1 Isolation and Phenotypic Characterization of Marine Phage

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Isolation and Phenotypic Characterization of Marine Phage…
Abstract
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 Klebsiella pneumoniae from the sewage of Don Ramon Village, San Agustin, 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 K. pneumoniae 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 K. pneumoniae using a Transmission Electron Microscopy (TEM) to observe the
morphology of the phage; this study focuses on the lytic phages. Quantitation will be observed
from a Plaque assay and PCR which determined the specific gene present in the phage virus this
will perform the qualitative approach. 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 Extended Spectrum β-Lactamase (ESBL) infection is
increasing. The study aims to give an idea and knowledge for future researchers of treatment against
bacterial infection caused by ESBL (K. peumoniae).
Keywords Antimicrobial Resistance, PCR (Polymerase Chain Reaction), TEM (Transmission

Electron Microscopy)
OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

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1.0 Introduction
Klebsiella pneumoniae is a standout amongst the most widely recognized Gram-negative

microscopic organisms experienced by clinicians worldwide as a reason for diseases in human. The
greater part of the diseases are obtained in health center setting therefore, it is accounted for to be
the among the 10 most basic nosocomial pathogen in different investigations. Nowadays, K.
pneumoniae diseases are entangled by increment in Extended-spectrum Beta Lactamase (ESBL)
producing separates. In this manner, this investigation is being directed with the goal to find out
the predominance of ESBL delivering K. pneumoniae in different clinical examples and to discover
there affectability design.
A recent disturbance in the uprising rates of the anti-infection opposition has now turned
into a genuine and an undeniably deficit worry, with extreme ramifications, particularly in the
concentrated consideration units. An assortment of β-lactamases which incorporate ESBLs, AmpC
β-lactamases and metallo-βlactamases, have risen as the most troubling system of opposition
among the gram negative microorganisms, which represent a helpful test to the human services
settings. Reported the increased rate of Extended-spectrum β-lactamases (ESBLs) enzymes which
causes the high number of diseased and death in intensive care units. The individual that commonly
acquire this is those who stays longer in health care facilities.
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

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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 in K. pneumoniae.
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

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through fine porcelain filters and is heat labile) was responsible for this phenomenon and for
checking the spread of cholera epidemics (Sulakvelidze etal., 2011).
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).

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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).
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

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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).
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.

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Second is where the organic material in the aquatic ecosystem (eukaryotes) is eaten by the bacteria
(Abendon, S. C., 2008).
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 virion particles exceeding the numbers of bacteria in the world's oceans for about 10:1
in ratio and thought to be enforce variety at the base of the microbiological community toward to
restraining outgrowth of a particular success microbial species and bring back to their biomolecules
as the nutrients goes by the water throughout the lysis. The specific host virus has critical roles of
interaction. Metagenomics where estimated to indicate an extraordinarily high numbers of viral
species, up until the present the total number of the estimated total number of marine bacteriophages
as of the last published was only 17. Now, the marine phages are expected to go higher to only a
few hundred (Hardies et al., 2013).
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

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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 bacterias found to
promote the protection of public health. (T. Prado et al, 2007)
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

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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, phagegenome 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 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).

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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
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. Likewise, staphylococcal phages have other
potential medicinal services utilizes, for example, application to catheter material to control biofilm
arrangement or close by wash answers for bring down CFUs of S. aureus on the skin of social
insurance laborers (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).

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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)
2.6.1 ESBL- K.pneumoniae
Klebsiella pneumoniae is the commonly isolated specie of Klebsiella. It is a nosocomial

