UNIVERSITI PUTRA MALAYSIA
DETECTION OF VIBRIO CHOLERAE AND VIBRIO PARAHAEMOLYTICUS IN SEAFOOD USING MPN-PCR TECHNIQUE, AND THEIR MOLECULAR
CHARACTERISTICS
VENGADESH S/O LETCHUMANAN
FPSK(m) 2013 35
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DETECTION OF VIBRIO CHOLERAE AND VIBRIO PARAHAEMOLYTICUS
IN SEAFOOD USING MPN-PCR TECHNIQUE, AND THEIR
MOLECULAR CHARACTERISTICS
By
VENGADESH S/O LETCHUMANAN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfillment of the Requirements for the Degree of Master of Science
July 2013
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DETECTION OF VIBRIO CHOLERAE AND
VIBRIO PARAHAEMOLYTICUS IN SEAFOOD
USING MPN-PCR TECHNIQUE, AND THEIR
MOLECULAR CHARACTERISTICS
By
VENGADESH S/O LETCHUMANAN
MASTER OF SCIENCES
UNIVERSITI PUTRA MALAYSIA
July 2013
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COPYRIGHT
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icons, photographs and all other artwork, is copyright material of Universiti Putra
Malaysia unless otherwise stated. Use may be made of any material contained within
the thesis for non-commercial purpose from the copyright holder. Commercial use of
material may only be made with the express, prior, written permission of Universiti
Putra Malaysia.
Copyright © Universiti Putra Malaysia
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment
of the requirement for the degree of Master of Science
DETECTION OF VIBRIO CHOLERAE AND VIBRIO PARAHAEMOLYTICUS
IN SEAFOOD USING MPN-PCR BASED TECHNIQUE, AND THEIR
MOLECULAR CHARACTERISTICS
By
VENGADESH S/O LETCHUMANAN
July 2013
Chair: Associate Professor Cheah Yoke Kqueen, PhD
Faculty: Medicine and Health Sciences
Seafood is professed by consumers worldwide to be healthy and nutritious food due
to abundance of scientific and documented health benefits. Approximately 90% of
global aquaculture production is based in Asia. Nevertheless, recent food borne
outbreaks are closely associated with seafood consumption. Seafood is known as a
vehicle of transmission of food borne bacteria and causes human illness worldwide.
In Malaysia, statistics in year 2009 have shown among the food and water borne
diseases, food poisoning has the highest incidence rate of 36.17 per 100,000
populations and with a mortality rate of 0.01 per 100,000 populations. The purpose
of this study is to apply and enumerate Vibrio cholerae and Vibrio parahaemolyticus
from seafood samples by utilizing the Most Probable Number Method (MPN) and
several molecular typing methods including polymerase chain reaction (PCR),
enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-
PCR) and plasmid profiling.
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The application of conventional method using the Most Probable Number (MPN)
method with selective enrichment broth and agar medium is very useful in isolating
Vibrio cholerae and Vibrio parahaemolyticus. This method is coupled with PCR
based method to obtain specific, sensitive and precise results. The densities
enumerated by the MPN-Real Time PCR targeting epsM gene and the MPN-PCR
targeting toxR gene is higher than those by MPN-Plate.
Genomic DNA of 104 Vibrio cholerae isolates was confirmed by a specific
optimized multiplex PCR program targeting hem gene, hlyA gene, ctx gene and zot
gene. 10 isolates were tested positive for cholera toxin gene (ctx) gene. All the
isolates were positive to hem gene at 519bp and hlyA gene at 738bp but none was
tested positive for zot gene. On the other hand, all the 100 Vibrio parahaemolyticus
isolates were tested positive for regulatory gene, toxR yielded 368bp. The PCR
amplification of the respective genes is a rapid and reliable method of detecting
Vibrio cholerae and Vibrio parahaemolyticus isolates from seafood samples.
ERIC sequences are short, highly conserved 126 bp non-coding regions found in the
Enterobacteriaceae. Its location in bacterial genomes allows discrimination at the
genus, species and serovars levels. Dendrogram of ERIC-PCR were analyzed using
the Bionumerics Version 6.0 (Applied Maths, Germany) software. From 104 isolates
of Vibrio cholerae, ERIC-PCR with primers ERIC-1 and ERIC-2 produced 15
clusters and 9 single isolates at 40% similarity. Where else, 100 isolates of Vibrio
parahaemolyticus produced 15 clusters and 6 single isolates at 40% similarity. The
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results demonstrated that ERIC-PCR is a excellent tool for differentiation and
characterization of Vibrio species.
