. aeruginosa strains by disc diffusion

method.
The susceptibility pattern of P. aeruginosa strains to different antimicrobial

agents was determined by Kirby-Bauer standard disc diffusion method (Patel,
2015). The bacterial suspensions density was adjusted to match that of 0.5
McFarland standards (approximately 1.5×108 colony forming units (CFU)/mL).
Bacterial suspension was swabbed over the surface of MH agar plates in different
directions. Antibiotic discs were placed on the agar plates using sterile forceps.
Discs were pressed lightly against the agar surface to ensure antimicrobial
diffusion. Plates were incubated inverted at 37ºC for 16-18 hr. Diameters of
inhibition zones around the discs were measured in millimeter (mm). P. aeruginosa
susceptibility to antibiotics was interpreted as resistant (R), intermediate (I) and
susceptible (S) according to guidelines recommended by Clinical Laboratory
Standards Institute CLSI, (2018).
5. Quantitative assessment of biofilm formation
The ability of P. aeruginosa isolates to form biofilm was assayed

spectrophotometrically as reported by Stepanovic et al. (2007). A 1:100 dilutions
of P. aeruginosa overnight culture were prepared in TS broth to form biofilms into
96-well polystyrene U-shaped microtiter plate and incubated for 24 hr at 37°C.
Wells contained TS broth only were included as negative control. Formed biofilms
were fixed using 150 µL/well of 99% methanol for 20 min. Methanol was removed
and wells were left to air dry. Fixed biofilms were stained by using 150 µL/well of
1% crystal violet for 15 min and the bound dye was dissolved by aliquots of 150
µL/well of 33% glacial acetic acid. The optical density (OD) was measured
spectrophotometrically at 570 nm using Bio-Tek synergy HT microplate reader,
48
Materials & Methods
USA. The experiment was repeated three times. The cut-off optical density (ODc)
was calculated as three times standard deviations above the mean OD of the
negative control. Bacterial isolates were categorized based on biofilm forming
capacity as follows: non-biofilm producer (OD ≤ ODc), weak biofilm producer
(OD > ODc, but ≤ 2x ODc), moderate biofilm producer (OD > 2x ODc, but
≤ 4x ODc) and strong biofilm producer (OD > 4x ODc).
6. Phage isolation
The phage was isolated from sewage water sample obtained from Zagazig
city, Egypt by the enrichment technique (Didamony et al., 2015; Chen et al.,
2021). The sewage sample was clarified through centrifugation at 6000 rpm for 20
min using Hermle cooling centrifuge (Germany) and filtered through a 0.45 μm
membrane filter (Millipore, USA). The filtrate was added to an equal volume of
double concentrated TS broth medium containing aliquots of different cultures of
P. aeruginosa isolates grown to the exponential phase. Suspensions were incubated
in a benchtop shaker incubator at 37ºC for 24 hr with shaking at 120 rpm. The
cultures were centrifuged at 6000 rpm for 10 min and the supernatants were
filtered through 0.22 μm membrane filters to remove bacteria. The filtrates were
checked for presence of phages by performing spot assay on Bacterial lawns.
6.1. Spot assay
The double agar overlay method was used to prepare the bacterial lawn as
described by Mazzocco et al. (2009). Briefly, a mixture of 3 mL of pre-warmed
soft TS agar (0.6% agar) and 100 µL of P. aeruginosa culture at exponential phase
were poured over solid bottom TS agar plate. After solidifying, 10 μL of the filtrate
49
Materials & Methods
was spotted onto the lawns and left to dry. Plates were incubated overnight at 37ºC.
Appearance of clear zone (plaques) in the plate indicates presence of phages.
6.2. Plaque assay (double agar overlay technique)
This technique was used for both detection and determination of the
concentration of phage particles by employing double agar overlay technique as
described by Adams (1959). Briefly, 100 μL of tenfold serial diluted phage
suspension was added to 100 μl of exponential phase of bacterial culture in a test
tube containing 3 ml pre-warmed soft TS agar and poured over solid TS agar
plates. After solidification, the plates were incubated overnight at 37ºC. The
resulting plaques were counted to determine phage titer and expressed as plaques
forming units per mL (PFU/mL) (Bonilla et al., 2016; Gencay et al., 2017). The
phage titer was determined as following: PFU per mL = (number of plaques ×
dilution factor) / volume plated in mL.
7. Phage purification and propagation
Bacteriophage purification was done by picking a well isolated single plaque

and resuspended in SM buffer. Then, aliquots of 100 μL of tenfold serial diluted
phage were mixed with 100 μL of P. aeruginosa culture and plated by double agar
overlay technique. This process was performed successive rounds in order to
obtain uniform plaque morphology. Phage propagation was done by incubating the
phage with P. aeruginosa as host with shaking at 120 rpm for 24 hr. The culture
was centrifuged at 6000 rpm for 20 min and the supernatant was filtered through
0.22 μm membrane filters. Finally, the purified phage stock was stored at 4°C and
the phage titer was evaluated before further analysis (Kumari et al., 2009; Russell
and Sambrook, 2001).
50
Materials & Methods
8. Preparation of high titer phage stock
To obtain a high titer phage stock, purified phage was propagated as

described by Kumari et al. (2009). Phage suspension was tenfold serially diluted
and plated by double agar overlay technique as described above. Plates with almost
confluent lysis were chosen and 5 mL of SM buffer was added to plates to elute
phages. The liquid was decanted and centrifugated at 6000 rpm for 15 min. The
supernatant was then passed through 0.22 μm membrane filter and kept at 4°C.
9. Morphological characteristics of isolated phages
The phage morphology was visualized using the transmission electron

microscope (TEM) as described by Shen et al. (2016). A drop of high titer purified
phage [1012 PFU/mL] was applied to carbon-coated copper grid (200 mesh). Phage
particles were negatively stained with 2% phosphotungstic acid (pH 7) and excess
liquid was removed using filter paper. Finally, the grid was air-dried and phage
particles were examined using TEM (Hitachi H600A, Japan) at the Electron
Microscopy Unit, Mansoura University, Egypt. The phage dimensions were
measured based on electron micrographs.
10. Characterization of physical characteristics of isolated phages
10.1. Characterization of thermal stability of isolated phages
For thermal stability evaluation, aliquot of 100 µL of purified phage

particles was mixed with 900 µL of SM buffer and placed in an adjusted water bath
incubator at various temperatures (4, 40, 50, 60, 70, 80, 90 and 100°C) for 1 hr.
51
Materials & Methods
The phage titer was determined after incubation at each specified temperature by
the double agar overlay technique (Shahin et al., 2018).
10.2. Characterization of isolated phages stability at various pH
Similarly, the impact of pH on phage survival was assessed. Purified phage

particles were incubated for 1 hr in SM buffer at different pH (3, 4, 5, 6, 7, 8, 9, 10,
11 and 12) adjusted using either 1M HCL or 1M NaOH followed by determining
the phage titer. The phage titers were estimated by the double agar overlay
technique as described above. These assays were carried out in triplicate and the
results were expressed as means ± standard errors (Asif et al., 2020).
11. Determination of phage host range
The host range of isolated phages against various clinical P. aeruginosa

strains, P. aeruginosa reference strains and other bacterial species (E. coli, S.
typhimurium, K. pneumonia, Serratia marcescens and S. aureus) to test cross
reactivity was performed using the spot testing method. Bacterial lawns were
prepared by double agar overlay technique. After solidification of top agar layer,
10 μL of purified phage stock was spotted on the bacterial surfaces. The plates
were incubated at 37°C for 24 hr and checked for presence of lytic inhibition area.
Clear inhibition zone was considered as evidence for bacterial susceptibility to
phage (Adnan et al., 2020).
52
Materials & Methods
12. Efficiency of plating (EOP) analysis
The efficiency of plating (EOP) of isolated phages was evaluated against P.

aeruginosa isolates that showed lysis in the spot assay. Aliquots of 100 µL of
bacterial cultures grown to the exponential phase were co-cultured with 100 µL of
tenfold serially diluted phage in soft agar layer and overlayed on surface of TS
agar plates. The plates were incubated overnight at 37°C and the PFUs were
counted for each phage-bacterium combination. The EOP values were estimated by
dividing the total number of PFUs obtained by the target bacteria to the total
number of PFUs obtained by host bacteria. Assays were repeated three times, the
results were recorded as mean ± SD and interpreted according to the follow; High
production if the EOP ratio was ≥ 0.5; Medium production if 0.5 > EOP ratio ≥
0.1; Low production if 0.1 > EOP > 0.001 and inefficient if EOP ≤ 0.001 (Khan
Mirzaei and Nilsson, 2015).
13. Phage growth kinetics analysis
One-step growth curve for isolated phages was carried out to detect
phage burst size and latent period as reported by Cao et al. (2015). Bacterial host
strain was cultured to reach exponential phase (108 CFU/mL) and mixed with
isolated phage at multiplicity of infection (MOI) of 0.1. Then, the mixture was
centrifugated at 6000 rpm for 15 min to remove non absorbed phages and the pellet
was resuspended in 10 mL fresh TS broth. Simultaneously, samples of 100 µL
were collected at time intervals of 5 min over and subjected to phage titration by
the double agar overlay method. The assay was evaluated in triplicate to estimate
phage latent period and burst size. The average burst size is defined as the average
number of new phages released per infected bacterium.
53
Materials & Methods
14. In vitro killing assay
In vitro bacteriolytic activity of isolated phages against their host strain and
other P. aeruginosa strains with high EOP ratio was determined by measuring
optical density (OD600) as mentioned by Peng et al. (2020). Phage suspensions at
different MOIs (0.1, 1 and 10) were co-cultured with bacterial suspension at (108
CFU/mL) and incubated with shaking at 37°C. Bacterial culture without phage was
used as a control. The inhibitory effect of isolated phages on bacterial growth was
observed by measuring change in OD600 at 2 hr, 4 hr, 6 hr, 8 hr, 12 hr and 24 hr
post inoculation and compared with the control bacterial culture without phage
treatment. The assays were performed in triplicate and results were expressed as
means ± standard errors.
15. Biofilm inhibition assay
The ability of isolated phages to eradicate already established biofilms

formed by P. aeruginosa was determined (Liu et al., 2020). Bacterial cultures were
allowed to form biofilm onto the surface of sterile polystyrene microtiter plate
exactly as described above. Following incubation, broth culture was gently
decanted and wells were washed with sterile PBS to remove the planktonic cells.
Next, aliquots of about 200 μL of phage suspension at various MOIs (0.1, 1 and
10) in TS broth were added to each well and incubated overnight at 37°C. Control
wells received sterile TS broth only without phage. The plates were washed three
times with sterile PBS. The formed biofilms were fixed using methanol for 20 min,
stained by 1% crystal violet for 15 min and the bound dye was dissolved by 33%
glacial acetic acid. The absorbance was measured spectrophotometrically at 570
nm. The experiment was performed in triplicate and results were expressed as
means ± standard errors.
54
Materials & Methods
16. Phage DNA extraction
The genome of isolated phages was extracted from high titer phage lysate
(2.5 x 1012 PFU/mL) using QIAamp1 DNA Mini kit (QIAGEN, Germany)
following the manufacturer guidelines and DNA pellet was stored at -20˚C until
use. The DNA library was prepared using the Nextera XT DNA Library
preparation kit (Illumina, USA). The DNA was fragmented then tagged utilizing
the transposome in the Nextera XT Kit (Jakočiūnė and Moodley, 2018).
17. Phage genome sequencing and data analysis
The whole genome sequencing was done by Illumina Miseq next-generation

sequencing at Genomics and Epigenomics Program, Children’s Cancer Hospital
Egypt, Cairo, Egypt. All preparations and the sequencing run were performed
according to Illumina manufacturing instructions. Quality of paired-end DNA
reads were evaluated using FASTQC (Brown et al., 2017). Low-quality bases were
trimmed using Trimmomatic v0.36 (Bolger et al., 2014). The generated sequences
were assembled using Unicycler v0.4.8 to assemble the reads into contigs (Wick et
al., 2017). Assembly quality was checked using Quality Assessment Tool for
Genome Assemblies (QUAST v 5.0.2) (Gurevich et al., 2013). The number of
open reading frames (ORFs) in phage genome was predicted and putative functions
of predicted ORFs were annotated using Prokka v 1.14 (Seemann, 2014). The virus
circular genome map was created using the CGView (Stothard and Wishart, 2005).
The online tool tRNAscan-SE 1.21 was used to look for tRNA genes in the phage
sequences (Chan and Lowe, 2019). The phylogenetic analysis of phage whole
genome was performed using the Molecular Evolutionary Genetics Analysis
(MEGA) X program v10.2.2. (Kumar et al., 2018). Additionally, the nucleotide
55
Materials & Methods
sequences of the phage major capsid protein, terminase large subunit genes and
RNA polymerase large subunit were compared with their corresponding sequences
of reference bacteriophages deposited in the National Center for Biotechnology
Information (NCBI) database to determine the phylogenetic position of isolated
phages (Altschul et al., 1990). Comparison of phage genome with similar phages
deposited in the NCBI database was accomplished using the EasyFig program
(Sullivan et al., 2011). A dot plot was constructed using the Gepard-2.1 program
(Krumsiek et al., 2007).
18. Assessment of the influence of isolated phages on P. aeruginosa

pathogenesis in vivo using mice infection model
The influence of isolated phages on P. aeruginosa pathogenesis was

characterized in vivo using mice infection model (Alvi et al., 2021). All procedures
in animal infection experiment were performed according to ethical standards of
the Zagazig University Institutional Animal and Use Committee (ZU IACUC),
which was granted approval number (ZUIACUC/3/F/72/2022). Briefly, five
groups (15 mice each) of 4-weeks old albino mice were included in the
experiment. The first group contained mice inoculated intraperitoneally (IP) with
P. aeruginosa at (2.5 × 107 CFU/mL), the second group contained Pseudomonas-
inoculated mice and treated intraperitoneally with isolated phage at MOI = 100
(2.5 × 109 PFU/mL). The third group contained mice inoculated with isolated
phage (2.5 × 109 PFU/ml) only. In addition, both non-injected and PBS-injected
mice groups were included as controls. Mice survival in each group was monitored
daily over 4 days-period and plotted using Kaplan–Meier method using Log-rank
test for statistical analysis. In addition to mice survival, three mice from each group
were anesthetized and sacrificed at 24, 48 and 72 hr post inoculation. Mice liver
and spleen were aseptically obtained for determination of both bacterial burden and
56
Materials & Methods
phage titer. Isolated organs were homogenized, serially diluted in PBS and plated
on cetrimide agar plates for enumeration of bacterial CFUs. In addition, the
homogenate was filtered, serially diluted and overlaid by the double layer agar
method to determine the phage titer in treated mice and expressed as (PFUs). Both
bacterial load and phage titer were determined and expressed as means ± standard
errors. The statistical analysis was performed by Mann–Whitney U analysis with P
value < 0.05 was considered as statistically significant, GraphPad Prism 5.
57
Results
Results
1. Bacterial isolation
A total of 50 P. aeruginosa isolates were recovered from 120 clinical

specimens during the period from April 2021 to August 2021. P. aeruginosa isolates
were collected from patients admitted to Zagazig University Hospitals and El-Ahrar
Educational Hospital, Zagazig, Sharkia, Egypt. P. aeruginosa were isolated from
different clinical sources including burns, surgical wounds, urine, ear infections and
endotracheal aspirates (Table 2).
Table 2: Source and number of P. aeruginosa clinical isolates
Isolation source Number (NO) of P. aeruginosa isolates

Burn 10
Surgical wounds 10
Urine 11
Ear infections 2
Endotracheal aspirates 17
Total 50
2. Identification of P. aeruginosa isolates
All P. aeruginosa isolates were identified as Gram-negative single rods,

lactose non-fermenters on MacConkey agar and showing bluish-greenish
pigmentation on nutrient agar with characteristic grape juice-like odor. P. aeruginosa
colonies on nutrient agar were smooth, translucent, large (2-4 mm in diameter) and
convex with irregular edges. On blood agar, most P. aeruginosa isolates produced β-
58
Results
hemolysis. Complete identification of recovered P. aeruginosa isolates based on

their biochemical characteristics is shown in table 3.
Table 3: Biochemical identification of P. aeruginosa isolates
Test Result
Greenish pigmentation (pyocyanin
± (Variable)*
production)
Oxidation-Fermentation (O/F) O+/F- (Oxidative)
Oxidase +
Motility Motile
Citrate utilization test +
Growth on triple sugar iron agar K/K (Red/Red)
Growth at 42°C +
Blood hemolysis β-hemolysis (Clear zone around
colonies)
* Variable: most P. aeruginosa produce pyocyanin pigment and others are non-pigmented.
3. Antimicrobial susceptibility testing of P. aeruginosa strains by disc diffusion

method.
The antibiotic susceptibility of 50 P. aeruginosa isolates was performed using

Kirby-Bauer disc diffusion method. The diameters of inhibition zones were
determined according to Clinical Laboratory and Standards Institute Guidelines
CLSI, (2018). The patterns of P. aeruginosa antimicrobial susceptibility against
different classes of antibiotics including β-lactams (penicillins, carbapenems,
cephalosporins, monobactams), aminoglycosides, lipopeptide, and fluroquinolones
were determined.
59
Results
P. aeruginosa showed varying resistance patterns to different antibiotics as shown in

table 4 and figure 4. All P. aeruginosa isolates were sensitive to colistin while high
bacterial resistance was observed towards gentamicin (60%). Majority of P.
aeruginosa isolates exhibited high resistance to fluoroquinolone and carbapenems
(58% and 54%; respectively). Intermediate bacterial resistance was observed against
pipracillin (46%), ceftazidime (40%) and piperacillin/tazobactam (38%). Low
bacterial resistance was recorded against aztreonam (18%). The results demonstrate
that about 56% of P. aeruginosa isolates were resistant to at least one antibiotic from
three or more antimicrobial classes and therefore were described as multi-drug
resistant (MDR).
Table 4: Antibiotic susceptibility of P. aeruginosa isolates
Antibiotic
CFP CAZ FEP TZP PRL CT CN TOB AK MEM CIP GAT ATM*
Isolate NO
1B** S S S S S S R R R S R R S
2B R R R R R S R R R R R R R
4B S S S S S S R R R S R R S
6B S S S S S S S S S S S S S
8B R R R I R S R R R S R R R
11W S S S S S S S S S S S S S
12W R I R I R S R R R R R R S
13W R R R R R S R I R R R R I
14W S S I S S S I S I I S S I
15W S S S S S S S S S S S S S
16W R R R R R S R R R R R R S
17W R R R I I S R R R R R R S
18W R S I I R S R R S S S S S
19W R S I I R S R R S S S S S
20W S S S S S S I S S I S S S
21U R S R R R S R R R R R R S
22U R R R I I S R R R R R R S
60
Results
25U S S S S S S S S S S S S S
26U S S S S S S S S S S S S S
27U R R R R I S R R R R R R S
28U R R R R R S R R R R R R S
29U R S R R R S R R R R R R R
30U S S I S S S R R R R R R S
31U R R R I I S R R R R R R I
32SP R R R R R S R R R R R R S
33SP S S S S S R S S S S S S S
34SP S S S S S S S S S S S S S
40SP R R R I I S R R R R R R S
44SP R R R R R S R R R R R R R
45SP S S S S R S S S S S S S S
48SP I S S S I S S S S R S S S
49E S S S S S S S S S R S S S
50E S S S S S S I S S I S S S
*
: Cefoperazone (CFP, 75µg), ceftazidime (CAZ, 30µg), cefepime (FEP, 30µg), piperacillin/tazobactam
(TPZ, 110μg), pipracillin (PRL, 100µg), colistin (CT, 10µg), gentamicin (CN, 10µg), tobramycin (TOB,
10µg), amikacin (AK, 30µg), meropenem (MEM, 10µg), ciprofloxacin (CIP, 5µg), gatifloxacin (GAT,
5µg) and aztreonam (ATM, 30µg).
**
: Burn (B); surgical wound (W); urine (U); endotracheal aspirate (SP); ear (E)
61
Results
100
90
80
70
% of resistance
60
50
40
30
20
10
T)
)
Pi ime P)
)
tin )
ef e ( )
Am n (T )
at ikac B)
)
zo me L)
T)
)
ac Ce cil EP
ci AK
yc (CN
M
M
op em IP
Az cta AZ
am Z
C
Ta idi PR
M flox GA
F
O
on (TP
az (ME
C AT
C pe n (C
ep C
a (F
(C
(
n
(
am n
(
i
lin
br ici
is
tre m
n
i
ac
on
i
ol
To tam
C xa
ba
n/ taz
o
pr
en
er
ifl
ro
C
G
f
er
ip
G
ef
illi
pr
Pi
Figure 4: Percentage of P. aeruginosa isolates resistance against tested antibiotics. The

antibiotic susceptibility test was performed using Kirby-Bauer disc diffusion method. The
diameters of inhibition zones were measured and susceptibility of P. aeruginosa isolates to various
antibiotics was determined.
4. Quantitative assessment of P. aeruginosa biofilm formation by

spectrophotometry
The biofilm forming capacity of P. aeruginosa clinical isolates and references

strains (P. aeruginosa ATCC 27853, ATCC 9027 and PAO1) were assessed
spectrophotometrically at 570nm by the crystal violet assay. The experiment was
carried out in triplicate and the mean optical density (OD) was calculated. The cut-
off OD (ODc) was calculated as three times standard deviation above the mean OD
62
Results
of the negative control which was found to be 0.091. Bacterial isolates were
classified into four categories according to their capacity to form biofilm (Table 5).
These four categories include; non-biofilm forming (OD ≤ 0.091), weak biofilm
forming (OD > 0.091, but ≤ 0.182), moderate biofilm forming (OD > 0.182, but ≤
0.364), strong biofilm forming (OD > 0.364).
Table 5: Classification of P. aeruginosa according to biofilm forming capacity
Isolate No Mean ± SD Biofilm forming capacity*

