Tahani's20Proposal-V720(1)

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‫دولة ليبيا‬
‫وزارة التعليم العالي‬
State of Libya
Ministry of High Education
Misurata University
Faculty of Science
Department of Biology / Microbiology Program
Comprehensive Molecular Characterization of Therapeutic Lytic
Bacteriophages Against Multidrug-Resistant Pseudomonas aeruginosa
Submitted by:
Tahani M. Ramadhan
A Thesis Proposal Submitted in Partial Fulfillment of the Requirements for
Doctor of Philosophy (PhD) Degree in Microbiology
Supervised by:
Prof. Dr. Farag Bleiblo
Co-Supervised by
Assoc.Prof. Dr. Mohamed A. Ganim
Academic Year 2024 / 2025

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Abstract
The global spread of multidrug-resistant (MDR) Pseudomonas aeruginosa has become a
significant concern for public health, particularly in developing countries, where urgent
alternative approaches to combating the disease are needed. The objective of this study was to
isolate and characterize lytic bacteriophages that target MDR P. aeruginosa strains from
clinical and environmental specimens in Benghazi, Libya. Phages will be isolated by direct
plating and enrichment, purified using plaque assays, and characterized both phenotypically
and molecularly. The analysis will include host range, thermal and pH stability, SDS-PAGE
analysis, PCR for lysogeny-related genes, whole-genome sequencing, and bioinformatics
annotation. The new, lytic phages that we expect to isolate shall exhibit large host ranges,
consistent phenotypic profiles, and genomes devoid of virulence and resistance genes. The
results will fill a regional information gap and aid in the development of phage-based
therapeutics for multi-resistant P. aeruginosa, thereby contributing to the combat against
antimicrobial resistance through an integrated approach that combines classical and genomic
methodologies.

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Table of Contents
1. Introduction
2. Literature Review
2.1 Overview of Pseudomonas aeruginosa
2.2 Emergence of Multidrug Resistance in Pseudomonas aeruginosa
2.3 Bacteriophage Therapy and Host Specificity
2.4 Isolation and Characterization of Bacteriophages
2.5 Gaps in Current Research
3. Research Objectives and Questions
3.1 Primary Objective
3.2 Secondary Objectives
3.3 Research Questions
4. Research Methodology
4.1 Study Design
4.2 Sample Collection and Isolation (Environmental Sources)
4.3 Bacterial Host Preparation (MDR P. aeruginosa Strains)
4.4 Bacteriophage Isolation and Purification
4.5 Host Range Determination
4.6 Bacteriophage Stability Testing (Thermal and pH Stability)
4.7 Molecular Characterization (DNA Extraction, RFLP, SDS-PAGE, PCR)
4.8 Bioinformatics and Genome Annotation
5. Data Analysis
5.1 Descriptive Statistics
5.2 Inferential Statistics
5.3 Molecular and Phylogenetic Analysis
5.4 Host Range Spectrum Interpretation

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6. Ethical Considerations
7. Timeline
7.1 Gantt Chart for 18 Months
8. Budget
8.1 Budget Summary
9. Expected Outcomes
9.1 Isolation of Novel Lytic Phages
9.2 Broad Host Range Candidates
9.3 Data on Phage Efficacy and Stability
9.4 Molecular and Genomic Insights
9.5 Contribution to Knowledge Gaps
9.6 Foundation for Phage Therapy Applications
10. Conclusion
11. References
12. Appendices
12.1 Gantt Chart
12.2 Budget Tables
12.3 Sample Forms (Ethical Approvals, Consent Forms, etc.)

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1. Introduction
Background
Multidrug-resistant (MDR) bacterial infections represent one of the world’s most pressing
health challenges. In 2019, there were 4.95 million deaths related to AMR, including 1.27
million attributable to AMR (Murray et al., 2022). P. aeruginosa, a Gram-negative opportunist,
is one of the leading AMR-related pathogens, contributing to ~19% of AMR-related deaths
(Murray et al., 2022). It is commonly responsible for nosocomial infections, especially in ICUs,
causing pneumonia (including ventilator-associated), sepsis, UTI, and wound infections
(Murray et al., 2022; Moradali & Rehm, 2020). It exhibits considerable versatility and can
produce biofilms with enhanced antibiotic resistance on medical devices (Moradali & Rehm,
2020). Infection with MDR P. aeruginosa is associated with substantially increased mortality;
for example, ventilator-associated disease is reported to result in mortality upwards of 40 to
80% when treated (Moradali & Rehm, 2020). According to the WHO, carbapenem-resistant P.
aeruginosa is a Priority 1: Critical pathogen, and novel treatments are urgently warranted
(Murray et al., 2022).
Significance of the Study
P. aeruginosa also possesses the ability to develop both intrinsic and acquired resistance, such
as natural reduced membrane permeability, efflux pumps (Roberts et al., 2006), β-lactamase
production, and biofilm formation (Pang et al., 2019; Moradali & Rehm, 2020). In Libya, this
is especially serious, as a study from Benghazi found resistance rates to major antipseudomonal
agents ranging from 49 to 76%, and blaNDM and blaGES-1 genes were detected in 26.7% and
17.8% of the isolates, respectively (Gadaime et al., 2024). All of these strains were able to form
biofilms, which made the treatment difficult. These MDR infections are difficult to treat, and
usually require colistin or combination therapy, and even then, may not respond. Increasing
rates of MDR P. aeruginosa from Benghazi are indicative of an emerging problem which will
put more pressure on alternative treatment strategies.