Plasmids of Vibrio cholerae and Vibrio parahaemolyticus vary in size from 2.2 Kb to
more than 7.4 Kb. Despite limited knowledge on their function, their presence is
frequently used for strain differentiation in epidemiological studies. Plasmid
profiling of 104 Vibrio cholerae isolates clustered into 19 groups based on the
number and pattern of the bands. Where else, plasmid profiling of 100 Vibrio
parahaemolyticus isolates clustered into 11 groups based on the number and pattern
of the bands.
As a conclusion, the concern about possible health illness from Vibrio species,
especially when seafood remains as a vehicle of transmission of Vibrio, will continue
into the likely future. Therefore, to establish effective control measures to reduce the
risk of this bacterium infection and to ensure the safety of foods, surveillance and
epidemiology, the employment of molecular methods for the detection of Vibrio
cholerae and Vibrio parahaemolyticus in food and environment is important. The
results from this study could serve as vital information in Vibrio spp. epidemiology,
surveillance, better infection control measures and support of public health policy.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk Ijazah Master Sains
PENGESANAN VIBRIO CHOLERAE AND VIBRIO PARAHAEMOLYTICUS
DALAM MAKANAN LAUT DENGAN MENGGUNAKAN TEKNIK MPN-
PCR, DAN MOLEKUL PENCIRIAN
Oleh
VENGADESH A/L LETCHUMANAN
July 2013
Pengerusi: Associate Professor Cheah Yoke Kqueen, PhD
Fakulti: Perubatan dan Sains Kesihatan
Makanan laut dikatakan oleh pengguna di seluruh dunia sebagai makanan yang sihat
dan berkhasiat kerana mengandungi banyak manfaat kesihatan yang saintifik dan
didokumenkan. Kira-kira 90% daripada global pengeluaran akuakultur berpusat di
Asia. Walau bagaimanapun, baru-baru ini penyakit keracunan makanan berkait rapat
dengan pengambilan makanan laut. Di Malaysia, statistik pada tahun 2009 telah
menunjukkan antara keracunan makanan mempunyai kadar insiden tertinggi 36.17
setiap 100,000 penduduk dan dengan kadar kematian 0.01 setiap 100,000 populasi.
Tujuan kajian ini adalah untuk mencirikan dan menghitung Vibrio cholerae dan
Vibrio parahaemolyticus dalam sampel makanan laut dengan menggunakan kaedah
‘Most Probable Number (MPN)’, dan beberapa kaedah molekul yang termasuk
‘polymerase chain reaction (PCR)’, ‘enterobacterial repetitive intergenic consensus-
polymerase chain reaction (ERIC-PCR)’ dan profil plasmid.
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Penggunaan kaedah konvensional menggunakan kaedah ‘Most Probable Number
(MPN)’ dengan selektif agar media adalah sangat effektif dalam mengasingkan
Vibrio cholerae dan Vibrio parahaemolyticus. Kaedah ini ditambah pula dengan
kaedah berasaskan PCR bagi mendapatkan keputusan, sensitif dan tepat. Kepadatan
disenaraikan oleh MPN-Real Time PCR mensasarkan gen epsM dan MPN-PCR
mensasarkan gen toxR adalah lebih tinggi daripada MPN-Plate.
DNA genomik 104 Vibrio cholerae pencilan telah disahkan oleh suatu multipleks
yang telah dioptimasi mensasarkan gen tertentu mengepung, gen hlyA, gen hem, gen
ctx dan gen zot. 10 pencilan telah diuji positif untuk ‘cholera toxin (ctx)’gen. Semua
pencilan adalah positif untuk gen hem mengepung di 519bp dan gen hlyA di 738bp
tetapi tiada sebarang pencilan yang menunjukkan keputusan positif apabila diuji
dengan gen zot. Sebaliknya, semua 100 Vibrio parahaemolyticus pencilan telah diuji
positif untuk gen ‘regulatory’, toxR menghasilkan 368bp. Amplifikasi PCR daripada
gen masing-masing adalah kaedah yang pantas dan boleh dipercayai untuk mengesan
Vibrio cholerae dan Vibrio parahaemolyticus pencilan daripada sampel makanan laut.
