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UNIVERSITI PUTRA MALAYSIA
MOLECULAR CHARACTERIZATION, PLASMID PROFILING AND ANTIBIOTIC SENSITIVITY OF
VIBRIO PARAHAEMOLYTICUS FROM SHELLFISH, HOSPITAL WASTEWATER AND HUMAN STOOLS SAMPLES
IN PADANG, WEST SUMATERA, INDONESIA
MARLINA
FSTM 2007 14
MOLECULAR CHARACTERIZATION, PLASMID PROFILING AND ANTIBIOTIC SENSITIVITY OF
VIBRIO PARAHAEMOLYTICUS FROM SHELLFISH, HOSPITAL WASTEWATER AND HUMAN STOOLS SAMPLES
IN PADANG, WEST SUMATERA, INDONESIA
By
MARLINA
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of
Philosophy
May 2007
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Dedicated to my loving husband, Ir. Adly Havendri, MSc and my children Aditya Rahmat, Adrian Faisal, Arief Darmawan and Putri Nadhira Adelina
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy
MOLECULAR CHARACTERIZATION, PLASMID PROFILING AND
ANTIBIOTIC SENSITIVITY OF VIBRIO PARAHAEMOLYTICUS FROM SHELLFISH, HOSPITAL WASTEWATER AND HUMAN STOOLS IN
PADANG, WEST SUMATERA, INDONESIA
By
MARLINA
May 2007
Chairman : Professor Son Radu, PhD Faculty : Food Science and Technology Vibrio parahaemolyticus, a gram-negative marine bacterium, is an important
food borne pathogen causing gastroenteritis, particularly in areas with high
seafood consumption.
A total of 29 human stools, 20 hospital wastewater and 120 shellfish samples
from Padang area in Sumatera, Indonesia were examined in this study.
Purple coloured colonies presumptive of V. parahaemolyticus from
preliminary screening process on CHROMagarTM Vibrio were selected and
confirmed as V. parahaemolyticus using polymerase chain reaction by the
amplification of the toxR fragment at 368 bp. Of the 169 samples, 42 (24.8%)
from shellfish (13 from B. violacae; 20 from C. moltkiana; 9 from F. ater), 13
(7.7%) from hospital wastewater and 12 (7.1%) from human stool samples
were found to be contaminated with V. parahaemolyticus. The presence of
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virulence genes (tdh and trh) of all toxR positive isolates were carried out
using PCR. None of the isolates possesed the trh gene. However, 18 isolates
from the human stools, hospital wastewater and shellfish (raw B. violacae and
cooked C. moltkiana) samples harboured the tdh gene.
A total of 97 V. parahaemolyticus isolates from human stools, hospital
wastewater and shellfish samples were examined for their resistance to 15
antibiotics. Majority of the isolates (70%) were resistant to more than nine
antibiotics in this study. The general, a V. parahaemolyticus isolates are
resistant to sulfamethoxazole (100%), rifampin (95%) and tetracycline (75%)
and sensitive to norfloxacin (96%). None of the isolates from human stools
were resistant to ampicillin. Overall, all isolates were sensitive to
chloramphenicol and floroquinolones (ciprofloxacin and norfloxacin agents).
Eighty three isolates were examined for the existence of plasmids. A total of
61 (74.7%) V. parahaemolyticus isolates were plasmid-free. Nine isolates
(11.8%) harboured the 9.4 kb plasmid. All of the remaining isolates carried
plasmid DNA with sizes ranging from 2.3 kb to >23 kb. A large plasmid of 23
kb was evident in the plasmid harboring strains and appeared as the only
plasmid in three isolates.
RAPD profiling with three primers (OPAR3, OPAR4 and OPAR8) produced
four major clusters (R1, R2, R3 and R4) and 7 minor clusters (I, II, III, IV, V,
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VI and VII). ERIC PCR using primer 1R and 2R produced two major clusters
(E1 and E2) and ten minor clusters (A, B, C, D, E, F, G, H, I and J). All the
dendrograms were being constructed utilizing the RAPDistance software
package based on the data retrieved from the presence or absence of banding
pattern. Both the molecular techniques of RAPD and ERIC genotyping
showed most strains (? % and ?% respectively) from different samples and
different locations revealed very high genetic variability in the microbial
population studied. Combining the results of RAPD with ERIC apparently
provides a degree of discrimination that should be adequate for identifying
possible origins of V. parahaemolyticus contamination and for establishing
relationships between isolates. Both methods showed a great diversity
among the isolates of this species and could represent useful tools for the
epidemiological typing of V. parahaemolyticus from Indonesia.
