1 Isolation and Phenotypic Characterization of Bacteriophage

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Isolation and Phenotypic Characterization of Bacteriophage…
Abstract Formatted: Centered
As bacteria mutated and became resistant to the antibiotics, even combining different drug
components it will continue to develop and improve its survival rate. Having an ineffective
treatment, patient’s condition becomes worse and leads to increase of morbidity and mortality. This
study aims to isolate and characterize the bacteriophage against Extended Spectrum β-lactamase-
producing Escherichia coli from the sewage of Mary-Grace Height Subdivision Main Road Dela
Paz, San Fernando, Philippines. Other bacteria that are immune to antibiotics are normal flora
inside the body making it opportunistic when the host’s immunity is compromise. The Escherichia
coli is a normal flora of the gastro intestinal tract that can cause an infection when introduced to
other organs. The research is set to examine the phenotypic characteristic of the isolated phage of
ESBL-producing E. coli using a Transmission Electron Microscopy (TEM) to observe the
morphology of the phage; this study focuses on the lytic phages. Qualitation will be observed from
a Plaque assay. The interest of this study is to introduce the use of bacteriophage that is not only
applicable in the European countries but also in other developing countries like the Philippines
where in Multi – Drug Resistance infection is increasing. The study aims to give an idea and
knowledge for future researchers of treatment against bacterial infection caused by Escherichia coli
Keywords Antimicrobial Resistance, Sewage water, TEM (Transmission Electron Microscopy)
OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

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1.0 Introduction
Escherichia coli producing extended-spectrum beta lactamases (ESBL) and/or

carbapenemases are of extraordinary concern globally in sufferers and a chance to all
modern healthcare. Further, community carriage of ESBL-producing E. coli, the
emergence of ESBL-producing E. coli in the farm animals’ population as properly as more
than one reports of ESBL-producing E. coli in wildlife exhibit that transmission and
persistence of such microorganism also happens backyard of clinical settings. (2017
Atterby et al.)
It is usually universal that traditional wastewater therapy reduces the numbers of

enteric bacteria. However, the extent to which this occurs can fluctuate appreciably relying
on the remedy process. Sewage handled by the activated sludge approach or different
biological tactics frequently nevertheless includes fecal microorganism or pathogens. To
limit the chance of environmental release of fecal bacteria, effluents are disinfected the use
of oxidative procedures to ruin or deactivate these organisms. (Anatasi et al.)
Bacteriophage is a method for bacteria eradication. Phages are said to be natural antibiotics
that have the ability to kill a pathogenic bacteria and it is capable of preventing infectious diseases
which is also used to cure infections caused by major pathogens in the agricultural. Phages are very
specific and do not harm the useful bacteria that live in or on the body that result in no side effects
like diarrhea or secondary infections which occur in treatment with antibiotics. The advantage of
phages is that they are self-replicating but also self-limiting because they only multiply as long as
sensitive bacteria are present.
The determination of this study will greatly contribute benefiting the society in
consideration of increasing infection in the Philippines that are caused by Extended-spectrum β-
lactamases (ESBLs) pathogens. Giving the readers the idea of the development and enhancement
in isolating phage that inhibits both pathogenic and non-pathogenic bacteria proposing its
alternative treatment for patients infected. This study will help them understand that phage therapy
will be an effective way in the inhibition of ESBL that are commonly detected.

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2.0 Review of Related Literature
2.1 History of Bacteriophage Discovery
On the introduction launched in early 2011 of new Bacteriophage journal by Alexander

Sulakvelidze, He described bacteriophage as “the most ubiquitous organism on Earth” which plays
a role for maintaining microbial balance. It can be found everywhere, along with other bacterial
host.
Bacteriophage was first discovered by William Twort, followed by Felix d’Herelle who is
also considered to be the founder of bacteriophages and its therapeutic implication of it (The Phage
Theory). Phages are small viruses that have the ability to kill bacteria while they do not affect cell
line from other organism. Due to its specificity to target cellular host, they proposed the application
of phages considering its beginning as a therapy in treating acute and chronic infections. In its
initial success it was first described in the department of dermatology, ophthalmology, urology,
stomatology, pediatrics, otolaryngology, and even in surgery. Their emotions over the first phage
therapy as a treatment for bacterial diseases in the era of pre biotic were tremendous (Sulakvelidze
et al., 2011).
In 1920’s and most of 1930’s the only available was serum therapy which is only for
selective pathogens such as diphtheria and pneumococci. They even described the use of
bacteriophage with considerable fanfate when Arrowsmith, the main protagonist in the Sinclair
Lewis’s Pulitzer Price-winning novel used it as a treatment to fight a bubonic plague outbreak on
a Caribbean island. A British bacteriologist in 1896 named Ernest Hanburry Hankin who worked
as an Chemical Examiner and Bacteriologist to the Government of the United Provinces and of the
Central Provinces of India, reported for the presence of antibacterial activity against Vibrio
cholerae in the waters from the Indian rivers Ganga and Yamuna that contains a biological principle
which destroys cultures of cholera-inducing bacteria. His work was published in French in the
Annals of the Pasteur Institute. He suggested that there is an unidentified substance (which passed
through fine porcelain filters and is heat labile) was responsible for this phenomenon and for
checking the spread of cholera epidemics (Sulakvelidze etal., 2011).

