UNIVERSITY OF DAR ES SALAAM
COLLEGE OF NATURAL AND APPLIED SCIENCE
DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHINOLOGY
RESEARCH PROJECT REPORT
ISOLATION OF BACTERIOPHAGES CAPABLE OF LYSING PATHOGENIC
ENTERIC ESCHELICHIA COLI
STUDENT NAME: GODFREY DAUDI
REG NO: 2012-04-02821
SUPERVISOR: PROF. MTUI
ISOLATION OF BACTERIOPHAGES CAPABLE OF
LYSING PATHOGENIC ESCHELICHIA COLI
By
GODFREY DAUDI
A Report Submitted in (Partial) Fulfillment of the
Requirements for Degree of Bachelor of Science
(Molecular Biology and Biotechnology) of the
University of Dar es Salaam
3
July 2015
CERTIFICATION
The undersigned certify that they have read and hereby
recommend for acceptance by University of Dare es Salaam a
Project Report entitled:
Isolation of bacteriophages capable of lysing pathogenic enteric
Escherichia Coli in (Partial) fulfillment of the requirements for
the degree of Bachelor of Science (Molecular Biology and
Biotechnology) of the University of Dar es Salaam.
PROF. G. Y. S. MTUI
(Supervisor): _____________________
Date:
ASSESSMENT
Proposal presentation………………………………………………………… (………../20)
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Final report oral presentation…………………………………………………
(……....../20)
Final report assessment……………………………………………………….. (………. /60)
Total marks…………………………………………………………………… (………/100)
Grade………………………………………………………………………….. (………….)
DECLARATION
AND
COPYRIGHT
I GODFREY DAUDI declare that this is my work and has not been
presented and will not be presented to this and any other
University for similar or any other degree award.
Signature:
5
This Report is the copyright material protected under the
Berne Convention, the copyright Act of 1999 and other national
and international enactments, in that behalf, on intellectual
property. It may not be reproduced by any means, in full or in
part, except for short extracts in fair dealing; for research
or private study, critical scholarly review or discourse with
an acknowledgement, without the written permission of the
Department of Molecular Biology and Biotechnology, on behalf
of both the candidate and the University of Dar es Salaam.
ACKNOWLEDGEMENTS
I wish to acknowledge the University of Dar es Salaam for giving
me the opportunity to advance in my education. I extend my
thanks to the staff Molecular Biology and Biotechnology
Department for preparing me to be competent in academic matters
related to life sciences and research.
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My sincere thanks go to Prof. G. Mtui, my academic supervisor,
Mrs. Ernest and Mrs. Henry, my research technical supervisors
for all their indispensable support they gave me in during my
research project.
My appreciation goes to my family; my father, mother, brother
and sisters for all their love. My special thanks go to my
father for his moral support.
Lastly I am thankful to all my colleagues, Molecular Biology and
Biotechnology students (2012/2015) for their cooperation and
support. God bless you all.
7
LIST OF ABBREVIATIONS/ACRONYM USED
DNA Deoxyribonucleic acid
E. coli Escherichia coli
ICTV International Committee on taxonomy of viruses
RNA Ribonucleic acid
EPEC Enteric pathogenic E. coli
IBC Intracellular Bacteria communities
UDSM University of Dar es salaam
MBB Molecular Biology and Biotechnology
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ABSTRACT
Bacteriophages, also called phages, or bacterial viruses, are
viruses that infect bacteria. They are classified according to
the International Committee on Taxonomy of Viruses (ICTV)
ending up with 19 families. A research project was conducted
to determine lytic effect of bacteriophage on enteric Escherichia
coli (EPEC), a causative agent of gut infection aiming to
develop a bio control technology for eradication of the EPEC.
All necessary requirements were settled in the laboratory. E.
coli strain was provided at the University of Dar es Salaam’s
MBB Department. Sewage water from The University of Dar es
salaam oxidation ponds was used to isolate bacteriophages.