P. aeruginosa ATCC 27853 0.43 ± 0.05 +++
P. aeruginosa ATCC 9027 0.53 ± 0.06 +++
PAO1 0.28 ± 0.03 ++
1B 0.26 ± 0.03 ++
2B 0.307 ± 0.01 ++
3B 0.448 ± 0.07 +++
4B 0.172 ± 0.04 +
5B 0.242 ± 0.05 ++
6B 0.356 ± 0.05 ++
7B 0.183 ± 0.01 ++
8B 0.23 ± 0.01 ++
9B 0.268 ± 0.1 ++
10B 0.31 ± 0.08 ++
11W 0.2 ± 0.08 ++
12W 0.297 ± 0.03 ++
13W 0.29 ± 0.06 ++
14W 0.26 ± 0.03 ++
15W 0.505 ± 0.01 +++
16W 0.19 ± 0.05 ++
17W 0.355 ± 0.06 ++
18W 0.213 ± 0.01 ++
19W 0.255 ± 0.07 ++
20W 0.17 ± 0.02 +
21U 0.156 ± 0.01 +
22U 0.373 ± 0.04 +++
23U 0.194 ± 0.04 ++
24U 0.376 ± 0.03 +++
63
Results
25U 0.306 ± 0.05 ++

26U 0.196 ± 0.06 ++
27U 0.286 ± 0.06 ++
28U 0.517 ± 0.07 +++
29U 0.31 ± 0.03 ++
30U 0.163 ± 0.01 +
31U 0.31 ± 0.03 ++
32SP 0.38 ± 0.05 +++
33SP 0.4 ± 0.06 +++
34SP 0.226 ± 0.05 ++
35SP 0.172 ± 0.02 ++
36SP 0.114 ± 0.02 +
37SP 0.338 ± 0.05 ++
38SP 0.38 ± 0.05 +++
39SP 0.267 ± 0.03 ++
40SP 0.247 ± 0.03 ++
41SP 0.257 ± 0.03 ++
42SP 0.31 ± 0.03 ++
43SP 0.184 ± 0.05 ++
44SP 0.11 ± 0.02 +
45SP 0.139 ± 0.01 +
46SP 0.339 ± 0.03 ++
47SP 0.271 ± 0.06 ++
48SP 0.25 ± 0.01 ++
49Ear 0.372 ± 0.04 +++
50Ear 0.114 ± 0.05 +
* +; Weak, ++; Moderate, +++; Strong
As shown in figure 5, P. aeruginosa isolates were categorized into 3 groups

according to biofilm formation; strong (20.4% of bacterial isolates), moderate
(64.8% of bacterial isolates) and weak biofilm forming (14.8% of bacterial isolates).
64
Results
Figure 5. Quantitative evaluation of P. aeruginosa biofilm formation using the crystal violet
(CV) assay. P. aeruginosa isolates were categorized into strong, moderate and weak biofilm
forming.
5. Isolation of P. aeruginosa phages
Sewage samples were collected from Zagazig city, Egypt and used for
isolation of phages targeting P. aeruginosa isolates by the enrichment technique.
The presence of lytic phages against P. aeruginosa in sewage samples was detected
by the spot assay (Figure 6) and the double agar overlay technique (plaque assay
method). Five different phages specific for P. aeruginosa were isolated and fully
characterized. These phages were designated as vB_PaeP_PS28, vB_PaeP_PS49,
vB_PaeP_PS14, vB_PaeM_PS3 and vB_PaeM_PS38 according to the
recommended nomenclature procedure (Kropinski et al. 2009). Based on the plaque
morphology, P. aeruginosa phage vB_PaeP_PS28 and vB_PaeP_PS49 produced
small circular plaques with no halo with diameter of 2-3 mm, vB_PaeP_PS14
produced large circular plaques with diameter of 3-4 mm with no halo,
65
Results
vB_PaeM_PS3 produced large clear circular plaques surrounded by halos with a

diameter of 4-5 mm and vB_PaeM_PS38 produced small, circular, clear plaque with
no halo with diameter of 1-2 mm in double layer agar method (Figure 7). Plaques
of homogenous morphology were selected, purified several rounds for further
analysis and the purified phage stock was stored in SM buffer at 4°C.
Figure 6. Detection of phages by the spot assay. Different phage lysates were spotted on surface
of bacterial lawns. A) Lysis: Indicate presence of phage. B) No lysis: Indicate absence of phages.
66
Results
Figure 7. Plaque morphology of isolated phages on different P. aeruginosa lawns by double agar
overlay technique. a) vB_PaeP_PS28, b) vB_PaeP_PS49, c) vB_PaeP_PS14, d)vB_PaeM_PS3
and e) vB_PaeM_PS38.
6. Morphological characteristics of isolated phages
The morphology of isolated phages was observed using TEM after staining
with 2% phosphotungstic acid (pH 7). TEM images revealed that the phage
vB_PaeP_PS28, vB_PaeP_PS49 and vB_PaeP_PS14 possess an icosahedral head
and short non-contractile tail which are closely related to phages belonging to
Podoviridae family and the order Caudovirales according to International
Committee Taxonomy of Viruses (ICTV) (Adriaenssens and Brister, 2017). The
67
Results
phage head diameters are 65.5, 54 and 97.9 nm, respectively. The tail length of
vB_PaeP_PS28, vB_PaeP_PS49 and vB_PaeP_PS14 are 37.8, 10 and 29.1 nm,
respectively. Meanwhile, vB_PaeM_PS3 and vB_PaeM_PS38 possess an
icosahedral head of 70 and 90 nm in diameter, respectively and contractile tails of
100 and 137 nm in length, respectively. These two phages were classified according
to ICTV guidelines and found to belong to the order Caudovirales and the family
Myoviridae (Figure 8).
Figure 8. Transmission electron microscope (TEM) images of a) vB_PaeP_PS28, b)

vB_PaeP_PS49, c) vB_PaeP_PS14, d) vB_PaeM_PS3 and e) vB_PaeM_PS38. Phage particles
were negatively stained by 2% phosphotungstic acid. Scale bar =100 nm.
68
Results
7. Physical characteristics of isolated phages
7.1. Thermal stability
The thermal stability of isolated phages was assessed by monitoring change

in phage titer upon incubation under different temperatures (4, 40, 50, 60, 70, 80, 90,
100°C). The results indicated that all isolated phages were tolerant to wide range of
temperatures. Both vB_PaeP_PS28 and vB_PaeM_PS3 could survive up to 60°C
with no significant reduction in phage titer. However, there was a significant
reduction in phage titer upon incubation of vB_PaeP_PS28 and vB_PaeM_PS3 at
higher temperatures (70, 80, 90 and 100°C) as shown in figure 9. On the other side,
while vB_PaeP_PS49, vB_PaeP_PS14 and vB_PaeM_PS38 were considerably
stable at temperatures range from 40°C to 70°C, their activity was entirely
diminished when incubated at 80, 90 and 100°C (Figure 9).
69
Results
Figure 9. Thermal stability of isolated phages. a) vB_PaeP_PS28; b) vB_PaeP_PS49; c)

vB_PaeP_PS14; d) vB_PaeM_PS3 and e) vB_PaeM_PS38. Purified phage particles were
incubated at various temperatures: 40, 50, 60, 70, 80, 90 and 100°C for 1 hr. Then, the phage titer
was determined after incubation at each specified temperature. Error bars represent mean ± SE
for three replicates.
7.2. pH stability
The impact of different pH on survival and infectivity of isolated phages was

determined using the plaque assay technique. Interestingly, all tested phages were
able to survive a wide range of pH as illustrated in figure 10. It was found that
70
Results
vB_PaeP_PS28, vB_PaeP_PS49, vB_PaeM_PS3 and vB_PaeM_PS38 could

survive and retain their infectivity at pH range from 4 to 10. There was a slight
reduction in phage viability at pH 11. Additionally, vB_PaeP_PS14 titer remained
relatively unchanged when incubated at pH values ranging from 5 to 8. However,
the phage infectivity slightly decreased at pH of 9 and 10. Of note that, no viable
phage particles were observed at extreme pH (3 and 12).
71
Results
Figure 10. pH stability of isolated phages. a) vB_PaeP_PS28; b) vB_PaeP_PS49; c)

vB_PaeP_PS14; d) vB_PaeM_PS3 and e) vB_PaeM_PS38. Purified phage particles were
incubated for 1 hr in SM buffer at different pH (3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) adjusted using
either 1M HCL or 1M NaOH. Then, the phage titer was determined after incubation at each
specified temperature. Error bars represent mean ± SE for three replicates.
8. Determination of phage host range
The host range of vB_PaeP_PS28, vB_PaeP_PS49, vB_PaeP_PS14,

vB_PaeM_PS3 and vB_PaeM_PS38 was determined against a total of 18 P.
aeruginosa strains (15 P. aeruginosa isolates from different clinical sources and 3 P.
72
Results
aeruginosa reference strains; P. aeruginosa ATCC 27853, ATCC 9027 and PAO1)
using the spot assay. The selected clinical P. aeruginosa isolates were chosen to be
representative for different clinical sources including; burn (4 isolates), wound (3
isolates), urine (4 isolates), ear infections (1 isolate) and endotracheal aspirates (3
isolate). In addition to P. aeruginosa, other bacterial species such as E. coli (4
isolates), S. Typhimurium (1 isolates), K. pneumoniae (1 isolates), Serratia
marcescens (1 isolates) and S. aureus (2 isolates) were included in host range
determination. Results show that isolated phages were able to infect most of tested
P. aeruginosa strains indicating a higher lytic efficiency of isolated phages.
However, no lytic activity was observed against other bacterial species by isolated
phages indicating that isolated phages possess broad spectrum lytic activity and high
specificity towards P. aeruginosa (Table 6). The phages vB_PaeP_PS14,
vB_PaeP_PS28 and vB_PaeP_PS49 exhibited a high bacteriolytic activity and were
able to infect 14 (77.7%), 13 (72.2%) and 12 (66.7%), respectively of the 18 tested
P. aeruginosa strains. On the other hand, vB_PaeM_PS38 and vB_PaeM_PS3
phages were able to infect 11/18 (61.1%) and 10/18 (55.5%) of tested P. aeruginosa
strains (Table 6).
Table 6: Host range of isolated phages
Infectivity b
Bacterial
isolate a vB_PaeP_PS28 vB_PaeP_PS49 vB_PaeP_PS14 vB_PaeM_PS3 vB_PaeM_PS38
PS 3B + + + + +
PS 6B + + + - +
PS 9B - - - + -
PS 10B + + + - -
PS 11W - + + - +
PS 13W + - + + +
PS 14W + + + + +
73
Results
PS 22U + - + - -
PS 23U + + + - -
PS 24U + + + + +
PS 28U + + + + +
PS 32SP + - + - +
PS 38SP + + + + +
PS 41SP - + - - -
PS 49E - + - + -
P. aeruginosa
+ + + + +
PAO1
P. aeruginosa
+ - + + +
ATCC 27853
P. aeruginosa
- - - - -
ATCC 9027
E. coli ATCC
- - - - -
10536
E. coli ATCC
- - - - -
O26
E. coli ATCC
- - - - -
O78
E. coli ATCC
- - - - -
O157
S.
Typhimuriu
- - - - -
m ATCC
14028
K.
pneumoniae
- - - - -
ATCC
700603
Serratia
- - - - -
marcescens
S. aureus
- - - - -
ATCC 6538
S. aureus
- - - - -
ATCC 9295
b+: indicates presence of clear zone (lysis) and -: indicates no lysis was observed.
a B; burn, W; surgical wound, U; urine, SP; endotracheal aspirates.
74
Results
9. Biofilm inhibition assay
The antibiofilm activity of isolated phages at different MOIs (0.1, 1 and 10)
against five strong biofilm forming P. aeruginosa clinical isolates from various
sources as well as the reference strains; ATCC 27853 and 9027 was evaluated by the
crystal violet assay. Isolated phages effectively degraded mature biofilms and
reduced biofilm biomass formed by all tested P. aeruginosa strains. As shown in
figure 11, the antibiofilm activity of isolated phages against P. aeruginosa was MOI
dependent where maximum biofilm inhibition was observed at a MOI of 10 as
compared with MOI of 1 and 0.1. In addition, there was a considerable decrease in
bacterial biomass in phage treated bacterial biofilm compared to the control
untreated bacteria.
75
Results
a)
P. aeruginosa ATCC 9027

28U
3B
33SP
0.8 15W
P = 0.004 49E
P = 0.002
0.7 P = 0.0006
P = 0.0001
P = 0.009 P = 0.03
P = 0.0004
0.6 P = 0.0003 P = 0.02
P = 0.0002
P = 0.005 P = 0.002 P = 0.04
P = 0.002
P = 0.01
0.5 P = 0.0003
P = 0.001
OD at 600nm
P = 0.003
P = 0.0001
P = 0.003
P = 0.0002
0.4
0.3
0.2
0.1
0.0
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
Control
Control
Control
Control
Control
Control
Control
b)

28U
3B
33SP
0.8 15W
P = 0.001 49E
P = 0.002
0.7 P = 0.001
P = 0.0001
P = 0.003 P = 0.0003
P = 0.005
0.6 P = 0.002 P = 0.002
P = 0.0001
P = 0.002 P = 0.001 P = 0.001
P = 0.002
P = 0.002
0.5 P = 0.006
P = 0.002
OD at 600nm
P = 0.02
P = 0.03
P = 0.001
P = 0.002
0.4
0.3
0.2
0.1
0.0
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
Control
Control
Control
Control
Control
Control
Control
76
Results
c)

28U
3B
33SP
0.8 15W
P = 0.002 49E
P = 0.001
0.7 P = 0.002
P = 0.0004
P = 0.0005 P = 0.0002
P = 0.0004
0.6 P = 0.0006 P = 0.001
P = 0.001
P = 0.001 P = 0.002 P = 0.0006
P = 0.003
P = 0.0003
0.5 P = 0.009
P = 0.002
OD at 600nm
P = 0.001
P = 0.04
P = 0.0009
P = 0.005
0.4
0.3
0.2
0.1
0.0
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
Control
Control
Control
Control
Control
Control
Control
d)

28U
3B
33SP
0.8 15W
P = 0.001 49E
P = 0.007
0.7 P = 0.004
P = 0.0007
P = 0.002 P = 0.0006
P = 0.0001
0.6 P = 0.002 P = 0.001
P = 0.0005
P = 0.005 P = 0.002 P = 0.0002
P = 0.001
P = 0.001
0.5 P = 0.005
P = 0.0008
OD at 600nm
P = 0.001
P = 0.01
P = 0.002
P = 0.0002
0.4
0.3
0.2
0.1
0.0
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
Control
Control
Control
Control
Control
Control
Control
77
Results
e)

28U
3B
33SP
0.8 15W
P = 0.001 49E
P = 0.002
0.7 P = 0.003
P = 0.0001
P = 0.001 P = 0.0005
P = 0.0002
0.6 P = 0.001 P = 0.001
P = 0.0007
P = 0.006 P = 0.002 P = 0.0004
P = 0.07
P = 0.003
0.5 P = 0.3
P = 0.003
OD at 600nm
P = 0.004
P = 0.3
P = 0.0003
P = 0.01
0.4
0.3
0.2
0.1
0.0
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
MOI = 10
MOI = 1
MOI = 0.1
Control
Control
Control
Control
Control
Control
Control
Figure 11. The antibiofilm activity of a) vB_PaeP_PS28, b) vB_PaeP_PS49, c)
vB_PaeP_PS14, d) vB_PaeM_PS3 and e) vB_PaeM_PS38 against various P. aeruginosa
isolates. Biofilms were formed in 96-well plates for 24 hr and treated with isolated phages at
different MOIs (0.1, 1 & 10) for 24 hr. Formed biofilms were stained by 1% crystal violet and
measured spectrophotometrically at OD600. The experiment was carried out at three independent
replicates and data was expressed as means ± SE with P < 0.05 was considered significant.
The work will be further continued on vB_PaeP_PS28 and vB_PaeM_PS3

only because they possessed considerable thermal and pH stability, unique lytic
activity against different P. aeruginosa isolates, greatest antibiofilm activity and
differ in morphological examination under TEM belonging to different families.
78
Results
10. Efficiency of plating (EOP) analysis
To further assess the phage infection capability of vB_PaeP_PS28 and

vB_PaeM_PS3, EOP analysis was performed for each susceptible strain that showed
lysis in the spot assay. Series of diluted phage were plated against susceptible strains,
the PFUs for each phage-bacterium combination were counted and the EOP was
determined (Table 7). Both vB_PaeP_PS28 and vB_PaeM_PS3 phages were able to
infect all susceptible P. aeruginosa strains with different infectivity patterns. EOP
values of vB_PaeP_PS28 phage-bacteria mixtures varied from low (n = 3); medium
(n = 4) to high (n = 6). While vB_PaeM_PS3 phage possessed low (n = 3), medium
(n = 2) and high affectivity (n = 5). Remarkably, P. aeruginosa PAO1 and PS14
exhibited an efficient production higher than the host strain (PS28) upon infection
with vB_PaeP_PS28 phage. On the other hand, only one P. aeruginosa isolate; PS49
exhibited an efficient production higher than that of the host strain (PS3) upon
infection with vB_PaeM_PS3 phage.
Table 7: Efficiency of plating (EOP) of vB_PaeP_PS28 and vB_PaeM_PS3

phages
vB_PaeP_PS28 vB_PaeM_PS3
Bacterial
EOP ratio Interpretation EOP ratio Interpretation
isolate
(Mean ± SD) (Mean ± SD)
PS 3B 0.03 ± 0.003 Low 1 High (host)
PS 6B 0.23 ± 0.05 Medium - -
PS 9B - - 0.41± 0.04 Medium
PS 10B 0.35 ± 0.05 Medium - -
PS 13W 0.62 ± 0.04 High 0.042 ± 0.005 Low
PS 14W 1.15 ± 0.1 High 0.8 ± 0.03 High
PS 22U 0.34 ± 0.04 Medium - -
PS 23U 0.06 ± 0.005 Low - -
PS 24U 0.43 ± 0.04 Medium 0.61 ± 0.05 High
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Results
PS 28U 1 High (host) 0.02 ± 0.003 Low