Hence, the problem of the contamination of foods by V. parahaemolyticus, like
many other food safety issues in diverse society, reflects the challenges of
larger public health systems of care. Addressing this problem will require a
combined approach including improved access, legislation, education, and
culturally relevant patient-provider interactions.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah
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PENCIRIAN SECARA MOLEKUL, PROFIL PLASMID DAN SENSITIVITI ANTIBIOTIK VIBRIO PARAHAEMOLYTICUS DARIPADA KERANG, AIR KUMBAHAN HOSPITAL DAN FECAL MANUSIA DARI
KAWASAN PADANG, SUMATERA BARAT, INDONESIA
Oleh
MARLINA
May 2007
Pengerusi : Profesor Son Radu, PhD Fakulti : Sains dan Teknologi Makanan
Vibrio parahaemolyticus, suatu bakteria marin gram negatif, adalah penyebab
gastroenteritis bawaan makanan sedunia, terutamanya di kawasan yang
pengambilan makanan lautnya adalah tinggi, dan merupakan patogen
bawaan makanan yang penting di kebanyakan negara Asia.
Sejumlah 29 sampel “fecal” manusia, 20 sampel kumbahan hospital, dan 120
sampel kerang dari kawasan Padang di Sumatera, Indonesia dikaji.
Beberapa teknik molekular dijalankan ke atas koloni berwarna ungu yang
dipilih dari CHROMagarTM Vibrio yang diandaikan sebagai V.
parahaemolyticus dengan menggunakan teknik reaksi rantai polymerase
(PCR) mensasari gen toxR yang menjangkakan fragmen toxR 368bp.
Kehadiran gen virulen (tdh dan trh) ke atas semua pencilan positif toxR
dijalankan menggunakan PCR. Tiada pencilan yang menghasilkan band
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yang dijangkakan untuk gen trh. Walau bagaimanapun, 18 pencilan daripada
sampel ”fecal” manusia, kumbahan hospital dan kerang (B. violacae mentah
dan C. moltkiana yang dimasak) menghasilkan band 250bp yang dijangkakan
daripada amplifikasi gen tdh. Keputusan daripada kajian ini menunjukkan
bahawa V. parahaemolyticus telah dikesan di dalam pencilan daripada kerang
(B. violacae, C. moltkiana dan F. ater), kumbahan hospital dan ”fecal” manusia.
Sembilan puluh tujuh pencilan V. parahaemolyticus tersebut diuji untuk
kesesuaiannya terhadap 15 antibiotik. 12 kluster sejajar dengan 97 pencilan
(C1 hingga C12) dapat ditakrifkan dalam dendrogram dengan tahap
kesamaan daripada 30% hingga 98% menggunakan perisian Bionumerics
versi 4.6. Kesemua pencilan tersebut didapati menunjukkan kerintangan
terhadap sekurang-kurangnya empat jenis antibiotik. Majoriti pencilan (70%)
mempunyai kerintangan terhadap sembilan jenis antibiotik.
Keseluruhannya, pencilan-pencilan V. parahaemolyticus berkelakuan rintang
terhadap sulphamethoxazole (100%), rifampin (95%), dan tetracycline (75%)
dan sensitif kepada norfloxacin (96%). Tiada pencilan daripada ”fecal”
manusia yang menunjukkan kerintangan terhadap ampicilin. V.
parahaemolyticus yang dipencilkan daripada ”fecal” manusia masih sensitif
kepada ampicillin. Keseluruhannya, semua pencilan sensitif kepada
chloramphenicol dan floroquinolones (agen ciprofloxacin dan norfloxacin).
Sejumlah 61 (74.7%) daripada 83 pencilan V. parahaemolitycus yang diuji
adalah bebas plasmid. 9 pencilan (11.84%) mempunyai 94 kb. Semua
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pencilan yang lain membawa DNA plasmid daripada 2.3 kb hingga >23 kb.
Plasmid besar 23 kb adalah nyata pada 3/22 pencilan yang mengandungi
plasmid dan muncul sebagai satu-satunya plasmid dalam 3 pencilan.