Phage therapy can be a solution. Phages belong to the virus family, which can only infect
bacteria, and are so common and specific to the bacteria they target that they are easily found
in nature. They multiply at the site of infection and are harmless to human cells and normal
flora. Phage therapy is experiencing a renaissance in response to the AMR emergency (Pires et
al., 2020; Abedon et al., 2017). Lytic phages have been effective in treating antibiotic-resistant
P. aeruginosa infections, which include biofilm infections (Aslam et al., 2023; Moradali &
Rehm, 2020). Examples include better treatment outcomes in cystic fibrosis and chronic
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wounds (Forti et al., 2018). Phages are readily available near their source and can be tailored
to infect modern strains, making them a sustainable treatment option.
Problem
Phage therapy is not perfect, however. Most phages have relatively narrow host ranges; thus,
phages need to be isolated against diverse or cocktail styles to target clinical strains (Kortright
et al., 2019). No phage products exist, and phage therapy has not been studied in Libya.
therapy for local P. aeruginosa. Given the growing MDR in Benghazi and scarce antibacterial
alternatives, investigation of phage therapies should be urgently considered. The proposed
study will isolate lytic phages from community sources (e.g., sewage) against MDR P.
aeruginosa and characterise them to determine their therapeutic potential, establishing the basis
for future phage-based therapeutics.
2. Literature Review
2.1 Overview of Pseudomonas aeruginosa
Pseudomonas aeruginosa is a ubiquitous, motile, Gram-negative rod bacterium that is often
isolated from soil, water, and plants. It is an opportunistic pathogen that generally infects
immunocompromised individuals with compromised barriers (Moradali et al., 2017). In
clinical practice, it leads to hospital-acquired infections such as ventilator-associated
pneumonia, bacteraemia, urinary tract infections (UTIs) (particularly catheter-associated),
surgical site infections, and burn wound infections (Moradali & Rehm, 2020). Pseudomonas
aeruginosa chronic lung colonization and biofilm formation in cystic fibrosis (CF) are
significant contributors to morbidity and mortality. It also results in chronic infections in those
with chronic obstructive pulmonary disease, bronchiectasis, and contact lens wearers with
keratitis.
This organism grows in nutrient-limited and challenging surroundings such as hospitals and
the environment due to its adaptability (Moradali & Rehm, 2020). They secrete a variety of
virulence factors, including exotoxins, proteases, and pyocyanin, that cause host tissue
destruction and immune evasion (Moradali et al., 2017). Its signature is biofilm, which helps
the infections become chronic and extremely antibiotic-resistant. Biofilm bacteria may be as
much as 1000-fold more tolerant than planktonic forms, which helps make P. aeruginosa a
healthcare “superbug.”
2.2 Development of Multidrug Resistance in Pseudomonas aeruginosa
P. aeruginosa has natural resistance due to low permeability of the outer membrane and
multidrug efflux pumps such as MexAB-OprM. It also expresses AmpC β-lactamase and
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quickly selects out resistant mutants upon antibiotic challenge (Pang et al., 2019). It acquires
new weapons via horizontal gene transfer (Breidenstein et al., 2011).
The prevalence of MDR P. aeruginosa—defined as resistance to ≥1 agent in ≥3 drug classes—
has been rising. An Ethiopian review from 2024 reported MDR in 80.5% of clinical isolates
with around 99% and 75% resistance to ceftiaxone and TMP-SMX, respectively (Sada &
Tessema, 2024). In Libya, a recent study's results demonstrate 48.9% resistance to piperacillintazobactam and 75.6% resistance to ciprofloxacin (Gadaime et al., 2024). Carbapenem
resistance is of particular concern, with NDM, VIM, and OXA variants being involved.
MDR isolates engender higher morbidity, mortality, and complexity of management. Colistin
or combination therapy is frequently needed, with a poor outcome. Across pneumonia, sepsis
and burn wound infections, MDR P. aeruginosa substantially increases the risk of death
(Moradali & Rehm, 2020), requiring emergent treatment alternatives.
2.3 Bacteriophage Therapy and Host Specificity
Bacteriophages (phages) are bacterial viruses with a narrow host range, which is conferred by
the recognition of bacterial ligands (Altamirano & Barr, 2019). This specificity spares human
cells and normal flora. At the site of infection, phages replicate and liberate lethal lytic enzymes
(endolysins) to kill the microbe (Oechslin, 2018).
But host specificity can be a constraint. A phage that works on one strain may fail to infect the
other. Therapy may be based on personalized cocktails or on personalized phages (Pires et al.,
2016). Trials demonstrated failures when phage mixtures didn’t match patient strains.
Therefore, it is desirable to provide broad-host-range lytic phages including those of Akremi
et al. (2022) that lysed multiple P. aeruginosa strains. Since bacteriophages are unable to infect
eukaryotic hosts, they are nontoxic. Instead of the temperate phages favored before, lytic
phages kill bacteria as they replicate. Nevertheless, they depend on priorogy at diagnosis and
real-time diagnosis is important.
The use of phages as therapy can be more specific against MDR bacteria; however, the success
of phage therapy hinges on the selection of phages against the pathogens. The former demand
for lytic phages with a broad strain coverage can be fulfilled in the context of this project.
2.4 Isolation and Characterization of Bacteriophages
Phages are often found in abundance in bacterial-rich environments such as sewage, hospital
wastewater, and natural waters (Forti et al., 2018; Wandro et al., 2022). Bacteria are filtered
out, and the filtrate is checked for phage activity by plaque assay. Firstly it is direct agar overlay
and enrichment procedures.