Sewage water was filtered to obtain viruses by using filter
paper. Nutrient broth and nutrient agar were the media used in
culturing EPEC. Double layer agar technique was applied in
9
phage plating by using two different concentrations, (28g/l
and 14g/l for bottom and top layer respectively). The top
semisolid media was mixed with both EPEC samples from broth
and phage filtrate, and then incubated for 24 hrs. The
expected plaques were not seen for all samples, indicating a
negative score. This may be caused by methodology constraints,
for example poor phage identification, inability to
concentrate the bacteriophage, inadequate laboratory equipment
and other supplies, time shortage and inability to go deeper
into other methodologies. Despite of the negative results
obtained, it is recommended that this study should be
continued until its goal is accomplished.
Key words: Bacteriophages, enteric E. coli, phage lytic effect
Table of ContentsCERTIFICATION......................................................3
10
The undersigned certify that they have read and hereby recommend foracceptance by University of Dare es Salaam a Project Report entitled:..........................................................3DECLARATION........................................................4
AND................................................................4COPYRIGHT..........................................................4
ACKNOWLEDGEMENTS...................................................5LIST OF ABBREVIATIONS/ACRONYM USED.................................6
ABSTRACT...........................................................7LIST OF FIGURES....................................................9
1.0 INTRODUCTION..................................................101.2 Problem Statement.............................................11
Justification.................................................111.3 Objectives of the Study.......................................11
1.3.1General objective............................................111.3.2 Specific objectives.........................................11
1.6 Literature Review.............................................122.0 MATERIALS AND METHODS.........................................16
2.4 Sampling......................................................17First session...................................................18
Second session..................................................19CHAPTER THREE.....................................................21
3.0 RESULTS.......................................................21CHAPTER FOUR......................................................23
4.0 DISCUSSION....................................................23CHAPTER FIVE......................................................26
5.0 CONCLUSION AND RECOMMENDATIONS................................265.1References.....................................................27
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LIST OF FIGURESFigure1. Cultured enteric E. coli strain……………………………………………………..17
Figure 2. Sewage water from UDSM oxidation pond
…………………………………….18
Figure.3. Plates containing enteric E. coli and expected
phage……………………………..20
Figure.4. Appearance of colonies on negative control
plate………………………………21
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CHAPTER ONE
1.0 INTRODUCTION1.1 BACKGROUND INFORMATION
Bacteriophage is a virulent infectious virus; this implies
that bacteriophage cause death to their host. The word
‘’phage’’ means ‘’eat’’ and so bacteriophages are viruses that
infect bacteria. Bacteriophages are obligate intracellular
parasites. They exclusively target and reproduce within
bacterial cells. Phages attach to the surface of their host
cell by specialized structures called tail fibers once
attached; the bacteriophage injects its nucleic acid into the
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bacterium. Using the host cells replication, translation, and
transcription machinery, the viral nucleic acid is replicated
and incorporated into its protein capsid. The escape of mature
viruses from the host cell places stress on the plasma
membrane resulting in the eventual death of the bacterium.
Phage therapy is the use of bacteriophages to treat pathogenic
bacterial infections. Phage therapy is an alternative to
antibiotics being developed for clinical use by research
groups in Eastern Europe and U.S. An important benefit of
phages therapy is that they are more specific than most
antibiotics that are in clinical use. Also, phages have few or
no side effects compared to drugs and do not stress the liver
and since phages are self-replicating in their target
bacterial cell, a single and small dose is theoretically
efficacious. Bacteriophage plaque is a clear circular zone in
an otherwise confluent growth of bacteria on an agar surface
resulting from bacterial lyses by bacterial viruses.
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1.2 Problem Statement
Nowadays many diseases associated with E. coli contaminations are
a problem in Tanzania where the rate of transmission is
increasing and the E. coli are becoming resistant against drugs.