Urutan ERIC adalah pendek, sangat dipelihara 126 bp bukan kod dalam lingkungan
dalam Enterobacteriaceae. Dendrogram daripada ERIC-PCR telah dianalisis
menggunakan Version Bionumerics yang 6.0 (Gunaan Matematik, Jerman) perisian.
Dari 104 pencilan Vibrio cholerae, ERIC-PCR dengan primers ERIC-1 dan ERIC-2
yang dihasilkan 15 kelompok dan 9 diasingkan tunggal pada 40% persamaan.
Sementara, 100 pencilan Vibrio parahaemolyticus mengahasilkan 15 kelompok dan
6 diasingkan tunggal pada 40% persamaan. Keputusan ini menunjukkan bahawa
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ERIC-PCR adalah alat yang sangat baik untuk pembezaan dan pencirian spesies
Vibrio.
Profil plasmid Vibrio cholerae dan Vibrio parahaemolyticus dalam kajian ini berbeza
dalam saiz daripada 2.2Kb kepada lebih daripada 7.4Kb. Walaupun pengetahuan
yang terhad kepada fungsi mereka, kehadiran mereka sering digunakan untuk
perbezaan tekanan dalam kajian epidemiologi. 104 Vibrio cholerae pencilan profil
plasmid berkelompok ke dalam 19 kumpulan berdasarkan bilangan dan corak band.
Manakala 100 Vibrio parahaemolyticus pencilan profil plasmid berkelompok kepada
11 kumpulan berdasarkan bilangan dan corak band.
Kesimpulannya, kebimbangan mengenai penyakit kesihatan yang mungkin daripada
spesies Vibrio, terutamanya apabila makanan laut kekal sebagai kenderaan
penghantaran Vibrio bakteria, akan berterusan pada masa depan yang mungkin. Oleh
itu, untuk mewujudkan langkah kawalan yang berkesan bagi mengurangkan risiko
jangkitan bakteria ini dan memastikan keselamatan makanan, pengawasan dan
epidemiologi, pengambilan kaedah molekul untuk mengesan Vibrio cholerae dan
Vibrio parahaemolyticus dalam makanan dan persekitaran adalah penting. Hasil
daripada kajian ini boleh dijadikan sebagai maklumat penting dalam Vibrio spp.
epidemiologi, pengawasan, langkah-langkah kawalan jangkitan yang lebih baik dan
menyokong dasar kesihatan awam.
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ACKNOWLEDGEMENTS
First and foremost, I would like to convey my greatest gratitude and appreciation to
the chairman of my supervisory committee, Associate Professor Dr. Cheah Yoke
Kqueen for his kind patience, priceless guidance, endless motivation, dedicated
efforts and continuous support given to me throughout the study. I am forever very
grateful to all his teachings and guidance along the path of research.
My special thanks to my co-supervisor, Professor Dr. Son Radu from Faculty of
Food and Technology, UPM, for his guidance and support given to make this project
and thesis a reality. I also would like to thank University Putra Malaysia for their
generosity in funding me with Graduate Research Fund (GRF) during my study at
the university.
My sincere gratitude to my dearest parents, Letchumanan, Gowri and my younger
brother Kanesh Babu for all their patience, support and full encouragement given
throughout my study. I also thank my friends for all the help and support given
along the journey of my master’s research. My sincere thanks to all the staff of the
Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, who has
contributed in somehow or other throughout the research project been conducted.
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I certify that a Thesis Examination Committee has met on 2nd
July 2013 to conduct
the final examination of Vengadesh a/l Letchumanan on his thesis entitled "Detection
of Vibrio cholerae and Vibrio parahaemolyticus in Seafood Using MPN-PCR Based
Technique, and Their Molecular Characteristics" in accordance with the Universities
and University Colleges Act 1971 and the Constitution of the Universiti Putra
Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the
student be awarded the Master of Science.