bacteria and it has a large polysaccharide capsule which protects the organism against phagocytosis
and antimicrobial absorption contributing to its virulence. It is also responsible for the moist,
mucoid colonies characteristic of K. pneumoniae. Occasionally, the capsule provides a presumptive
identification of the organism. (Mahon et al., 2015)
In 2016, there are 10, 310 isolates reported. The ESBL production was initially screened
using ceftazidine disk diffusion. There are 8,861 K. pneumoniae isolates tested, resulting 40%
positive for those suspected ESBL. The rates of suspected ESBL varies, from as low as 13.5% to
as high as 81.2%. (DOH, 2016) It emerged in the following year, the number of K. pneumoniae
isolates increases upto 12, 591. Primarily, isolated from respiratory specimen there are 57%
reported according to DOH, others were isolated from urine (15%), wounds(13%), cutaneous and
blood (9%), tissues (3%), other fluids (2%), other isolates (2%).K. pneumoniae isolates have the
highest rates of resistance to co-trimoxazole at 61% (n=7,444), followed by cefuroxime at 50%
(n=5,909; 95% CI: 48.9-51.5) and ceftriaxone at 46% (n=11,076; 95% CI: 45.2-47.0). Carbapenem
resistant are increasing with rates against ertapenem at 8% (n=6,637; 95% CI: 4.7-8.7); imipenem

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at 11% (n=10,496; 95% CI: 10.3-11.5) and meropenem at 11% (n=11,409; 95% CI: 10.3-11.5).
(DOH, 2017)
2.7 Application of Phage
Application of Phage display and typing techniques thus aid studies to characterize and
identify the structure of the unknown strain bacteriophage (Rami et al., 2015).
2.7.1 Phage Display
Phage display is the technology that inserts the DNA sequence of foreign protein or peptide
into the appropriate position of phage coat protein structural gene, so that the exogenous gene can
be expressed along with the expression of phage coat protein itself. Meanwhile, the foreign protein
also can be shown in the surface of phage coat protein following the reassembly of phage. In short,
a phage library expressing a wide diversity of peptides or proteins is used to select those binding
the desired targets (Kushwaha et al., 2014).
Phage display claims numerous unique advantages over the other methods of detecting
protein-ligand interactions including the following: First, Very wide repertoire of bait molecules.
The foremost reason is in the diversity of molecules capable of acting as bait in affinity selection.
Phage display is a very powerful means of isolating protein fragments that interact with other
proteins, nucleotides, carbohydrates, etc. Essentially, if a polymer/molecule can be attached to a
recoverable support, it can be screened for affinity with phage displayed proteins. Second, Phage
resistance to extraneous factors for some baits may require environmental stress such as heats, high
osmolyte concentrations and specific cofactors. Furthermore, the phage are usually resistant to
metabolic poisons and interfering small molecules that would, at the least, result in pleiotropic
effects on metabolically active test organisms. Following the stringency washes, the poison is
removed before a large volume of bacteria are introduced for infection so the poison is diluted to a
range innocuous to the bacteria or subsequent phage replication (Kushwaha et al., 2014).

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2.7.2 Phage Typing
Phage typing is method used to detect single strains of bacteria and to trace the source
outbreaks of infection. A culture of the strain is grown in the agar and dried. A grid is drawn on the
base of the petri dish or petri plate to mark out different regions. Inoculation of each square of the
grid is done by a different phage. The phage drops are allowed to dry and are incubated: The
susceptible phage regions will show a circular clearing where the bacteria have been lysed, and this
is used in differentiation (Kushwaha et al., 2014).
2.7.3 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)
2.8 Problem Statement
The study aims to isolate and to characterize the bacteriophage present from the sewage of Don
Ramon Village, San Agustin, San Fernando in Philippines.
Specifically, it will answer the following questions:
Research question 1: What is the characterization of isolated Extended Spectrum Beta-

Lactamse-Klebsiella pneumoniae?
Research question 2: What is the gene present in the bacteriophage?