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Following plaque visualization, phages are then purified by serial plating. Critical in
characterizing a phage is identifying host range, morphology, e.g., Myoviridae, or Podoviridae
by EM, ability to withstand extreme pH and temperature, and replication parameters such as
adsorption rate and burst size (Forti et al., 2018).
Safety (absence of virulence and resistance genes) and the lytic nature (lack of integrase genes)
are confirmed through genomic analysis. Taxonomic identification and protein function
prediction through full genome sequencing, as has been exploited by Akremi et al. (2022).
Recent samples are Sada & Tessema (2024) with 17 E. coli phages being isolated and 7
eventually chosen for analysis by these authors.31 In this paper, family identification was
performed by EM and PCR. Akremi et al. (2022) demonstrated that P. aeruginosa phages are
stable over pH 3–11 and 4–50 °C, which is suitable for multiple therapeutics delivery systems.
Complete characterisation is essential to determine phage sensitivity, stability, and suitability
as therapeutic agents.
2.5 Gaps in Current Research
Despite worldwide interest in phage therapy, there is considerable unfinished business. On the
one hand, Libyan phages against Libyan P. aeruginosa strains have not yet been the target of
study. Indeed, for genetic reasons, exogenous phages may be ineffective. Enrichment of local
phages raises the likelihood of success.
Second, clinical translation will need phages that are genome-safe and well-characterised. This
research will adhere to rigorous and transparent procedures, and the genomic data will be made
publicly available in repositories.
Third, the host range must be well-defined. Screening phages against various MDR strains is a
prerequisite to cocktail development, which is a major drawback with current approaches
(Khan Mirzaei & Nilsson, 2015).
Last, his project includes cutting-edge proteomics (such as SDS-PAGE) and genome-wide
methodologies, which tend to be lacking in the first rounds of the study. These activities will
result in fully characterized phages that are available for the next stage of clinical development.
In summary, literature upholds the environmental distinction of lytic phages for MDR P.
aeruginosa. This research is based on verifying techniques in Benghazi, and is geared towards
the discovery of new phages that have a broad host range, as well as leading to the development
of phage therapy in Libya, in furtherance of global AMR action.
3. Research Objectives and Questions
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3.1 Primary Objective:
Isolation and characterization of Pseudomonas aeruginosa lytic bacteriophages from sewage
and wastewater from the environment of Benghazi, Libya, that are active on a multidrugresistant clinical strain of P. aeruginosa.
3.2 Secondary Objectives:
O1: Recover and enrich phages isolated from local sewage and water, and screen for lytic
activity against MDR P. aeruginosa.
O2: Characterization of the host range of each phage tested against a panel of clinical P.
aeruginosa isolates with a range of resistance profiles.
O3: Characterize temperature and pH-stability of phages that are related to storage and therapy.
O4: Molecular characterization of phages (restriction digestion, sequencing) and safety
determination for broad classification.
O5: Genome annotations and phylogenetic analysis to compare isolates to known phage
lineages.
O6: Employ kit and package statistical and computational methods to analyze data and draw
powerful evidence-based conclusions.
By achieving these goals, the investigators hope to create biologically well characterized
phages suitable for future therapeutic development and deeper research.
3.3 Research Questions:
To streamline the analysis, the study focuses:
Which lytic bacteriophages infecting P. aeruginosa are present in wastewater and
environmental waters in Benghazi?
o (a) How many unique phages can be isolated and what are their plaque morphologies and
titers?
o (b) Are these phages non-lysogens?
How potent are these phages on diverse MDR P. aeruginosa isolates?
o (a) What fraction of MDR isolates are infected by each phage?
o (b) Is the phage’s host range broad or specific?
How stable are these phages under varying conditions?
o (a) What temperatures can they survive (4 °C to 50 °C, freeze-thaw cycles)?
o (b) At what pH levels do they function (e.g., pH 3 to 11)?
o (c) What are the best conditions for storage and use?
What are the molecular/genomic traits of the phages?
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o (a) What is the size and nature of their genomes (e.g., GC content, DNA/RNA)?
o (b) Are there bad genes (virulence, resistance, integrases) in genomes?
o (c) How closely are they related to known phages (BLAST homology searches)? Are they
novel?
What is the phylogenetic relationship of these phages to others?
o (a) What taxonomic family does each phage belong to (e.g., Myoviridae)?
o (b) What does phylogenetic analysis show them to be to each other and to known phages?
o (c) Are there genomics/proteomics correlates of host range or functional?
Through systematic resolution of these questions, the project will lead to a holistic portrait of
locally sourced phages useful for establishing a platform for region-specific phage therapy
against MDR P. aeruginosa, while informing preclinical or formulation endeavors to come.
4. Research Methodology
4.1 Study Design
This is an experimental laboratory study performing microbiological, molecular, and
bioinformatic methodologies and will be organized in eight phases as follows:
Environmental sample collection
Bacterial strain preparation
Isolation of phages (direct and enrichment procedure)
Phage purification and amplification
Biological characterization (host range, stability, and so on)
Molecular assays (DNA extraction, PCR)
Genome sequencing and annotation
Data analysis and interpretation
This is an exploratory study, however, which can reproduce its analyses (Kutter &
Sulakvelidze, 2005; Clokie & Kropinski, 2009). Descriptive (plaque morphology) and
quantitative (host range, titer) approaches will be used to construct comprehensive biological
and molecular descriptions of each phage.
All experiments will be performed in the BSL-2 Microbiology Laboratory of the University of
Benghazi (or an equivalent one). The lab is equipped with incubators (room temperature/37
°C), laminar flow hoods, autoclave, centrifuges, PCR thermocyclers, gel systems,
spectrophotometers, -20 C/-80 °C freezers, and microscopes. External access for phage
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morphology through an electron microscope will be organized. Media and reagents will be
covered as described in the budget.
