CHARACTERISATION OF Escherichia coli ISOLATED FROM THE AQUACULTURE ENVIRONMENT

SHEILA MARIA ANDREW STANLEY

(22283)

A Final Year Project Report submitted in partial fulfilment of

the requirement for the degree of Bachelor of Science with Honours (Resource Biotechnology)

Faculty of Resource Science and Technology UNIVERSITI MALAYSIA SARA W AK

2011

I ACKNOWLEDGEMENTS

T

Thank You.

Deep appreciation for Dr. Samuel Lihan, as my supervisor, for all the information and

guidance upon the completion of this research.

Heartfelt acknowledgement towards Kathleen Michelle Mikal, Velnetti Linang and Chen Yik

Ming, as the postgraduate students in the Microbiology Lab for the knowledge and assistance

in my labworks.

Big thanks to Nurismi bt. Ismaili for the superior contribution towards the samples and stock

cultures which were vastly useful for my research.

Sincere gratitude towards my labmates; Elsabet, Jackson, Nurfarhana, Ong Sue Ann, Mimi,

Sabatini, Ardi, Nurkhairunnisa and Nurul Ain; for the supports and facilitation during the

proj eet management throughout the semesters.

Last but not least, lots of love for my family for their unwinding care and supports that

strengthen me throughout the completion of this proj ect.

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Pusat Khidmat Maklumal Akademik ,JNIVEl-;'SlTI MAU',VSIA SARAWAK

TABLE OF CONTENTS

Acknow ledgements

Declaration II

Table ofContents III

List of Abbreviations v

List ofTables and Figures VII

Abstract

1.0 Introduction 2

2.0 Literature Review 5 2.1 Aquacultural Environment 5 2.2 Escherichia coli 6 2.3 Antibiotic Resistance Among Bacteria 7

2.3.1 Antibiotic Resistance 7

2.3.2 Types of Antibiotics 8

2.4 Bacterial Plasmid DNA lO 2.4.1 Alkaline Lysis 11 2.4.2 Plasmid DNA Profiling 12

2.5 Genetic Recombination as the Main Medium for Antibiotic 13 Resistance Transfer

2.5.1 Horizontal Gene Transfer 13

2.5.1.1 Conjugation 14 2.5.1.2 Transduction 15

2.5.1.3 Transformation 16

2.5.2 Vertical Gene Transfer 16

2.6 Polymerase Chain Reaction 17

2.6.1 (GTG)5 PeR 19

III

3.0 Materials and Methods 21 3.1 Bacteria Isolates Purification and Preparation of Stock Cultures 21

3.1.1 Preparation of Eosin Methylene Blue (EMB) Agar 21 3.1.2 Revival and Purification of the E. coli isolates from the 22

Glycerol Stocks

3.1.3 Preparation of Stock Cultures 22 3.2 Antibiotic Susceptibility Testing 23

3.2.1 Preparation of Bacterial Broth Culture 23 3.2.2 Preparation of Mueller-Hinton Agar 23 3.2.3 Antibiotic Susceptibility Testing 24

3.3 Plasmid DNA Extraction 25 3.3.1 Alkaline Lysis 25 3.3.2 Agarose Gel Preparation and Agarose Gel Electrophoresis 27

3.4 Polymerase Chain Reaction (PCR) 28 3.4.1 Genomic DNA Extraction by Boiling Method 28 3.4.2 (GTG)5 Polymerase Chain Reaction 29 3.4.3 (GTGh PCR Fingerprint Analysis 31

4.0 Results 32 4.1 Antibiotic Susceptibility Test and Plasmid DNA Profile Analysis 32 4.2 (GTG)s Polymerase Chain Reaction 42

5.0 Discussion 47 5.1 Antibiotic Resistance Patterns ofE. coli 47 5.2 Plasmid DNA Profile Analysis ofE. coli 50 5.3 (GTG)5 PCR for DNA Fingerprinting of E. coli 52

6.0 Conclusion 55 7.0 References 57

Appendices 60

Appendix I 61 Appendix II 62 Appendix III 63 Appendix IV 64

IV

.. "