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After two years, a Russian bacteriologist Gameleya discovered the same phenomenon
while studying with Bacillus subtilis and several investigators who also observed was thinking that
it is related to bacteriophage phenomenon but none of them continued their findings until Frederick
Twort, A British microbiologist who renowned or introduced the subject 20 years after Hankins
discovery by saying that they have similarities but then he made an advance hypothesis that it may
have been because of a virus. Due to financial and various reason, Twort was not able to continue
his study and it took another two years for bacteriophage to be discovered by Felix d’Herelle who
was a French-Canadian microbiologist at the institute Pastuer in Paris (Sulakvelidze etal., 2011).
Felix d’ Herelle alone made a same experiment result while he was studying a patient that is
suffering or recovering from bacillary dysentery. He used stool sample for isolation of recovering
shigellosis patient that is called “anti-shiga microbe” by incubating 18 hours of filtered stool
(Wittebole et al., 2014).
2.2 Bacteriophage in General
Bacteriophages are viruses that infect and use bacterial resources. There are two types of
bacteriophage reproduction. It can either by Lytic cycle, or lysogenic cycle. Its classification is
based on the bacteriophage morphology and nucleic property (Orlova, 2012).
2.2.1 Morphology of Bacteriophages
The morphology of phages can be based on pleomorphic, filamentous, capsids, with and
without tails. Pleomorphic phages are found in the family of Plasmaviridae, members of the
Fuselloviridae family and Guttavirus phage group. In Plasmaviridae, dsDNA phages are covered
with lipoprotein envelope called as nucleoprotein granule. The dsDNA in a lemon-shaped capsid
belongs to the Fuselloviridae and a droplet-shaped virus-like particles represents a Guttavirus phage
group (Orlova, 2012).
Filamentous or helical phages have Inoviridae, Lipothrixviridae & Rudiviridae family.

Lipothrixviridae has a Rod-like shape and lipoprotein envelope while Rudiviridae phages are
straight rigid rods and doesn’t have an envelope (Orlova, 2012).

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Phages with capsids belong to Leviviviridae and Cystoviridae. Leviviridae has ssRNA
genome with small capsids and looks like enterovirus. Phages also have icosahedral symmetry
which are found on these three families Microviridae has small virions, Corticoviridae are the
marine phages and Tectiviridae which has lipoprotein vesicle that surrounds the protein capsid.
Tail Phages are seen in three families, the myoviridae that contains a sheath and a central tube,
Siphoviridae has long non contractile tail, and the Podoviridae which has short tails (Orlova, 2012).
Electron Microscopy is best used in studying the morphology of the bacteriophage. Phages
basically consist of a nucleic acid molecule surrounded by a protein coat. A bacteriophage is tadpole
shape with a head and a tail. Head consists of tightly packed DNA covered by a protein coat and it
is bipyramidal hexagonal in shape and measures 950 x 650 Å. The contents of headare enclosed by
a membrane (capsid). The capsid is made up of morphological subunits called capsomeres
(proteins).The capsomeres are consist of a number of protein subunits or molecules called
protomers. The head encloses a linear double stranded DNA, which contains more than 75 genes
while the tail is hollow core surrounded by contractile protein sheath. The tail is in the form of a
hollow cylinder. It consists of a central hollow core surrounded by a spring-like contractile sheath.
The sheath is formed of 144 subunits which are arranged in a hollow cylinder consisting of 24 rays
of six subunits each. Through the central space of the core, the phage chromosome travels into the
host cell (Giuseppe et al., 2015).
2.2.2 Genomes of Bacteriophages
According to the International Committee on Taxonomy of Viruses (ICTV), phages

classification in families and genera are based on capsid and morphology, conserved genomic
synteny and homology in amino acid sequences of phages genetic material encoded proteins.
Phages are tremendously diversified, with genome sizes from as low as 17 kbp up to 0.5 Mbp, and
38 constitute a reservoir of great genetic diversity, which is supported by the high frequency of
novel 39 genes found in newly characterized phage genomes. The phage genomes are typically 46
mosaic, with individual genes, or groups of genes, shared among unrelated genomes as a result of
47 horizontal and vertical gene transfer during phage evolution, particularly as a result of 48
recombination events between temperate phages (Oliveira, H. et al., 2013).