Amplifikasi polimorfik DNA rawak (RAPD) PCR menggunakan tiga primer
(OPAR3, OPAR4 dan OPAR8) menghasilkan empat kluster besar (R1, R2, R3
dan R4), 7 kluster kecil (I, II, III, IV, V, VI dan VII). Konsensus intergenik
repetitif enterobakterial (ERIC) PCR menggunakan primer 1R dan 2R
menghasilkan dua kluster besar (E1 dan E2) dan sepuluh kluster kecil (A, B,
C, D, E, F, G, H, I dan J). Semua dendrogram dihasilkan menggunakan pakej
RAPDistance berdasarkan data yang diperolehi daripada kehadiran atau
ketidakhadiran paten band. Kedua-dua teknik molekular genotaiping RAPD
dan ERIC menunjukkan beberapa pencilan daripada sampel dan lokasi
berbeza dan menunjukkan variasi genetik yang sangat tinggi dalam populasi
mikrobial yang dikaji. Menggabungkan keputusan RAPD dan ERIC dengan
jelas memberikan darjah diskriminasi yang mencukupi untuk mengenalpasti
sumber yang mungkin bagi kontaminasi V. parahaemolyticus dan untuk
menetapkan hubungan antara pencilan. Kedua-dua kaedah menunjukkan
kepelbagaian yang tinggi antara pencilan spesis ini dan dapat dijadikan alat
berguna untuk taiping epidemiologikal V. parahaemolyticus di Indonesia.
Jadi, masalah kontaminasi makanan oleh V. parahaemolyticus, seperti
kebanyakan isu keselamatan makanan dalam masyarakat pelbagai,
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menggambarkan cabaran sistem penjagaan kesihatan awam yang lebih
besar. Penyelesaian maslah ini memerlukan pendekatan kombinasi antara
akses yang dikembangkan, legislasi, pendidikan dan interaksi pesakit-
pembekal yang relevan secara budaya.
ACKNOWLEDGEMENTS
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In the name of Allah, The Most Gracious and The Most Merciful First and
foremost, I would like to express my heartfelt thanks to Almighty for giving
me the will, strength and the perseverance to pursue and complete my PhD.
I would like to express my sincere gratitude and appreciation to the
chairman of my supervisory committee, Professor Dr. Son Radu, for his
invaluable guidance, dedicated efforts, supervision and continuous support
throughout the study.
Sincere thanks and appreciation are also go to my co-supervisory, Associate
Professor Dr. Suhaimi Napis, Dr. Sahilah Abdul Mutalib and Dr. Zunita
Zakaria for their advices, suggestions and supports.
Sincere gratitude is also extended to all the staff of the Faculty of Food
Science and Technology, Universiti Putra Malaysia, who contributed one
way or another towards the completation of my study.
Also, I would like to express and extended my thank to Profesor. Dr.
Mitsuaki Nishibuchi from Center for Southeast, Asian Studies, Kyoto
University, Japan for his support and contribution.
My special thanks also to all my students in Department of Pharmacy,
University of Andalas, Indonesia especially Haura, Budi, Sanil, Ndaru, Reti,
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Yerli, Winda, Yuni, Amel, Indah, Kakang, Ayip, Dona, Rini, Dayu, Ijul, Ica,
Sari, Ella, Imung and Eko, for assistance in sampling.
I am indebted to my dear friend, Cheah Yoke Kqueen for his willingness and
constant assistance and help in time when I was having difficulties with my
molecular works and help me to write scientific papers and poster. Words
cannot express my gratitude for his supports.
I am grateful to my friends and my housemate, Lesley Maurice and
Chandrika for their help, encouragement and for paying attention for all my
problems. I am also grateful to my best friends Aida and her family, for her
help, support and most of all a family to me during my study here. I love
you.
I would like to thank all my friends in the Laboratory of Food Safety and
Molecular Typing, Faculty of Food Science and Technology, UPM: Mas,
Kqueen, Lesley, Chandrika, Yousr, Tung, Gwen, Belinda, Tuan Zainazor,
Zila, Syila, Zul, Daniel, Rani, Sia Yen, Tosiah, Patrick, Azura, Henie and Jurin
and Chai.