E. coli

kb

ilL

Ilg

mL

%

°C

g

DNA

MAR

PCR

dNTPs

RAPD

EMB

LB

MHA

LIST OF ABBREVIATIONS

Escherichia coli

kilobase

microlitre

microgram

millilitre

percentage

degrees Celcius

gram

deoxyribonucleic acid

multiple antibiotic resistance

polymerase chain reaction

deoxynucleotide triphosphates

Rapid amplified polymorphic DNA

Eosin Methylene Blue

Luria-Bertani

Mueller-Hinton agar

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I

TSA Tryptose Soy agar

0 degrees

mm millimetre

rpm revolution per minute

MgCh Magnesium chloride

mM milli molar

tlM micro molar

Ji ~dH20 distilled water i' ~:

mms minutes I

bp base pairs

AGE agarose gel electrophoresis

VI

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LIST OF TABLES AND FIGURES

Table la: PCR mixture for (GTG)s PCR 30

Table I b: The conditions required for (GTG)s PCR 30

Table 2: Percentage of antibiotic resistance end susceptibility among E. coli isolates

35

Table 3: Antibiotic resistance patterns and plasmid profile analysis of E. coli 41

Table 4: E. coli isolates from aquaculture environment obtained from the stock cultures in Microbiology Lab

61

Table 5:

Figure I:

Figure 2:

Figure 3a:

Figure 3b:

Composition of resuspension, lysis and neutralizing solution for Alkaline Lysis

Overall steps involved in alkaline lysis for plasmid DNA extraction

Schematic diagram of Polymerase chain reaction

Antimicrobial susceptibility test for E. coli isolate B6-1-W -5

Positive control for antimicrobial susceptibility test by using E. coli A TCC 25922 strain

62

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17

34

34

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Figure 4a: Agarose 1 % gel electrophoresis of plasmid DNA from E. coli isolates. Lane M: E. coli V517 molecular mass standard of plasmids; 1: B5-1-S-4; 2: B5-1-W-5; 3: B6-1-F-l; 4: B6-1-F-2; 5: B6-I-F-3; 6: B6-1-W-4.

37

Figure 4b: Agarose 1% gel electrophoresis of plasmid DNA from E. coli isolates. Lane M: E. coli V517 molecular mass standard of plasm ids; I: B7-1-F-2; 2: B7-I-F-3; 3: B7-2-S-BC4; 4: B7-2-S-BC6.

38

Figure 4c: Agarose 1 % gel electrophoresis of plasmid DNA from E. coli isolates. Lane M: E. coli V 517 molecular mass standard of plasmids; 1: SB-I-F-l; 2: SB-I-F-2.

39

Figure 4d: Agarose 1 % gel electrophoresis of plasmid DNA from E. coli isolates. Lane M: E. coli V517 molecular mass standard of plasm ids; I: B6-1-W-5; 2: B6-I-W-6; 3: B6-1-W-7; 4: B6-1-W-8; 5: B6-1-W-IO; 6: SB-I-F-3; 7: SB-I-W-l.

40

VII

,...

Figure 5a: Agarose 1.5% gel electrophoresis of (GTG)s PCR analysis. Lane M: 1 kb DNA ladder; 1: B5-1-S-4; 2: B5-I-W-5; 3: B6-1-F-2; 4: B6-1­F-3; 5: B6-1-W-6; 6: SB-l 1; 7: SB-I-F-2; 8: SB-I-F-3; 9: SB-l-W -I; 10: negative control with (GTG)s primer.

44

Figure 5b: Agarose 1.5% gel electrophoresis of (GTG)s PCR analysis. Lane M: 1 kb DNA ladder; 1: B6-I-W-4; 2: B6-1-W-5; 3: B6-1-W-7; 4: B6-1­W-8; 5: B6-1-W-I0; 6: B7-1-F-2; 7: B7-I-F-3; 8: B7-2-S-BC4; 9: B7-2-S-BC6; 10: B6-I-F-I; 11: negative control with (GTG)s

primer.

45

Figure 6: Dendrogram illustrating the relationship between E. coli isolates analysed by repetitive sequence-based PCR using (GTG)5 primer.