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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 Don Ramon Village, San Agustin, San Fernando
for bacteriophage sample and the clinical isolated of K. pneumoniae is the initial step of the
experiment. Transporting immediately of the sample from the collection site to the laboratory is
needed.
Cultivating bacteria on a MAC and TSA agar will be followed then by incubate overnight.
Isolating the bacteriophage from the sewage sample using direct isolation method requiring
phage buffer, centrifugation to separate possible debris, filter out large particles from virus by using
special filter (0.45micro) on a conical tube container, then store on a micro tube containing the
bacterium.
Experimental phase will begin after collecting and preparing all the samples needed and
gathering materials.
Plaques Assay will be conduct to cultivate and to assess the presence of the bacteriophage
and also to quantify the presence of ESBL K. pneumoniae sensitivity using TAP agar (liquid) then
put it to the TSA agar then left overnight to grow.
Phenotypic is the assessment of the identification and the characterization of the

bacteriophage bonded to the K. pneumoniae using Transmission Electron Microscope (TEM) to
view the bacteriophage. Observing the pH and temperature stability of the phage isolated from the
seawater.

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Ethical Review
Preparation and Gathering of Materials
Collecting Sewage sample in Don Ramon Clinical isolation of ESBL-

Village, San Agustin, San Fernando Klebsiella Pneumoniae
Cultivating bacteria in TSA

Isolating Bacteriophage in Agar
Agarose Agar
Incubation for overnight
Experimental Phase
Plaque Assay
Phenotypic
Transmission pH Stability Temperature

Electron Stability
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 and quantitative 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 ESBL K. pneumonia. 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 will be conducted in Our Lady of Fatima University
(Pampanga and Valenzuela campus) except the phenotypic characterization of phage in which
Electron Microscopy is needed; it will be performed in University of the Philippines Los Baños
Campus. The duration of this study will start from the collection of the specimen along with the
isolation of the bacteriophage in Don Ramon Village, San Agustin, San Fernando, Philippines. The
procedures in this experiment are expected to be done in the span of 15 days.
3.3 Sample of the Study
The sewage sample that will be used for this study will be collected in the Don Ramon
Village, San Agustin, San Fernando in the Philippines and the sample of the Extended Spectrum
β-lactamase Klebsiella pneumoniae will be collected in pure isolated broth from laboratory.
3.3.1 Sewage Sample
The sample we are going to use will be collected from the seawater of Don Ramon Village,
San Agustin, San Fernando about 3 feet from the side of the sewage and 2 feet depth. These samples
are to be placed in a clean glass bottles where in the 20ml of the sample is to be processed as soon
as it arrived at the laboratory

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3.3.2 Extended Spectrum β-lactamase Klebsiella pneumonia Sample
Extended Spectrum β-lactamase Klebsiella pneumoniea will be collected from the

laboratory of Our Lady of Fatima University in Valenzuela City. We are going to cultivate the
bacteria using MacConkey agar at 37ºC for 24 hours 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. Rotary shaker is also
needed for the preparation of TSB to dislodge the bacteriophage from the host cell, micropipettes
and tips will be used to transport and to gather the exact amount of the sample or reagent needed
required and tips. Biosafety cabinet level 2 to control the contamination of the pathogenic bacteria.
PCR machine will be used to amplify segments of the DNA. Electron microscope for the
phenotypic of the specific phage present. Centrifuge will be used to separate the light and heavy
sediments in isolating phages. Hot plate will used to make a culture media for the phages.
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 will be needed for the collection
and transport of phage, test tubes. A magnifying glass will be needed to observe the formation
plaque, membrane filter for the filtration. Polymerase chain reaction machine (PCR) and
Transmission Electron Microscope (TEM) is also needed in this experiment Polymerase chain
reaction (PCR) requires a high number of thermal cycle steps to create thousands of strands of DNA