PS 32SP 0.8 ± 0.02 High - -
PS 38SP 0.04 ± 0.004 Low 0.75 ± 0.07 High
PS 49E - - 1.23 ± 0.07 High
P. aeruginosa 1.25 ± 0.05 High 0.034 ± 0.005 Low
PAO1
P. aeruginosa 0.7 ± 0.03 High 0.28 ± 0.03 Medium
ATCC 27853
-: indicates no lysis was observed
11. Phage growth kinetics analysis
The one-step growth curve was performed to characterize the phage infection
process including the latent period and burst size for vB_PaeP_PS28 and
vB_PaeM_PS3. The phage burst size was determined as the ratio of the average
number of free phage particles after the release phase to their number during the
latency phase. The time taken for new phage particles to be released after successful
adsorption is referred to the latent period. The PFUs per infected cell in P.
aeruginosa cultures were determined at different time points following infection.
The results revealed that vB_PaeP_PS28 has a latent period of 15 min and an average
burst size of 210 PFUs per infected bacterium (Figure 12a). The phage
vB_PaeM_PS3 exhibited a short latent period of 10 min and a burst size of 132 PFUs
per infected cell (Figure 12b).
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a)
10
8
Log (PFU/mL)
7
Burst size
Latent
period
6
4
0 10 20 30 40 50 60 70
Time (min)
b)
8
Log (PFU/mL)
Burst size
Latent
period
6
4
0 10 20 30 40 50 60 70
Time (min)
Figure 12. One-step growth curve of a) vB_PaeP_PS28 and b) vB_PaeM_PS3. Phage was
incubated with exponential culture of P. aeruginosa PS28 and PS3, respectively for 10 min,
centrifuged and pellet was resuspended in TS broth. Free phages were counted at 5 min interval
by the double agar overlay technique. Three biological replicates were performed and data were
presented as mean ± SE.
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Results
12. In vitro killing assay
The bacteriolytic activity of vB_PaeP_PS28 and vB_PaeM_PS3 phages was

determined against both PS28 and PS3; the host strains of isolated phage,
respectively. In addition, P. aeruginosa PAO1 and PS29 were chosen as they
possessed high EOP ratio to further confirm the bacteriolytic activity of
vB_PaeP_PS28 and vB_PaeM_PS3 phages. P. aeruginosa cultures were infected
with phage at various MOIs (0.1, 1 and 10) followed by measuring of bacterial
turbidity at OD600 over 24 hr. As shown in figure 13 and 14, control P. aeruginosa
culture without phage treatment continued to grow during the incubation period. On
the other hand, both vB_PaeP_PS28 and vB_PaeM_PS3 adversely affected bacterial
growth over 24 hr. Importantly, bacterial growth inhibition was found to be MOI-
dependent where growth inhibition was higher at MOI of 10 as compared to MOI of
1 and 0.1.
Figure 13. Bacteriolytic activity of phage vB_PaeP_PS28 against host strain (a) and PAO1
(b). Early exponential bacterial cultures were incubated with and without isolated phage
suspension at MOI of (0.1, 1 & 10) at 37°C for 24 hr. Bacterial growth was determined and
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measured spectrophotometrically at OD600. The results were expressed as means ± SE of three

independent experiments.
Figure 14. In vitro planktonic cell lysis assay of vB_PaeM_PS3 at different MOIs against host
bacteria (a) and PS49 (b). Bacterial growth inhibition was measured spectrophotometrically
(OD600) for bacterial cultures with and without phage over 24 hr. The results were expressed as
means ± SE of three independent experiments.
13. Phage genome characterization
13.1. Genomic characterization of vB_PaeP_PS28
The whole genome of vB_PaeP_PS28 was sequenced by Illumina Miseq and

assembly was performed by Unicycler v0.4.8. The genome sequence of the phage
vB_PaeP_PS28 has been deposited in the GenBank database under GenBank Acc.
No. OQ134474. The phage vB_PaeP_PS28 genome is composed of 72,283 base pair
(bp) of circular double-stranded (ds) DNA, with 54.75% G + C content. The entire
genome structure of vB_PaeP_PS28 is shown in figure 15a. In total, 94 ORFs were
recognized; of them, 32 ORFs (34%) were predicted to encode for functional
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proteins, whereas 62 ORFs (66%) were annotated as hypothetical proteins. The

predicted functions of 32 ORFs were divided into 5 major modules including
structure proteins (ORF 8, ORF 9, ORF 18, ORF 21, ORF 24, ORF 88, ORF 90 and
ORF 93), DNA metabolism and replication (ORF 1, ORF 11, ORF 34, ORF 35, ORF
37, ORF 52, ORF 56, ORF 59, ORF 69, ORF 71 and ORF 84), packaging and
assembly proteins (ORF 27, ORF 32, ORF 81, ORF 82, ORF 83 and ORF 85), host
cell lysis modules (ORF 25, ORF 38, ORF 42 and ORF 51) and additional functions
(ORF 30, ORF 31 and ORF 40) (Table 8). ORFs 30 and 31 play a role in cell lysis
inhibition and interference with cell metabolism. This would delay releasing of
phage holin enzyme until the phage particles are formed and accumulated within the
host. On the other hand, ORF 40 is involved in energy-requiring activities such as
phage DNA packaging replication. The phage genome did not reveal any lysogenic
genes or host genome-related sequences, so that vB_PaeP_PS28 referred to be a lytic
phage. The genes related to antibiotic resistance in P. aeruginosa, host virulence
factors and toxin genes were also absent in vB_PaeP_PS28 phage genome. The
phylogenetic analysis (Figure 15b) shows that vB_PaeP_PS28 is closely related to
Pseudomonas phage vB_PaeP_FBPa1 (GenBank Acc. No ON857943.1), which is a
member of the family Podoviridae and the genus Litunavirus. Additionally,
phylogenetic trees were created for some of the predicted essential phage proteins;
the terminase large subunit (Figure 15c) and RNA polymerase large subunit (Figure
15d). Importantly, the genomic sequence similarity of the phage vB_PaeP_PS28 to
other previously characterized phages infecting P. aeruginosa was determined. As
shown in (Table 9, Figure 16 and Figure 17), greatest similarity was observed
between the phage vB_PaeP_PS28 and Pseudomonas phage vB_PaeP_FBPa1
(GenBank Acc. No. ON857943.1, identity, 94.81%); Pseudomonas phage
VB_PaeS_VL1 (GenBank Acc. No. OK665488.1, identity, 94.37%); Pseudomonas
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phage YH6 (GenBank Acc. No. KM974184.1, identity, 94.07%); and Pseudomonas
phage PA26 (GenBank Acc. No. NC_041907.1, identity, 94.04%).
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a)
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Results
b)
c)
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Results
d)
Figure 15. Genomic characterization of vB_PaeP_PS28. (a) Circular genomic map of

vB_PaeP_PS28; from inside to outside, the first to third circles represent the scale, GC Skew [the
normalized excess of C over G in a given sequence, (C − G)/(C + G)], and GC content respectively;
the fourth represents the position of ORFs. The prediction and direction of ORFs are indicated by
arrow heads. The genomic map was generated and visualized using CGView (b) Phylogenetic
analysis of vB_PaeP_PS28 and other closely related sequences. (c) Phylogenetic tree analysis
based on the amino acid sequence of terminase large subunit. (d) Phylogenetic tree analysis based
on the amino acid sequence of RNA polymerase large subunit. Phylogenetic trees were constructed
using BLAST (Basic Local Alignment Search Tool) using neighbor-joining method
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Results
Table 8: Predicted ORFs identified in the phage vB_PaeP_PS28
Amino acid sequence

Coding GC Protein MW BLAST score
Start…..End Putative function identity/similarity Accession no
Sequence (%) length (KDa) Gene name (E-Value)
to best homologs
ORF1 51….9608 56.25 3185 347.48 RNA Transcription process Virion-associated RNA polymerase 0 QKE55114.1
polymerase and DNA replication [Pseudomonas phage PAP02]
ORF2 9624….9827 50.00 67 7.42 Hypothetical Unknown function Hypothetical protein FG40_gp65 2e-07 YP_009031842.1
protein [Pseudomonas phage vB_PaeP_C2-
10_Ab09]
ORF3 9837....1015 57.55 105 11.47 Hypothetical Unknown function Hypothetical protein PP-LIT1_gp69 3e-70 YP_003358466.1
4 protein [Pseudomonas phage LIT1]
ORF4 10151...103 55.27 78 8.88 Hypothetical Unknown function Hypothetical protein PAP02_040 1e-48 QKE55111.1
87 protein [Pseudomonas phage PAP02]
ORF5 10687....108 47.44 51 5.76 Hypothetical Unknown function Hypothetical protein [Pseudomonas 3e-27 AWY02728.1
42 protein phage LP14]
ORF6 10793....111 57.04 134 14.52 Hypothetical Unknown function Hypothetical protein ACQ34_gp70 7e-94 YP_009152570.1
97 protein [Pseudomonas phage YH6]
ORF7 11255....116 62.79 145 14.64 Hypothetical Unknown function Hypothetical protein vB_Pae575P- 1e-39 ANT44344.1
92 protein 3_65 [Pseudomonas phage
vB_Pae575P-3]
ORF8 11704....122 54.19 186 20.41 N4 gp46-like Structural protein N4 gp46-like protein [Pseudomonas 1e-126 YP_003358462.1
64 protein (tubular tail A phage LIT1]
homologue)
ORF9 12252....126 56.31 147 17.15 N4 gp48-like Structural protein N4 gp48-like protein [Pseudomonas 1e-87 YP_003358461.1
95 protein (major capsid) phage LIT1]
ORF10 12692....130 55.19 121 13.83 Hypothetical Unknown function Hypothetical protein BIZ95_gp65 2e-83 YP_009290599.1
57 protein [Pseudomonas phage
vB_PaeP_MAG4]
ORF11 13061....138 58.90 248 26.78 Putative DNA replication Putative single-stranded DNA- 1e-157 SBT96838.2
07 single- binding protein [Pseudomonas
stranded aeruginosa]
DNA-binding
protein
ORF12 13834....145 58.90 244 28.08 Hypothetical Unknown function Hypothetical protein FDH24_gp59 0.0 YP_009598413.1
68 protein [Pseudomonas phage PA26]
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Table8:8:Continued
Table Continued
ORF13 14617....167 58.90 722 82.15 Hypothetical Unknown function Hypothetical protein BIZ95_gp62 0.0 YP_009290596.1
vB_PaeP_MAG4]
ORF14 16800....178 54.60 336 37.8 Hypothetical Unknown function Hypothetical protein FBPa1_0077 0.0 UVN14432.1
vB_PaeP_FBPa1]
ORF15 17812....181 56.69 126 14.4 Hypothetical Unknown function Hypothetical protein [Pseudomonas 6e-81 UGL60992.1
92 protein phage vB_PaeS_TUMS_P81]
ORF16 18189....186 57.58 142 15.76 Hypothetical Unknown function Hypothetical protein ACQ34_gp60 4e-99 YP_009152560.1
17 protein [Pseudomonas phage YH6]
ORF17 18820....189 53.80 56 6.38 Hypothetical Unknown function Hypothetical protein FBPa1_0074 1e-30 UVN14429.1
vB_PaeP_FBPa1]
ORF18 19011....203 52.44 429 46.32 Tail fiber Phage assembly Tail fiber protein [Pseudomonas 0.0 ANT44334.1
00 protein (Tail morphogenesis) phage vB_Pae575P-3]
vB_PaeP_FBPa1]
vB_PaeP_FBPa1]
ORF21 21303....245 55.03 1085 118.09 Tail fiber Phage assembly Putative tail fiber protein 0.0 YP_009286275.1
60 protein (Tail morphogenesis) [Pseudomonas phage PEV2]
vB_PaeP_MAG4]
vB_PaeP_MAG4]
ORF24 25106....256 58.29 174 19.6 Major capsid Phage structural Major capsid protein [Pseudomonas 1e-123 UVN14423.1
30 protein assembly phage vB_PaeP_FBPa1]
(Head
morphogenesis)
ORF25 25627....261 56.14 170 18.68 Endopeptidas Host cell lysis gene Endopeptidase protein 1e-103 QKE55097.1
39 e protein [Pseudomonas phage PAP02]
ORF26 26202....267 52.51 185 21.36 Hypothetical Unknown function Hypothetical protein vBPaeSVL1_52 1e-128 UGV19848.1
59 protein [Pseudomonas phage VB_PaeS_VL1]
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Results
Table 8: Continued
ORF27 26752....269 50.00 63 7.2 HNH Phage DNA packaging HNH endonuclease [Pseudomonas 1e-32 YP_009226156.1
43 endonuclease phage YH30]
vB_PaeP_FBPa1]
ORF30 27666....294 60.17 589 63.4 Putative rIIB- Lysis inhibition, Putative rIIB-like protein 0.0 UGV19844.1
35 like protein interfere with cell [Pseudomonas phage VB_PaeS_VL1]
metabolism
ORF31 29447....319 55.36 842 95.11 Putative rIIA- Lysis inhibition, Putative rIIA-like protein 0.0 YP_009152547.1
75 like protein interfere with cell [Pseudomonas phage YH6]
metabolism
ORF32 31979....321 56.77 63 7.2 HNH Phage DNA packaging HNH endonuclease [Pseudomonas 5e-40 YP_009152546.1
70 endonuclease phage YH6]
vB_PaeP_FBPa1]
ORF34 32740....333 55.61 189 20.62 Putative Nucleotide Putative dCMP deaminase 2e-113 UGV19839.1
09 dCMP metabolism and DNA [Pseudomonas phage VB_PaeS_VL1]
deaminase replication
ORF35 33391....360 54.51 871 97.96 Putative DNA DNA replication Putative DNA polymerase 0.0 AWY02758.1
06 polymerase [Pseudomonas phage LP14]
ORF36 36006....365 54.92 175 19.98 Hypothetical Unknown function Hypothetical protein PAP02_016 2e-125 QKE55087.1
ORF37 36533....376 56.64 388 44.08 Putative DNA DNA replication Putative DNA helicase 0.0 YP_009148216.2
99 helicase [Pseudomonas phage Pa2]
ORF38 37789....390 56.40 408 45.58 Putative Lysis bacterial cell Putative metallopeptidase domain 0.0 YP_009152539.1
15 metallopeptid wall peptidoglycan [Pseudomonas phage YH6]
ase domain
vB_PaeP_FBPa1]
ORF40 39539....406 57.80 356 40.04 ATPase Energy production ATPase [Pseudomonas phage YH30] 0.0 YP_009226170.1
09 during phage DNA
packaging
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Results
Table 8: Continued
vB_PaeP_FBPa1]
ORF42 40880....417 57.67 277 30.99 ATP- Energy dependent ATP-dependent Clp protease ATP- 0.0 UGV19832.1
13 dependent Clp protein degradation binding subunit [Pseudomonas
protease ATP- phage VB_PaeS_VL1]
binding
subunit
ORF43 41772....419 49.46 61 7.07 Hypothetical Unknown function Hypothetical protein FDH24_gp30 4e-37 YP_009598384.1
ORF44 41966....421 44.16 76 8.4 Hypothetical Unknown function Hypothetical protein vB_pae575P- 2e-31 ANT44308.1
96 protein 3_30a [Pseudomonas phage
vB_Pae575P-3]
vB_PaeP_MAG4]
ORF46 42652....431 54.31 169 18.96 Hypothetical Unknown function Hypothetical protein [Pseudomonas 3e-65 QHZ59466.1
61 protein phage LY218]
vB_PaeP_MAG4]
vB_PaeP_FBPa1]
ORF51 45217....453 49.67 50 5.7 Membrane Lysis protein Membrane protein [Pseudomonas 2e-27 QKE55073.1
69 protein phage PAP02]
ORF52 45467....467 55.72 413 47.13 RNA Transcription and Putative RNA polymerase II (RNAP2) 0.0 ANT44300.1
08 polymerase mRNA processing [Pseudomonas phage Pa2]
large subunit
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Results
Table 8: Continued
vB_PaeP_FBPa1]
vB_Pae575P-3]
ORF56 47588....485 50.16 310 35.94 RNA Transcription of RNA polymerase small subunit 0.0 YP_009226181.1
20 polymerase phage protein [Pseudomonas phage YH30]
small subunit
vB_PaeP_FBPa1]
vB_PaeP_FBPa1]
ORF59 49355....496 54.37 83 9.62 Transcriptiona Transcriptional Transcriptional regulator 6e-50 YP_009226097.1
06 l regulator regulator [Pseudomonas phage YH30]
vB_PaeP_FBPa1]
ORF62 50557....508 61.86 96 10.65 Hypothetical Unknown function Hypothetical protein FG40_gp12 2e-60 YP_009031789.1
47 protein [Pseudomonas phage vB_PaeP_C2-
10_Ab09]
vB_PaeP_FBPa1]
ORF64 51197....514 53.60 73 8.03 Hypothetical Unknown function Hypothetical protein [Pseudomonas 5e-41 UNY40717.1
18 protein phage CMS1]
ORF65 51415....516 56.84 77 8.57 Hypothetical Unknown function Hypothetical protein [Pseudomonas 3e-44 AIZ94943.1
48 protein phage phi176]
vB_PaeP_FBPa1]
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Table 8: Continued
vB_PaeP_FBPa1]
ORF69 52382....526 50.81 102 12.08 Wall- DNA metabolism and Wall-associated receptor kinase-like 1e-65 YP_009226107.1
90 associated replication 20-like protein [Pseudomonas
receptor phage YH30]
kinase-like 20-
like protein
ORF70 52687....529 47.62 83 9.83 Hypothetical Unknown function Hypothetical protein P3P1_05 3e-35 SBT96754.2
38 protein [Pseudomonas aeruginosa]
ORF71 52935....531 49.15 77 8.95 Putative Transcriptional Putative transcriptional regulator 2e-28 UGV19802.1
68 transcriptional regulator [Pseudomonas phage VB_PaeS_VL1]
regulator
vB_PaeP_FBPa1]
vB_PaeP_FBPa1]
ORF74 54016....542 52.55 84 9.6 Hypothetical Unknown function Hypothetical protein FG40_gp01 1e-50 YP_009031778.1
70 protein [Pseudomonas phage vB_PaeP_C2-
10_Ab09]
ORF77 55827....561 53.62 91 10.68 Hypothetical Unknown function Hypothetical protein [Pseudomonas 4e-34 UEP18636.1
02 protein phage vB_PaeP_TUMS_P121]
vB_PaeP_FBPa1]
vB_PaeP_MAG4]
vB_PaeP_FBPa1]
ORF81 57534....591 53.18 550 62.77 Terminase Packing process and Terminase large subunit 0.0 UVN14366.1
86 large subunit phage assembly [Pseudomonas phage
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Table 8: Continued
vB_PaeP_FBPa1]
ORF82 59183....599 50.88 244 27.91 Putative tail Phage assembly Putative tail protein [Pseudomonas 8e-178 YP_009226120.1
17 protein (Tail morphogenesis) phage YH30]
ORF83 59950....602 56.00 99 11.1 ABC Mediate translocation ABC transporter-like protein 5e-63 YP_009226121.1
49 transporter- to cell surface [Pseudomonas phage YH30]
like protein
ORF84 60253....606 55.00 139 14.98 Putative DNA replication and Putative dUTPase [Pseudomonas 8e-94 YP_009286305.1
72 dUTPase metabolism phage PEV2]
ORF85 60707....628 54.20 726 81.66 Portal protein Translocation of Portal protein [Pseudomonas phage 0.0 YP_009206215.1
87 phage DNA and DL64]
packaging process
(virion assembly)
ORF87 63295....644 52.28 393 44.13 Hypothetical Unknown function Hypothetical protein vBPaeSVL1_82 0.0 UGV19878.1
76 protein [Pseudomonas phage VB_PaeS_VL1]
ORF88 64511....657 59.75 399 44.06 Major capsid Phage structural Major capsid protein [Pseudomonas 0.0 UGV19877.1
10 protein assembly phage VB_PaeS_VL1]
(Head
morphogenesis)
vB_Pae575P-3]
ORF90 66436....674 55.49 321 35.21 Structural Structural protein Structural protein [Pseudomonas 0.0 ANT44353.1
01 protein phage vB_Pae575P-3]
ORF91 67459....696 53.13 740 82.22 Hypothetical Unknown function Hypothetical protein [Pseudomonas 0.0 UGL60976.1
81 protein phage vB_PaeS_TUMS_P81]
vB_PaeP_FBPa1]
ORF93 70129....716 55.43 521 57.27 Putative lytic Structural protein Lytic tail fiber [Pseudomonas phage 0.0 YP_009226131.1
94 tail fiber (Tail fiber YH30]
protein morphogenesis)
ORF94 71674....721 57.02 158 16.78 Hypothetical Unknown function Hypothetical protein [Pseudomonas 4e-65 QHZ59508.1
50 protein phage LY218]
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Table 9: Homology of vB_PaeP_PS28 to other phages