The project does not include human or animal studies. The clinical strains of bacteria will be
employed with institutional consent (ethics).
The overall aim of PHEiRA is to generate comprehensive molecular and biological datasets for
each phage, thereby underpinning cross-phage comparisons and highlighting candidates for
therapeutic application.
4.2 Sample Collection and Isolation
Water and sewage swabs will be collected from select hot spots in Benghazi as follows:
(a) The primary wastewater-treating outlet,
(b) sewage from hospitals (Benghazi Medical Center, Al-Jalaa Hospital),
(c) Wet drainage dams.
(d) coastal-zone urban runoff sites.
Samples will be collected in sterile containers, with 1-2 liters per site. When available, samples
of sediment will also be collected. Samples will be transferred on ice (~4°C) and analyzed
between 6 and 12 hours.
In the laboratory, the samples will be clarified through centrifugation (10,000×g, 10 min, 4°C)
or sedimentation, followed by stepwise filtration with a final 0.22 μm filtration to obtain phagerich filtrates. The available vacuum or syringe filters will be used.
The filtrates will be the crude phage lysates for further detection. PEG 8000 and NaCl may
also be added for concentration, if necessary. Phages will be pelleted by centrifugation and
resuspended in SM buffer (50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO₄, pH 7.5).
Where possible, unrefined filtrates can be used directly for the primary isolation procedure as
sewage usually contains sufficient phage.
4.3 Preparation of Bacterial Host (MDR P. aeruginosa Strains)
A set of 10 to 20 MDR P. aeruginosa clinical isolates will be collected from hospital
microbiology laboratories of Benghazi (e.g., Benghazi Medical Center and University of
Benghazi). The focal strains for resistance to carbapenems, fluoroquinolones and
aminoglycosides, respectively, will be included.
Isolates from various sources (sputum, urine, or wounds) and different institutions should be
included. Isolates will be anonymized in code.
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One of the reference strains (e.g., PAO1 or PA14) will also be used as a control and in the
propagation thereof. Nevertheless, emphasis is given to the phages against MDR isolates. One
or two known well-characterized clinical isolates (e.g., strong biofilm formers) will be used as
our enrichment host, forming a stimulus to bias selection towards clinically relevant phage.
All strains will be maintained as glycerol stocks at –80C and grown on nutrient or tryptic soy
agar as working stocks. Fresh cultures will be grown in LB or TSB broth at 37°C with shaking
until mid-log phase (OD₆₀₀ ≈ 0.4–0.6) prior to experiments, to promote the best possible phage
infection (Forti et al., 2018).
For host range tests, all isolates will be tested against each phage in standard conditions to
verify cultivability and susceptibility.
Quality Control
All isolates will be confirmed as P. aeruginosa by standard biochemical tests (65), oxidase
reactivity, pyocyanin production on Pseudomonas P agar, and growth at 42°C [65, 66], as well
as molecular confirmation by PCR targeting a species-specific gene (e.g., ecfX) [67]. Antibiotic
resistance profiles will be re-validated using disk diffusion or MIC assays to correlate
resistance with phage susceptibility.
4.4 Bacteriophage Isolation and Purification
(a) Plaque Assay The method of plaque assay (double-layer agar)
was used as described by Reed and Muench with minor modifications.
PGL also will test for phages in filtered environmental samples using the DLA procedure. Midlog phase P. aeruginosa (100μL) will be combined with 3mL soft agar (0.6%) and 100μL of
filtrate or its serial dilutions and overlaid onto LB agar plates and incubated at 37°C and plaques
(lysis zones) will indicate phage presence. Clear plaques may indicate only lytic phages; turbid
plaques may be indicative of temperate activity (Wandro et al., 2022). Plates will be monitored
for 18–48 hours. If no plaques are formed, enrichment will be conducted.
(b) Enrichment Culture Method
For enhanced phage detection, 10–20mL of filtrate will be co-incubated with 1mL of host
culture (one or multiple strains) at 30–37°C for 18–24 hours. Following growth, cultures will
be spun and the sup collected and filter-sterilized to yield enriched lysates−tested with the DLA
assay. Using distinct host strains for each sample allows to broaden the recovered phage
diversity.
Isolation of Individual Phages
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Isolated plaques will be selected with sterile instruments and eluted in SM buffer or broth to
generate crude stocks. Three rounds of plaque purification will provide clonality.
Morphological characteristics (size and clarity of plaques) will be recorded. Each phage will
be assigned a code (e.g., “BGP1”), and the source and host will be indicated.
Phage Amplification and Storage
Phage particles are purified and propagated in liquid cultures at low MOI (~0.1) and cultured
until lysed. Lysates will be filtered and titered (PFU/mL). High-titer stocks (>10⁹ PFU/mL)
will be maintained in SM buffer with chloroform at 4°C, and for long-term storage, kept at –
80°C in the presence of cryoprotectants. We anticipate 5–15 unique lytic phages based on other
environmental reports (Wandro et al., 2022).
Host Range Determination
Each purified phage will do subsequent adsorption tests with all available P. aeruginosa strains
(clinical MDR isolates and reference strain) to test its host range. Two overlapping assays will
be used:
Host Range Determination
Spot Test
This one-step screening methodology consists of flooding the lawns of each P. aeruginosa strain
cultured on LB agar overlaid with top agar. After the plates have solidified, 5 to 10 μL of each
high-titer phage lysate (~10⁸ PFU/mL) will be spotted onto the plate.
Lysis will be assessed following an overnight incubation at 37°C:
· (+) clear lysis,
· (+/–) incomplete/turbid lysis,
· (–) no lysis.