18
sequencing for analysis. This is a harsh environment for standard thermoelectric modules due to
the mechanical stresses that occur during the heating and cooling stage. Laird’s power cycling (PC)
thermoelectric modules (TEMs) enhance reliability of PCR cyclers. The PC Series TEMs are tested
to over 1 million temperature cycles, offering a significantly longer operating life and lower overall
cost of ownership compared to standard TEMs. The Laird PC Series TEMs are designed for high
reliability applications like PCR thermal cycling, which require extended mean time between
failures. The polymerase chain reaction is a biochemical technology that amplifies a single copy or
a few strands of DNA across several orders of magnitude, generating several hundred thousands of
copies of a particular DNA sequence.
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).For PBS each tubes was adding a drop
of 1M of HCL or 0.5 M of NaOH . And 10Ul of phage lysate with 10^8 PFU / mL.
3.5.2 Research Procedures
3.5.2.1 Collection of Sewage Sample
Check and note the temperature and pH. The specimen collected is placed on a light-proof
box containing ice packs with water for rapid cooling. Samples without ice are not allowed to
exceed 2 hours for the test to be reliable and accurate. 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 samples will be stored at 4ºC then phage enrichment TSB broth will be added to
dehydrate the samples thoroughly vortex to dislodge the phage particles. TSB broth would be added
to liquid samples and will be incubated for 24 hours at room temperature. Then, the sample will be
placed in a rotary shaker and will be shaken at 60RPM for the enrichment of the phage. Then, after

19
24 hours, samples will be transferred in clean test tubes and will then centrifuged to separate the
supernatant. 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 ESBL- Klebsiella Pneumoniae. 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).
3.5.2.3 Spot Testing
Overnight the cultures and prepare for TSB and the sub cultured by the addition of 100 µl
to 3ml TSB and growth at 37ºC for 90 minutes. 100 µl of subculture was inoculated into 3ml of
molten TSB top agar and overlaid into TSA plates. Each overlay was allowed to solidify for 15
minutes. All phages lysates (original titer approximately 10’8 PFU/mL) were diluted in 10 fold
increments and 10 µl of each dilution was spotted on the bacterial overlay, dried and incubate at
37ºC overnight. As a control, each bacterial strain was also mock infected with sterile phage buffer.
Results were analyzed by detection of any lysis and further dilutions were check for single plaques
to ensure phage lysis rather than bacteriocin induced lysis (Jensen et al ,. 2015).
3.5.2.4 pH Stability Test
For the pH stability, the procedure was done by Jonczk et al., (2011) was follow with some
modifications. A stock of PBS was prepare and the pH for each tubes was adjust to 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12 and 13 by adding drop wise of 1 M of HCl or 0.5 M of NaOH until the desired
pH was achieve. The 10 µL of the phage lysate with 108 PFU/mLtiter were mixed together with
90 µL pH adjusted PBS and store at 2-8ºC for 24 hours. Each of the lysate exposed for different
pH were then subjected to plaque assay and incubated for 18-24 hours at 37ºC. Then after
incubation, the number of plaques will be counted and phage titer will be calculated. Then changes
in titer were recorded. All assays were carried out in triplicates. (Mercines et al,. 2017).
3.5.2.5 Temperature Stability Test
For the temperature stability, the procedure was done by Jonczk et al., (2011) will be
followed by some modifications. Phage lysate aliquot with 107 PFU/mL titer were obtained and

20
incubated at various temperature. 1 mL of phage solution will be distributed to each of the micro
centrifuge tubes labeled with their corresponding incubation temperature. The tubes were incubated
for 1 week at the following temperatures: -20ºC and 2-4ºC; 24 hours incubation period: 25ºC and
37ºC; 10 minutes incubation period for 45ºC and 70ºC. After incubation, the lysates were subjected
to plaque assay and the number of plaques was counted and the phage titer was calculated. All
assays were carried out in triplicate (Mercines et al,. 2017).
3.5.2.6 Transmission 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).
3.6 Data Analysis
For gathering data analysis One-way ANOVA assess by post-hoc test as Tukey's test will
be used to this study. The important part is the hypotheses that will be statistically tested. The
software’s that may be used for this study are MS excel, SPSS or SAS. This will determine the
significant difference of the variable of the sample while post hoc which means “after the fact”,
assess the particular important differences after the first statistically significant results.