Accession
Scientific name Percent identity Accession
length (bp)
Pseudomonas phage vB_PaeP_FBPa1 94.81% 72814 ON857943.1
Pseudomonas phage VB_PaeS_VL1 94.37% 73308 OK665488.1
Pseudomonas phage YH6 94.07% 73050 KM974184.1
Pseudomonas phage PA26 94.04% 72321 NC_041907.1
Pseudomonas phage LP14 94.02% 73080 MH356729.1
Pseudomonas phage DL64 94.01% 72378 KR054032.1
Pseudomonas phage vB_Pae1396P-5 94.00% 72508 KX171210.1
Pseudomonas phage vB_Pae575P-3 94.00% 72728 KX171209.1
Pseudomonas phage PAP02 93.97% 73345 MT080102.1
Pseudomonas phage vB_PaeP_DEV 93.90% 72697 MF490238.1
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Figure 16. Comparative genomic analysis between phage vB_PaeP_PS28 and related
sequences. Pseudomonas phage vB_PaeP_FBPa1 (GenBank Acc. No. ON857943.1),
Pseudomonas phage VB_PaeS_VL1 (GenBank Acc. No. OK665488.1), Pseudomonas phage YH6
(GenBank Acc. No. KM974184.1) and Pseudomonas phage PA26 (GenBank Acc. No.
NC_041907.1). Sequence similarity is represented by the gray scale bar. The coding sequences are
represented by directional arrows. Predicted ORFs in vB_PaeP_PS28 genome are listed below.
Comparative analysis was performed using Easyfig.
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Figure 17. Dot Plot comparisons of the genomic nucleotide sequences of vB_PaeP_PS28
and related bacteriophages infecting P. aeruginosa. a) Pseudomonas phage vB_PaeP_FBPa1
(GenBank Acc. No. ON857943.1). b) Pseudomonas phage YH6 (GenBank Acc. No.
KM974184.1). c) Pseudomonas phage PA26 (GenBank Acc. No. NC_041907.1). d)
Pseudomonas phage VB_PaeS_VL1 (GenBank Acc. No. OK665488.1).
13.2. Genomic characterization of vB_PaeM_PS3
The genome of vB_PaeM_PS3 comprises 93,922 bp of dsDNA with 49.39 %

G + C content and was found to be a member of the Myoviridae family and the genus
Pakpunavirus. The genome sequence of the phage vB_PaeP_PS28 has been
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deposited in the GenBank database under GenBank Acc. No OQ411628. As shown

in table 10 and figure 18a, a total of 171 ORFs were identified, of which 27 (15.8%)
were assigned as functional proteins whereas 144 (84.2%) ORFs were annotated as
hypothetical proteins. The functional proteins were divided into four major classes;
structure proteins (ORF 17, ORF 20, ORF 31, ORF 35, ORF 39 and ORF 41); DNA
metabolism, repair and replication which involves components that help in viral
replication and enzymes responsible for modification of infected cell surface
polysaccharides (ORF 51, ORF 61, ORF 64, ORF 66, ORF 67, ORF 68, ORF 75,
ORF 86, ORF 88, ORF 90, ORF 91, ORF 157, ORF 159, ORF 161, ORF 165, ORF
170 and ORF 171); packaging and assembly proteins (ORF 15) and finally host cell
lysis (ORF 10, ORF 42, ORF 167 and ORF 168). Interestingly, a cluster of 14 tRNA
genes was predicted in the phage vB_PaeM_PS3 genome and listed in Table 11 that
enhance phage protein translation that make the phage independent on the host. Of
note that neither lysogenic genes nor host related sequences were identified in
vB_PaeM_PS3 genome, confirming the lytic nature of vB_PaeM_PS3. In addition,
the genes related to P. aeruginosa antibiotic resistance as well as toxins and
virulence proteins production were absent in vB_PaeM_PS3 genome. Phylogenetic
analysis based on the overall similarity with completely sequenced phage genomes
in database shows that vB_PaeM_PS3 shares greatest nucleotide similarity with
Pseudomonas phage vB_PaeM_SCUT-S2 (GenBank Acc. No MK340761.1),
Pseudomonas phage vB_PaM_EPA1 (GenBank Acc. No MN013356.1),
Pseudomonas phage PaYy-2 (GenBank Acc. No MH725810.1) and Pseudomonas
phage SRT6 (GenBank Acc. No MH370478.1) representing percent identities of
96%, 95.2%, 95% and 94.9%, respectively (Table 12, Figure 18b, c and Figure
19). Moreover, phylogenetic trees were constructed for the phage major capsid
protein and terminase large subunit to better illustrate the evolutionary relationships
of vB_PaeM_PS3 with previously characterized phages. As shown in Figure 20a
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and b, vB_PaeM_PS3 displayed close relation to other Caudovirale related

pseudomonas phages, specifically, Pseudomonas phage PAK P1 and Pseudomonas
phage PaYy-2, respectively. These results support and are in accordance with the
whole genome phylogeny.
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a)
b)
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c)
Figure 18. General features of vB_PaeM_PS3 genome. (a) Schematic genomic map of
vB_PaeM_PS3 phage. The inner rings represent genome location, GC skew + (green) and GC
skew (purple) and GC content (black). ORFs are represented by colored arrows. Functional ORFs
were classified into four groups: yellow; DNA packaging, red; DNA metabolism, repair and
replication, blue; structural proteins and purple; lysis proteins. Hypothetical proteins are indicated
in grey. The figure was generated using CGView program. (b) Phylogenetic tree of vB_PaeM_PS3
using whole genome sequence and other closely related sequences. Phylogenetic tree was
generated using MEGA-X computer program. (c) Comparative genomic analysis of the phage
vB_PaeM_PS3 with homologous phages. Similarity level among phage sequences is represented
by the colored scale bar from 20% to 100%. The coding sequences are represented by directional
arrows. Predicted ORFs in vB_PaeM_PS3 genome are listed below. Genomic comparison was
performed and plotted using Easyfig program.
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Figure 19. Dot Plot comparisons of the genomic nucleotide sequences of vB_PaeM_PS3 and
related bacteriophages infecting P. aeruginosa. a) Pseudomonas phage vB_PaeM_SCUT-S2
(GenBank Acc. No. MK340761.1). b) Pseudomonas phage vB_PaM_EPA1 (GenBank Acc. No.
MN013356.1). c) Pseudomonas phage PaYy-2 (GenBank Acc. No. MH725810.1). d)
Pseudomonas phage SRT6 (GenBank Acc. No. MH370478.1).
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Table 10: Predicted ORFs found in vB_PaeM_PS3
Amino acid sequence BLAST

Coding GC Protein MW
Start…..End Gene name Putative function identity/similarity score Accession no
Sequence (%) length (KDa)
to best homologs (EValue)
Hypothetical protein
ORF1 180......392 49.77% 71 7.48 Hypothetical protein Unknown function IttPL_0040 [Pseudomonas 9e-41 QBP28054.1
phage ITTPL]
ORF2 389......649 47.13% 87 9.66 Hypothetical protein Unknown function [Pseudomonas phage PaGz- 2e-53 QAX98104.1
1]
ORF3 662......1321 45.45% 220 25.39 Hypothetical protein Unknown function X831_gp162 [Pseudomonas 9e-163 YP_008857202.1
phage PAK_P2]
ORF4 1323......1670 49.43% 116 13.1 Hypothetical protein Unknown function PAK_P400162 [Pseudomonas 1e-78 YP_008859373.1
phage PAK_P4]
phage PAK_P4]
phage PAK_P4]
ORF7 2239......2424 54.30% 62 7.12 Hypothetical protein Unknown function 8e-31 WP_016064770.1
[Pseudomonas aeruginosa]
BN405_2-10_Ab1_orf_38
ORF8 2425......2718 54.42% 98 11.41 Hypothetical protein Unknown function 2e-66 YP_007236859.1
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
ORF9 2715......2900 46.77% 62 7.16 Hypothetical protein Unknown function PJG4_041 [Pseudomonas 3e-35 YP_007002487.1
phage JG004]
ORF10 2961......3509 48.27% 183 20.4 putative protease Protein degradation putative protease subunit 8e-110 YP_007002486.1
104
Results
Table 10: Continued
subunit [Pseudomonas [Pseudomonas phage JG004]
phage JG004]
phage JG004]
phage JG004]
ORF13 4852......5040 47.09% 63 6.93 Hypothetical protein Unknown function PaP1_gp044 [Pseudomonas 8e-39 YP_007236455.1
phage PaP1]
phage PAK_P1]
putative terminase
putative terminase large
large subunit Packaging process
ORF15 8877......10397 47.53% 507 57.09 subunit [Pseudomonas phage 0.0 YP_004327194.1
[Pseudomonas phage and phage assembly
PAK_P1]
PAK_P1]
BN405_2-10_Ab1_orf_48
ORF16 10410......11849 47.29% 480 54.33 Hypothetical protein Unknown function 0.0 YP_007236869.1
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
putative capsid and Phage structural
putative capsid and scaffold
scaffold [Pseudomonas assembly
ORF17 11859......12329 49.47% 157 17.16 [Pseudomonas phage 2e-98 YP_009623460.1
phage vB_PaeM_C2- (Head
vB_PaeM_C2-10_Ab02]
10_Ab02] morphogenesis)
ORF18 12326......13243 48.37% 306 33.07 Hypothetical protein Unknown function PaYy2_161 [Pseudomonas 0.0 AXY86955.1
phage PaYy-2]
phage JG004]
Phage structural
major capsid protein major capsid protein
assembly
ORF20 13725......14759 53.24% 345 39.38 [Pseudomonas phage [Pseudomonas phage 0.0 YP_004327199.1
(Head
PAK_P1] PAK_P1]
morphogenesis)
105
Results
Table 10: Continued
ORF21 14810......15286 48.01% 159 18.16 Hypothetical protein Unknown function PaoP5_059 [Pseudomonas 1e-112 YP_009224750.1
phage PaoP5]
BN405_2-10_Ab1_orf_54
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
ORF23 15737......16117 46.19% 127 14.35 Hypothetical protein Unknown function [Pseudomonas phage 1e-80 UGL61562.1
phipa10]
BN405_2-10_Ab1_orf_56
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
ORF25 16690......17976 51.20% 429 46.31 Hypothetical protein Unknown function Kat_gp015 [Pseudomonas 0.0 UQS93423.1
phage vB_Pae_Kat]
BN405_2-10_Ab1_orf_59
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
phage PAK_P1]
phage JG004]
phage JG004]
phage PaP1]
106
Results
Table 10: Continued
Tail and base plate
putative tape measure putative tape measure
structural
ORF31 20227......22593 50.99% 789 85.95 protein [Pseudomonas protein [Pseudomonas phage 0.0 ATI16080.1
component and
phage YS35] YS35]
assembly
phage vB_Pae_Kat]
ORF33 23357......23713 46.70% 119 13.99 Hypothetical protein Unknown function Kat_gp007 [Pseudomonas 1e-81 UQS93415.1
phage vB_Pae_Kat]
ORF34 23710......24627 45.64% 306 33.77 Hypothetical protein Unknown function X831_gp020 [Pseudomonas 0.0 YP_008857060.1
phage PAK_P2]
Tail and base plate
baseplate protein
structural baseplate protein
ORF35 24624......25364 48.72% 246 26.69 [Pseudomonas phage 0.0 YP_007236476.1
component and [Pseudomonas phage PaP1]
PaP1]
assembly
BN405_2-10_Ab1_orf_68
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
phage vB_Pae_Kat]
phage JG004]
tail fiber protein
Phage assembly tail fiber protein
(Tail morphogenesis) [Pseudomonas phage PaoP5]
PaoP5]
phage JG004]
putative tail fiber
Phage assembly putative tail fiber protein
ORF41 30461......31960 51.53% 500 53 protein [Pseudomonas 0.0 YP_009186965.1
(Tail morphogenesis) [Pseudomonas phage C11]
phage C11]
107
Results
Table 10: Continued
endolysin
endolysin [Pseudomonas
ORF42 31977......32537 47.24% 187 20.93 [Pseudomonas phage Cell lysis 1e-134 UQS93578.1
phage vB_Pae_Kat]
vB_Pae_Kat]
BN405_2-10_Ab1_orf_75
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
1]
phage JG004]
phage PAK_P2]
phage PaoP5]
phage PaoP5]
ORF49 34694......35506 51.54% 271 30.44 Hypothetical protein Unknown function [Pseudomonas phage PaZq- 0.0 QJC44187.1
1]
phage PaoP5]
Repair, splicing and
putative RNA ligase
editing pathways of putative RNA ligase
broken RNAs (RNA [Pseudomonas phage PaoP5]
PaoP5]
repair)
phage JG004]
108
Results
Table 10: Continued
ORF53 37111......37467 47.34% 119 13.17 Hypothetical protein Unknown function FDH21_gp131 [Pseudomonas 9e-70 YP_009598139.1
phage Zigelbrucke]
BN405_2-10_Ab1_orf_84
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
Hypothetical protein S2_091
ORF55 38001......38486 51.44% 162 18.92 Hypothetical protein Unknown function [Pseudomonas phage 7e-115 QAU05363.1
vB_PaeM_SCUT-S2]
phage vB_Pae_Kat]
1]
phage vB_Pae_Kat]
1]
1]
anaerobic NTP
anaerobic NTP reductase
reductase large subunit DNA replication and
ORF61 40259......40681 48.94% 141 16.51 large subunit [Pseudomonas 3e-101 QBJ04655.1
[Pseudomonas phage repair
phage JHP]
JHP]
phage PAK_P1]
109
Results
Table 10: Continued
phage PaP1]
putative DNA putative DNA
primase/helicase primase/helicase
ORF64 41006......41251 52.44% 82 9.21 DNA replication 2e-53 QAU05373.1
[Pseudomonas phage [Pseudomonas phage
vB_PaeM_SCUT-S2] vB_PaeM_SCUT-S2]
1]
putative DNA putative DNA
primase/helicase primase/helicase
ORF66 41487......43349 51.26% 621 70.57 DNA replication 0.0 YP_004327242.1
PAK_P1] PAK_P1]
putative DNA
putative DNA polymerase
polymerase
ORF67 43410......45419 50.05% 670 77.47 DNA replication [Pseudomonas phage 0.0 YP_008869170.1
[Pseudomonas phage
PAK_P1]
PAK_P1]
DNA polymerase A
DNA polymerase A family
family protein
ORF68 45704......46393 51.30% 230 25.44 DNA replication protein [Pseudomonas phage 4e-152 YP_009200042.1
[Pseudomonas phage
K8]
K8]
ORF69 46483......46881 51.63% 133 14.21 Hypothetical protein Unknown function [Pseudomonas phage PaZq- 1e-87 QAX99843.1
1]
phage PaP1]
phage JG004]
ORF72 47898......48902 51.74% 335 37.15 Hypothetical protein Unknown function [Pseudomonas phage PaZq- 0.0 QAX99846.1
1]
110
Results
Table 10: Continued
phage PAK_P1]
phage PAK_P1]
Putative Putative
DNA replication,
exodeoxyribonuclease exodeoxyribonuclease
ORF75 49471......50523 50.90% 351 40.03 modification and 0.0 YP_007236926.1
regulation
vB_PaeM_C2-10_Ab1] vB_PaeM_C2-10_Ab1]
phage PaoP5]
phage PaoP5]
ORF78 51463......51693 47.19% 77 8.61 Hypothetical protein Unknown function [Pseudomonas phage PaZq- 5e-46 QJC44194.1
1]
phage PaoP5]
phage PaoP5]
ORF81 52291......53070 51.03% 260 29.34 Hypothetical protein Unknown function PJG4_124 [Pseudomonas 0.0 YP_007002416.1
phage JG004]
phage PAK_P2]
[Pseudomonas phage
ORF83 53261......53470 45.71% 70 7.5 Hypothetical protein Unknown function 9e-31 QYC95157.1
PhL_UNISO_PA-
DSM_ph0034]
111
Results
Table 10: Continued
phage PAK_P1]
phage JG004]
3'-phosphatase, 5'- 3'-phosphatase, 5'-
polynucleotide kinase DNA metabolism and polynucleotide kinase
ORF86 54033......54989 48.38% 319 35.56 0.0 QAX99857.1
[Pseudomonas phage replication [Pseudomonas phage PaZq-
PaZq-1] 1]
phage vB_Pae_Kat]
FAD-dependent
Nucleotide FAD-dependent thymidylate
thymidylate synthase
ORF88 55193......55906 48.88% 238 27.17 metabolism and DNA synthase [Pseudomonas 3e-131 HCI1709201.1
[Pseudomonas
replication aeruginosa]
aeruginosa]
ORF89 56160......56504 48.99% 115 13.19 Hypothetical protein Unknown function Y35_GM000048 4e-80 ATI16021.1
[Pseudomonas phage YS35]
ribonucleotide-
ribonucleotide-diphosphate
diphosphate reductase
DNA replication and reductase beta subunit
ORF90 56521......57567 47.76% 349 40.34 beta subunit 0.0 YP_007236938.1
repair [Pseudomonas phage
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
vB_PaeM_C2-10_Ab1]
ribonucleoside-
ribonucleoside-diphosphate
diphosphate reductase
DNA replication and reductase alpha chain
ORF91 57560......59305 51.32% 582 66.64 alpha chain 0.0 QAX98182.1
repair [Pseudomonas phage PaGz-
[Pseudomonas phage
1]
PaGz-1]
1]
1]
112
Table 10: Continued Results
1]
1]
1]
1]
1]
1]
1]
phage PAK_P1]
1]
phage PaP1]
ORF104 62677......63663 49.24% 329 37.56 Hypothetical protein Unknown function [Pseudomonas phage PaGz- 0.0 QAX98195.1
1]
phage PAK_P1]
113
Results
Table 10: Continued
[Pseudomonas phage
PhL_UNISO_PA-
DSM_ph0034]
ORF107 65240......65506 50.56% 89 10.3 Hypothetical protein Unknown function BI047_gp158 [Pseudomonas 7e-58 YP_009291099.1
phage phiMK]
phage vB_Pae_Kat]
phage PaP1]
Hypothetical protein K8_149
[Pseudomonas phage K8]
phage PAK_P1]
phage PAK_P2]
phage PaP1]
phage JG004]
1]
phage PAK_P2]
1]
114
Results
Table 10: Continued
phage Zigelbrucke]
phage JG004]
1]
ORF121 69770......69994 54.67% 75 8.38 Hypothetical protein Unknown function AU075_gp065 [Pseudomonas 3e-38 YP_009187045.1
phage C11]
phage PAK_P1]
phage PaP1]
1]
phage PaP1]
1]
1]
1]
phage phiMK]
115
Results
Table 10: Continued
ORF130 73844......73960 48.72% 39 4.43 Hypothetical protein Unknown function PaSzw1_62 [Pseudomonas 1e-18 QAY01625.1
phage PaSzW-1]
MULTISPECIES: DUF551
ORF131 74066......74260 53.85% 65 7.6 Hypothetical protein Unknown function domain-containing protein 4e-39 WP_088172792.1
[Pseudomonas]
phage phiMK]
phage phiMK]
phage phiMK]
phage vB_Pae_Kat]
ORF136 75590......75820 53.68% 77 8.19 Hypothetical protein Unknown function FBPa2_0081 [Pseudomonas 2e-39 UVN13020.1
phage vB_PaeM_FBPa2]
[Pseudomonas phage
PhL_UNISO_PA-
DSM_ph0034]
phage Zigelbrucke]
phage PAK_P1]
ORF140 77422......78015 48.82% 198 22.68 Hypothetical protein Unknown function [Pseudomonas phage 1e-143 CEF89317.1
vB_PaeM_C2-10_Ab08]
116
Results
Table 10: Continued
BN405_2-10_Ab1_orf_02
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
phage PAK_P2]
ORF143 79167…….79583 46.28% 139 16.01 Hypothetical protein Unknown function FDH21_gp004 [Pseudomonas 8e-98 YP_009598056.1
phage Zigelbrucke]
ORF144 79606…….80292 50.51% 229 27.13 Hypothetical protein Unknown function PaP1_gp005 [Pseudomonas 2e-153 YP_007236416.1
phage PaP1]
BN405_2-10_Ab1_orf_06
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
phage Zigelbrucke]
phage PAK_P4]
PAK_P100139c
[Pseudomonas phage
PAK_P1]
phage JG004]
BN405_2-10_Ab1_orf_12
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
ORF151 82339......82716 49.47% 126 14.99 Hypothetical protein Unknown function Henu5_gp69 [Pseudomonas 2e-87 QAU05099.1
phage Henu5]
117
Results
Table 10: Continued
phage vB_Pae_Kat]
phage vB_Pae_Kat]
phage PaP1]
phage vB_Pae_Kat]
1]
nicotinamide nicotinamide
phosphoribosyltransfer phosphoribosyltransferase
ORF157 84472......86160 50.98% 563 62.92 DNA modification 0.0 UQS93463.1
ase [Pseudomonas [Pseudomonas phage
phage vB_Pae_Kat] vB_Pae_Kat]
phage vB_Pae_Kat]
ribose-phosphate ribose-phosphate
pyrophosphokinase DNA metabolism and pyrophosphokinase
ORF159 86432......87307 49.20% 292 31.71 0.0 UQS93461.1
[Pseudomonas phage replication [Pseudomonas phage
vB_Pae_Kat] vB_Pae_Kat]
1]
putative RNA ligase/tail
putative RNA ligase/tail
attachment protein DNA modification
ORF161 87748......88665 49.24% 306 34.82 attachment protein 0.0 YP_007002506.1
[Pseudomonas phage and repair
[Pseudomonas phage JG004]
JG004]
ORF162 88677......89084 47.55% 136 15.13 Hypothetical protein Unknown function [Pseudomonas phage 9e-59 AZF89735.1
vB_PaeM_LCK69]
ORF163 89081......89356 45.65% 92 10.42 Hypothetical protein Unknown function Hypothetical protein 5e-61 YP_007002504.1
118
Results
Table 10: Continued
PJG4_024 [Pseudomonas
phage JG004]
phage JG004]
Splicing of t-RNA
putative
introns and cellular putative phosphoesterase
phosphoesterase
ORF165 89607......90161 47.93% 185 21.93 processes [Pseudomonas phage 4e-119 YP_007236845.1
[Pseudomonas phage
Or, DNA metabolism vB_PaeM_C2-10_Ab1]
vB_PaeM_C2-10_Ab1]
and replication
ORF166 90161......90592 48.61% 144 17.02 Hypothetical protein Unknown function PJG4_027 [Pseudomonas 3e-98 UQS93454.1
phage JG004]
putative
phosphohydrolase Cell lysis (hydrolyzing putative phosphohydrolase
ORF167 90582......91142 49.02% 187 21.08 5e-126 YP_007002500.1
[Pseudomonas phage peptidoglycan) [Pseudomonas phage JG004]
JG004]
putative cell wall
putative cell wall hydrolase
hydrolase
ORF168 91144......91704 50.45% 187 21.48 Bacterial cell lysis [Pseudomonas phage 1e-137 YP_007236848.1
[Pseudomonas phage
vB_PaeM_C2-10_Ab1]
vB_PaeM_C2-10_Ab1]
ORF169 91769......92230 48.05% 154 17.26 Hypothetical protein Unknown function Henu5_gp86 [Pseudomonas 3e-110 QAU05115.1
phage Henu5]
putative DNA ligase
DNA replication and putative DNA ligase
repair [Pseudomonas phage PaoP5]
PaoP5]
putative CMP/dCMP putative CMP/dCMP
Nucleotide
deaminase, zinc-binding deaminase, zinc-binding
ORF171 93448......93864 50.60% 139 15.41 metabolism and DNA 2e-95 YP_004327179.1
replication
PAK_P1] PAK_P1]
119
Results
Table 11: tRNAs identified in the vB_PaeM_PS3 genome
tRNA No. Location (bp) Length (bp) Amino acid Anticodon