JustificationThis study was intended to address the problem of E. coli
infections by introducing phage therapy application using
phages instead of antibiotics so as to reduce problem.
1.3 Objectives of the Study1.3.1General objective
Isolation of bacteriophage and use it as a bio control tool in
remediating pathogenic E. coli in habitats where they pose health
problems to both human and animals.
1.3.2 Specific objectives
To determine the presence of phages in sewage.
To establish the lytic effect of phages on E. coli.
1.4 Significance of the Study
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This study is useful in the application of phages as a
therapeutic tool to eradicate problems caused by enteric E. coli
1.5 Research Questions
(i) What is the magnitude of phages in sewage?
(ii) How effectives are phages in lysing enteric E. coli
1.6 Literature Review1.6.0 Pathogenic Enteric E. coli
Escherichia coli are Gram-negative, facultative anaerobic and non-
sporulation bacteria (Evans et al., 2007). They are typically rod-
shaped, and are about 2.0 micrometers (μm) long and 0.25-
1.0 μm in diameter, with a cell volume of 0.6–0.7 μm3. They can
live on a wide variety of substrates. In anaerobic conditions
E. coli uses mixed-acid fermentation, producing lactate,
succinate, ethanol, acetate and carbon dioxide (Todar, 2007).
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Optimal growth of E. coli occurs at 37 C (98.6°F) but some
laboratory strains can multiply at temperatures of up to 49°C
(120 °F). They grow in both aerobic and anaerobic condition,
using a large variety of redox pairs. They may oxidize pyruvic
acid, formic acid, hydrogen and amino acids. Also, they reduce
oxygen, nitrate, fumarate, dimethyl sulfoxide and
trimethylamine N-oxide. (Todar, 2007)
1.6.1 Pathogenesis of E. coli
Serotypes of E. coli are recognized based on O, H, and K
antigens. Serotyping helps in distinguishing the small number
of strains that cause diseases. The serotype O157:H7 (O refers
to somatic antigen; H refers to flagellar antigen) is uniquely
responsible for causing hemolytic uremic syndrome (HUS). Those
that cause diarrhea (pathogenic E. coli) are classified based on
their unique virulence factors and can only be identified by
these traits. Hence, analysis for pathogenic E. coli usually
requires that the isolates first be identified as E. coli before
being tested for virulence (Todar, 2007).
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Pathogenic E. coli are responsible for three types of infections
in humans: urinary tract infections (UTI), neonatal
meningitis, and intestinal diseases (gastroenteritis). The
diseases caused by a particular strain of E. coli depend on
distribution and expression of virulence determinants,
including adhesins, invasins, toxins, and abilities to
withstand host defenses (Todar, 2007)
In the IBC, bacteria find safe haven, are resistant to
antibiotics and subvert clearance by host innate immune
responses (Todar, 2007)
1.6.2 Bacteriophages
Viruses that attack bacteria were observed by Twort and
d'Herelle in 1915 and 1917. They observed that broth cultures
of certain intestinal bacteria could be dissolved by addition
of a bacteria-free filtrate obtained from sewage. The lysis of
the bacterial cells was said to be brought about by avirus
which meant a "filterable poison" ("virus" is a Latin word for
"poison"). Probably every known bacterium is subject to
infection by one or more viruses or "bacteriophages" as they
are known ("phage" for short, from Greek Word "phagein"18
meaning "to eat" or "to nibble"). Most research has been done
on the phages that attack E. coli, especially the T-phages and
phage lambda. Like most viruses, bacteriophages typically
carry only the genetic information needed for replication of
their nucleic acid and synthesis of their protein coats. When
phages infect their host cell, the order of business is to
replicate their nucleic acid and to produce the protective
protein coat. But they cannot do this alone. They require
precursors, energy generation and ribosome’s supplied by their
bacterial host cell. Bacterial cells can undergo one of two
types of infections by viruses termed lytic infections and
lysogenic (temperate) infections. In E. coli, lytic infections
are caused by a group seven phages known as the T-phages,
while lysogenic infections are caused by the phage lambda.
