ISOLATION AND CHARACTERIZATION OF LYTIC …
Transcript of ISOLATION AND CHARACTERIZATION OF LYTIC …
ISOLATION AND CHARACTERIZATION OF
LYTIC BACTERIOPHAGES INFECTING SOME
BATHOGENIC BACTERIA
BY
DINA GAMAL ELSAYED ABED B.Sc. Agric. Sci. (Food Science), Fac. Agric., Cairo Univ., 2011
THESIS Submitted in Partial Fulfillment of the
Requirements for the Degree of
MASTER OF SCIENCE
In
Agricultural Sciences (Agricultural Microbiology)
Department of Agricultural Microbiology
Faculty of Agriculture
Cairo University
EGYPT
2017
APPROVAL SHEET
ISOLATION AND CHARACTERIZATION OF
LYTIC BACTERIOPHAGES INFECTING SOME
BATHOGENIC BACTERIA
M.Sc. Thesis
In
Agric. Sci. (Agricultural Microbiology)
By
DINA GAMAL ELSAYED ABED B.Sc. Agric. Sci. (Food Science), Fac. Agric., Cairo Univ., 2011
APPROVAL COMMITTEE
Dr. El-SHAHAT MOHAMED RAMADAN ………....................... Professor of Microbiology, Fac. Agric., Ain-Shams University
Dr. HUSSEIN EMAM MAKBOUL ……………..…………………… Professor of Microbiology, Fac. Agric., Cairo University
Dr. HANAN ABDEL LATIEF GODA ………………………….…. Associate Professor of Microbiology, Fac. Agric., Cairo University
Dr. NADIA FAHMY AMIN EMAM ……………………………… Professor of Microbiology, Fac. Agric., Cairo University
Date: 11/ 01/2017
SUPERVISION SHEET
ISOLATION AND CHARACTERIZATION OF
LYTIC BACTERIOPHAGES INFECTING SOME
BATHOGENIC BACTERIA
M.Sc. Thesis
In
Agric. Sci. (Agricultural Microbiology)
By
DINA GAMAL ELSAYED ABED B.Sc. Agric. Sci. (Food Science), Fac. Agric., Cairo Univ., 2011
SUPERVISION COMMITTEE
Dr. NADIA FAHMY AMIN EMAM
Professor of Microbiology, Fac. Agric., Cairo University
Dr. HANAN ABDEL LATIEF GODA Associate Professor of Microbiology, Fac. Agric., Cairo University
Name of Candidate: Dina Gamal El-Sayed Abed Degree: M.Sc.
Title of Thesis: Isolation and Characterization of Lytic Bacteriophages
Infecting some Pathogenic Bacteria.
Supervisors: Dr. Nadia Fahmy Amin Emam
Dr. Hanan Mohamed Abdel Latief Goda
Department: Agricultural Microbiology
Approval: 11/01/2017
ABSTRACT
This study was aimed to isolate and characterize bacteriophages infecting some
pathogenic bacteria. The E. coli O157:H7 wild type strain 93111 and B. cereus ATCC
33013 - specific phages were successfully isolated. Four B. cereus phages (BCP1, BCP2,
BCP3 and BCP4) were isolated from clay soil samples, whereas four E. coli phages (ECP1,
ECP2, ECP3 and ECP4) were isolated from sewage and Nile water. The morphological
characterization of isolated phages suggests that the phages of infecting B. cereus could
be considered as members of Siphoviridae or Myoviridae families. The morphology of E.
coli phages confirms their classification to Tectiviridae, Corticoviridae, Siphoviridae or
Podoviridae families. The genome size of the isolated B. cereus and E. coli phages
ranged between 28.7 - 33.6 kbp and 34.5- 41.5, respectively. The four isolated B. cereus
phages exhibited very limited host spectrum, infecting only 2 of 9 Bacillus spp. The ECP2
has the ability to infect eight strains of Escherichia coli including five strains of shiga
toxin producing E. coli and three nontoxigenic E. coli strains. The SDS-PAGE analysis of
phage proteins revealed that the BCP3 and ECP1 had five protein bands with molecular
weights ranging from 37.0 to 110.0 and from 34.0 to 112.0 kDa, respectively. The effect
of some stress conditions on the BCP1 and ECP2 survivability was evaluated. These
conditions comprised UV radiation, high and low temperatures, acidic and alkaline pH,
and some essential oils. After 75 min exposure to the UV light with a wavelength of
254.0 nm, the BCP1 and ECP2 retained 58.7 and 10.89% of the lytic activity, respectively.