1 5512-5585 74 Gln TTG
2 5892-5966 75 Arg TCT
3 5976-6052 77 Lys TTT
4 6327-6411 85 Leu TAG
5 6620-6695 76 Ile GAT
6 6705-6783 79 Asp GTC
7 7195-7270 76 Cys GCA
8 7281-7356 76 Asn GTT
9 7419-7496 78 Pro TGG
10 7667-7742 76 Gly TCC
11 8263-8339 77 Phe GAA
12 8346-8421 76 Glu TTC
13 8482-8556 75 His GTG
14 8743-8817 75 Thr TGT
120
Results
a)
b)
121
Results
Figure 20. Phylogenetic trees of vB_PaeM_PS3 based on the amino acid sequences of major
capsid protein (a) and terminase large subunit (b). Phylogenetic trees were constructed using
MEGA-X using neighbor-joining method.
Table 12. Homology of phage vB_PaeM_PS3 to other phages genomes

Percent Accession
Scientific Name Accession No.
Identity Length (bp)
Pseudomonas phage vB_PaeM_SCUT-S2 96% 94434 MK340761.1
Pseudomonas phage vB_PaM_EPA1 95.20% 91394 MN013356.1
Pseudomonas phage PaYy-2 95.00% 92348 MH725810.1
Pseudomonas phage SRT6 94.90% 91364 MH370478.1
Pseudomonas phage PaoP5 94.50% 93464 KU297675.1
Pseudomonas phage C11 94.08% 94109 KT804923.1
Pseudomonas phage phiMK 94.52% 93129 KU761955.1
Pseudomonas phage PhL_UNISO_PA- 96.77% 92408 MW526259.2
DSM_ph0034
Pseudomonas phage Zigelbrucke 96.04% 92338 NC_041904.1
Pseudomonas phage PaGz-1 94.00% 93975 MH791399.2
14.1. Characterization of the influence of both vB_PaeP_PS28 and

vB_PaeM_PS3 on P. aeruginosa pathogenesis in vivo using mice infection model
The effect of vB_PaeP_PS28 on P. aeruginosa pathogenesis was evaluated in

vivo using mice infection model. Mice survival as well as both bacterial and phage
counts were monitored in infected mice. Importantly, phage-injected mice and
negative control (non-infected and PBS-injected) mice exhibited 100% survival. On
the other hand, all mice infected with P. aeruginosa died at 24 hr post inoculation.
However, the mortality rate of mice infected with P. aeruginosa and treated with
122
Results
vB_PaeP_PS28 dramatically decreased as compared to mice infected with bacteria

alone (Figure 21a). In addition, bacterial loads were determined in organs isolated
from infected mice. As shown in (Figure 21b, c), the number of viable bacteria in
the liver and spleen isolated from mice infected with P. aeruginosa and treated with
vB_PaeP_PS28 (3193 ± 12, 5134 ± 13 CFUs/g, respectively) was significantly lower
than that of mice infected with P. aeruginosa alone (36 x 104 ± 23, 88 x 104 ± 15
CFUs/g, respectively). Of note that, the phage titers were determined in isolated
organs from phage-injected mice as well as mice infected with P. aeruginosa and
treated with phage. The phage titer in liver and spleen isolated from P. aeruginosa
infected mice and treated with vB_PaeP_PS28 was significantly higher (10241 ± 23,
8520 ± 13, PFU/mL, respectively) as compared to phage injected mice (1250 ± 11,
1005 ± 10, PFU/ mL, respectively). Importantly, the phage vB_PaeP_PS28 was
rapidly cleared from the liver and spleen isolated from phage-injected mice.
Importantly, the phage-injected mice did not develop any abnormal symptoms over
the experiment course. These findings clearly demonstrate that vB_PaeP_PS28
phage is promising for therapeutic use and would be cleared from the body without
any harmful effects on the patient.
123
Results
a)
100
*** P < 0.001
Control mice (PBS-injected)
80 Control mice (Uninfected)
P. aeruginosa infected
Percent survival
vB_PaeP_PS28 injecetd
60 P. aeruginosa and vB_PaeP_PS28 injected
40
* P < 0.05
20
0
24 48 72 96
Time (hr)
Figure 21. In vivo characterization of the influence of phage vB_PaeP_PS28 on P. aeruginosa

pathogenesis in mice infection model. (a) Survival curves of mice infected with P. aeruginosa
124
Results
and treated with isolated phage. Mice in first group were infected with P. aeruginosa (2.5x107
CFU/mL), mice in second group were injected with vB_PaeP_PS28 (2.5 × 109 PFU/mL) and mice
in third group were infected with P. aeruginosa and treated with the phage vB_PaeP_PS28.
Uninfected and PBS-injected mice served as negative controls. Mice survival was monitored in
each group daily for 4 days and plotted using Kaplan-Meier survival curve. Bacterial burden and
phage titers were determined in liver (b) and spleen (c) of infected mice. Mice were
anesthetized, liver and spleen were obtained and homogenized for enumeration of CFU and PFU
at 24, 48, 72 hr post infection. Of note that, bacterial load in P. aeruginosa infected mice was
determined at 24 hr post infection only as all mice in this group died after 24 hr. Bacterial and
phage load were represented on left and right y axis, respectively. Data are expressed as means ±
SE of three independent experiments.
Similarly, the impact of vB_PaeM_PS3 on P. aeruginosa virulence was

assessed in vivo. The mice infected with P. aeruginosa showed 100% mortality rate
at 24 hr. On contrast, the survival rate of P. aeruginosa infected mice was
significantly improved (66.7%) following treatment with vB_PaeM_PS3 (Figure
22a). Importantly, no death was recorded either in mice injected with vB_PaeM_PS3
alone or negative control mice and they remained healthy over the course of
experiment. Additionally, both bacterial burden and vB_PaeM_PS3 titer were
determined in isolated mice organs. Interestingly, treatment of P. aeruginosa-
infected mice with vB_PaeM_PS3 effectively lowered Pseudomonas colonization
in mice organs. The results revealed that bacterial loads in liver and spleen isolated
from P. aeruginosa mice were markedly higher (27 x 104 ± 28, 52 x104 ± 21 CFUs/g,
respectively) compared to those of phage-untreated mice (4837 ± 16, 9210 ± 17
CFUs/g, respectively) (Figure 22b, c). The phage titer in liver and spleen isolated
from P. aeruginosa infected mice and treated with vB_PaeM_PS3 was significantly
higher (11056 ± 27, 16405± 35, PFU/mL, respectively) as compared to phage
125
Results
injected mice (4029 ± 15, 5560 ± 18, PFU/ mL, respectively). It is worth mentioning
that the phage vB_PaeM_PS3 was completely eliminated and not detected in organs
isolated from mice at 72 hr post-inoculation.
a)
100
*** P < 0.001
Control mice (PBS-injected)
80 Control mice (Uninfected)
P. aeruginosa infected
Percent survival
vB_PaeM_PS3 injecetd
60 P. aeruginosa and vB_PaeM_PS3 injected
40 * P < 0.05
20
0
24 48 72 96
Time (hr)
126
Results
Figure 22. Impact of phage vB_PaeM_PS3 on P. aeruginosa pathogenesis in vivo. a) Survival

rate of mice infected with P. aeruginosa and treated with isolated phage. Mice survival was
plotted using Kaplan-Meier survival curve and statically analyzed using Log-rank test. Bacterial
count and phage titer in liver (b) and spleen (c) isolated from mice at 24, 48 and 72 hr after
infection. Bacterial and phage load were represented on left and right y axis, respectively. Data are
presented as means ± SE of three independent experiments
127
Discussion
Discussion
P. aeruginosa is a serious nosocomial pathogen which causes a variety of
health illnesses due to increased prevalence of resistant strains (Gellatly and
Hancock, 2013). P. aeruginosa is responsible for a majority of infections including
pneumonia, urinary tract and lung infections (Litwin et al., 2021). Numerous
virulence factors contribute to P. aeruginosa pathogenesis including hemolysins,
rhamnolipids, proteases and biofilms (Lee and Zhang, 2015). The extensive use of
antibiotics and improper antibiotic dispensing policy could lead to continuous
increase of antibiotic resistance strains and infectious diseases (Nitsch-Osuch et
al., 2016). Over the past few decades, there has been a lack of novel antibiotic
discoveries (Ventola, 2015). Development of new antibiotics takes a long time and
is extremely cost-effective. In addition, fast antibiotic resistance progress shortens
their lifespan (Fernandes and Martens, 2017). The lack of therapeutic alternatives
means that infections caused by antibiotic-resistant bacteria pose a considerable
threat regarding morbidity and mortality worldwide (Tacconelli et al., 2018).
Therefore, bacteriophages are seemed to be efficient alternatives in management of
infections caused by P. aeruginosa (Pires et al., 2015).
In the current study, a total of 50 P. aeruginosa isolates were obtained from

patients admitted to Zagazig University Hospitals and El-Ahrar Educational
Hospital in the period from April 2021 to August 2021 with a prevalence rate of
41.6%, which is in parallel with other studies in Egypt that reported prevalence rate
of 35% and 32.8% (Mansour et al., 2013; Raouf et al., 2018). However, other
studies in Iraq and Egypt by Hasan et al. (2020) and Gad et al. (2007), respectively
have found a lower prevalence rate of 20.1 and 18.6%, respectively than that
reported in the present study. Variations in the prevalence rates of P. aeruginosa in
various studies could be attributed to the type of clinical specimens, studied
128
Discussion
population, geographical locations and differences in hygienic practices (Hasan et

al., 2020).
P. aeruginosa causes a wide range of serious infections mainly in

immunocompromised hosts such as those with severe burns or wound surgery as
well as in those with indwelling devices (Bezalwar and Charde, 2019; Shidiki et
al., 2019). In this study the highest number of P. aeruginosa was isolated from
endotracheal aspirates followed by urine, burn and surgical wound infections. This
is in agreement with similar observation conducted in Saudi Arabia (Mansour et
al., 2013) and in Brazil (Lima et al., 2017). Most of the other studies showed that
the highest isolation source was from pus specimens (Mahmoud et al., 2017;
Corehtash et al., 2015).
Bacterial resistance to antibiotics is a critical problem worldwide and has

extensively increased among pathogenic bacteria (Weber and Rutala, 2006). P.
aeruginosa isolates were tested for their susceptibility to a panel of antimicrobial
chemotherapeutic agents. P. aeruginosa isolates exhibited a higher resistance
towards multiple antibiotics including aminoglycosides, fluoroquinolones and
monobactam; revealing that 56% of isolated P. aeruginosa strains were MDR
which is in accordance with Inan et al. (2000) who reported that 60% of P.
aeruginosa isolates were MDR. The increase in MDR has been observed in
many studies in Egypt (Gad et al., 2007; Hassuna et al., 2015). Additionally,
MDR rate of 19.6%, 89.4% and 100% among P. aeruginosa isolates had been
reported from studies conducted elsewhere including; Malaysia (Pathmanathan et
al., 2009), Nepal (Bhandari et al., 2012) and Iran (MOAZAMI and Eftekhar,
2013), respectively.
129
Discussion
This high level of resistance could be explained by the lack of proper