(Todar, 2007)
1.6.3 Bacteriophage and E. coli
During infection a phage attaches to a bacterium and inserts
its genetic material into the cell, the initial step of viral
infection is the binding of a virus onto the host cell
19
surface. λ phages uses its g3p protein to bind to specific
receptors, LamB, on the host cell surface during the infection
(Brock Biology of Microorganisms), resulting in wealth of
information on the structure, biochemical properties and
molecular biology of this system. After this, a phage follows
one of two life cycles, lytic (virulent) or lysogenic
(temperate). Lytic phages take over the machinery of the cell
to make phage components. They then destroy, or lyse, the
cell, releasing new phage particles. Lysogenic phages
incorporate their nucleic acid into the chromosome of the host
cell and replicate with it as a unit without destroying the
cell. Under certain conditions lysogenic phages can be induced
to follow a lytic cycle.
1.6.4 Phage therapy
Phage therapy is the therapeutic use of lytic bacteriophages
to treat pathogenic bacterial infections. Phage therapy is an
alternative to antibiotics being developed for clinical use by
research groups in Eastern Europe and the U.S. after having
been extensively used and developed mainly in the former
Soviet Union countries for about 90 years. An important20
benefit of phage therapy is derived from the observation that
bacteriophages are much more specific than most antibiotics
that are in clinical use. Theoretically, phage therapy is
harmless to the eukaryotic host undergoing therapy, and it
should not affect the beneficial normal flora of the host.
Phage therapy also has few, if any, side effects, as opposed
to drugs, and does not stress the liver. Since phages are
self-replicating in their target bacterial cell, a single,
small dose is theoretically efficacious. On the other hand,
this specificity may also be disadvantageous because a
specific phage will only kill a bacterium if it is a match to
the specific subspecies. Thus, phage mixtures may be applied
to improve the chances of success, or clinical samples can be
taken and an appropriate phage identified and grown.
Phages are currently being used therapeutically to treat
bacterial infections that do not respond to conventional
antibiotics, particularly in the country of Georgia. They are
reported to be especially successful where bacteria have
constructed a biofilm composed of a polysaccharide matrix that
antibiotics cannot penetrate (Todar, 2007)
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CHAPTER TWO
2.0 MATERIALS AND METHODS2. 1 Materials used
Petri dishes
Inoculating loops
Nutrient broth
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Incubator
Nutrient agar
Epperndorf tubes
Phosphate buffered saline (PBS )
Test tubes
Centrifuge machine
Agar powder
Conical flasks
Pipette
Enteric E. coli strain
2. 2Study site
The study was carried out at the University of Dar es Salaam
MBB department and sewage was collected from the University of
Dar es Salaam oxidation ponds.
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2.3 Sampling
Sample of sewage water was collected from the oxidation
ponds of the University of Dar es Salaam.
Enteric pathogenic E. coli strain specimen were provided by
the University of Dar es Salaam MBB Department.
2.4 Sampling
S/N SAMPLE TYPE QUANTITY
1 Sewage water 6 Litres
2 Pure water (control) 6 Litres
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First session
E. coli strains were cultured on nutrient agar plates and
incubated overnight at 37oC
Figure 2: Obtained E. coli colonies
grown on petri dishes using nutrient agar.
Second session
ISOLATION OF BACTERIOPHAGE
26
Day 1: 5 Ml nutrient broth was inoculated with E. coli. Then
it was incubated overnight at 37oC for 24 hours.
Day 2: 4.5mL sewage was inoculated with 0.5mL of your
overnight E. coli culture and 0.5mL nutrient
broths. Incubation was carried out for 24-48 hours at
37oC.
Day 3: The tubes were centrifuged for 10 minutes at
2500rpm.
Then, 3mL of the supernatant was taken and filtered into
a sterile tube. Then, the filtrate was labeled as
"Enriched phage”.
