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

July 2015

2

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.

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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

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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

11

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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Figure 1. Sewage water collected from UDSM oxidation pond

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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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CHAPTER THREE

29

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.

35

36

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.

37

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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