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Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome. There
are basically four types of Genetic material comprising bacteriophages genome, Single-stranded
and double-stranded DNA and RNA (ssDNA, dsDNA, ssRNA, dsRNA). Phages have relatively
simple and elaborate structures. Their genomes may encode as few as four genes and as many as
hundreds of genes. Phages replicate within the bacterium following the injection of their genome
into its cytoplasm. The genomes of bacteriophages contain unusual or modified bases that protect
them from degradation by host nucleases during phage infection (Pardini et al., 2017).
2.2.3 Life Cycle of Bacteriophages
There are two cycles used in production of Bacteriophage lytic cycle or lysogenic cycle
(Orlova, 2012). It starts with insertion of prophage into the host genome. Then cell division after
that they will release prophages due to environmental signal the DNA must replicate and undergo
synthesis of new phages. The lytic cycle undergo phage assembly. The host cell will lyse and the
phage will be release. The host is absorbed and recognized. Penetration and DNA injection will be
done (Doss et al., 2017).
2.2.4 Phage Ecology
Phage ecology is the study of how organisms interact with their environment; it can be
biotic or abiotic. Organismal ecology, evolutionary ecology, physiological ecology, behavioural
ecology, population ecology, community ecology, ecosystem ecology, and landscape ecology are
some of the ecological subdisciplines according to Abendon. The said subdisciplines studies the
intracellular and extracellular adaptation of phage survival and acquiring of bacteria to infect, also
the evolution of how they adapt on the community (Abendon, 2008).
In 1990s, with the use of electron microscope they have observed plethora of aquatic
virions that gives arise to the interest in aquatic phage ecology. There are two cases in aquatic
environment. First is where photosynthesis happens, prokaryotes carries the aquatic ecosystem.
Second is where the organic material in the aquatic ecosystem (eukaryotes) is eaten by the bacteria
(Abendon, S. C., 2008).

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Bacteriophages mostly replicate through the lysis of bacterial host cell hence, phages do
not only participate in bacterial population. Freshwater environment of phages hadn’t been well
studied despite of their numerical dominance. The role of phages in controlling bacterial genetic
diversity is by mediating horizontal gene transfer through carriage of host gene fragments while
currently infecting one host after the other. Phages influences on bacterial behaviors. Phage
infection has been found to modulate and include motility, biofilm formation, defense, toxicity,
replication, metabolism, sporulation, stress response, and quorum sensing. (Hargreaves et al.,
2014).
2.3 Sewage Phage
Bacteria's may be seen in their planktonic forms in the environment where they are infected
with faecal waste. Genes are transfered by horizontal transduction where the toxin genes are found
showing advantages of positive transfer. Recent studies shows that Staphylococcus aureus to
Listeria monocytogenes Toxins are transferred successfully. (Maite, 2011)
The studies about the incidence of phages in the water environment had been reported in
decades of most of the world now. In addition the data are not particularly consistent and it will not
always have a meaningful comparison. There are several ways, techniques and approaches in
recovering phage, unfortunately much of works along in these ways are still in research or
development stage. One of the reasons for discrepancy is because the result of the host bacteria was
used for the detection of various groups of bacteriophages. However, numerous parts of the world
and international collaboration are conducting into a much meaningful, universally accepted
guideline for the detection and recovery of bacteriophages in the water environments (Grabow et
al. 2001).
Improper handling and disposal in inappropriate locations may infect the public health.
Contamination of soil and water may lead to high risk of Spreading pathogens in the community.
Final disinfection in sewage water may be used to minimize the effect of this bacteria found to
promote the protection of public health. (T. Prado et al, 2007)

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2.4 Temperate Virus
Bacteriophages are also called phage for short. They are classified generally as template or
virulent. When a bacterial cell has been infected by a virulent virus, it will replicate then the release
viral progeny then eventually the cell host will die. In contrary, if the phage is used to be a temperate
virus, there are two possible outcomes after the bacterium has been infected. The production of
viral progeny and the host cell the lysis was similar of what happen to virulent viruses. Significance
of the phage genome is to stably incorporated as prophage in the host cell and in chromosome as
or as an episome (Gama et al., 2013).
Temperate bacteriophages have several ecological roles, because the infection does not
immediately can have caused cell death. The viral genetic of material will remain temporary or
either integrate through the genome of the host. The standard view can either be they are parasites
because the host for reproduction will act, or the predator because the phage replication is usually
kills the host. Recently, it shows bacteriophages and the bacterial cells may also be mutual in
relationship given that a lot of phages that may code for the virulence factors and it allow the
bacteria to be successful to infect the host and it will enlarge the distribution (Yosef et al., 2015).
The Modes of transmission of temperate phage is infection from the cell to the community.
There are two ways temperate virus can infect, the first one is through virion productive, where it
hijacks the metabolism of the host to create and produce a new virion progeny. And the second one
is through lysogenic cycles where it copies the genome of the host without producing a new virion.
The production of virion particles occur by following phage adsorption or instead by to a productive
cycle from a lysogenic cycle. Details can change with specific phage-host types, it ranges from
efficient to inefficient infections, the final results and the dynamics of the infection may change, in
spite of the fact that these infections are generalized dynamics. During the lysogenic cycle, the
persistence describe the prophage stage, during the production cycles, phage genome state
describes as the replication, and the release refers to the impacts of productive cycles on the
bacterium that was infected by the phage, not just to the means by which progeny phage transition
from the intracellular to extracellular state. This implies the modes of temperate phage infection on
the community of bacteria. The phenotypic effects of prophage carriage to its host cell is because
of its lysogenic conversion constitute. By combining the cell where few lysogens are removed
through lysis it will result the lysogenic to lytic switch changes community structure and will