With the HTST pasteurization, the BCP1 and ECP2 retained 81.9% and 90.4% lytic
competence, respectively. On the other hand, the two phages lost their infection activity
entirely after boiling for 10, 20 and 30 minutes. The survivability of BCP1 and ECP2 was
not affected significantly under refrigeration conditions at 4.0ºC for nine months. During
the storage period, the BCP1 and ECP2 counts were fluctuated between 8.70 ± 0.37 to
9.79 ± 0.55 log10 and 9.023 ± 0.27 to 10.02 ± 0.41, respectively. The BCP1 appeared to be
extremely unstable at very vigorous acidic and alkaline environments as its infection
ability was lost completely at pH 1, 3 and 13. On the other hand, the ECP2 lost its activity
entirely at pH 1 and 79.96% at pH 13. The greatest inhibition of the BCP1 lytic activity
was recorded with the garlic oil (0.004 mg/mL) followed by ginger oil (0.11 mg/mL) as
the phage activity has reduced by 11.12 and 9.64%, respectively. The clove, garlic and
ginger oils diluted by 50% had weak significant antiviral effect against ECP2, whereas
only clove oil with a concentration of 20 % had a significant effect.
Keywords: Bacillus cereus, Escherichia coli O157:H7, Bacteriophage, Characterization,
Host range, Phage stability
DEDICATION
I dedicate this work to my family specially to my lovely mother and my dear father who supported and helped me along my entire life, and gave me endless love that made me able to overcome difficulties I faced during the completion of this work.
ACKNOWLEDGEMENT
First and forever I feel always indebted to Allah the most beneficent and merciful. Praise Allah for all the gifts which he has given me.
I wish to express my sincere thanks, deepest gratitude and appreciation to Dr.Nadia Fahmy Emam Professors of Microbiology, Faculty of Agriculture, Cairo University Dr. Hanan Abdel Latif Goda Associate Professors of Microbiology, Faculty of Agriculture, Cairo University for their Keen interest, continued assistance and helpful revision of the manuscript of this thesis.
Grateful appreciation is also extended to all staff members of Microbiology Department, Faculty of Agriculture, Cairo University. Specially to Mrs. Ayatollah S. El-Zayat and Mrs. Rasha Samir Ahmed
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CONTENTS
Page
INTRODUCTION........................................................................... 1
REVIEW OF LITERATURE.............................…................. 4
1. Definition and incidence of bacteriophage ……........... 4
2. Infection and reproduction of bacteriophage……………….. 5
3. Characterization and structure of bacteriophage………… 6
4. Host range …………………………………………………….….. 8
5. Factors affecting the phage stability …………………………. 9
a. Temperature …………………………………………………….. 9
b. pH…………………………………………………………………... 12
c. Salt concentration ………………………………………………. 14
d. UV Radiation …………………………………………………… 15
e. Chemicals……………………………………………………….. 15
f. Essential oils …………………………………………………… 16
6. Bacteriophages as biocontrol agent………………………… 17
MATERIALS AND METHODS………................................ 19
1. Bacterial strains and growth conditions……………………. 19
2. Detection of lysogenic phages ………………………………. 19
3. Sampling…………………………………………………………. 20
4. Bacteriophage enrichment and isolation …………………… 20
5. Plaque assay …………………………………………………….. 21
6. Bacteriophage purification ………………………………….. 22
7. Characterization of bacteriophages ………………………… 23
a. Transmission electron microscopy …………………….. 23
b. Phage host range determination …………………………. 23
c. DNA and protein analysis ……………………………….. 25
(1) Preparation of phage sample for DNA and protein
analysis ……………………………………………………. 25
(2) DNA extraction ……………………………………….. 25
(3) Genome size estimation ……………………………… 27
(4) Preparation of phage structural proteins and SDS-
PAGE ……………………………………………………… 27
8. Factors affecting the stability of isolated phages ….…....... 27
a. UV radiation …………………...……………………………… 27
b. Temperature …………………………………………………… 28
(1) High temperature ………………………………………… 28
(2) Low temperature…………………………………………. 28
c. pH ……………………………………………………………… 29
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d. Essential oil …………………………………………………..