The controls will be SM buffer and a sensitive host. The spot test is very sensitive, and even
subtle lytic activity is detected.
Efficiency of Plating (EOP) Assay
For spot-positive strains, EOP will provide a quantitative measure of infectivity. Serially
diluted phage suspensions will be plated using the DLA method on the test strain and on the
original isolation host. EOP is calculated as:
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EOP=PFU/mL on test strainPFU/mL on original host\text{EOP} = \frac{\text{PFU/mL on
test strain}}{\text{PFU/mL on original
host}}EOP=PFU/mL on original hostPFU/mL on test strain
An EOP near 1.0 indicates similar efficiency; lower values (e.g., 0.001) suggest limited
infectivity. Phages with high EOP on clinical strains will be prioritized for therapeutic
potential.
Data Interpretation and Additional Testing
4.5 Host Range Determination
Host range data will categorize phages as broad- or narrow-spectrum. Associations between
phage infectivity and bacterial traits (e.g., O-antigen types) will be explored.
If resources permit, select phages will be tested against non-P. aeruginosa species (e.g., E. coli,
A. baumannii) to confirm specificity and biosafety.
Phage combinations with complementary host ranges (e.g., Phage A covers isolates 1–5; Phage
B covers 6–10) will be identified to guide potential cocktail formulation.
4.6 Bacteriophage Stability Testing
Thermal Stability
Phage aliquots in SM buffer will be incubated at set temperatures (4 °C, 25 °C, 37 °C, 42 °C,
50 °C, 60 °C) for 1 hour. Post-incubation, samples will be diluted and titrated via plaque assay
to assess residual infectivity. Survival fractions will be calculated relative to control samples
stored at 4°C or 25 °C.
Phages will be characterized by their thermal inactivation point (e.g., temperature at which
99% titer loss occurs). Some may tolerate temperatures of up to 60°C briefly, but most will be
inactivated above 50°C (Wandro et al., 2022).
For long-term evaluation, phages will be incubated at 37 °C for 7 days with daily titer
measurements. Phages retaining high titers at both 4 °C and 37 °C over time will be considered
more suitable for clinical storage and delivery.
pH Stability
Phage lysates will be tested across a range of pH values using appropriate buffers:
pH 2: Glycine-HCl
pH 4: Acetate or citrate
pH 7: Phosphate
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pH 9: Tris-HCl or carbonate
pH 11: CAPS or NaOH
Subsamples will be incubated at room temperature for 1–2 h, neutralized (if necessary) and
titrated by plaque assay. The majority of phages are stable from pH 6 to 8, but activity
diminishes at extremes. However, while some of them tolerate acidic or alkaline conditions
(Forti et al., 2018), others do not end sporulate when exposed to these same conditions. These
findings will aid in evaluating the applicability of phages through various therapeutic means.
For example, phage with instability at pH 2 would be unsuitable for oral administration, but
appropriate for inhalation or injection.
Data Analysis and Presentation
Stability will be visualized by plotting phage titers against temperature and pH. Total
inactivation (none, no plaques) will also be explicitly reported. The profile of each of the
phages will be described as follows:
"Phage BGP2 is active at pH 5-10 with a 100-fold decrease in phage activity at pH 4, and the
phage becomes inactivated at pH 3. It resists at 50°C for 1 h but is inactivated at 60°C.
Such data will facilitate formulation readiness, and the choice of which phages could be applied
clinically or in the industry.
4.7 Molecular Characterization
Phage DNA Extraction
High-titer lysates are treated with DNase and RNase to eliminate host nucleic acids. Next,
phages will be lysed with proteinase K and SDS. DNA extraction will be carried out using
either phenol–chloroform and ethanol precipitation (Forti et al., 2018; Adriaenssens & Brister,
2017) or commercial extraction kits. Yield and integrity will be evaluated using gel
electrophoresis.
Restriction Fragment Length Polymorphism (RFLP)
Phage DNA will be cut with restriction enzymes such as EcoRI, HindIII or BamHI. The
products will be fractionated on agarose gels for the detection of characteristic banding
patterns. RFLP is a quick technique to classify phages before they are sequenced.
SDS-PAGE (Protein Profiling)
SDS-PAGE analysis will separate the phage structural proteins following PEG or
ultracentrifugation concentration. Denature and load proteins onto 12% gels, stain (e.g.,
Coomassie), and compare among phages. Higher molecular weight bands (30-60kDa)
represent capsid or tail proteins. In case of need, bands can also be cut for mass spectrometry
from external collaborations.
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Polymerase Chain Reaction (PCR)
Targeted PCR assays will assess:
Lysogeny markers (eg integrase genes) - adequate representation of lysogenic phages to be
deprioritised.
Virulence/resistance genes: for the safety of therapy.
Family-specific markers such as (Capsid or DNA polymerase genes): for taxonomic
assignment-31.
(Optional) P. aeruginosa strain typing if applicable during the study
The PCR result will be visualized by gel electrophoresis. Those phages with undesirable genes
will not be used in therapy but will still be catalogued. This set of molecular tools will provide
a complete fingerprint for each phage, which will include genome size, restriction profile,
protein composition, and the presence of hallmark genes.
4.8 Bioinformatics and Genome Annotation
Phage isolates of the broadest range and most stable will also be whole-genome sequenced. At
least 3–5 genomes will be sequenced to take advantage of recent sequencing cost reductions.
Sequencing Strategy
DNA libraries will be generated for the:
Illumina (short-read, high-accuracy)
Oxford Nanopore (long-read, few assembly gaps)
Hybrid methods utilize both techniques with high completeness.