Day 4: Six tubes 1–6 were labeled. 0.9 ml of sterile PBS
was pipetted.0.1 ml of phage suspension was transferred
into tube 1, and mixed. Using the same pipette, 0.1 ml of
the sample was transferred from tube 1 to tube 2, and
tube 2 to tube 3.
0.5 ml of E. coli was distributed into each of the six
tubes, labeled 1–6.
To each tube of bacteria, 0.1 ml was added to the
corresponding phage dilution (0.1 ml of dilution 1 to
cell tube 6, and so forth).
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The tubes were incubated at 37°C for 10 minutes to allow
the phage to adsorb (attach) to the bacteria.
Then, six warm, dry, nutrient agar plates were labeled
(bactephage₊E. coli) for each infection). Then, plates were
kept at 37°C in the incubator until when they were ready
to be used.
The labeled plates from the incubator were collected and
the contents of cell-phage tube 1 were added to a vial
containing 3 ml of top agarose (molten, at 45°C) and
quickly the contents were mixed and poured onto warmed
plates.
The above step was repeated for each of the remaining
five samples, 2–6.
The plates were allowed to cool without being disturbed
for approximately 10 minutes. When the agar had
solidified, the plates were inverted, at 37°C for 24
hours.
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3.0 RESULTS
The results below indicate the plates where Enteric PEC was
grown together with samples suspected to contain
bacteriophages. These were supposed to have plaques if phages
are there, but unfortunately there were no plaques at all the
plates. The bacteria grew rapidly in a double layered nutrient
agar. Results of all the plates showed no formation of any
plaque in all tested bacterial colonies. Morphologically, what
was grown in the plates were E. coli and not zone of clearance.
30
F
Figure 3: Plates showing growth of E. coli but without formation
of plaques.
Figure 4: Control plate showing growth of E. coli in nutrient
agar using distilled water instead of sewage.
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CHAPTER FOUR
4.0 DISCUSSION
The bacteria species are always infected by phages. The plan
of this study was to isolate bacteriophages which are capable
of lysing EPEC. Sewage samples were collected from the
University of Dar es Salaam oxidation ponds. These were
suspected to contain the EPEC- specific phages although there
was no any preliminary test conducted to test the presence of
phages in the collected samples. The formation of plaques was
the only expected indication of phage infection in the sample.
All the samples were treated as per the specified protocol.
The results obtained in this study showed that no plaques were
formed in any plate. It seems the isolated bacteriophages (if
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any)were not able to lyse EPEC. Morphological examination of
the bacteria on plates indicated that they were E. coli species
as shown in Figure 3 and 4 above. They grew on both nutrient
broth and nutrient agar. In nutrient broth they caused
turbidity. Their appearances give a clue that they are E. coli.
Then, the bacteria were mixed with the phage containing
samples by a double layer technique, which was the right way
to observe the suspected plaques. After incubation, the
results showed no plaques in any plate. An experiment was
repeated two times but the results remained the same.
Literature suggests that bacteriophages have ability to lyse
EPEC .The negative results obtained could be caused by some
unforeseen constraints.
The methodology used could be one of the most probable cause
of unexpected results. Lack of methodology that could help to
prove the presence of bacteriophages in collected sewage
samples before mixing with bacterial strains could be one of
the reasons. On the other hand, sewage water collected might
not have contained phages. This is because no test was
conducted to prove the presence of phages in samples although
33
it is known from the literature that phages are abundant in
the environment. It was very difficult to prove the quality of
methodology before applying it.
Due to shortage of time to concentrate to the techniques,
many procedures were conducted within a short period of time,
thus no intense study was conducted. This study, probably,
requires a much more research time for repeating and changing
experimental conditions such as, time, materials, techniques
and location. However, this study was conducted in a very
limited time.
The study was not conducted using molecular techniques due to
lack of funds. Some important equipment such as 0.25
micrometer filters were not available.