Lastly, heartfelt appreciation to be my loved family, my husband, Abang
Adly, my children Adit, Yayan, Arief and Dhira, my mother Ibunda Hajjah
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Syarifah and mother in law mama Hajjah Mahyar for their love and support,
encouragement who share much of my joy and sorrow.
MARLINA
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I certify that an Examination Committee has met on _________to conduct the final examination of Marlina on her Doctor of Philosophy thesis entitled “Isolation and Molecular Characterization of Vibrio parahaemolyticus from Shellfish, Environmental and Clinical Samples in Padang, West Sumatera Indonesia” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Chairman, PhD Associate Professor Fatimah Abu Bakar, PhD Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Examiner 1, PhD Associate Professor Mariana Nor Shamsudin, PhD Faculty of Universiti Putra Malaysia (Internal Exminer) Examiner 2, PhD Associate Professor Shuhaimi Mustafa, PhD Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) External Examiner, PhD Professor Thong Kwai-Lin, PhD Faculty of Science Universiti of Malaya (External Examiner)
_________________________________ HASANAH MOHD GHAZALI, PhD
Professor / Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:
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This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: Son Radu, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Suhaimi Napis, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Zunita Zakaria, PhD Lecturer Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Sahilah Abdul Mutalib, PhD Lecturer Faculty of Science University Malaya (Member) _______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date: 13 September 2007
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DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions. __________________________ MARLINA
Date: 1 August
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TABLE OF CONTENTS Page
DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xii DECLARATION xiv LIST OF TABLES xviii LIST OF FIGURES xx LIST OF ABBREVIATIONS xxiii CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW 5 2.1 Vibrio parahaemolyticus 11 2.1.1 Taxonomy of V. parahaemolyticus 12 2.1.2 Morphology and Physiology of V. 13 parahaemolyticus 2.1.3 V. parahaemolyticus Serotypes 15 2.1.4 Virulence factor of Vibrio parahaemolyticus 17 2.1.5 Pathogenicity of V. parahaemolyticus 21 2.1.6 Occurrence and outbreak of V. parahaemolyticus 22 2.2 Molecular Biology of V. parahaemolyticus 27 2.2.1 Plasmid of V. parahaemolyticus 27 2.2.2 Antibiotic Resistance of V. parahaemolyticus 30 2.3 Analysis of V. parahaemolyticus Using PCR 36 2.3.1 Polymerase Chain reaction 36 2.3.2 RAPD (Random Amplified Polymorphic DNA) PCR 38 2.3.3 ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR 40 2.4 Foodborne Disease 42 2.4.1 Types of Microbial Foodborne Diseases 45 2.4.2 Foodbone Disease Worldwide 46 2.4.3 Foodbone Disease in Asia and Indonesia 47 Shellfish 2.4.4 Types of Shellfish 51 2.4.5 Shellfish used in this Study 53 2.4.6 Gastroenteritis related to shellfish 56 2.4.9 Gastroenteritis related to hospital wastewater 59
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III MATERIAL AND METHODS 62 3.1. Samples Collection and Preparation 3.1.1 Shellfish 62 3.1.2 Hospital wastewater 63 3.1.3 Human stools 63 3.2.Isolation of Vibrio parahaemolyticus Using Selective Media
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3.3.Confirmation of the V. parahaemolyticus isolate by amplification of the toxR gene and screening for the tdh and trh genes
65
3.3.1 DNA Extraction 65 3.3.2 PCR Amplification Procedure 66 3.3.3 Agarose Gel Electrophoresis 66 3.3.4 Sequencing of the toxR amplicon 68 3.4. Antibiotic Susceptibility Testing 70 3.5. Plasmid Isolation 72 3.5.1 Agarose Gel Electrophoresis 73 3.6 Random Amplified Polymorphic DNA Fingerprinting 74 3.6.1 Genomic DNA Extraction for RAPD 74 Fingerprinting 3.6.2 RAPD PCR Amplification 75 3.6.3 Agarose Gel Electrophoresis 76 3.6.4 Selection of Primers 76 3.6.5 Analysis of RAPD Fingerprinting Pattern 76 3.7 Enterobacterial Repetitive Intergenic Consensus Fingerprinting (ERIC-PCR)
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3.7.1 ERIC-PCR Protocol 78 3.7.2 ERIC PCR Primers 78 3.7.3 Analysis of ERIC Fingerprinting Pattern 78 IV. RESULTS 80 4.1 Distribution of Vibrios 80
4.2 Molecular Identification of the V. parahaemolyticus 84 isolates 4.3 Sequencing of the toxR amplicon 93