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release virions that can infect the cells surrounding it. When a bacterium possesses a numerous
numbers prophages, polylysogeny will occur (Varona et al., 2017).
2.4.1 Lysogenic Phage
Bacteriophages (phages), little infections of around 20– 200 nm in estimate, are likely the
most old and pervasive existing life forms on Earth. They go back 3 billion years, and they
particularly contaminate microscopic organisms to reproduce, hence assuming an imperative job
in keeping up the balance of each biological community where microorganisms exist (Criscuolo et
al., 2017).
Phages may go about as vectors for hereditary trade by means of summed up or

concentrated transduction, whereby a hereditary quality is conveyed by phage particles from a
benefactor bacterial cell to a beneficiary cell. Summed up transduction embroils the exchange of
any part of the contributor genome to the beneficiary cell by either a lytic or lysogenic phage, while
particular transduction includes just mild phages, in which a couple of particular giver qualities can
be exchanged to the beneficiary cell. A particular transducing phage produces particles that convey
both chromosomal DNA and phage DNA and contains just particular areas of the bacterial
chromosome found neighboring the prophage connection site. Some calm phages may likewise
actuate an adjustment in the phenotype of the contaminated host, through a procedure known as
lysogenic change (Balcazar et al., 2014).
An answer for the issue of anti-infection opposition may not involve finding totally novel
medications, but rather thinking once more into history, to bacteriophage treatment. Phage
treatment bridles the executing intensity of microscopic organisms' common predators, lytic
bacteriophages, to battle infection. The idea of phage treatment originates before the utilization of
customary anti-infection agents, at first set forth by Felix d' Herelle, co-pioneer of phages, in the
mid 1920's. In spite of the fact that this was trailed by an underlying blast of intrigue and research,
phage treatment blurred from utilize following the revelation of anti-infection agents. Be that as it
may, both phage treatment and research have proceeded in Eastern Europe and previous Soviet
states, revealing achievement in treating an assortment of bacterial diseases, including those by S.
aureus. The rising issue of anti-microbial obstruction and the requirement for elective medications
have prompted a resurgence of enthusiasm for phage treatment. Nonetheless, numerous

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investigations to date need sufficient controls and are ineffectively planned; extra research is
expected to substantiate the wellbeing and viability of these medications previously they can be
incorporated into standard clinical consideration (Pincus et al., 2015).
2.5 Virulent Phage
Virulent phage is a type of bacteriophage differs on its way of attacking and its mechanism
to reproduce inside the host cell. This phage is also known as lytic phage, it inject the chromosomes
on the sensitive host cell where it reproduce eventually will be fill of new phages inside the cell
that will lead to burst causing the leakage of the contents of the cell. This is the edge of this type of
bacteriophage from the temperate or lysogenic phage (Rossman, F. S. et al., 2015). Different types
of virulent are not all similar in terms of period of time to burst to lyse, where as in the investigation
of Eriksen, R. S. et al (2018) that T3 and T7 are specifically create a substantial amount in plaques,
while T4 will begin in few plaques because of lysis inhibition. (Eriksen et al., 2018).
A lytic phage has the ability of using a lytic life cycle. Vertical transmission is selected in
using virulent phage. The intensity of virulence depends on the transmission output. The growth of
virulent phage is done thru diffusion of not infected bacteria, going to the attachment on host,
intracellular maturation, releasing of progeny to the environment (Abendon, 2008).
This phage may trigger the release of elements. An example given according to the
stoichoimetry of bacterial biomass N and P are absorbed by the bacteria’s and C attaches to the
bacteria. Lytic phage goal is to completely isolate the host cell and replicate in its surroundings.
(Abendon, 2008).
2.6 Extended Spectrum β-lactamase
Extended Spectrum Beta Lactamase are enzymes that mediates plasmid. It hydrolyzes
oxyimino-Beta lactam agents such as third-generation cephalosporins and aztreonam. They are also
a resistance genes carrier against other antibiotics including aminoglycosides, chloramphenicol,
sulfonamides, trimethoprim, and tetracycline. According to Gupta et al., during a 30-month
outbreak of ESBL-producing K. oxytoca in an NICU; K. oxytoca spread to K. pneumoniae, E. coli,
Enterobacter cloacae, and Citrobacter freundii. (Gupta et al., 2003)