46

Figure 7: Graphical method of relating the molecular size (kb) of plasmid DNA to its electrophoretic mobility (mm). E. coli V517 were used as standards for calibrating the size of plasmid molecules.

63

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VIII

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Characterisation of Escherichia coli isolated from the Aquaculture Environment

Sheila Maria Andrew Stanley

Resource Biotechnology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Aquaculture environment is often exposed to contamination from the sewage and organic pollutants which are the main habitat for E. coli. Pathogenic E. coli can cause severe disease outbreaks through food consumption from aquaculture sources. Currently, antibiotics are used in the aquaculture industries for treatment of diseases among aquatic organisms, as a growth promoter, water treatment and pollution control. Nineteen E. coli isolates from the aquaculture environment were tested for their antibiotic resistance by disc diffusion method. The plasmid DNA extraction of the isolates was performed by alkaline lysis method. The genetic variations among the isolates were analysed by (GTG)5 PCR. Based on antibiotic susceptibility test, 100% of the isolates were resistant towards erythromycin while the lowest level of resistance was against chloramphenicol and tetracycline (31.58%). Plasmid analysis revealed that 63.16% isolates contain plasmid DNA, whereas 36.84% are plasmidless. (GTG), PCR analysis indicated that E. coli isolated were grouped into 2 main clusters and 6 minor clusters. This study proved that E. coli isolates from the aquaculture environment were highly diverse, as shown by their differences in antibiotic resistance patterns, plasmid profiles and (GTG)s PCR analysis. The multiple antibiotic resistance of the isolates indicates the potential hazard associated with antibiotic resistance bacteria from the aquaculture environment.

Keywords: Escherichia coli, antibiotics, plasmid profiles, (GTG)s PCR

ABSTRAK

Persekitaran akuakultur sering terdedah kepada pencemaran daripada sistem kumbahan dan bahan organik tercemar yang merupakan habitat utama untuk coli. Patogen E. coli boleh menyebabkan penyebaran wabak penyakit melalui pengambilan makanan dari sumber akuakultur. Kini, antibiotik telah luas digllnakan dalam industri akuakultur untuk perawatan penyakit di kalangan organisma akuatik, sebagai penggalak pertumhuhan, pera,vatan air dan kawalan pencemaran. Semhi/an helas coli dari kawasan akuakultur telah diuji untuk kepekaan terhadap antihiotik melalui kaedah penyerapan cakera. Pengekstrakan DNA plasmid telah dijalankan melalui kaedah pemecahan alkali. Kepelhagaian genetik antara E. coli telah dianalisa melalui (GTG)5 peR. Berdasarkan ujian kerintangan antihiotik. 100% daripada E. coli rintang terhadap eritromisin manakala tahap terendah adalah kerintangan terhadap kloramfenikol dan tetrasiklin (31.58%). Analisis plasmid mendedahkan 63.16% daripada E. coli mengandungi DNA plasmid manakala 36.84% tidak mengandungi plasmid. (GTGh peR memmjllkkan coli telah dihahagikan kepada 2 kllmpulan utama dan 6 kumplilan minor. Kajian ini memhuktikan coli daripada perseltitaran akllakultur mempunyai variasi genetik yang tinggi, sebagaimana kepelhagaian yang ditun}ukkan dalam pola kelintangan antihiotik. profil plasmid dan ana/isis (GTGh peR. Kerintangan antihiotik herganda dari kalangan coli memtfljllkkan potensi yang tinggi lintuk kehadiran hakteria yang rintang antihiotik dwi persekitaran akuakultur.

Kala kunci: Escherichia antibiotik. profil plasmid. (GTGh peR

1.0 INTRODUCTION

The emergence of several antibiotic resistances among different bacteria species has been a

great concern of public health in the world. Antibiotic resistance can be acquired from the

widespread use of antibiotics among human which may lead to the transfer of resistance

gene among the same or different bacteria strains (Wan et aI., 2003). As proposed by

Sunde and Norstrom (2006), the distribution of antibiotic resistant bacteria between human

and environment has been increased in food and water which may severely affect the

national public health and development if the management of antibiotic usage is not being

done properly.

Aquaculture industry is one of the main concerns as it can be an important medium

for the emergence of antimicrobial-resistant E. coli and also other pathogenic bacteria.