infection control measures and the extensive abuse of different antimicrobials. For
instance, a study carried out in Minia showed that β-lactams and macrolides were
commonly prescribed both by doctors and pharmacists for common cold (Dooling
et al., 2014). Importantly, in various low-income countries, antibiotics are readily
available over the counter in community pharmacies (Mukonzo et al., 2013;
Najjuka et al., 2016) and this portends high rates of antimicrobial resistance in
low-income countries (Kateete et al., 2017). MDR P. aeruginosa develops
resistance by various mechanisms like multidrug resistance efflux pumps,
production of β-lactamases, aminoglycoside modifying enzymes, and decrease
outer membrane permeability (Abaza et al., 2017).
In the current study, the resistance rates against different antibiotics were
variable. Generally, P. aeruginosa isolates showed highest resistance towards
gentamicin, which agreed with a previous study that showed high aminoglycoside
resistance among MDR and extensively drug-resistant (XDR) P. aeruginosa
clinical isolates in Egypt (El-Far et al., 2021). Another study showed that the
primary mechanism of resistance against aminoglycoside is methyltransferases
responsible for changing the drug-binding sites (Valderrama-Carmona et al., 2019).
Besides, the antibiotic susceptibility profile of P. aeruginosa isolates indicated that
majority of P. aeruginosa isolates were more resistant to fluoroquinolone, which
agreed with Zhao et al. (2020). Fluoroquinolones are the most commonly used
antibiotics against P. aeruginosa infections. However, P. aeruginosa acquires
resistance to fluoroquinolones through overexpression of efflux pumps and
mutations in target site genes (Agnello et al., 2016). Moreover, P. aeruginosa
isolates exhibited a high resistance to β-lactams which owes to many reasons
including; production of β-lactamase, especially the AmpC β-lactamase, efflux
130
Discussion
pump expression, permeability changes, and changes in the target site (Torrens et
al., 2019; Sid Ahmed et al., 2020). As revealed herein, colistin and aztreonam
exhibited the most potent activity against P. aeruginosa isolates. However,
continuous survey of the antimicrobial susceptibility of P. aeruginosa isolates is
important in order to establish control strategies for this pathogen. In addition,
monitoring antibiotic use could help prevent the development of resistance
(Masterton, 2008).
Biofilms play a vital role for bacterial pathogens both as a virulence factor
and in resistance to antimicrobial therapy. Bacterial ability to form biofilm is
considered a big challenge that enhances bacterial capacity to resist antimicrobial
agents making bacterial eradication is very difficult (Basanisi et al., 2017). In
addition, biofilm formation is one of the key strategies for bacterial survival during
unexpected changes such as temperature fluctuation and nutrient availability
(Rollet et al., 2009). Of note that, bacteria within a biofilm can escape host
immune responses and resist antimicrobial treatments up to 1000 times more than
their planktonic counterparts (Lewis, 2001). P. aeruginosa is a well-known biofilm
former, which protect them from surrounding environmental stresses, impede
phagocytosis and thereby confer capacity for colonization and long-term
persistence (Moradali et al., 2017). P. aeruginosa also effectively colonizes a
variety of surfaces including medical materials (urinary catheters, implants and
contact lenses) and food industry equipments (mixing tanks, vats and tubing)
(Ghafoor et al., 2011; Coughlan et al., 2016). Therefore, understanding of the
composition biofilm and the molecular mechanisms underlying the antimicrobial
tolerance of bacteria growing within a biofilm are vital for the design of effective
strategies to manage and eradicate biofilm-associated infections (Amankwah et al.,
2021).
131
Discussion
The biofilm forming potential of P. aeruginosa isolates was determined

herein. The potential of P. aeruginosa isolates to form biofilm was evaluated
quantitatively by the microtiter plate method (Mathur et al., 2006). Results indicate
that almost all P. aeruginosa isolates dominant biofilm formers with variable
degrees and 20.4% of isolates were strong biofilm producers. These findings could
account for the higher resistance rates developed by P. aeruginosa isolates to
various antibiotics tested herein. Similar findings were reported by Nagaveni et al.
(2010) and Ansari et al. (2014) who indicated that 25% and 18.2% of P.
aeruginosa isolates were strong biofilm formers, respectively.
Increasing incidence of acute and persisting infections worldwide also

highlights the need to develop therapeutic strategies as an alternative to traditional
antibiotics. Therefore, phage therapy could be a promising alternative strategy to
control the alarming increase in bacterial resistance of P. aeruginosa.
Bacteriophages are supposed to have several advantages compared to antibiotics as
being safer and best tolerated without affecting mammalian cells (Kakasis and
Panitsa, 2019). Bacteriophages are in abundance in aquatic environments and play
useful role in controlling their host populations (Maal et al., 2015). Hence, it could
be used to eliminate microbial contaminants that pose threats to public health.
Phages have been successfully demonstrated for therapy in several bacterial
diseases such as chronic otitis (Wright et al., 2009), burn wounds (Rose et al.,
2014), chronic lung infections and biofilm-associated cystic fibrosis (Hirsch and
Tam, 2010). Furthermore, there is no need for repeated administration of phage
doses which commonly known for antibiotics. There is a significant increase in
phage concentration at infection site via auto “dosing” that results in greater
bacterial killing following only a single dose (Abedon and Thomas-Abedon, 2010).
132
Discussion
One of the major benefits of phage therapy over antibiotics is host

specificity without influencing human microbial flora (Drulis-Kawa et al., 2012).
Consequently, isolation and characterization of new lytic phages targeting
antibiotic resistant P. aeruginosa would be helpful to combat these infections that
threaten human healthcare.
Lytic phages are more preferred for therapeutic purposes as compared with
temperate ones. Temperate phages exhibit drawbacks including the transfer of
virulence genes that could lead to increased antibiotic resistance within bacteria
(Monteiro et al., 2019). In this context, five novel virulent phages designated as
vB_PaeP_PS28, vB_PaeP_PS49, vB_PaeP_PS14, vB_PaeM_PS3 and
vB_PaeM_PS38 targeting P. aeruginosa were isolated from sewage samples
collected from different localities in Zagazig city, Egypt and fully characterized
herein. Phages are isolated from different environments including sewage,
freshwater and soil (Hyman, 2019). The most common habitats for P. aeruginosa
are water, soil, plants, and animals (Chatterjee et al., 2017). These sources are
natural environments from which Pseudomonas specific phages can be isolated, as
phages coexist with their host cells (Shaburova et al., 2006; Krylov, 2014).
Spot test and plaque assay were performed to isolate and purify phages
which is considered as traditional viable approaches in bacteriophage isolation (Gu
et al., 2016). In plaque assay, the phages produced circular clear plaque on
bacterial lawn indicating potential lytic activity of isolated phages. Plaque assay is
extremely valuable as a first selection method, as it is commonly believed that the
shape, size and outline of the plaques are characteristic for each phage (Marei and
Elbaz, 2013; Elmaghraby et al., 2015). In this study, isolated phages exhibited a
different plaque morphology based on plaque size, turbidity and presence of a
surrounding halo. Both vB_PaeP_PS28 and vB_PaeP_PS49 produced small
133
Discussion
circular plaques with no halo with diameter of 2-3 mm; a finding that agrees with
Barazandeh et al. (2021) who isolated a lytic phage vB-PaeP-007 that produced
clear and circular plaques with a diameter of 2 mm. The phage vB_PaeP_PS14
produced large circular plaques with diameter of 3-4 mm with no halo which are
similar to plaques produced by bacteriophage RLP (Alvi et al., 2020). On the other
hand, vB_PaeM_PS3 produced large clear circular plaques surrounded by halos
with a diameter of 4-5 mm and vB_PaeM_PS38 produced small, circular, clear
plaque with no halo with diameter of 1-2 mm in double layer agar method. These
data are similar to the findings reported by Guo et al. (2019) and Jiang et al.
(2020). Halos around plaques are formed because phages produce depolymerases
enzymes that can degrade capsules of bacteria, prevent biofilm formation and
facilitate the access of phages to the bacterial surface (Li et al., 2016; Pires et al.,
2016).
Tailed phages with double stranded DNA are classified into three different
morphological families; Siphoviridae, Podoviridae and Myoviridae within the
order Caudovirales. P. aeruginosa phages have covered all families, and
importantly, phages belonging to Podoviridae and Myoviridae families were found
to be of major importance and are highly preferred for phage therapy (Garbe et al.,
2011; Alemayehu et al., 2012). The morphological characters of isolated phages
against pathogenic P. aeruginosa were characterized by transmission electron
microscope (TEM) as it is considered the most widely used for phage classification
and taxonomy (LaBauve and Wargo, 2012). TEM analysis revealed that
vB_PaeP_PS28, vB_PaeP_PS49 and vB_PaeP_PS14 are members of Podoviridae
family with icosahedral heads and short non contractile tails. The structures and
dimensions of vB_PaeP_PS28, vB_PaeP_PS49 and vB_PaeP_PS14 are identical to
many previously mentioned phages including; Pseudomonas Phage_AUS034
134
Discussion
which was isolated from wastewater sample, (Brisbane, Australia) (Namonyo et

al., 2022), Pseudomonas phage TC6 which was isolated from the sewage of
Southwest Hospital (Chongqing, China) (Tang et al., 2018) and Pseudomonas
phage Pa33 which was isolated from sewage samples collected either from
drainage of different localities in the town or from fresh water lakes around
Chandigarh, India (Kumari et al., 2009). All these phages were assigned to the
Podoviridae family. Meanwhile, vB_PaeM_PS3 and vB_PaeM_PS38 were found
to be members of Myoviridae family with icosahedral heads and contractile tails
exhibit similarity with isolated phages in other studies such as Pseudomonas phage
vB_PaeM_USP_18 which was isolated from domestic Sewage Treatment Station,
Ribeirão Preto, Brazil (Oliveira et al., 2020) and vB_PaeM_SCUT-S2 phage which
was isolated from water samples collected from a small pond in Guangzhou, China
(Guo et al., 2019).
Importantly, the isolated phages vB_PaeP_PS28, vB_PaeP_PS49,

vB_PaeP_PS14, vB_PaeM_PS3 and vB_PaeM_PS38 fulfill the requirements of
pH and temperature stability and showed a higher stability over a wide temperature
and pH ranges. It is well known that stability of phage over a range of temperatures
and pH has been found to influence phage survival and persistence (Anand et al.,
2020). Temperature plays a fundamental role in phage attachment, genetic material
ejection and phage multiplication (Olson et al., 2004). Phage tolerance to various
environmental conditions is very relevant if phage would be involved in
therapeutic applications as well as during storage and production process to
overcome environmental changes during therapeutic applications (Capra et al.,
2006). In agreement with present findings, tailed phages have been shown to be
more stable under harsh conditions including temperature and pH changes
(Ackermann et al., 2004). Current findings are similar to previous studies reporting
135
Discussion
the isolation of highly thermal stable phages infecting P. aeruginosa such as

vB_PaeM_GUMS6, vB_PaeM_GUMS32, and vB_PaeM_GUMS45 phages
isolated by Aghaee et al. (2021). Similarly, Danis-Wlodarczyk et al. (2015)
reported the isolation of two phages; KTN6 and KT28 that could infect P.
aeruginosa and show a higher pH stability ranging from 3 to 12. These findings
agree with earlier studies reporting that majority of phages survive well at pH
range of 5 to 9 (Jamalludeen et al., 2007). Phages are less stable in acidic
environments since it denatures their proteins (Laemmli, 1970). Determination of
phage stability at various temperatures and pH ranges is useful in phage therapy
and for the purpose of biocontrol which allows for potential application within a
variety of industrial environments and room temperature conditions (Jończyk-
Matysiak et al., 2019).
The results of host range and EOP analysis are very important parameters
that should be determined when selecting bacteriophages for therapeutic purposes
(Viscardi et al., 2008). The isolated phages herein exhibit a broad host range and
were able to lyse majority of tested P. aeruginosa strains. Most of these phage-
susceptible strains were highly resistant to traditionally used antibiotics to control
P. aeruginosa infections including β-lactams (penicillins, carbapenems,
cephalosporins, monobactams), aminoglycosides and fluroquinolones. These
findings support the effectiveness of isolated phages and their suitability for
application in phage therapy. It is well-known that broad host range phages are
considered as efficient biocontrol and more preferable for therapeutic application
in phage therapy (Fernández Llamas et al., 2019). These findings were similar to
those obtained by Miyata et al. (2014) who reported that podophage KPP25 had a
broad host range and infected 24 of 38 (63.2%) of isolated P. aeruginosa strains.
136
Discussion
Moreover, Garbe et al. (2011) isolated JG004 phage that was able to infect around
50% of the tested clinical P. aeruginosa isolates.
These findings were further confirmed by EOP analysis which is considered

a rigorous test for evaluating phage infectivity indicated the productive infectivity
and releasing of new progeny rather than by non-productive infection or lysis from
without (Turner et al., 2012). Changes on surface molecules that serve as phage
receptors can significantly affect EOP and reduce phage adsorption (Filippov et al.,
2011; Denes et al., 2015). The majority of susceptible P. aeruginosa strains were of
medium or high efficiency according to the criteria proposed by Khan Mirzaei and
Nilsson, (2015). Ideally, every viral particle attaching to a host cell can enter and
result in a plaque on the appropriate bacterial strains under optimum conditions.
However, there are a number of factors that affect plating efficiency, such as the
specific host strain, thus highlighting the importance of investigating the relative
EOP on a variety of susceptible hosts (Kutter, 2009).
The EOP and host range experimental results emphasize the importance of
using these techniques when selecting phages for therapeutic or antimicrobial
purposes (Viscardi et al., 2008). Efficiency of plating assays indicated a
considerable variation from low to high effectivity between the different
bacteriophages on the different strains. Differences in EOP and absence of lysis
can be explained by the recognition process, which can diverge in different strains
according to the expression of bacteriophage receptors on the bacterial outer
membrane (Uchiyama et al., 2016). Furthermore, the localization, volume, and
density of surface molecules can affect bacteriophage adsorption (Rakhuba et al.,
2010). Considering the mentioned variation in molecules for bacteriophage
adsorption, the application of a bacteriophage cocktail, which retains distinct
adsorption mechanisms, is important to maintain bacteriophage infectivity (Forti et
137
Discussion
al., 2018). Apart from the recognition process, well-known bacterial resistance
systems against bacteriophages could have led to low EOP values. For instance,
many antiviral mechanisms may be employed by the host cell to evade infection
from bacteriophages, such as restriction nucleases and Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR) (Seed, 2015).
Full characterization of newly isolated bacteriophages is essential in

order to achieve the maximum benefits of bacteriophage-based therapies. It was
shown that bacteria killing during infection with bacteriophages largely depends on
phage multiplication process (Manohar et al., 2019). Characterization of phage
growth dynamics such as latent period and burst size is considered as very critical
parameters for phages used in therapy. The phages with a short latent period and
higher burst size are considered efficient antimicrobial agents (Drulis-Kawa et al.,
2012). The isolated phage vB_PaeP_PS28 possesses a latent period of 15 min and
burst size of 210 virions per infected bacterium that was similar to PaBa3 phage
targeting P. aeruginosa which had a latent time of 38 min and burst size
approximately of 217 virion per infected cell (Marashi et al., 2022). On the other
hand, previously isolated phages ɸParuNE1 and JG024 showed latent periods of 28
and 50 min and burst sizes of 300 and 180 virions per infected bacterium,
respectively (Enwuru et al., 2021; Garbe et al., 2011). The isolated phage
vB_PaeM_PS3 has a latent period of 10 min and burst size of approximately 132
virions per infected cell with a higher likelihood of previously isolated phage Ps12-
on-D with latent period of 10 min and burst size of 115 virions per infected cell
(Akremi et al., 2022).
These various values in both latent time and burst size reported for P.
aeruginosa phages could be related to envelope nature and variation in their size
(Enwuru et al., 2021). Phages that have short latent period and large burst size
138
Discussion
have been reported to be efficient antimicrobial agents (Khan Mirzaei and Nilsson,
2015). Additionally, Hyman, (2019) reported that phages with a long latent period
were less preferred for phage therapy. The findings of growth characteristics
further support the potential incorporation of vB_PaeP_PS28 and vB_PaeM_PS3
in treatment of P. aeruginosa infections. Both vB_PaeP_PS28 and vB_PaeM_PS3
could be considered as good candidates for phage therapy and biocontrol of P.
aeruginosa infections.
A number of in vitro studies have shown that bacteriophages have the

potential to lyse targeted bacterial pathogens (Wang et al., 2016). Assessing the
efficacy of vB_PaeP_PS28 and vB_PaeM_PS3 phages in reducing P. aeruginosa
growth in planktonic form was characterized herein. Phages were added at MOIs of
0.1, 1 and 10 to exponential-phase cultures of P. aeruginosa followed by
measuring of bacterial turbidity at OD600 over 24 hr. There was a significant
reduction in bacterial growth and this growth inhibition was found to be MOI-
dependent in all investigated strains. These results were in agreement with those
found by Imam et al. (2019) who observed that there was a significant inhibition in
P. aeruginosa PAO1 growth infected with vB_PaeM_MIJ using different MOI
values (10, 1 and 0.1). Oliveira et al. (2020) reported that five isolated phages
reduced bacterial growth of 13 P. aeruginosa strains up to 8 hr following
incubation together. This reduction in bacterial growth of tested starins was above
60% after 6 and 8 hr of incubation. Current results were similar to previous
findings of Jamal et al. (2017) who reported that phage ZCKP1 was highly
effective in inhibition of bacterial planktonic cells at exponential phase at MOI of 1
and no bacterial resistance was observed during period of treatment. On contrary, a
previously isolated phage BrSP1 showed reduction in P. aeruginosa bacterial
growth for the first 12 hr after phage infection. However after 24 hr of incubation,
139
Discussion
there was a little difference in growth between infected and non-infected bcateria
which could be attributed to the emergence of phage-resistant mutant strains (de
Melo et al., 2019). This could be explained by blocking of bacteriophage receptors
on bacterial hosts, production of extracellular matrix, production of competitive
inhibitors, prevention of bacteriophage DNA entry, slicing of bacteriophage nucleic
acids, and abortive infection mechanisms (Labrie et al., 2010).
The most critical aspect associated with P. aeruginosa infections is biofilm

formation. Biofilms play an important role in bacterial pathogenesis and could lead
to persistence of infections and increase resistance towards external environmental
stresses such as disinfectants, antibiotics and evasion of immune system (Mah et
al., 2003). Bacteria within biofilm encased themselves with extracellular
polysaccharide matrix called glycocalyx which hinder penetration of antibiotics so
that biofilm forming cells more resistant to antimicrobial agent than their
planktonic cells (Ma et al., 2009). Thus, therapy using antibiotics alone is not
effective in controlling this pathogen. Fortunately, phages have been found to be
highly effective in treatment of recalcitrant infections and eradicating P.
aeruginosa biofilms (Tian et al., 2021). In the current study, the influence of phage
treatment on biofilm formation by P. aeruginosa was investigated using the crystal
violet assay. Current results indicate a potent antibiofilm activity of isolated phages
against P. aeruginosa biofilms and significant decrease in biofilm biomass when
treated with isolated phages at different MOIs (0.1, 1 and 10) confirming their
suitability for treatment of Pseudomonas infections. The current data was in
agreement with the findings of Yuan et al. (2019) who reported that
vB_PaeM_LS1 phage prevented biofilms formed by P. aeruginosa and greatly
reduced the number of biofilm cells. In another study, JHP phage exhibited a great
antibiofilm activity on P. aeruginosa biofilms of different ages as JHP could
140
Discussion
effectively reduce biofilm biomass (60–90 %). In addition, pretreatment by JHP

reduced the bacterial load up to 9 logs (>95 % bacterial load reduction) as
compared with untreated control (Shafique et al., 2017).
Bacteriophages have showed promising effects on eradicating P. aeruginosa

biofilms. The antibiofilm activity of bacteriophages could be related to that
bacteriophage can easily penetrate the P. aeruginosa biofilm and destroy its
structure by inducing the synthesis of enzymes such as polysaccharide
depolymerase (Hanlon, 2007). Polysaccharide depolymerase, a polysaccharide
hydrolase encoded by bacteriophages, can specifically degrade the macromolecule
carbohydrates of the host bacterial envelope. This enzyme helps the bacteriophage
to adsorb, invade, and disintegrate the host bacteria (Yan et al., 2014).
Furthermore, bacteriophages could generate peptidoglycan hydrolases known as
endolysins at the end of the lytic cycle that decompose peptidoglycan and assist in
the release of new forming progeny phages from the host cell (Briers et al., 2009).
Endolysins are always proposed as antibacterial agents because of their higher
specific activity and unique mode of action against bacteria.
It should be noted that endolysins activity is independent of antibiotic