(1) Antibacterial susceptibility …………………………..
(2) Determination Minimum Inhibitory Concentration
(3) Antiviral assay …………………………………………
9. Statistical analysis……………………………………………….
10. Culture media and reagents……………………………………
a. Culture media………………………………………...............
b. Reagents…………………………………………….................
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RESULTS AND DISCUSSION ….......................….......... 33
1. Lysogenic test for the host bacterial strains ……………… 33
2. Isolation lytic of phages …………………………………….. 34
3. Phage morphology ……………………………………………. 37
4. Phage host range ………………………………………………. 45
5. Genome size estimation ……………………………………….. 49
6. Phage structural proteins analysis ……………………………
7. Factors affecting the stability of isolated phages ………… 50
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a. Effect of UV radiation ………………………………………
b. Effect of high and low temperatures ……………………
c. Effect of the pH ……………………………………………….
d. Antiviral effect of the essential oils……………………….
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CONCLUSION……………………………………............................ 70
SUMMARY………………………………………...………................ 72
REFERENCES …………….…………….………………………… 78
ARABIC SUMMARY…………………………………………….
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INTRODUCTION
The bacterial viruses or bacteriophages are extremely
abundant and exert enormous influence on the biosphere.
Bacteriophages, kill about 4-50% of the bacteria produced every
day, are a driver of global geochemical cycles and a reservoir of the
greatest genetic diversity on earth (Suttle, 2005).
The different researches suggested that there are globally
approximately 100 million phage species, but only small number of
these phages has been characterized with around 6000 have been
identified at the end of last century (Zaman, 2014).
Bacteriophages can be defined as obligatory intracellular
bacterial parasites which lack an independent metabolism
exploiting the bacterial cells for their reproduction. Bacteriophages
are typically highly specific, often being restricted to particular
strains within a single bacterial species. However, some
bacteriophages have a relatively broad host range, infecting
multiple species within a genus closely related to their host.
A bacteriophage particle or virion consists of single or
double strands (ss or ds) DNA or RNA molecule encapsulated
inside a protein or lipoprotein coat. According to the International
Committee on the Taxonomy of Viruses (ICTV), over 95% of all
phages described in the literatures belong to the tailed dsDNA
phages (Ackermann, 2007).
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The potential profiteering of phages as therapeutic agents to
control human and animal disease has been recognized especially
they are natural, non-toxic and specific to their bacterial hosts.
Recently, the extension of phage biocontrol to food
applications has been investigated (Coffey et al., 2011;
Sulakvelidze, 2013; Tan et al., 2014 and Bhardwaj et al.,2015). In
2006, for the first time, FDA announced that it had approved the
use of a bacteriophage preparation made from six individually
purified phages to be used on Ready-to-Eat (RTE) meat and poultry
products as an antimicrobial agent against 170 strains of Listeria
monocytogenes.
Initial studies on the application of bacteriophage for control
of pathogens require the isolation and identification of an
appropriator phage from the multitude of phages that exist in the
environment.
The isolation and characterization of phages specific for
pathogens are necessary to determine the most promising candidate
for pathogen control. Therefore, the intentional objectives of the
present study were:
1- Isolation of bacteriophages infecting some pathogenic and
toxigenic bacteria from different samples under Egyptian
conditions.
2- Characterization of the isolated phages morphologically and
genetically.
3- Evaluating the stability of isolated phages infecting E. coli O
157:H7 and B. cereus ATCC 33018 under numerous
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conditions representing UV radiation, different levels of
temperature and pH.
4- Assessment of the antiviral effect of some essential oils.