Laboratory setting and equipment arrangement were not good to
support the methodology by 100%. Many types of equipment were
shared by almost all students, a situation which could have
contributed high chances of cross contamination and this could
34
have resulted to more E. coli colonies in plates than correlated
amount of bacteriophages.
Furthermore, the bacteriophages isolated (if any) could not
be specific to EPEC, thus they may have failed to lyse the
bacteria tested.
Differences in temperature, and other environmental conditions
for phage infectivity to bacteria, may have also contributed
to the negative results obtained. , For example, since sewage
stream temperature is different from that tested in the
laboratory, it was rather difficult to maintain the
bacteriophages’ natural conditions.
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CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATIONS
Expected results were not realized, so the ability of phage to
lyse PEC was found to be negative as they were not able to
lyse enteric E. coli. However, some literature suggests that
phages have the ability to lyse pathogenic E. coli. If the
constraints explained in the Discussion section will be
monitored and corrected by other coming scientists, the
positive results may be obtained and the objectives of this
study will be fulfilled. There is a need to repeat this kind
of study in order to identify which kind of bacteriophage
predominates in the sewage water system at the University of
Dar es Salaam oxidation ponds.
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5.1References
Brzuszkiewicz, E. Thürmer, A.Schuldes, J.Leimbach,
A.Liesegang, H. Meyer,F.D.Boelter, J. Petersen,H.
Gottschalk, G. Daniel, R. (2011). "Genome sequence
analyses of two isolates from the recent Escherichia coli
outbreak in Germany reveal the emergence of a new
pathotype: Entero-Aggregative-Haemorrhagic Escherichia
coli (EAHEC)". Arch. Microbiology. Vol 193 (12): 883–91
pp.
38
Carlton, R. M. (1999). “Phage Therapy: Past History and Future
Prospects.” Archivum ImmunologiaeetTherapiaeExperimentalis.
47: 267-74.
Dhakal B. K., R. R. Kulesus and M. A. Mulvey: Mechanisms and
consequences of bladder cell invasion by pathogenic
Escherichia coli. European Journal of Clinical Investigation Volume 38,
Issue s2, Pages 2-11.
Evans, J.r. Doyle, J. Dolores, Evans, G. (2007) "Escherichia Coli".
Medical Microbiology, 4th edition. The University of Texas Medical
Branch at Galveston.
Grahn, A. M. Haase, J. Lanka, E. Bamford, D. H. (1997).
“Assembly of a functional phage PRD1 receptor depends on
11 genes of the Inc. plasmid mating pair formation
complex,” Journal of Bacteriology, vol. 179, no. 15, 4733–
4740. pp.
Guttman, B. Raya, R. Kutter, E. (2005). Basic Phage Biology.
In: Bacteriophages: Biology and Applications, CRC Press, Boca Rutan
FL, pp. 29-66K. Parent and I. D. Wilson,
“Mycobacteriophage in Crohn's disease,” Gut, vol. 12, no.
12, 1019–1020, pp.
39
Lin, X.-m.; Yang, M.-j.; Li, H.; Wang, C.; Peng, X.-X (2014).
Decreased expression of LamB and Odp1 complex is crucial
for antibiotic resistance in Escherichia coli. Journal of Proteomics.
98, 244.
Sulakvelidze, A. Barrow, P. (2005). Phage Therapy in Animals
and Agribusiness. In: Bacteriophages: Biology and Applications, CRC
Press, Boca Rutan FL, 335-380. pp.
Sulakvelidze, A. Kutter, E. (2005). Bacteriophage Therapy in
Humans. In: Bacteriophages: Biology and Applications, CRC Press,
Boca Rutan FL, 381-436.pp.
Todar, K. (2007). “E. coli”. Online Textbook of Bacteriology.
University of Wisconsin–Madison Department of
Bacteriology. Site visited on 20/4/2015.
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