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



BATHOGENIC BACTERIA

M.Sc. Thesis

In

Agric. Sci. (Agricultural Microbiology)

By


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



BATHOGENIC BACTERIA

M.Sc. Thesis

In

Agric. Sci. (Agricultural Microbiology)

By


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

i

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

ii

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…………………………………………….................

29

29

30

30

31

31

31

32

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

55

a. Effect of UV radiation ………………………………………

b. Effect of high and low temperatures ……………………

c. Effect of the pH ……………………………………………….

d. Antiviral effect of the essential oils……………………….

55

58

62

65

CONCLUSION……………………………………............................ 70

SUMMARY………………………………………...………................ 72

REFERENCES …………….…………….………………………… 78

ARABIC SUMMARY…………………………………………….

1

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).

2

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

3

conditions representing UV radiation, different levels of

temperature and pH.

4- Assessment of the antiviral effect of some essential oils.

4

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)

5

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).

6

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.

7

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

isolation and characterization of lytic …

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