21
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Hardies, S. C., Hwang, Y. J., Hwang, C. Y., Jang, G. I., & Cho, B. C. (2013). Morphology,
Physiological characteristics, and complete sequence of marine bacteriophage
φ{phonetic}RIO-1 Infecting pseudoalteromonas marina. Journal of Virology, 87(16),
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Hargreaves, K. R., Kropinski, A. M., &Clokie, M. RJ. (2014) Bacteriophage behavioural ecology.
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Jensen, K., Hair, B., Wienclaw, T., Murdock, M., Hatch, J., Trent, A., White, T., Haskell, K., &
Berges, B. (2015). Isolation and Host Range of Bacteriophage with Lytic Activity against
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Mahon, C. R., Lehman, D. C. and Manuselis, G., 2015 Textbook of Diagnostic Microbiology 5 th
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25
Appendix A
The Research Budget
Items Unit Price
Reagents and chemicals
Tryptone Soy Agar 500 grams 3,200.00
Trytone Soy Broth 500 grams 1,900.00
DNA Extraction KIT 5,000.00
Distilled Water 2 Litres 60
Antibiotic (Oxacillin and Penicillin) 200
Materials
Cotton 50 g 33
Micropipettes tips 10-100ul (1 box) 375
100-1000ul (1 box) 485
Pasteur Pipette 1ml (15pcs) 150
Syringe (Plastic) 20cc (5pcs) 50
Petri dish (20 pcs) 400
Bottle Container 20 ml (5pcs) 150
Microtubes (5pcs) 2500 (1box)
Unground Glass Slides (1 Box) 75
Cover slips 35
Filter membrane 0.22 um (5pcs) 545
0.45 um (5pcs) 545
Amican ultra-5 centrifugal filter unit (5pcs) 900
Cooler 150
Glass wares Materials and others
Erlenmeyer Flask 1000 ml 400
Beaker 100 ml 180
Test tubes 10 ml 10
Gauze wire 290
Stirring rod 60
Alcohol lamp 50
Forceps 150
Tong/test tube clamp 150
Inoculating loop 1200
Other Test
Transmission Electron Microscope 3,500
Other
Ice for cooler 200
Printing materials 500
Travel expenses 5000
Marker 75
Masking Tape 100
GRAND TOTAL 23868

26
Appendix B
Timeline
Gantt Chart
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Activity/ies
Purchasing of
materials and
Reagents
Collection of Phage in
Calatagan, Batangas
Collection of Bacterial
Sample (Klebsiella
pneumoniae)
Media Preparation
Phenotypic
identification of phage
using TEM
Use of PCR in OLFU
Quezon City campus
Reporting and analysis
of data
Reviewing and editing
of Final Data
Final Defense

27
Appendix C
Certifications
Molecular Diagnostic Seminar Workshop

28
Appendix C
Certifications
Animal Handling Seminar Workshop

29
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.

30
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.

31
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.

32
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.

33
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.

34
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.

35
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.

36
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.

37
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.

38
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.

39
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.

40

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Isolation and Phenotypic Characterization of Marine Phage…

Keywords Antimicrobial Resistance, PCR (Polymerase Chain Reaction), TEM (Transmission

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

Klebsiella pneumoniae is a standout amongst the most widely recognized Gram-negative

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

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

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

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

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.6 Extended Spectrum β-lactamase

2.6.1 ESBL- K.pneumoniae

Klebsiella pneumoniae is the commonly isolated specie of Klebsiella. It is a nosocomial

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.7 Application of Phage

2.7.1 Phage Display

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.7.2 Phage Typing

2.7.3 Phage Therapy

2.8 Problem Statement

Specifically, it will answer the following questions:

Research question 1: What is the characterization of isolated Extended Spectrum Beta-

OUR LADY OF FATIMA UNIVERSITY MEDICAL LABORATORY SCIENCE

2.9 Discussion of Flow Chart

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 Don Ramon Clinical isolation of ESBL-

Cultivating bacteria in TSA

Incubation for overnight

Transmission pH Stability Temperature

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 Extended Spectrum β-lactamase Klebsiella pneumonia Sample

Extended Spectrum β-lactamase Klebsiella pneumoniea will be collected from the

3.4 Research Ethics

3.5 Research Instruments