Some aquaculture products such as prawns, fish and oysters may contain their own normal

microflora which can cause some negative effects towards the consumers. These products

can be pathogenic when their micro flora have possessed certain antibiotic resistance gene

which can be directly transferred to human and it will possibly cause epidemic such as

diarrhoea or food-borne disease (Sunde & Norstrom, 2006). Besides, food contamination is

also one of the factors that enable the bacteria to exchange their genetic materials with one

another (Kelly et al., 2009).

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Antimicrobial drugs have been widely used in the aquaculture environment.

Despite of being used as a disease control, they are also required for the fisheries growth

promoter in order to enhance meat production and nutritional value (Yoo et al., 2003).

However, the prolonged use of these drugs may lead to severe health problem due to the

emergence of food-borne disease and thus, the production of agricultural products will be

declining (Bischoff et aI., 2005).

E. coli is one of the common bacteria that can be isolated from the aquaculture

environment. Enterotoxigenic E. coli (ETEC) is one of the major E. coli strains that affect

the gastrointestinal tracts of the human by releasing its toxin which causes diarrhoea

(Jayaratne et aI., 1987). As well as the other pathogenic strains of E. coli, this strain is also ill,

able to confer plasmid that codes for antibiotic resistance and enterotoxin production which

can be transferred to the same or the other bacterial species through genetic recombination.

The recombination of gene between two species has enabled the massive amount of

bacterial population to acquire multiple resistances towards antibiotics.

The resistance gene may be transferred through conjugation, transduction or

transformation (Dzidic & Bedekovic, 2003). The most common genetic transfer among

bacteria is through conjugation which involves the cell-cell interaction. Conjugation

between the same and different bacteria species is a type of horizontal gene transfer

involving a communication between two bacterial cells which are the donor cell and

recipient cells (Somkiat et al., 2007). The recombination of the resistance gene from the

donor and the antibiotic susceptible recipient will occur when the gene is integrated in the

chromosomes or plasmid through conjugation process. Plasmid is a mobile element that

3

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can increase the ability of bacteria to spread their resistance gene (Rijavec et al., 2006).

This has caused some changes in the recipient resistance phenotype when the new

resistance gene from the donor integrates in their chromosomes. Therefore, the frequency

of bacterial antimicrobial resistant will increase and it will lead to a serious global health

problem.

The genetic diversity of coli isolates can be determined by several DNA

fingerprinting methods. One of the DNA fingerprinting methods that can be used is the

repetitive sequence-based peR which is easy to perform based on repetitive sequenced

primer complementary to the bacterial genome (Svec et aI., 2005).

The widespread of antibiotic resistance among bacteria in aquaculture environment

has caused some major illness and problems in the pharmaceutical industries. This study

will further investigate the risks associated with antibiotic resistance bacteria from the

aquaculture environment.

The main objectives of this study were:

1) To determine the patterns of antibiotic resistance among the E. coli isolates.

2) To analyse plasmid DNA among the E. coli isolates.

3) To determine the genetic diversity among the E. coli isolates. .

4

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2.0 LITERATURE REVIEW

2.1 Aquaculture Environment

Aquaculture environment has been one of the most important medium in the agricultural

industries for economic development (Sahu et aI., 2008). It provides the powerful source of

protein from different species of aquatic organisms such as fish, prawns, oyster and also

crabs. Cole et al. (2009) studied that approximately 40% of fish food is supplied by the

aquaculture industries. However, these organisms are exposed to pathogens and other

harmful microorganisms which lead to the massive loss of aquatic sources due to

infections and diseases. Therefore, antimicrobial drugs have been extensively used in the

aquaculture environment in order to control and prevent the spread of diseases.

The widespread use of antimicrobial drugs in the aquaculture environment has

resulted in the serious concern towards the public medical and health sectors as the

antimicrobial residues in the water and food sources may increase the population of multi-

drug resistant microorganisms (Lu et al., 2009).