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2.6.1 ESBL- E. coli
The odor Escherich was the first one to isolate and describe Escherichia spp. These
organisms are Gram negative bacilli which are commonly found on feces. Escherichia coli infect
the intestine and urethra that causes the UTI’s to women. E. coli bacterimia is high on babies and
adults aging 65 and above, it is caused by improper hygiene (Sue, 2015).
The production of ESBL differs its resistance to Beta lactam antibiotics except
carbapenems and cephamycin. They have proved its resistance to penicillin,sulfamethoxazole,
trimethroprime and cephalothin results in 100%, 30.89%, 16.26% and 20.32%. It was said that in
99 E.coli pathotypes only 35% of it were found they have plasmids that ranges 1.7 kb to 4.5 kb
(Talat et.al, 2017)
2.7 Phage Therapy
Phage therapy is a practice that uses bacterial viruses (phage) to treat bacterial infections.
Frederick Twort first described the characteristic zone of lysis associated with phage infection in
1915, but it was Felix d’Herelle who identified the source of this phenomenon, attributed the
plaques to bacterial viruses, and coined the term “bacteriophage” (literally “bacteria-eater”). It was
also d’Herelle who conceived of the idea to use phages therapeutically and is responsible for the
first documented clinical use of phage in 1919 at the Hôpital des Enfants-Malades in Paris where
phages were successfully used to treat four pediatric cases of bacterial dysentery. The generated
renewed interest in revisiting this practice is due to universal decline in the effectiveness of
antibiotics. This therapy relies on naturally occurring phages to infect lyse the bacteria present at
the site of infection. Most phages are infectious only to the bacteria that carry their complementary
receptor, which effectively determines lytic phage host range (Lin et al., 2017)

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2.8 Problem Statement
The study aims to isolate and to characterize the bacteriophage present from the sewage of Mary-
Grace Height Subdivision Main Road, San Fernando, Pampanga in the Philippines.
Specifically, it will answer the following questions:
Research question 1: What is the characterization of isolated bacteriophage against

Extended Spectrum β-lactamase Escherichia coli?
Research question 2: What is the significance of the isolation?
2.9 Discussion of Flow Chart
Initial procedure will be the review of Ethics assuring that there will be no violation will
be made conducting this experiment.
Preparation and gathering of materials is done before the beginning of the experiment.
Collecting of the samples in the sewage in Mary-Grace Height Subdivision Main Road
Dela Paz, San Fernando, Philippines for bacteriophage sample and the clinical isolated of E. coli is
the initial step of the experiment. Transporting immediately of the sample from the collection site
to the laboratory is needed.
Cultivating bacterial suspension will be followed then by incubate overnight.
Isolating the bacteriophage from the sewage sample using direct isolation method requiring
centrifugation to separate possible debris, filter out large particles from virus by using special filter
(0.45micro) to a conical tube container.
Experimental phase will begin after collecting and preparing all the samples needed and
gathering materials.

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Plaques Assay was conducted to cultivate and to assess the presence of the bacteriophage
against the E. coli on a TSA agar that was overlay with the 7% soft agar then left overnight.
Phenotypic is the assessment of the identification and the characterization of the

bacteriophage bonded to the E. coli using Transmission Electron Microscope (TEM) to view the
bacteriophage. Observing the pH and temperature stability of the phage isolated from the sewage
water.
Vitek test to identify the organism that was used in the study and also the antibiotic
sensitivity test (AST).

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Ethical Review
Preparation and Gathering of Materials
Collecting Sewage sample in Mary-Grace Clinical isolation of

Height Subdivision Main Road Dela Paz, Escherichia coli
San Fernando, Philippines
Cultivating bacteria in MAC

Isolating Bacteriophage in and 1 hour bacterial
Agarose Agar suspension
Experimental Phase
Plaque Assay
Phenotypic
Transmission Vitek ID of organism

Electron and AST
Microscope
Analysis and Interpretation of Results
Conclusion
Figure 1.0 Research Flowchart

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3.0 Research Methods
3.1 Research Design
This research will apply the qualitative approach to the study. This study will be dealing
with an experimental method in gathering data and isolating the phage virus from the sewage
sample then applied to Escherichia coli. Whereas, will evaluate the lytic activity of the phage by
plaque assay and observe the phenotype of the phages isolated.
3.2 Research Locale
The experimentation of this research was conducted in Our Lady of Fatima University
(Pampanga campus) except the phenotypic characterization of phage in which Electron Microscopy
is needed was performed in Research Institute for Tropical Medicine (RITM) in Muntinlupa,
Philippines. The Vitek test was sent to Hi-Precision Laboratory in Angeles City. The duration of
this study will start from the collection of the specimen along with the isolation of the bacteriophage
in Mary-Grace Height Subdivision Main Road Dela Paz, San Fernando, Pampanga, Philippines.
The procedures in this experiment are expected to be done in the span of 30 days.
3.3 Sample of the Study
The sewage sample used for this study is collected in Mary-Grace Height Subdivision
Main Road Dela Paz, San Fernando, Philippines and the organism of Escherichia coli isolated
using the selective media Mac Conkey plate from the school laboratory.
3.3.1 Sewage Sample
The sample used in this study was collected from the stagnant water in the area of Mary-
Grace Height Subdivision Main Road Dela Paz, San Fernando, Philippines less than 1 feet from
the side of the sewage and about 2 feet depth. These samples are to be placed in a clean glass bottles
where in the 80ml of the sample is to be processed as soon as it arrived at the laboratory.