If in-house sequencing is not available, external sequencing service providers will be
employed, targeting ~100× genome coverage.
Bioinformatic Analyses
Genome Assembly
Reads will be quality- screened, trimmed, and assembled Singh tools such as SPAdes or
Unicycler. Quality of the assembly will be evaluated in terms of coverage depth, contiguity
and lack of host DNA. The average size of lytic P. aeruginosa phages is ~90–100kb (Forti et
al., 2018).
Genome Annotation
Annotations will be done using:
RAST, PHANOTATE and PHASTER
BLASTp/BLASTn search against NCBI viral database
Annotation targets include:
Genes of the structure (capsid, tail fibers)
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Genes of DNA replication and lysis
Regulatory elements
Genomes will be searched for toxin genes, antibiotic resistance genes, and lysogeny markers
(for example, integrases). Only phages that are free of those will progress towards therapy
development.
Genome Features Analysis
Comparing the GC content with that of P. aeruginosa (≈66%)
Topology of genome (linear/circular/repeats), derived from read mapping patterns
Host adaptation by analysis of tRNA genes
Comparative Genomics and Phylogenetics
Comparisons to other known phages (e.g., PAK_P1, PB1-like) will be made against the phage
genomes. A novel classification is attributed to phages with <95% identity to defined species.
Phylogenetic reconstruction Phylogenetic trees will be estimated based on:
Clustal Omega or MUSCLE for alignment
MEGA X (whatever method you like for generating the tree, e.g., NJ or ML, but with bootstrap
support)
Clustering with known genera will enable taxonomic assignment.
Genome Visualization and Data Sharing
Genome maps will be constructed with SnapGene or BRIG to illustrate the modular
organization (replication, structure, lysis).
All annotated sequences will be deposited to NCBI GenBank with accession number(s)
reported in the thesis and/or publication(s) for transparency and accessibility.
Upon finishing this pipeline, every phage will have a complete genomic profile that includes:
Therapeutic safety (no toxins/AMR genes)
Evolutionary lineage
Unique genetic characteristics
This not only finalizes molecular analysis but also meets the goal of the study to screen phage
candidate that could control MDR P. aeruginosa.
5. Data Analysis
5.1 DescriptiveStatistics
This investigation will provide qualitative sightings, quantitative (such as plaque count, titer),
and genomic sequences. Descriptive and inferential statistics will be used (p < 0.05). Statistical
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analysis all will be performed using SPSS software (v28) and R statistical software (v4. x),
and GraphPad Prism for graphing.
Stability: Titer stability under different conditions (log₁₀ PFU/mL).
Genomic Contents: Total size of genome, GC content, predicted ORFs, tRNA figures.
Continous data (titers, burst size) will be summarized as means ± SD, and categorical data (e.g.
lysis presence) as frequencies and percentages.
5.2 Inferential Statistics
Proper tests will be implemented to justify conclusions:
Stability Determination (Thermal/pH stability): One way analysis of variance -ANOVA test,
follow by Tukeys significant test.
Proximal/Distal Comparisons: Paired t-tests (For example paired before and after pH
exposure).
Host Range Associations: Test for association with resistance profiles by Chi-square.
Comparisons of Growth Curves: ANOVA on per cent burst size and latency.
Phylogenetic Trees: Bootstrap support (≥70%) will validate clades.
Sequence-based comparisons (e.g., ANI) will be computed and are NOT proposed to be tested
for statistically.
Software and Visualization Tools
SPSS : t- tests, chi- square, ANOVA.
R: advanced analytics, clustering, heatmaps.
GraphPad Prism Graphs (bar graphs, line plots etc).
SnapGene: Genome map construction.
Graphs will adhere to the best practice guidelines for clarity, labeling, and error bars.
5.3 Molecular and Phylogenetic Analysis
RFLP: Gel bands are compared; dendrograms may indicate clusters of similarity.
Phylogenetic Trees: Constructed by MEGA X from sequence alignments (Clustal, MUSCLE);
examined for conserved motifs, divergence etc.
5.4 Host Range Spectrum Interpretation
A matrix of phage vs. strains (lysis/EOP data) will be used.
Heatmaps (e.g., R's pheatmap) will show patterns of infectivity.
Cluster analysis that defines phage groupings or aids cocktail design.
Synthesis of Results
The results will be combined to:
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Prioritize the top phage candidates.
Support efficacy, stability and host range conclusions.
Flag any anomalies for further investigation.
The findings will be presented in APA format: test statistic, degrees of freedom, and the exact
p-value. Careful data interpretation will lead to strong, replicable conclusions based on
evidence that will be applicable to all research questions.
6. Ethical Considerations
Since it is a laboratory work without participants, neither in human nor in animals, it will have
to comply, however, with specific ethical and biosafety provisions.
Use of Clinical Isolates
MDR strains of Pseudomonas aeruginosa will be collected from diagnostic laboratories in
Benghazi using anonymized remnant clinical specimens. No patient information will be
retrieved and only metadata information including isolate source and resistance data will be
collected. Appropriate hospital committee ethical review or material transfer agreements will
be obtained. Patient consent is not applicable because of complete de-identification of cases,
however institutional approvals will be reported for the sake of transparency.
Biosafety Measures
All work with MDR strains and phages will be house according to BSL-2 guidelines, which
include:
· PPE practise (lab coats, gloves, eye protection)
· All open manipulation to be performed using a biosafety cabinet
Surface disinfection (10 % bleach or 70 % ethanol)
· Autoclaving all biohazardous waste
· Actions to avoid phages losing at the environment
Antibiotic Screening
Susceptibility testing to antibiotics (discs, MIC) shall be performed responsibly to prevent
resistance emergence. Antibiotics-containing waste will be discarded by chemistry safety
regulations.