5

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2.2 Escherichia coli

Escherichia coli (abbreviated as E. coli) is a type of gram negative bacilli which uses its

own flagella to move. It exists as a normal micro flora in the digestive tract of human and

the other warm-blooded organisms. Most of E. coli strains are considered as harmless but

there are certain types of E. coli that are pathogenic and may cause severe effects towards

the environment. E. coli is classified into six classes which are enteropathogenic (EPEC),

enterotoxigenic (ETEC), enteroinvasive (EIEC), Shiga-toxin producing (STEC),

enteroaggregative (EAEC) and diffuse adhering (DAEC) (Blanco et at., 2006). All of these

six classes of E. coli are categorised as pathogenic as they are able to infect their host and

exhibit some antibiotic resistance through genetic transfer inside the host.

Most of E. coli strains carry resistance factors (R factors) in their plasmid which

encodes for antibiotic resistance (Wan et a!., 2003). These R factors are transferable among

the same E. coli species or to the other kinds of bacteria in order to establish multiple

antibiotic resistances through genetic transfer. Antibiotic resistance develop when the

resistant strains infected their host and transfer their plasmid "into the host's DNA These

strains may also interact with the normal micro flora in the gastrointestinal tract. This will

cause the normal flora in the human body to establish certain resistance towards antibiotics

which increase their pathogenicity towards the host. Pathogenic E. coli strains can also

encode for various virulence determinants on their individual chromosomes and plasmids

(Ambrozic et at., 1998). Therefore, their resistance towards multiple drugs will increase

6

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and cause failure to treat the disease which will lead to a major outbreak (Dzidic &

Bedekovic, 2003).

E. coli strains are reported to have increase in their resistance which cause many

disease outbreaks in countries such as clinical diarrhoea due to their presence in the

contaminated food and water (Wan et al., 2003). Previous study investigated that E. coli

have a great activity against tetracycline, sulphonamide and amynoglycoside in farm

animals (Yang et al., 2004). Hence, this has cause a concern towards the treatment for

infected farm animals, especially in the aquaculture environment which provides the

highest protein source across the countries. Consequently, the emergence of multiple ~"

antibiotics resistance among the E. coli isolates will greatly influence the economy and

farm industries.

2.3 Antibiotic Resistance among Bacteria

2.3.1 Antibiotic Resistance

Antibiotic resistance is an ability of bacteria species to resist certain drugs in specific

concentration which will indirectly increase the pathogenicity of the bacteria (Varaldo,

2002). The increase of pathogenicity of these species may be due to the transferable

resistance genes that occur progressively within the environment. As reported by Varaldo

(2002), the antimicrobial resistance among bacteria which is related particularly towards

7

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the pathogenicity has been emerged over the last few years. Antibiotic resistance gene can

be transferred to a wide varieties of bacteria species, regardless of the gram negative or

gram positive strains. These genes can be transferred directly from the gram negative

donor to the gram positive recipient or to the same group of bacteria strains.

The resistance of bacteria towards certain antibiotics can be tested by antimicrobial

susceptibility test through disc diffusion method (Wan et aI., 2003). Bacterial strains will

be cultured in an agar media, such as commonly used Mueller-Hinton agar to investigate

the susceptibility of certain bacterial strains against antimicrobial drugs. The zone diameter

interpretive standards obtained from the test are the standard measurement applied to

determine the lowest concentration of antibiotics used for bacterial growth inhibition

against the drugs (Wikler et ai., 2006).

2.3.2 Types of Antibiotics

Antibiotic is a type of antimicrobial drugs that is able to kill or inhibit the bacterial growth.

Antibiotics are chemically derived from microorganisms which enable them to eliminate

pathogens that surround them. Antibiotics can be bactericidal or bacteriostatic. Bactericidal

antibiotics act by killing bacteria, whereas bacteriostatic antibiotic acts by inhibiting the

bacterial growth (Pankey & Sabath, 2004). Every antibiotic has their own spectrum range,

which indicates their antimicrobial activity towards certain bacteria. Antibiotics with a

broader spectrum range will be able to inhibit a large population of gram positive and gram

8

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negative bacteria. Meanwhile, a narrow spectrum range of antibiotics are only able to

eliminate a few types of bacteria (Perlman., 1977).