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3.3.2 Escherichia coli Sample
Escherichia coli will be collected from the laboratory of Our Lady of Fatima University in
Pampanga. We are going to cultivate the bacteria using Mac Conkey agar at 35ºC for overnight and
it will be used for the study.
3.4 Research Ethics
Ethical standards promote the values that are essential to conduct this experiment. The
study also ensures that there is no participants will be used during the experimental phase.
Researchers held liable and accountant for any misconducts that will happen during the study.
3.5 Research Instruments
An incubator is needed for the phage's temperature requirement. Digital rotator will be
needed to properly agitate the sample that is to be enriched. The centrifuge machine will be used
to separate the light and heavy sediments in isolating phages. Electron microscope for the
phenotypic characterization of the specific phage present. Centrifuge will be used to separate the
light and heavy sediments in isolating phages. Hot plate will used to prepare media for the
cultivation and assays. Autoclave will be used to ensure to sterile the materials that will be used in
the experiment.
3.5.1 Research Materials

Water samples tested for physiochemical analysis are placed on a glass or polyethylene
bottle; it must be clean, but not sterile (WHO, 2015). A pure culture of ESBL(Klebsiella
pneumoniae), TSA plate and TSB, sterile phage buffer, micropipette and pipet tips are also needed.
Inoculating loop and also used for the lawn of the bacterium in the agar, alcohol lamp is also used
for the purpose of sterilization of loops. A sterile glass container was needed for the collection and
transport of phage, test tubes. A colony counter was used to observe the formation plaque,
membrane filter for the filtration. Transmission Electron Microscope (TEM) is also needed in this
experiment analysis. Vitek test was use to confirm the organism and to identify the specific strain
present.

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3.5.1.1 Reagents and Chemicals
TSA contained (10 gtryptone, 5 yeast extract, 5 NaCI, 1 mL 2N NaOH, 12g/L agar). TSB
contain (10g tryptone, 5g yeast extract, 5g NaCI, and1mL 2N NaOH per liter. Top Agar contained
4g/ agar, supplemented with 4mM MgCI2 and 4mM CaCl).
3.5.2 Research Procedures
3.5.2.1 Collection of Sewage Sample
Disturbing the stagnant waste water before submerging the 550ml of bottle to collect
sample, maintaining the room temperature in transporting the sample is necessary to keep the
organisms alive. There are a lot of methods used in isolation of organisms from the water this
includes the Membrane-filtration method, Multiple-tube method, and Presence-absence test (WHO,
2015).
3.5.2.2 Isolation of Virulent Phage
The sample will be stored at room temperature then phage enrichment which made into Set
A and Set B. For set A, a 60mL of samples a 10 mL of TSB will be added to dehydrate the samples
thoroughly agitate to dislodge the phage particles using a digital rotator. 10mL to 15mL of 1 hour
bacterial suspension of E. coli would be added to liquid samples and continue to agitate at 126
RPM for 2 hours for the enrichment of the phage that will be incubated for overnight at 35ºC. For
set B sample a 50mL of sewage is centrifuged at 1, 800 RPM for 15 minutes, transferred samples
into sterile test tubes and then centrifuged to separate the supernatant again repeated this for three
times. Then, the supernatant will be filtered using a membrane filter (0.45μm) to eliminate the
bacteria present. The supernatant will be then check if there’s a presence of lytic phages by spotting
a drop lawn of Escherichia coli. After overnight of incubation, the formation of a clear zone
(plaques) suggested the presence of lytic phage specific for each strain. (Jensen et al., 2015).

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3.5.2.3Transmission Electron Microscopy
Negative staining will be used for visual observation under the Transmission Electron
Microscope to view the physical structure of the phages (Mohammed-Ali, MN. and
Jamalludeen,NM., 2015). The stained grid were observed in JEOL JEM 1220 under direct
magnification of 30,000x and 40,000x.
3.5.2.4 Vitek Test

Organism that was used in the study was subjected for the identification and antibiotic
sensitivity test using Vitek service in Hi-Precision Laboratory in Angeles City.
3.6 Data Analysis

The viruses that were isolated from the sewage water have the length of the phage of 50
nm to 100 nm.

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4.0 Results
A B
Figure 2: Isolated bacteriophage from the sewage water against E. coli

The phenotypic characterization of the isolated phage is belong to the Siphoviridae a
dsDNA virus according to the ICTV, it is describe with a long, non – contractile, thin tails range
with 65 – 570 x 7-10 nm. Heads and tails are assembled separately. Known as the “Lambda-like
virus. Phage head is icosahedra, about 60 nm in diameter, and consist of 72 capsomeres. Tails are
flexible, 150 x 8 nm and have a short terminal fiber and four long, jointed fibers attached
subterminally, (figure 2. D). the latter fibers are mostly absent.

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5.0 Discussion
-major findings
-rationalize the results / explain the major finding (depth explanation from related literature
and journals).
- cite the study regarding the major findings.
1st paragraph discussed the problem of the study
2nd paragraph import procedure
3rd import finding
MINIMUM OF 3 PAGES

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6.0 Conclusion
Therefore we conclude that the phage isolated from the random collection of the
sewage water at the Mary-Grace Height Subdivision in San Fernando, Pampanga is belong
to the family of Siphoviredae that can lysate the pathogenic organism Escherichia coli and
it head is icosahedra, about 60 nm in diameter, and consist of 72 capsomeres. Tails are
flexible, 150 x 8 nm and have a short terminal fiber and four long, jointed fibers attached
subterminally, (figure 2. D). the latter fibers are mostly absent.
7.0 Recommendation
The researchers were able to isolate and characterized the phenotypic of a bacteriophage
from a sewage water against an E. coli a pathogenic organism to man, nevertheless, further study
should be recommend to be able to understand more, there are areas that needed to be improved.
8.0 Acknowledgement
The researchers would like to express the gratitude and appreciation to the faculty in CMLS
department in Our Lady of Fatima (Pampanga) for guiding and providing us materials and organism
to be able to conduct our research and Electron Microscope Department in Research Institute for
Tropical Medicine (RITM) in the microscopy.