Environmental Sampling and Permissions
Sampling will be done from sewage and natural waters, but only upon approval from municipal
and hospital authorities. Collection will be completed with little to no environmental impact
using sealed spill/leak proof containers to prevent contamination during shipping.
Dual-Use Risk Awareness
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Although phages are harmless to people, pathogen use requires dual-use consideration.
Mitigation strategies include:
Non-GMO (Not genetically modified)
Exclusive of naturally harvested strains
Transparent measure for public health purposes
Compliance with biosafety and biosecurity best practices at the institution
Data Integrity and Scientific Conduct
All findings, whether positive or negative, will be accurately recorded and published. Images
(e.g., gels, plaques) will be processed as little as possible for enhancement. For adapted
protocols and past results we will give appropriate citation and attribution.
Compliance and Oversight
Although no GMO’s will be employed, National and international biosafety laws, will be fully
adhered to during handling and transportation of biological materials for the study. In the event
that DNA is sent (to a third-party sequencer) the DNA shipments also will include any
necessary biosafety documents.
University IBC and human [ if necessary ( our committee is in place, just have to fill the app.)
] approval will also be garnered. The total number of certificates will be added to the final
proposal.
7. Timeline
The research is to be conducted over a period of 18 months and will be phased with the
expectation of overlapping work tasks for more effective design. Appendix A contains the Gantt
chart.
7.1 Gantt chart for 18 Months
Months 1–2: Preparation and Review of Literature
Complete the research plan, seek approvals, get lab supplies, and do technical training with
model phages.
Milestones: Institutional approval; construction of lab; literature review finished.
Months 2–4: Sample Collection
Collecting and Analysing environmental samples from various Locations in Benghazi. Obtain
and characterise ~15 MDR P. aeruginosa isolates.
Targets: Infrequently feed 8–10 samples; set up strain bank.
Months 3–6: Phage Isolation
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Start phage screening for direct and enrichment protocols. Conduct iterative plaque
purification.
Benchmarks: First phage isolation by Month 4; library of ~10 phages by Month 6.
Months 5–7: Phage Amplification
Sequential Purification and Amplification to High-Titer Stocks.
Milestones: Hig h-titer phages that have been purified by Month 7.
Months 7-9: Host Range & Stability Testing.
Perform spot, EOP and temperature or pH stability assays; test growth parameters (burst size).
Milestones: Host range matrix - completed (M8); Stability profiles (Month 9).
Months 8–10: Molecular Characterization
Make phage DNA, and do RFLP, SDS-PAGE, and PCR for lysogeny, toxin, and resistance
genes.
Milestones: Molecular results ready by Month 10; candidates selected for sequencing.
Months 10–12: Sequencing & Analysis of Genome
Assemble
a
large
number
of
selected
complete
genomes,
annotate
with
RAST/PHASTER/BLAST and make phylogenetic trees.
Milestones: Draft genome annotations and submission to GenBank by Month 12.
Months 13–15: Data Analysis
Perform statistical analyses (such as ANOVA and chi-square), creating heatmaps and
phylogenetic trees, and merge results.
Timelines: Data: Month 14; interpretation/conclusions written by Month 15.
Months 15–17: Thesis Writing
Draft MSc thesis, prepare figures and tables and revise according to in-house feedback.
Milestones: Draft finalized in mid-Month 17.
Month 18: Thesis Submission and Defence
Minor revisions, thesis submission, and defense presentation.
Milestones: Thesis submitted, and defense completed at month 18.
8. Budget
8.1 Budget Summary
Item
Quantity/Duration
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Estimated
Cost (LYD)

22.

Laboratory
Consumables
Culture media &
reagents
Disposable supplies
Filtration units (0.22
μm)
PCR primers &
reagents
DNA extraction kits
Restriction enzymes &
gel items
Culture media, reagents, disposables, PCR
primers, DNA kits, filtration units, restriction
enzymes, SDS-PAGE supplies
18 months
Continuous use
~100 filters
For multiple screenings
~10 phage samples
Agarose, enzymes, ladders
Sequencing (service)
5 phage genomes (Illumina)
SDS-PAGE reagents
Acrylamide, markers, stains
Equipment Usage &
Services
Electron microscopy
External service for 2 samples
Computer/software
Workstation + required licenses
Field Sampling
Transportation
Sample containers &
ice
Total Estimated Cost
Local travel to sites
20 bottles + coolers
Final total to be detailed in Appendix
9. Expected Outcomes
9.1 Isolation of Novel Phages
A minimum of 5–10 individual lytic phages may be present in wastewater in Benghazi against
MDR P. aeruginosa, thereby contributing to phage diversity globally, particularly with exotic
Libyan isolates such as “BGP-1”.
9.2 Broad Host Range Candidates
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We anticipate that 1 or more phages will infect >80% of tested MDR isolates, and/or discover
mutually complementing, narrow-host-range phages for cocktail design—meeting the need for
flexible therapy alternatives.
9.3 .Efficacy & Stability Profiles
Efficacy: Strong infectivity (EOP > 0.5) against MDR strains is expected.
Stability Most likely stable between 4°C–37°C, pH 5–9, some may tolerate extremes.
Optional replication (performed): Anticipated burst size 100 PFU/cell and latency 20 min.
9.4 Molecular & Genomic Insights
Genomic sequencing will provide phage taxonomy, and will verify safety (no toxins, AMR
genes).