Various groups of antibiotics have been identified according to their spectrum

range and their effectiveness towards certain infections in the medical treatments. The

main groups of antibiotics are macrolides, aminoglycosides, penicillins, tetracyclines,

fluoroquinolones and cephalosporins. Macrolides are a type of erythromycin antibiotics

which acts by binding upon the the ribosomes of the bacteria to inhibit protein productions

(Leclercq, 2002). Meanwhile, aminoglycosides are broad spectrum antibiotics which act

towards gram negative bacteria by inhibiting their protein synthesis through binding to 30S

ribosomes (Mingeot-Leclercq et al., 1999). Penicillins have the ability to kill susceptible

bacteria by preventing the final stage of bacterial cell wall synthesis which causes fatality

towards the bacteria. Besides that, tetracyclines are also widely used as the anti-

inflammatory broad spectrum drugs which are able to inhibit the tumour progression

(Amin et al., 1996). Other broad spectrum antibiotics that are extensively used in medical

term are fluoroquinolones which kill bacteria by disrupting the bacterial DNA synthesis

and cephalosporins which inhibit the synthesis of bacterial cell walls.

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2.4 Bacterial Plasmid DNA

In spite of bacterial DNA, plasmid DNA also exists in some bacterial strains and it may

contain information about the diversity of bacterial genetics. According to Jan et al.

(2009), plasmid is one of the main medium for the transfer of antibiotic resistance among

bacteria. As well as bacterial DNA, plasmid has its own replication system which encodes

for the plasmid behaviour and characteristics for bacterial pathogenicity because one of

them might contain several plasmids that code for different antimicrobial resistance.

Plasmid DNA is also a mobile element that enables the bacteria to spread its

resistance potential to the other bacterial strains through genetic recombination (Rijavec et

al., 2006). This mobile element can encodes for both antibiotic resistance and virulence

factor that can cause the replication of more virulent antibiotic resistant strains. Rijavec et

al. (2006) also reported that the most prevalent strains of E. coli are resistance towards

tetracycline and chloramphenicoL These antibiotic resistances are found to be associated

with the behaviour of conjugal plasmid of Enterobacteriaceae which primarily promotes

the transferable plasmids into other strains in the environment.

Currently, plasmids are used as a vector for genetic engineering because of its

capability of transferring a specific gene from one cell to another. Besides, plasmids have

, I different sizes that enable differentiation between two bacterial strains which is crucial for

plasmid profiling.

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2.4.1 Alkaline Lysis

Alkaline lysis is one of a method used in the plasmid DNA extraction. It was first proposed

by Bimboim and Doly (1979). This step involves the differential denaturation and

reannealing of plasmid DNA, as compared to the chromosomal DNA (Ehrt &

Schnappinger, 2003). Alkaline lysis consists of three main steps, which are resuspension,

lysis and neutralisation. As stated by Kado and Liu (1981), chromosomal DNA and

plasmid DNA can be obtained directly through the disruption of cell, or as well known as

lysis. Lysis can be done by the mean of lysozyme action and detergent. Therefore, a series

of centrifugations will be performed to assist in a better plasmid DNA extraction.

Resuspension [",___LY_S_iS__..",) Neutra Ii zatio n J ~'

Figure 1: Overall steps involved in alkaline lysis for plasmid DNA extraction

1

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... r 2.4.2 Plasmid DNA Profiling

The study of prevalence of bacterial strains towards antibiotic resistances can be further

investigated through plasmid profiling. Antibiotic resistance gene among bacteria can be

carried within the plasmid DNA which contributes to the increased populations of bacteria

that confers multiple drug resistance (Rijavec et al., 2006). Plasmid identification and

characterisation act as the main role for epidemiology investigation as this mobile element

has created a great genetic diversity, primarily towards the bacterial expressions against

antibiotics and pathogenicity (Jan et al., 2009).

Plasmid profiling also enables the measurement of antibiotic resistance and

virulence factors of some bacteria as plasmid may contain the R factor carrying the

resistance gene which can be distributed in different sizes as well in the chromosomal

DNA (Alam et al., 2010). Therefore, different sizes of plasmid DNA in every bacterial

strain will enable the identification and characterisation of plasmids for profiling purpose.