24
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28
Appendix A
The Research Budget
Items Unit Price
Reagents and chemicals Formatted: Justified
Tryptone Soy Agar 500 grams 3,200.00 Formatted: Justified
Trytone Soy Broth 500 grams 1,900.00 Formatted: Justified
Agar Agar 50 grams 550.00 Formatted: Justified
Distilled Water 7 Liters 141.00 Formatted: Justified
Lysol 170grams (2 pcs) 460.00 Formatted: Justified
Tissue Towel 1 roll 35.00 Formatted: Justified
Safeguard soap 25grams 7.00 Formatted: Justified
Materials
Formatted: Justified
Cotton 50 g 33.00
Micropipettes tips 10-100ul (1 box)
100-1000ul (1 box)
Syringe (Plastic) 10cc (70 pcs) 420.00
5 cc (30 pcs) 90.00 Formatted: Justified
1 cc (50 pcs 175.00 Formatted: Justified

Disposable Petri dish (105 pcs) 2, 080.00 Formatted: Justified
Bottle Container 550 ml (10pcs) 550.00 Formatted: Justified
Unground Glass Slides (1 Box) 75.00 Formatted: Justified
Filter membrane 0.45 um (pcs) 12, 370 Formatted: Justified
Centrifuge tubes 10 mL(15pcs) 325.00 Formatted: Justified
13 mL (10 pcs) 250.00
Inoculating loop 50 pcs 700.00
Other Test
Transmission Electron Microscope 7,000.00
Vitek Organism ID and AST 1,050.00 Formatted: Justified
Other Formatted: Justified
Ice for cooler 200.00 Formatted: Justified

Printing materials 1,000.00 Formatted: Justified
Travel expenses 8,000.00 Formatted: Justified
Marker 75.00 Formatted: Justified
Masking Tape 100.00 Formatted: Justified
GRAND TOTAL 40,786.00

29
Appendix B
Timeline
Gantt Chart Formatted: Justified
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Activity/ies
Purchasing of Formatted: Justified
materials and Reagents
Collection of water Formatted: Justified

sample in Dela Paz,
San Fernando,
Pampanga
Collection of Bacterial Formatted: Justified
Sample (Escherichia
coli)
Media Preparation
Phenotypic Formatted: Justified
identification of phage
using TEM
Reporting and analysis Formatted: Justified
of data
Reviewing and editing Formatted: Justified
of Final Data
Final Defense Formatted: Justified

30
Appendix C
Certifications
Molecular Diagnostic Seminar Workshop

31
Appendix C
Certifications
Animal Handling Seminar Workshop

32
Appendix D
The Authors
Rosa Milma M. Dagdag

#09 Villa Ocampo Brgy. San Juan Mexico Pampanga
mendozarosamilma@gmail.com
Rosa Milma Dagdag graduated highschool in San Juan High School in San Juan Mexico Pampanga.
She was a Campus President during the school year of 2013-2014. Currently, she is aiming to finish
her Bachelor’s degree in Medical Laboratory Science at Our Lady of Fatima University, Pampanga
Campus. To complete her degree she’ll be taking the board exam on March 2020.

33
Appendix D
The Authors
Janine Kate M. David

Maligaya Floridablanca, Pampanga
Janine_kate01@yahoo.com
Janine Kate M. David graduated high school in Saint Augustine Academy of Pampanga. Currently,
she is studying in Our Lady of Fatima University, San Fernando Pampanga. She is aiming to finish
her bachelor’s degree in Medical Laboratory Science.

34
Appendix D
The Authors
Katrina Jasmin D. Gomez

109 Ma.Aurora Rd. Villa Gloria Angeles City Pampanga
katrina.jasmin.gomez@gmail.com
Katrina Jasmin D. Gomez graduated high school in Holy Family Academy Barangay Cutcut
Angeles City. Currently, she is studying in Our Lady of University, San Fernando Pampanga. She's
aiming to finish her bachelor's degree in Medical Laboratory Science.

35
Appendix D
The Authors
Irene C. Hernandez
Block 2 lot 4 homesite subdivision Caduang tete, Macabebe, Pampanga
pringhernandez@gmail.com
Irene C. Hernandez graduated high school in St. Nichols Academy: Centre of Catholic Education
Inc. in Macabebe, Pampanga. Currently, she is studying in Our Lady of Fatima University,
Pampanga. She is aiming to finish her bachelor’s degree in Medical Laboratory Science.