susceptibility patterns and therefore bacteriophages have advantages over
antibiotics to inhibit infections caused by bacterial biofilms (Jun et al., 2017). For
example, unlike antibiotics, bacteriophages have the capacity to penetrate the inner
layers of biofilms. Furthermore, bacteriophages can dissolve the biofilm matrix
and are capable of infecting persister cells and destroying them if they are
reactivated (Lewis, 2007; Hraiech et al., 2015). Bacteriophages can also produce
enzymes that inhibit quorum sensing in P. aeruginosa and therefore suppress
biofilm formation (Whiteley et al., 2017). Therefore, bacteriophages seem to be a
141
Discussion
suitable option for the fight against persistent biofilms (Fernández-Barat et al.,
2012).
Current requirements for therapeutic drugs prescribe describing them in

detail. In the case of bacteriophages, it is necessary to determine their genomic
sequences and thus confirm the virulent nature through showing that there are no
integrase genes in their genomes. Temperate phages are not used for therapy
because they can facilitate transfer of genes of bacterial toxins and determinants of
antibiotic resistance in the bacterial population. Besides, in order to assess the
therapeutic safety of a bacteriophage its genome is searched for genes of known
toxins (Principi et al., 2019). Accordingly, the genome of vB_PaeP_PS28 was
sequenced and gene annotation confirmed that vB_PaeP_PS28 is a member of
Podoviridae family and the genus Litunavirus of subfamily Migulavirinae. The
Litunavirus is a member of a well-characterized N4-like phage. Almost all of N4-
like phages exhibit highly conserved gene organization and expression (Wittmann
et al., 2020; Menon et al., 2021). The vB_PaeP_PS28 genome encodes ORF
1which is highly similar to the virion-associated RNA polymerase, a remarkable
protein that is characteristic of N4-like viruses and unique among all other phages.
N4-like viruses co-inject this enzyme with DNA during infection and is
responsible for the transcription of early genes (Farmer et al., 2013). The genomes
of the N4-like phages from the Pseudomonas group did not reveal any tRNA genes
which is in accordance with the annotated vB_PaeP_PS28 phage genome
(Wittmann et al., 2020). Other genes within phage genome with functional
annotation such as ORF 8 and ORF 9 showed a higher similarity to previously
annotated N4-like proteins which further support the designation of
vB_PaeP_PS28 as an N4-like phage.
142
Discussion
Comparative analysis was performed between vB_PaeP_PS28 phage and

other phages infecting P. aeruginosa available in the NCBI. The genomic
comparison shows that isolated phage has best similarities with previously isolated
and characterized phages; Pseudomonas phage vB_PaeP_FBPa1 (GenBank Acc.
No. ON857943.1), Pseudomonas phage vB_PaeS_VL1 (GenBank Acc. No.
OK665488.1) and Pseudomonas phage YH6 (GenBank Acc. No. KM974184.1)
with percent identity of 94.81, 94.37 and 94.07 %; respectively. These findings
were further confirmed upon performing a phylogenetic analysis based on the
genes encoding for the terminase large subunit and DNA polymerase that are
conserved in different classes of bacteriophages (Shapiro and Putonti, 2018;
Akhwale et al., 2019). These proteins are considered as helpful phylogenetic
markers and routinely used in the investigation of several phage groups and
describing the phylogenetic positioning of newly isolated phage (Casjens et al.,
2005; Wittmann et al., 2014).
Regarding vB_PaeM_PS3, the genomic analysis revealed that it belongs to

the family Myoviridae and the genus Pakpunavirus. In addition, the phylogenetic
analysis based on the overall similarity with completely sequenced phage genomes
in database shows that vB_PaeM_PS3 shares greatest nucleotide similarity with
Pseudomonas phage vB_PaeM_SCUT-S2 (GenBank Acc. No MK340761.1),
Pseudomonas phage vB_PaM_EPA1 (GenBank Acc. No MN013356.1),
Pseudomonas phage PaYy-2 (GenBank Acc. No MH725810.1) and Pseudomonas
phage SRT6 (GenBank Acc. No MH370478.1) representing percent identities of
96%, 95.2%, 95% and 94.9%, respectively. These findings were in accordance
with obtained data by neighbor-joining (NJ) phylogenetic trees based on phage
major capsid protein and terminase large subunit. Phylogenetic analysis was
performed based on the major capsid proteins since related phages are considered
143
Discussion
to have similar head structural components and therefore could be clustered based
on their major capsid proteins (Bamford et al., 2005).
Importantly, no tRNA was found in the vB_PaeP_PS28 genome which suggests

that upon entry into the host, the phage vB_PaeP_PS28 is completely dependent on
the host tRNA for its translation machinery. Meanwhile, vB_PaeM_PS3 phage
genome analysis allowed the detection of 14 tRNAs. The presence of tRNAs is
often found in myoviruses with large genomes and is common in strictly virulent
or lytic phages (Bailly-Bechet et al., 2007; Santos et al., 2011). The phage-encoded
tRNA genes are generally present in clusters and promote an efficient and more
rapid translation rate (Lu et al., 2013). In addition, it is thought that tRNAs are
responsible for phages have short latent period and large burst size because. It has
been reported that tRNAs enable phages propagation and enhance viral replication
kinetics (Jun et al., 2014).
A critical aspect related to bacteriophage therapy is the possibility of transduction

where bacteriophages could transfer bacterial virulence genes among bacteria
leading to increased bacterial resistance (Mahichi et al., 2009; Sillankorva et al.,
2010). Importantly, the genome annotation of both vB_PaeP_PS28 and
vB_PaeM_PS3 phages indicated the absence of genes related to lysogenic cycle,
antibiotic resistance and virulence encoding genes. These findings confirm that
vB_PaeP_PS28 and vB_PaeM_PS3 phages are virulent phages and further support
their suitability for therapeutic applications to combat P. aeruginosa infections.
The above findings become more convincing in light of previous reports that
documented the effectiveness of local and systemic application of phages in
controlling infections by P. aeruginosa (Hagens et al., 2004). In the current study,
144
Discussion
the efficiency of vB_PaeP_PS28 and vB_PaeM_PS3 phages to reduce

Pseudomonas pathogenesis was assessed in vivo using mice infection model.
Intriguingly, there was a significant reduction in the mortality of mice infected
with P. aeruginosa and treated with the phages; vB_PaeP_PS28 and
vB_PaeM_PS3 as compared to bacteria-inoculated mice without phage treatment.
Moreover, phage treatment effectively reduced bacterial colonization in the organs
isolated from Pseudomonas-infected mice relative to mice injected with bacteria
alone. Interestingly, the vB_PaeP_PS28 and vB_PaeM_PS3 phages have been
found to be highly efficient in attenuation of P. aeruginosa virulence in mice,
which further encourages their future application in phage therapy. It is worth
mentioning that no detectable side effects or harmful signs were observed in mice
upon phage treatment over the experiment course. Current results are in
accordance with previous in vivo studies reporting similar survival rates in
Pseudomonas-infected mice following the administration of lytic phages (McVay
et al., 2007; Watanabe et al., 2007). Additionally, KPP12 phage successfully
treated Pseudomonas-induced keratitis and markedly reduced bacterial load in
infected mice (Fukuda et al., 2012). Rezk et al. (2022) reported that topical
application of phage ZCPA1 resulted in complete bacterial eradication in skin
wounds and led to efficient wound closure. P. aeruginosa bacterial load in mice
lungs with pneumonia was markedly diminished following treatment with phage
nasal inhalation (Jeon and Yong, 2019). Similarly, other successful clinical trials
revealed the efficacy of phage therapy against ear and burn infections caused by P.
aeruginosa (Wright et al., 2009; Jault et al., 2019). Given the promising results of
current study, it is recommended to incorporate vB_PaeP_PS28 and
vB_PaeM_PS3 phages in treatment of P. aeruginosa infections.
145
Conclusion & Recommendations
Conclusion and Recommendations
In this study, five novel lytic phages specific for P. aeruginosa were isolated
and characterized. These phages were designated as vB_PaeP_PS28,
vB_PaeP_PS49, vB_PaeP_PS14, vB_PaeM_PS3 and vB_PaeM_PS38
vB_PaeP_PS28 and vB_PaeM_PS3. The isolated phages exhibit a broad lytic
activity as well as higher stability under various environmental conditions. Both
vB_PaeP_PS28 and vB_PaeM_PS3 phages showed a promising in vitro stability and
antibacterial activity against P. aeruginosa. In addition to their inhibitory activity
against P. aeruginosa planktonic cells, both phages exhibit a pronounced antibiofilm
potential. Therefore the genomic characterization of these two phages as well as their
in vivo effect on P. aeruginosa pathogenesis were further conducted in the present
study. Genomic analysis revealed that vB_PaeP_PS28 and vB_PaeM_PS3 phages
do not contain any toxic genes or integrases confirming the lytic nature of both
phages. Additionally, the therapeutic efficacy of vB_PaeP_PS28 and vB_PaeM_PS3
were investigated in vivo using mice infection model. Interestingly, treatment with
both phages markedly reduced mortality in P. aeruginosa-infected mice and lowered
bacterial colonization in isolated organs. Furthermore, in vivo results proved the
safety and efficiency of isolated phages against P. aeruginosa as an alternative to
traditionally used antibiotics. Based on these findings, the vB_PaeP_PS28 and
vB_PaeM_PS3 phages fulfill the requirements of effective phages for further
application as innovative candidates against P. aeruginosa infections.
146
Summary
Summary
P. aeruginosa is a multidrug resistant opportunistic pathogen which causes
acute or chronic infection in immunocompromised individuals, cystic fibrosis,
burns and sepsis. P. aeruginosa is able to form biofilm, which helps bacteria
survive in a hypoxic atmosphere or other extremely harsh environments. Treatment
of P. aeruginosa infection is extremely difficult due to its rapid mutations and
adaptation to gain resistance to antibiotics and consequently, it has been listed
among the critical group of pathogens which urgently need new strategies to
control. One of these strategies that has been recently employed is phage therapy
using bacteriophages to limit bacterial infections. Phage therapy has received a
significant interest as an alternative to antibiotics and to alleviate antimicrobial
resistance. Bacteriophages are seemed to be efficient alternatives in management
of infections caused by P. aeruginosa. The current study aimed to isolate and
characterize new lytic phages targeting antibiotic resistant P. aeruginosa that would
be helpful to combat these infections that threaten human healthcare.
A total of 50 P. aeruginosa isolates in this study were recovered from 120

clinical specimens during the period from April 2021 to August 2021 from patients
admitted to Zagazig University Hospitals and El-Ahrar Educational Hospital,
Zagazig, El Sharkia, Egypt. P. aeruginosa were isolated from different clinical
sources including burns, surgical wounds, urine, ear infections and endotracheal
aspirates. Antibiotic susceptibility testing was done by the standard disc diffusion
method. All P. aeruginosa isolates were sensitive to colistin while higher bacterial
resistance was observed towards gentamicin, fluoroquinolone and carbapenems
(60, 58 and 54%; respectively). Intermediate bacterial resistance was observed
against pipracillin (46%), ceftazidime (40%) and piperacillin/tazobactam (38%).
Conversely, low bacterial resistance was recorded against aztreonam (18%). It is
147
Summary
worth mentioning that 56% of P. aeruginosa isolates in this study were MDR. The
biofilm forming capacity of P. aeruginosa clinical isolates in addition to P.
aeruginosa reference strains; P. aeruginosa ATCC 27853, P. aeruginosa ATCC
9027 & P. aeruginosa PAO1 were assessed spectrophotometrically by the crystal
violet assay. Almost all P. aeruginosa isolates were found to be capable of forming
biofilm with variable degrees; 20.4% were strong biofilm producers, 64.8% were
moderate biofilm producers and 14.8% were weak biofilm producers.
Five lytic phages targeting P. aeruginosa isolates were isolated and fully
characterized from sewage. These phages were designated as vB_PaeP_PS28 &
vB_PaeP_PS49 & vB_PaeP_PS14 & vB_PaeM_PS3 and vB_PaeM_PS38. Phage
particles morphology was characterized using the transmission electron
microscope (TEM) after staining with 2% phosphotungstic acid (pH 7). TEM
images revealed that the phage vB_PaeP_PS28 & vB_PaeP_PS49 and
vB_PaeP_PS14 possess an icosahedral head and short non-contractile tail which
are closely related to phages belonging to the family Podoviridae and order
Caudovirales according to International Committee Taxonomy of Viruses (ICTV).
Meanwhile, vB_PaeM_PS3 and vB_PaeM_PS38 possess an icosahedral head and
contractile tails and therefore were found to belong to the family Myoviridae and
the order Caudovirales.
Physical stability of isolated phages was determined against wide range of

pH and temperature degrees. Isolated phages were tolerant to wide range of
temperatures. However, isolated phages; vB_PaeP_PS28 and vB_PaeM_PS3 could
survive up to 60°C and lost their infectivity at higher temperatures (70, 80, 90 and
100°C). On the other hand, vB_PaeP_PS49 & vB_PaeP_PS14 and
vB_PaeM_PS38 were considerably stable at temperatures range from 40°C to
70°C. However, their activity was entirely diminished when incubated at 80-
148
Summary
100°C. Furthermore, the impact of different pH on survival and infectivity of all

isolated phages was determined. It was found that all phages were able to retain
their infectivity and could survive at wide pH ranges (4-10). There was a slight
reduction in phage viability at pH 11. Of note that, no viable phage particles were
observed at extreme pH (3 and 12) which means that the phages completely lost
their infectivity.
The host range of vB_PaeP_PS28 & vB_PaeP_PS49 & vB_PaeP_PS14 &

vB_PaeM_PS3 and vB_PaeM_PS38 phages was determined against a total of 18 P.
aeruginosa strains (15 P. aeruginosa isolates from different clinical sources in
addition to 3 P. aeruginosa reference strains; P. aeruginosa ATCC 27853, P.
aeruginosa ATCC 9027 & P. aeruginosa PAO1) using the spot assay. Moreover,
other bacterial species such as E. coli, S. Typhimurium, K. pneumoniae, Serratia
marcescens and S. aureus were included in host range determination. Results show
that isolated phages have a unique lytic profile and were able to infect most of
tested P. aeruginosa strains indicating a higher lytic efficiency of isolated phages.
However, no lytic activity was observed against other bacterial species indicating
that the isolated phages possessed broad spectrum lytic activity and high specificity
towards P. aeruginosa.
The antibiofilm activity of isolated phages at different MOIs (0.1, 1 and 10)
P. aeruginosa was evaluated by the crystal violet assay. The isolated phages
effectively degraded mature biofilms and reduced biofilm biomass formed by all
tested P. aeruginosa strains. The antibiofilm activity of isolated phages against P.
aeruginosa was MOI-dependent where maximum biofilm inhibition was observed
at MOI of 10 as compared with MOI of 1 and 0.1. Moreover, the efficiency of
plating (EOP) of two phages; vB_PaeP_PS28 and vB_PaeM_PS3 was performed
for each susceptible strain that showed lysis in the spot assay. Both phages were
149
Summary
able to infect all susceptible P. aeruginosa strains with different infectivity

patterns. Furthermore, one-step growth curve was performed to characterize the
phage infection process including the latent period and burst size. The phage
vB_PaeP_PS28 has a latent period of 15 min and an average burst size of 210
virions per infected bacterium. On the other hand, vB_PaeM_PS3 exhibited a short
latent period of 10 min with host-cell lysis releasing about 132 new virions per
infected cell. Furthermore, the bacteriolytic activity of both vB_PaeP_PS28 and
vB_PaeM_PS3 was determined against P. aeruginosa strains at different MOIs
(0.1, 1 and 10) and bacterial turbidity was measured at OD600 over 24 hr. Both
vB_PaeP_PS28 and vB_PaeM_PS3 were found to adversely affect bacterial
growth over 24 hr. Of note that, bacterial growth inhibition was found to be dose-
dependent where growth inhibition was higher at MOI of 10 as compared to MOI
of 1 and 0.1.
Importantly, the whole genome of both vB_PaeP_PS28 and vB_PaeM_PS3

were sequenced by Illumina Miseq next-generation sequencing. In brief, the
genome of vB_PaeP_PS28 consists of 72,283 bp circular double-stranded DNA,
with G+C content of 54.75 %. The phage genome harbors 94 open reading frames
(ORFs); 32 for known functional proteins and 62 for hypothetical proteins and no
tRNA genes. Meanwhile the genome of vB_PaeM_PS3 consists of 93,922 bp of
dsDNA with 49.39% G+C content. A total of 171 ORFs were identified, of which
27 were assigned as functional proteins whereas hypothetical proteins were
encoded by 144 ORFs and 14 genes as tRNA. Moreover, the annotation of both
vB_PaeP_PS28 and vB_PaeM_PS3 genomes showed the absence of genes related
to lysogenic cycle, antibiotic resistance and virulence encoding genes. These
findings confirm that vB_PaeP_PS28 and vB_PaeM_PS3 are virulent phages and
150
Summary
further support their suitability for therapeutic applications to combat P.

aeruginosa infections.
Finally, the influence of both vB_PaeP_PS28 and vB_PaeM_PS3 on P.