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2.5 Genetic Recombination as the Main Medium for Antibiotic Resistance

Transfer

2.5.1 Horizontal Gene Transfer

Horizontal gene transfer of two bacteria of the same and different species involves the

transfer of genetic material fonning a transconjugant bacterial species that encounter a new

phenotype. As stated by Somkiat et al. (2007), conjugation between different bacteria

strains involved a horizontal gene transfer process by which the donor possessing

conjugative plasmid will transfer its resistance to the plasmid-free recipients. The transfer

of antibiotic resistance between two bacteria enables the donor cell to transfer certain

antimicrobial resistant gene to the antibiotic susceptible recipient while the donor interacts

closely with the recipient. This phenomenon has cause a major increase in antimicrobial

resistance in some nonnal flora in the environment which makes them become pathogenic

towards human and the other organisms.

Horizontal gene transfer can be divided into three mechanisms which are

conjugation, transduction and transfonnation (Hassan & Amin, 2010). These mechanisms

have been reported as one of the means that contributed into the evolutionary of

recombinant bacteria which is useful for the agricultural and bioremediation purpose.

However, bacteria pathogens also may emerge due to some mutation during the

recombination and it will attract some major attention around the world. Furthennore,

13

bacteria that have acquired certain antibiotic resistance will be able to spread their

pathogenicity to the other bacteria and may create pandemic.

2.5.1.1 Conjugation

Conjugation is a type of horizontal gene transfer that involves the transfer of conjugative

plasmid which carries certain resistance gene through cell-cell interaction (Kelly et al.,

2009). Certain genes that encode for antibiotic resistance are often linked to mobile genetic

elements such as transposon and plasmid which primarily occur in the bacterial

chromosomes (Sunde & Norstrom, 2006). During conjugation, the resistance gene will be

transferred by forming a sex pilus which is made by the donor cell (NCBE, 2009). Sex

pilus appears in a form of tube which will provide a pathway for the plasmid to be

transferred from the donor strain to the recipient strain. Plasmid DNA from the donor will

pass through the sex pilus as a single stranded DNA and when it is arrived upon the

recipient cell, the single-stranded DNA will either replicate to form the previous plasmid

circular shape or integrate into the recipient's chromosome (NCBE, 2009).

14

1

2.5.1.2 Transduction

Transduction is a process whereby the DNA of the bacteria is being transferred to another

cell through the replication of virus, known as bacteriophage (Dzidic & Bedekovic, 2003).

Transduction may occur between eukaryotes and prokaryotes. An example of virus that

involved in transduction is bacteriophage. Transduction may arise in two different cycles

which are lytic cycle and lysogenic cycle.

During lytic cycle, the bacteriophage will come in contact with the cell and injects

its viral genome into the cell. The viral genome will replicate themselves within the cell

through transcription process in the presence of reverse transcriptase. After transcription

process is done, the viral genome will assemble together with the cell genome forming new

groups of bacteriophages. When the phages are matured, the cell will burst and the phages

are released to surroundings, which are ready to infect new cells (Kelly et ai., 2009).

The mechanism oflysogenic cycle is quite similar with lytic cycle. After the phages

have penetrated the cell, the viral genome will be integrated with the cell genome, instead

of undergoing reverse transcription as in the lytic cycle. Therefore, during this mechanism,

the phenotype of the cell will be altered and the cell will carry virulence factor from the

transduction process (Kelly et ai., 2009).

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2.5.1.3 Transformation

Transformation is a system by which the recipient cell takes up DNA from its surrounding,

regardless of a living cell or a dead cell. The recipient cell has to be competent to enable

the extracellular DNA integrates with its DNA (Kelly et ai., 2009). This mechanism also

may increase the virulence of the recipient when the recipient possesses any antibiotic

resistance genes from the other pathogenic bacteria.

2.5.2 Vertical Gene Transfer

Vertical gene transfer involves the transfer of genetics materials from the parents to the

offspring that can be passed through generations. Vertical gene transfer is one of the

evolutionary mechanisms among bacterial strains which explain the origin of bacterial

existence from a single lineage of bacterial generation (Lawrence, 2005). This type of

genetic transfer enables a great diversity among bacteria as the offspring obtained their

resistance characteristics from both of the parents.

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