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REVIEW OF LITERATURE
1. Definition and incidence of bacteriophage
Bacteriophages (bacterial viruses) are viruses that infect and
lyse the bacterial cells and they are widely present in the
environment, wherever the host bacterial is expected to be found
(Hendrix et al., 1999).
Bacteriophages are diverse in shape and size (Fig.1). They
can pass through bacteriological filters with a pore size of 0.2 µm
(Prescott et al., 2002b). Like all viruses, phages are metabolically
inert in their extra cellular form (Sulakvelidze et al., 2001), they
are obligate intracellular parasites and lack their own metabolism
(Zink and Loessmer, 1992).
Fig .1. Basic bacteriophage morphology (Ackermann, 2009)
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Phages have been detected at high levels in the
environments where the host bacteria present. A single
environment sample may be containing different phage species
specific for different bacteria and also contain multiple phages
specific for the same bacteria (Wichels et al., 1998 and Heringa et
al., 2007).
Phages specific for E. coli O157: H7 have previously been
isolated from human faecal materials as animal manures (Morita et
al., 2002 and O’Flynn et al., 2004 ), lack or pond water
(Shahrbabak et al., 2013).
Specific phages of E. coli O157: H7 were isolated from the
environment where both acidity and salinity were high (Lu and
Breidt, 2015).
The isolation and characterization of cold-active
bacteriophages specific for Bacillus cereus from low temperature
environments contributes the understanding of cold-adaptation
mechanisms and co-evaluation of bacteriophages and their hosts (Ji
et al., 2015).
2. Infection and reproduction of bacteriophage.
Bacteriophages are adsorbed or attached to the bacterial cells
which have a specific chemically complementary site. Viral
nucleic acid enters the bacterial cell and replicates intercellulary,
where viral genes are transcribed and translated and proteins are
synthesized. The nucleic acid and proteins assemble to form a
complete phage particle (Budzik, 2003).
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There are two types of bacteriophages could be distinguished
according to the infection way of the bacterial cells, lytic and
temperate phages. Lytic phages or virulent phages multiply in the
host bacteria and lyse it at the end of the cycle. The lysogenic
cycle is a cycle in which bacteriophages reproduce without killing
the host. The temperate or lysogenic phages which may take the
form of a ʺprophageʺ by integrating with the viral DNA in the host
chromosome. The bacterial host cell continues to replicate and
grow and the phage genes also replicate as a part of the bacterial
chromosome. After induction by UV or mitomycin c the
prophages become lytic and lysis the host cells and release new
phage particles into the environment (Lu et al., 2003)
3. Characterization and structure of bacteriophage
Characterization has traditionally been based on physical
properties of virion such as capsid size, shape, genome size,
resistance to organic solvents and host range. According to the
International Committee for Taxonomy of Viruses (ICTV), the
phages are classified into 13 families based on their shape, size,
type of nucleic acid and presence/absence of envelope. ICTV
requires phage particles to be observed by electron microscopy
and capsid morphology to be established for their formal
classification (Rohwer and Edwards, 2002).
The basic structure features of bacteriophage are coats or
capsid that protect the genome (DNA or RNA) hidden inside a
capsid (Fig.1). Phage virions can be tailed, polyhedral, filamentous
and pleomorphic and most of them contain double –stranded DNA.
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About 5568 bacterial viruses have been examined by electron
microscopy since 1959, at least 96% of these are tailed and 3.7%
are polyhedral, filamentous or pleomorphic (Ackermann, 2007).
Fig. 2. Structure of bacteriophage (Birge, 2006).
Many, but not all phages, have tails attached to the phage
head, the tail is hollow tube through which the nucleic acid passes
during infection, at the end of the tail, some phages have a base
plate and one or more tail fibers attached to it (Fig. 2) (Ginoza,
1967).
The majority of phages described were belonged to the order
caudovirals, tailed phages with isometric heads containing double-
stranded DNA (Hagens and Loessner, 2007).
Tailed phages are characterized by contractile or non-
contractile tail. The phage length generally ranges from 50-200 nm
and the head averages from 50-90 nm in width. The genetic