Some of the phages might be categorized into Pakpunavirus or PB1-like groups; while the rest
might be new.
SDS-PAGE will demonstrate capsid proteins (~45 kDa) and could show novel proteins.
9.5 Knowledge Contribution
Regional: The first Phage investigation targeting P. aeruginosa in Libya.
Pragmatic: How we tailored methods to a resource-poor context will inform other researchers.
Therapeutic Design: Equips rationally effective design of phage cocktails with strong empirical
data.
9.6 Foundation for Phage Therapy
At the conclusion of this project, we hope to provide the following:
A bacteriophage lysing ≥90% of local MDR isolates.
A validated data set for pre-clinical trials.
10. Conclusion
Multidrug Resistant (MDR) Pseudomonas aeruginosa is an increasing problem affecting
healthcare in Libya and around the globe. This MSc project provides a rigorous methodology
to deal with this hazard as we will isolate and characterize lytic bacteriophages for medical
exploitation as alternative treatment.
Through the isolation of environmental samples in Benghazi and the focus on indigenous MDR
isolates the present study is intended to provide a initial platform for the objective of phage
therapy. It includes major sections on sample processing and plaque assays through host range
profiling, stability testing and analysis of genomes with modern bioinformatics analysis tools.
Key outcomes include:
Kill curves for previously isolated lytic phages toward local P. aeruginosa strains.
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Understanding of phage potency, stability, and genomic stability.
Design of a putative MDR isolates targeted phage cocktail.
This study has practical and scientific significance:
Clinical Effect: Provides a potential phage-based therapeutic approach for multidrug-resistant
infections in Libya.
Value: Introduces genomic and phenotypic information from an underrepresented population
to the global phage community.
Through maintaining strong ethical and biosafety practices, this project is doing its part to keep
its findings ethical and reproducible. It provides a local but globally relevant response in the
face of a global AMR challenge. In the final analysis, this research returns phages to their place
as “living antibiotics” that are natural enemies of MDR bacteria, and partners of physicians in
antibiotic-treated infection.
11. References
1. Akremi, I., Merabishvili, M., Jlidi, M., Haj Brahim, A., Ben Ali, M., Karoui, A., ... & Ben Ali, M.
(2022). Isolation and characterization of lytic Pseudomonas aeruginosa bacteriophages from
sewage samples in Tunisia. Viruses, 14(11), 2339.
2. Aslam, S., Lampley, E., Wooten, D., Karris, M., Abdel-Mohsen, M., & Simon, D. (2024).
Pseudomonas aeruginosa ventricular assist device infections: Findings
from ineéective phage therapies in five cases. Antimicrobial Agents and Chemotherapy, 68(4),
e01728-23.
3. Asmare, Z., Reta, M. A., Gashaw, Y., Getachew, E., Sisay, A., Gashaw, M., ... & Abate, B. B. (2024).
Antimicrobial resistance profile of Pseudomonas aeruginosa clinical isolates from healthcareassociated infections in Ethiopia: A systematic review and meta-analysis. PLoS ONE, 19(8),
e0308946.
4. Eiselt, V. A., Bereswill, S., & Heimesaat, M. M. (2024). Phage therapy in lung infections caused
by multidrug-resistant Pseudomonas aeruginosa – a literature review. European Journal of
Microbiology and Immunology, 14(1), 1–10.
5. Gadaime, N. K., Haddadin, R. N., Shehabi, A. A., & Omran, I. N. (2024). Antimicrobial resistance
and carbapenemase dissemination in Pseudomonas aeruginosa isolates from Libyan hospitals: A
call for surveillance and intervention. Libyan Journal of Medicine, 19(1), 2344320.
6. Jault, P., Leclerc, T., Jennes, S., Pirnay, J. P., Que, Y. A., Resch, G., ... & Damas, P. (2019). Eéicacy and
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tolerability of a bacteriophage cocktail in treating burn wounds infected by Pseudomonas
aeruginosa (PhagoBurn): A randomised, controlled, double-blind phase 1/2 trial. The Lancet
Infectious Diseases, 19(1), 35–45.
7. Kim, M. K., Sacher, J. C., Bollyky, P. L., & others. (2025). Bacteriophage therapy for multidrugresistant infections: Current technologies and therapeutic approaches. Journal of Clinical
Investigation, 135(5), e187996.
8. Onallah, H., Negedu, M., Diallo, K., Miambi, B., Ba, B., & Lagneaux, D. (2023). Refractory
Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series.
Med, 4(9), 600–611.
9. Sada, T. S., & Tessema, T. S. (2024). Isolation and characterization of lytic bacteriophages from
various sources in Addis Ababa against antimicrobial- resistant diarrheagenic Escherichia coli
strains and evaluation of their therapeutic potential. BMC Infectious Diseases, 24(1), 310.
12. Appendices
Appendix A – Gantt Chart (Research Timeline): A Ganttchart is provided illustrating the project
schedule over 18 months. It outlines each major task (literature review, sample collection, phage
isolation, characterization experiments, analysis, writing) along a timeline broken into months,
highlighting overlapping activities and key milestones (see attached chart).
Appendix B – List of Samples and Isolates: Detailed table of environmental sample sites (sewage
and water sources in Benghazi) and the MDR P. aeruginosa clinical isolates (with source and
resistance profile) to be used in the study. This appendix includes anonymized strain IDs and
antibiogram summaries for reference.
Appendix C – Ethical Approval and Supporting Documents: Includes the ethics committee
approval letter for use of clinical bacterial isolates, and a biosafety
compliance form for handling pathogenic organisms. Also provided is a letter of collaboration
from the Benghazi wastewater management authority permitting sewage sample collection.
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