aeruginosa pathogenesis was investigated in vivo using mice infection model.
There was a significant reduction in the mortality of mice infected with P.
aeruginosa and treated with phages; vB_PaeP_PS28 and vB_PaeM_PS3 as
compared to bacteria-inoculated mice without phage treatment. Moreover, phage
treatment effectively reduced bacterial colonization in the organs isolated from
Pseudomonas-infected mice relative to those isolated from mice injected with
bacteria alone. Interestingly, vB_PaeP_PS28 and vB_PaeM_PS3 have been found
to be highly efficient in attenuation of P. aeruginosa virulence in mice, which
further encourages their future application in phage therapy. It is worth mentioning
that no detectable side effects or harmful signs were observed in mice upon phage
treatment over the experiment course.
In conclusion, current study highlights the influence of phage therapy as a

promising tool for management P. aeruginosa infections. Isolated phages showed a
pronounced bacteriolytic activity against P. aeruginosa as well as an efficient
antibiofilm potential. In addition, the virulence capacity of P. aeruginosa in mice
was significantly diminished upon treatment with isolated phages.
Present data further support the efficiency of isolated phages as a novel

candidate, either alone or to be incorporated in a phage cocktail therapy, to control
P. aeruginosa infections.
151
References
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‫الملخص العربي‬
‫تعتبر بكتريا السودوموناس ايروجينوزا بكتريا انتهازية ومقاومة لعديد من االدوية وتسبب عدوى حادة أو مزمنة‬
‫لدى األفراد الذين يعانون من نقص المناعة والتليف الكيسي والحروق وتسمم الدم‪ .‬إن بكتريا السودوموناس‬
‫ايروجينوزا قادرة على تكوين غشاء حيوي‪ ،‬مما يساعد البكتيريا على البقاء على قيد الحياة عندما ينقص‬
‫األكسجين أوفي ظروف بيئية أخرى قاسية للغاية‪ .‬أصبح عالج األمراض التي تسببها بكتريا السودوموناس‬
‫ايروجينوزا صعب للغاية بسبب طفراتها السريعة وقدرتها على التكيف الكتساب مقاومة للمضادات الحيوية‬
‫وبالتالي فقد تم إدراجها ضمن المجموعة الحرجة (الشرسة) من مسببات األمراض التي تحتاج بشكل عاجل‬
‫إلى استراتيجيات جديدة للسيطرة عليها‪ .‬ومن إحدى هذه االستراتيجيات التي تم استخدامها أخيرا هي العالج‬
‫بواسطة البكتريوفاجات للحد من االلتهابات البكتيرية حيث تلقي اهتماما كبيرا كبديل للمضادات الحيوية ولتقليل‬
‫المقاومة العالية ضدها‪ .‬يبدو العالج بواسطة البكتريوفاجات بديل فعال لعالج األمراض التي تسببها بكتريا‬
‫السودوموناس ايروجينوزا‪ .‬ولهذا تهدف الدراسة الحالية الي عزل وتوصيف البكتريوفاجات التي تصيب بكتريا‬
‫السودوموناس ايروجينوزا والتي قد تكون مفيدة لمكافحة العدوى التي تهدد الرعاية الصحية البشرية‪.‬‬
‫وفي الدراسة الحالية تم عزل خمسون عزلة من بكتريا السودوموناس ايروجينوزا من مائة وعشرون عينة‬
‫خالل الفترة من أبريل ‪ 2021‬الي أغسطس ‪ 2021‬من المرضي المصابين بعدوي الحروق‪ ،‬الجروح‪ ،‬الجهاز‬
‫البولي‪ ،‬األذن وعدوي الجهاز التنفسي والذين كانوا في مستشفي األحرار التعليمي ومستشفي الزقازيق الجامعي‪.‬‬
‫تم اجراء اختبار الحساسية للمضادات الحيوية باستخدام طريقة انتشار القرص‪ .‬وقد أظهرت بكتريا‬
‫السودوموناس ايروجينوزا الحساسية الكاملة للكوليستين بينما أعلي مقاومة تجاه الجينتاميسين‪ ،‬الفلوروكينولون‬
‫والكاربينم بنسبه (‪ %54 ،58 ،60‬على التوالي)‪ .‬يليهم في المقاومة البيبراسيلين بنسبه (‪ ،)%46‬سيفتازيديم‬
‫بنسبه (‪ )%40‬والبيبراسيلين‪/‬تازوبكتام بنسبه (‪ .)%38‬وعلي النقيض وجد أقل مقاومة تجاه أزيترونام بنسبه‬
‫(‪ .)%18‬كما أوضحت هذه الدراسة أن ‪ %56‬من بكتريا السودوموناس ايروجينوزا كانوا متعددي المقاومة‬
‫للمضادات الحيوية‪ .‬تم قياس قدرة العينات القياسية من بكتريا السودوموناس ايروجينوزا الي جانب جميع‬
‫العزالت االكلينيكية على تكوين الغشاء الحيوي عن طريق قياس التحليل الضوئي‪ .‬وأظهرت النتائج أن معظم‬
‫عزالت السودوموناس ايروجينوزا قادره على تكوين الغشاء الحيوي بنسب مختلفة حيث أن ‪ %20.4‬كانوا من‬
‫العينات التي تتميز بقدرتها العالية علي تكوين الغشاء الحيوي بينما كان ‪ %64.8‬ذات قدره متوسطة وحوالي‬
‫‪ %14.8‬من العينات ذات قدرة ضعيفة علي تكوين الغشاء الحيوي‪.‬‬
‫‪1‬‬
‫في هذه الدراسة تم عزل وتوصيف خمس بكتريوفاجات متخصصه لبكتريا السودوموناس ايروجينوزا من‬
‫الصرف الصحي‪ .‬وتم تسميتها كالتالي ‪vB_PaeP_PS14 ، vB_PaeP_PS49،vB_PaeP_PS28‬‬
‫‪ . vB_PaeM_PS38 ،vB_PaeM_PS3‬تم وصف الخصائص الخارجية للبكتريوفاجات باستخدام المجهر‬
‫االلكتروني وقد أظهرت الصور المجهرية ‪vB_PaeP_PS14، vB_PaeP_PS49،vB_PaeP_PS28‬‬
‫لهم رأس متعددة األوجه وذيل قصير وينتمون لعائله البودوفيريدي بينما البكتريوفاجات ‪vB_PaeM_PS3‬‬
‫و‪ vB_PaeM_PS38‬ينتمون لعائله الميوفيريدي حيث ان لهم راس متعددة األوجه وذيل منقبض وفقا للتصنيف‬
‫الدولي للفيروسات (‪.)ICTV‬‬
‫وتم دراسة تأثير درجات مختلفة من الحرارة وكذلك األس الهيدروجيني على حساسية البكتريوفاجات المعزولة‬
‫وأوضحت النتائج ثبات هذه البكتريوفاجات في ظل ارتفاع درجات الحرارة حيث وجد أن البكتريوفاجات‬
‫‪vB_PaeP_PS28‬و‪ vB_PaeM_PS3‬تزال قادرة على اإلصابة حتى درجه الحرارة ‪ 60‬وتفقدها عند‬
‫درجات حرارة أعلي منها (‪ 90،80،70‬و‪ .)100 °C‬علي الناحية األخرى البكتريوفاجات ‪vB_PaeP_PS49‬‬
‫و ‪vB_PaeP_PS14‬و‪ vB_PaeM_PS38‬لها ثبات عالي وقادرة على اإلصابة حتي درجة الحرارة ‪70°C‬‬
‫بينما تتأثر بارتفاع درجة الحرارة (‪ )100-80‬وتفقد قدرتها علي اإلصابة بالبكتريا‪ .‬وباإلضافة الي ذلك أظهرت‬
‫النتائج قدرة جميع البكتريوفاجات المعزولة البقاء في مستوي واسع من درجات األس الهيدروجيني (‪.)10-4‬‬
‫بينما كان يوجد انخفاض طفيف عدد البكتريوفاجات عند األس الهيدروجيني ‪ 11‬وتفقد جميع البكتريوفاجات‬
‫فاعليتها وثباتها وقدرتها على االصابة عند األس الهيدروجيني (‪ 3‬و‪.)12‬‬
‫تم تحديد نطاق فاعلية البكتريوفاجات ‪vB_PaeP_PS28‬و‪vB_PaeP_PS49‬و ‪vB_PaeM_PS14‬‬

‫و ‪vB_PaeM_PS3‬و‪ vB_PaeM_PS38‬على ‪ 18‬عزلة من بكتريا السودوموناس ايروجينوزا من بينهم‬
‫‪ 15‬عزلة من العزالت االكلينيكية المعزولة من مصادر مختلفة باإلضافة الي ‪ 3‬عينات قياسية لبكتريا‬
‫السودوموناس ايروجينوزا (‪ ATCC 9027 ،ATCC 27853‬و‪ )PAO1‬باستخدام اختبار البقعة‪ .‬وتم أيضا‬
‫ادراج أنواع مختلفة من البكتريا مثل ايشريشا كوالي‪ ،‬السالمونيال‪ ،‬الكليبسيال نيومونيا‪ ،‬السيراتيا والمكورات‬
‫العنقودية الذهبية‪ .‬وأظهرت النتائج أن البكتريوفاجات المعزولة لها نطاق تحللي فريد وقادرة على اصابة معظم‬
‫بكتريا السودوموناس ايروجينوزا المختبرة مما يفيد الفاعلية الكبرى لهذه البكتريوفاجات‪ .‬وعلي الرغم من ذلك‬
‫لم تظهر أي فاعليه أو تأثير تجاه األنواع األخرى من البكتريا مما يشير الي أن البكتريوفاجات المعزولة لها‬
‫نشاط تحللي كبير ومدي تخصصي عالي تجاه بكتريا السودوموناس ايروجينوزا‪.‬‬
‫‪2‬‬
‫كما تم دراسة تأثير البكتريوفاجات على منع تكوين الغشاء الحيوي عند ‪ MOIs‬مختلفة (‪ )10،1،0.1‬عن طريق‬
‫قياس التحليل الضوئي‪ .‬وقد وجد أن البكتريوفاجات لها قدره فائقة على تدمير ومنع تكوين الغشاء الحيوي‬
‫اعتمادا على جرعته حيث وجد أن أعلي انخفاض في تكوين البيوفيلم عند ‪ 10MOI‬مقارنه ب ‪ 1 MOI‬و‪.0.1‬‬
‫كما أنه تم دراسة قدرة ‪vB_PaeP_PS28‬و‪ vB_PaeM_PS3‬على تكوين بالكات على كل بكتريا حساسة‬
‫في اختبار البقعة‪ .‬ووجد أن كل من البكتريوفاجين قادرين على إصابة كل عزالت بكتريا السودوموناس‬
‫ايروجينوزا بأنماط مختلفة‪ .‬وتم اجراء منحني الخطوة الواحدة للبكتريوفاجات للتعرف على مرحلة العدوي‬
‫داخل البكتريا بما في ذلك تحديد فترة الكمون وعدد البكتريوفاجات المنطلقة بعد تحلل البكتريا‪ .‬حيث كان‬
‫‪ vB_PaeP_PS28‬له فترة كمون ‪ 15‬دقيقة ومتوسط تحرر الفيروسات هو ‪ 210‬لكل خلية مصابة وعلى‬
‫الناحية األخرى ‪ vB_PaeM_PS3‬لديه فترة كمون أقصر وهي ‪ 10‬دقائق وتخرج حوالي ‪ 132‬فيروس جديد‬
‫من كل خلية بكتيرية مصابة‪ .‬وعالوة على ذلك تم اختبار النشاط البكتيري التحللي للبكتريوفاجات ‪vB-‬‬
‫‪PaeP_PS28‬و‪ vB_PaeM_PS3‬ضد عزالت مختلفة من بكتريا السودوموناس ايروجينوزا عند جرعات‬
‫(‪ )MOIs‬مختلفة (‪10‬و‪1‬و‪ )0.1‬عن طريق قياس العكارة البكتيرية ‪OD600‬علي مدار ‪ 14‬ساعة‪ .‬حيث وجد أن‬
‫تعرض واصابة البكتريا للبكتريوفاجات يؤثر سلبا على نموها وانخفاض كبير في عدد خاليا البكتريا لمدة ‪24‬‬
‫ساعة وان هذا التأثير يعتمد على جرعة وتركيز البكتريوفاج بالنسبة للبكتريا حيث يتم منع نمو البكتريا بصورة‬
‫فائقة عند ‪ 10 MOI‬مقارنة ب ‪ 1 MOI‬و‪.0.1‬‬
‫تم عزل الحامض النووي لكل من ‪vB_PaeP_PS28‬و ‪vB_PaeM_PS3‬ومعرفة التسلسل النيوكلوتيدي‬

‫لها بالكامل عن طريق ‪ .Illumina Miseq‬وأظهرت النتائج أن الحامض النووي ‪ vB_PaeP_PS28‬دائري‬
‫ومزدوج يتكون من ‪ 72283‬قاعدة نيتروجينية ونسبة ‪ G+C‬هي ‪ .%54.75‬ويحتوي الحامض النووي علي‬
‫‪ ORF 94‬من بينهم ‪ 32‬للبروتينات معروفة الوظيفة و ‪ 62‬للبروتينات االفتراضية وعدم وجود جينات ‪tRNA‬‬
‫بينما الحامض النووي ‪ vB_PaeM_PS3‬يتكون من ‪ 93922‬قاعدة نيتروجينية ونسبة ‪ G+C‬هي ‪%49.39‬‬
‫وتم إيجاد‪ ORF 171‬من بينهم ‪ 27‬للبروتينات معروفة الوظيفة و ‪ 144‬جين للبروتينات االفتراضية وحوالي‬
‫‪. tRNA 14‬كما أظهرت النتائج عدم احتواء الحامض النووي للبكتريوفاجات علي أي جينات سامة أو عوامل‬
‫ضراوة وأيضا الجينات المسؤولة عن االلتحام الذى يساعد على اتصال الحامض النووي للبكتريوفاج مع‬
‫الحامض النووي الخاص بخلية العائل‪ .‬وهذه النتائج تؤكد أن البكتريوفاجات هي بكتريوفاجات محللة للبكتريا‬
‫وتدعم مالءمة البكتريوفاج واستخدامه لمكافحة األمراض التي تسببها بكتريا السودوموناس ايروجينوزا‪.‬‬
‫‪3‬‬
‫وأخيرا تم فحص تأثير كل من ‪ vB_PaeP_PS28‬و‪ vB_PaeM_PS3‬على قدرة بكتريا السودوموناس‬

‫ايروجينوزا الممرضة باستخدام الفئران كنموذج‪ .‬وقد وجد أن هناك انخفاض كبير في معدل وفيات الفئران‬
‫المصابة ببكتريا السودوموناس ايروجينوزا والمعالجة بالبكتريوفاجات مقارنة بالفئران المصابة ببكتريا‬
‫السودوموناس ايروجينوزا فقط دون عالج كما أنها قللت االستعمار البكتيري في األعضاء المعزولة من الفئران‬
‫المصابة بالبكتريا وحقنت بالبكتريوفاج على غير الفئران المصابة بالبكتريا فقط‪ .‬ولهذا وجد أن‬
‫‪vB_PaeP_PS28‬و ‪ vB_PaeM_PS3‬لها دور فعال في تقليل شراهة بكتريا السودوموناس ايروجينوزا‬
‫في الفئران مما يشجع استخدامها للعالج في المستقبل‪ .‬ومن الجدير بالذكر أن العالج بواسطة البكتريوفاجات‬
‫ليس له أي آثار جانبية أو اعراض ضارة للفئران خالل فترة التجربة‪.‬‬
‫واجماال تسلط هذه الدراسة الحالية الضوء على استخدام البكتريوفاجات كنهج جديد لعالج عدوي بكتريا‬
‫السودوموناس ايروجينوزا‪ .‬حيث لقد أظهرت البكتريوفاجات المعزولة نشاط تحللي كبير ضد السودوموناس‬
‫ايروجينوزا الي جانب دورها الفعال في منع تكوين الغشاء الحيوي‪ .‬باإلضافة الي انها أضعفت القدرة المرضية‬
‫وشراسة السودوموناس ايروجينوزا في الفئران‪ .‬كما أن النتائج الحالية تدعم فاعلية البكتريوفاجات المعزولة‬
‫كبديل جديد للعالج اما بفردها او حين استخدامها مع بكتريوفاجات أخري للتحكم في العدوي التي تسببها بكتريا‬
‫السودوموناس ايروجينوزا‪.‬‬
‫‪4‬‬
‫جامعة الزقازيق‬ ‫كلية الصيدلة‬
‫عزل وتوصيف البكتريوفاجات التي تصيب السودوموناس إيروجينوزا‬
‫رسالة مقدمه كمتطلب لستيفاء الحصول على درجة دكتوراه الفلسفة في العلوم الصيدلية‬
‫(ميكروبيولوجي ومناعة)‬
‫مقدمة من‬
‫الماجستير‪ /‬علياء عاطف طه عبد الغفار‬

‫مدرس مساعد بقسم الميكروبيولوجي والمناعة‬
‫كلية الصيدلة ‪ -‬جامعة الزقازيق‬
‫تحت إشراف‬
‫األستاذ الدكتور‪ /‬غادة حامد شاكر‬

‫أستاذ الميكروبيولوجي والمناعة‬
‫عميد كلية صيدلة‬
‫جامعة سيناء – فرع القنطرة‬
‫األستاذ الدكتور‪ /‬أميرة محمد الجنايني‬

‫أستاذ الميكروبيولوجي والمناعة‬
‫األستاذ المساعد‪ /‬مؤمن محمود عز العرب عسكورة‬

‫أستاذ مساعد الميكروبيولوجي والمناعة‬
‫قسم الميكروبيولوجي والمناعة‬

‫كلية الصيدلة‬
‫جامعة الزقازيق‬
‫(‪)2023‬‬

Final Aliaa-PHD Thesis

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Final Aliaa-PHD Thesis

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Zagazig University Faculty of Pharmacy

Isolation and characterization of bacteriophages infecting

Prof. Dr. Ghada Hamed Shaker

Prof. Dr. Amira Mohamed El-Ganiny

Ass. Prof. Dr. Momen Mahmoud Ez Elarab Askoura

Department of Microbiology and Immunology

First and foremost, I thank ALLAH for helping me to complete this

I would like to express my sincere gratitude from my heart to Prof. Dr.

I owe my deepest thanks to my adviser Ass. Prof. Dr. Momen Mahmoud Ez

I would also like to express my deepest appreciation to the staff members at

Aliaa Atef Taha Abdelghafar

This work is lovely dedicated to my dear father

and my beloved mother who supported me

through work and always there whenever I

To my lovely husband Abdelfattah and my

Special thanks for my lovely sisters Alshimaa,

Doaa, Lamiaa and Mariam.

Pseudomonas aeruginosa (P. aeruginosa) is a widely distributed

adaptability encoded in its genome. Furthermore, P. aeruginosa is the causative

threatening ones including bacteremia and pneumonia. P. aeruginosa could grow on

in immunocompromised patients and those suffering from severe burns or cystic

Numerous virulence factors contribute to P. aeruginosa pathogenesis including

hemolysins, rhamnolipids, proteases and biofilms. P. aeruginosa is capable of

forming biofilms which protect bacteria from environmental stresses and

phagocytosis. Additionally, it has been suggested that biofilm formation markedly

contributes to bacterial resistance to various antibiotics, including quinolones,

aminoglycosides and β-lactams leading to long term persistence. P. aeruginosa is

intrinsic resistance to various antibiotics including beta-lactams. Therefore,

treatment of P. aeruginosa infections by conventional antibiotics has become a

major global challenge. Recently, due to increased antimicrobial resistance, there is

an urgent necessity for the development of alternative antimicrobial approaches to

efficiently control bacterial infections.

Latterly, researchers have indicated that phage therapy could be considered as an

effective approach to control P. aeruginosa infections and biofilms. Bacteriophages

the biosphere. Phage therapy refers to the use of bacteriophages in treatment of

bacterial infections as potential alternatives to overcome limitations of antibiotics.

by antibiotics Hence, phage therapy possesses a great potential to replace antibiotic

treatment in treatment of various bacterial infections.

The aim of work:

aeruginosa isolated from various clinical sources. The physical properties,

antibiofilm activity as well as whole genome sequencing of isolated phages will be

determined herein. Moreover, the influence of isolated phages on P. aeruginosa

findings of present study would be of great importance and helpful in treatment of

P. aeruginosa related infections.

For fulfillment of this purpose, the following aspects will be investigated:

1- Isolation and identification of P. aeruginosa from different clinical sources.

2- Determination of susceptibility of P. aeruginosa isolates to antimicrobial agents.

3- Isolation and propagation of bacteriophages targeting P. aeruginosa isolates.

4- Using electron microscopy for characterization of the morphology of isolated

5- Determination of pH and thermal stability of isolated bacteriophages.

6- Determination of host range of isolated bacteriophages.

7- Investigating the role of isolated bacteriophages in controlling biofilm formation

8- Genomic characterization of isolated bacteriophages.

9- Characterization the influence of isolated bacteriophages on P. aeruginosa

pathogenesis in vivo using mice infection model.

Pseudomonas aeruginosa is a widespread Gram-negative, aerobic, rod-

P. aeruginosa is an opportunistic pathogen and a major cause of nosocomial

1. Structure and growth conditions

P. aeruginosa is a heterotrophic, motile, rod-shaped bacterium about 1–5 µm

cytoplasmic membrane with a phospholipid bilayer and outer membrane with a

2. Pathogenesis and diseases caused by P. aeruginosa

P. aeruginosa is a causative agent of a diverse array of infections including

2.1. Burn and wound infections (soft tissue infections)