Members of the Thesis Examination Committee were as follows:
Mohamad Aziz bin Dollah, PhD
Associate Professor
Faculty of Medicine and Health Science
Universiti Putra Malaysia
(Chairman)
Mohd Nasir bin Mohd Desa, PhD
Associate Professor
Faculty of Medicine and Health Science
Universiti Putra Malaysia
(Internal Examiner)
Shuhaimi bin Mustafa, PhD
Professor
Institut Penyelidikan Produk Halal
Universiti Putra Malaysia
(Internal Examiner)
Thong Kwai Lin, PhD
Professor
Institute of Biology Science, Faculty of Science
Universiti Malaya
Malaysia
(External Examiner)
NORITAH OMAR, PhD
Associate Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 19 September 2013
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Cheah Yoke Kqueen, PhD
Associate Professor
Faculty of Medicine and Health Science
Universiti Putra Malaysia
(Chairman)
Son Radu, PhD
Professor
Faculty of Food Sciences and Technology
Universiti Putra Malaysia
(Member)
NORITAH OMAR, PhD
Associate Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously, and is
not concurrently, submitted for any other degree at Universiti Putra Malaysia or at
any other institution.
_________________________________
VENGADESH S/O LETCHUMANAN
Date: 2 July 2013
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK v
ACKNOWLEDGEMENTS viii
APPROVAL ix
DECLARATION xi
LIST OF TABLES xiv
LIST OF FIGURES xvi
LIST OF ABBREVIATIONS xvii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 8
2.1 Vibrio-The Genus 8
2.2 Vibrio cholerae 9
2.2.1 Pathogenesis of Vibrio cholerae 11
2.2.2 Epidemiology and Incidence of Vibrio cholerae 13
2.3 Vibrio parahaemolyticus 15
2.3.1 Pathogenesis of Vibrio parahaemolyticus 16
2.3.2 Epidemiology and Incidence of Vibrio parahaemolyticus 18
2.4 Source and Transmission 20
2.5 Purification of Vibrio cholerae and Vibrio parahaemolyticus 21
2.6 Molecular Typing Method 23
2.6.1 Polymerase Chain Reaction (PCR) 25
2.6.2 Specific Gene 27
2.6.4 Enterobacterial Repetitive Intergenic Consensus (ERIC-
PCR)
29
2.7 Most Probable Number (MPN) Method 30
2.8 Plasmid Profiles 33
3 MATERIALS AND METHODS/METHODOLOGY 35
3.1 Sampling 35
3.2 Most Probable Number (MPN) 36
3.2.1 Vibrio cholerae MPN-Plate 35
3.2.2 Vibrio parahaemolyticus MPN-Plate 37
3.3 Molecular Most Probable Number (MPN) 38
3.3.1 DNA Extraction by Direct Boiled Cell Lysate Method 38
3.3.2 Vibrio cholerae MPN-Real Time PCR 39
3.3.3 Vibrio parahaemolyticus MPN-PCR 41
3.4 Polymerase Chain Reaction (PCR) 42
3.4.1 Phenol-Chloroform DNA Extraction Method 42
3.4.2 Multiplex PCR detection of Vibrio cholerae 43
3.4.3 Singleplex PCR detection of Vibrio parahaemolyticus 44
3.5 Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) 46
3.6 Plasmid Profiling 47
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4 RESULTS 49
4.1 Identification of Vibrio cholerae and Vibrio parahaemolyticus 49
4.1.1 Conventional Most Probable Number (MPN)-Plate
Method
49
4.1.2 Most Probable Number (MPN)-Real Time PCR of Vibrio
cholerae
55
4.1.3 Most Probable Number (MPN)-PCR of Vibrio
parahemolyticus
59
4.2 Polymerase Chain Reaction (PCR) 61
4.2.1 Multiplex PCR detection of Vibrio cholerae 61
4.2.2 Singleplex PCR detection of Vibrio parahaemolyticus 64
4.3 Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-
PCR)
66
4.4 Plasmid Profiles of Vibrio cholerae and Vibrio parahaemolyticus 73
5 DISCUSSION 83
5.1 Identification of Vibrio cholerae and Vibrio parahaemolyticus 83
5.1.1 Conventional Most Probable Number (MPN)-Plate
Method
83
5.1.2 Molecular Most Probable Number (MPN) Method 87
5.2 Polymerase Chain Reaction (PCR) 90
5.3 Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-
PCR)
93
5.4 Plasmid Profiling of Vibrio cholerae and Vibrio parahaemolyticus 97
6 CONCLUSION AND FUTURE RECOMMENDATION 100
REFERENCES 102
APPENDICES 119
BIODATA OF STUDENT 133
LIST OF PUBLICATIONS 135