36
Appendix D
The Authors
Carrol B. Montenegro
Zone 6 San Basilio, Santa Rita, Pampanga, Philippines 2002
carrolmontenegro@gmail.com
Carrol B. Montenegro graduated high school in Santa Rita College in San Jose, Santa Rita,
Pampanga. She was a Brigade Captain of their CAT. Currently, she is studying in Our Lady of
Fatima University, Pampanga. She is aiming to finish her bachelor’s degree in Medical Laboratory
Science.

37
Appendix D
The Authors
Andrew Villamor C. Ong

0809 Casmor Phase 2 Mabiga Mabalacat City Pampanga
AndrewOng@gmail.com
Andrew Villamor Ong graduated high school in the school of Don Bosco Academy of Pampanga.
He is now studying in Our Lady of Fatima University, San Fernando Pampanga. He is aiming to
finish his bachelor’s degree in Medical Laboratory Science.

38
Appendix D
The Authors
Gian Karla T. Rivera

#084 Sampaguita Street. Sindalan City of San fernando Pampanga
riveragiankarla@yahoo.com
Gian Karla Rivera graduated high school in New Era University in Dela Norte, City of San
Fernando Pampanga. Currently, she is studying in Our Lady of University, San Fernando
Pampanga. She's aiming to finish her bachelor's degree in Medical Laboratory Science.

39
Appendix D
The Authors
Paulo Louise Salalila

Block 5a Fulton St. Timog Residences, Angeles City, Pampanga
Paulolouise.salalila@gmail.com
Paulo Louise Salalila graduated high school in Dee Wa Liong College in Magalang, Pampanga.
Currently, He is studying in Our Lady of Fatima University, San Fernando Pampanga. He is aiming
to finish his bachelor’s degree in Medical Laboratory Science.

40
Appendix D
The Authors
Joy Sairah B. Talib

2278 San Vicente, Apalit, Pampnga
talibsairah@gmail.com
Joy Sairah B. Talib graduated high school in Saint James School in Santiago, Apalit, Pampanga.
Currently, she is studying in Our Lady of Fatima University, Pampanga. She is aiming to finish her
bachelor’s degree in Medical Laboratory Science.

41
Appendix D
The Authors
Aliana Marie F. Tiongson

#991 waling waling street Villa Corazon San Agustin City of San Fernando Pampanga
YhannaTiongson@gmail.com
Aliana Marie Tiongson graduated high school in University of the Assumption in Unisite Subd.
Del Pilar, San Fernando Pampanga. Currently, she is studying in Our Lady of Fatima University,
San Fernando Pampanga. She is aiming to finish her bachelor’s degree in Medical Laboratory
Science.

42
Appendix D
The Authors
Yukie T. Tsumura
Lot block 30 bridge Pointe subdivision, Del Rosario, San Fernando, Pampanga
YukieTsumura@yahoo.com
Yukie T. Tsumura Graduated high school in San Roque Catholic School de Alabang. Currently, he
is studying Our Lady of Fatima University Pampanga aiming to finish his bachelor’s degree in
Medical Laboratory Science.

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Isolation and Phenotypic Characterization of Bacteriophage…

Abstract Formatted: Centered

Keywords Antimicrobial Resistance, Sewage water, TEM (Transmission Electron Microscopy)

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

Escherichia coli producing extended-spectrum beta lactamases (ESBL) and/or

It is usually universal that traditional wastewater therapy reduces the numbers of

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.0 Review of Related Literature

2.1 History of Bacteriophage Discovery

On the introduction launched in early 2011 of new Bacteriophage journal by Alexander

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.2 Bacteriophage in General

2.2.1 Morphology of Bacteriophages

Filamentous or helical phages have Inoviridae, Lipothrixviridae & Rudiviridae family.

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.2.2 Genomes of Bacteriophages

According to the International Committee on Taxonomy of Viruses (ICTV), phages

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.2.3 Life Cycle of Bacteriophages

2.2.4 Phage Ecology

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.3 Sewage Phage

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.4 Temperate Virus

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.4.1 Lysogenic Phage

Phages may go about as vectors for hereditary trade by means of summed up or

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.5 Virulent Phage

2.6 Extended Spectrum β-lactamase

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.6.1 ESBL- E. coli

2.7 Phage Therapy

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.8 Problem Statement

Specifically, it will answer the following questions:

Research question 1: What is the characterization of isolated bacteriophage against

2.9 Discussion of Flow Chart

Cultivating bacterial suspension will be followed then by incubate overnight.

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

Phenotypic is the assessment of the identification and the characterization of the

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

Preparation and Gathering of Materials

Collecting Sewage sample in Mary-Grace Clinical isolation of

Cultivating bacteria in MAC

Transmission Vitek ID of organism

Analysis and Interpretation of Results

Figure 1.0 Research Flowchart

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

3.0 Research Methods

3.1 Research Design

3.2 Research Locale

3.3 Sample of the Study

3.3.1 Sewage Sample

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

3.3.2 Escherichia coli Sample

3.4 Research Ethics

3.5 Research Instruments

3.5.1 Research Materials

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

3.5.1.1 Reagents and Chemicals

3.5.2 Research Procedures