4.4 Antibiotic Susceptibility Testing 94 4.5 Plasmid Profiling 102 4.6 Random Amplified Polymorphic DNA Fingerprinting 104 4.7 Enterobacterial Repetitive Intragenic Concensus Fingerprinting
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V. DISCUSSION 115 5.1 Distribution of V. parahaemolyticus 115 5.2 Molecular identification of V. parahaemolyticus isolates 121 5.3 Antibiotic Susceptibility Testing 127 5.4 Plasmid Profiling 136
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5.5 Random Amplified Polymorphic DNA Fingerprinting 139 5.6 Enterobacterial Repetitive Intragenic Concensus (ERIC) PCR for Characterization of V. parahaemolyticus Isolates
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VI. CONCLUSION 151 REFERENCES 156 APPENDICES 183 BIODATA OF THE AUTHOR 184
LIST OF TABLES Table Page
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2.1 Relationship between O and K Antigens of V. parahaemolyti- cus strains
16
2.2 Type of shellfish used in this study 52
3.1 Number and origin of samples 63
3.2 Primer pairs utilized in the Specific PCR 65
3.3 Program for Specific PCR (toxR, tdh and trh genes) 66
3.4 Response-related groups of antibiotics 71
4.1 Number of samples positive for V. parahaemolyticus 82
4.2 Characterization of V. parahaemolyticus from shellfish samples
86
4.3 Characterization of V. parahaemolyticus from hospital wastewater
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4.4 Characterization of V. parahaemolyticus from human stools samples
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4.5 Distribution of antimicrobial resistance of V. parahaemolyticus isolated from shellfish, hospital wastewater and human stools
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4.6 Characterization of V. parahaemolyticus clustered defined in hierarchic analysis performed with antibiotic resistance data
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4.7 Plasmid profile patterns of V. parahaemolyticus isolated from
shellfish, hospital waste water and human stools 104
LIST OF FIGURES
Figure Page
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2.1 Polymerase Chain Reaction (PCR) technique (Adopted from Kimball, 2005)
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2.2 Map of Indonesia Country and West Sumatera Province (Adopted from Archipelago Indonesia).
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4.1 V. parahaemolyticus colonies on CHROMAgarTM Vibrio 80
4.2 V. cholerae colonies on CHROMAgarTM Vibrio 81
4.3 V. alginolyticus colonies on CHROMAgarTM Vibrio 81
4.4 Histogram of prevalence V. parahaemolyticus , V. cholerae and V. alginolyticus in hospital wastewater samples
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4.5 Representative specific PCR detection of toxR gene from V. parahaemolyticus on agarose gel electrophoresis (1.2%). Lanes 1, control positive; lanes 2 to 5, toxR gene from shellfish (B. violacae); lane 6 to 8, toxR gene from C. moltkiana; lane 9 to 10, toxR gene from F. ater; lane11 and 12, toxR gene from hospital wastewater; lane 13 and 14, toxR gene from human stool samples; lane 15, 1 kb DNA ladder (Bioron)
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4.6 Representative specific PCR detection of tdh gene from V. parahaemolyticus on agarose gel electrophoresis (1.2%). Lane 1, 1 kb DNA ladder (Bioron); lanes 2 to 4, tdh gene from shellfish (B. violacae); lane 5 to 6, tdh gene from C. moltkiana; lane 7 tdh gene from F. ater; lane 8 tdh gene from hospital wastewater; lane 9 tdh gene from human stool samples; lane 10, positive control
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4.7 The partial sequence of toxR gene producing significant alignments compared with in database through BLASTQ2 Program
93
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4.8 Comparison of partial sequence toxR gene in V. parahaemolyticus isolates from Padang compared with toxR gene from other countries in database through CLUSTALW Program (1.83) Multiple Sequence Aligments
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4.9 Dendrogram showing the clustering of antibiotics patterns for V. parahaemolyticus isolates from shellfish, hospital waste water and human stools using BioNumeric Software Version 4.6 (Applied Maths, Kortrijk, Belgium), computed with Pearson correlation coefficient and agglomerative clustering with UPGMA. Black box: resistant response and white box: sensitive response
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4.10 Representative plasmid profile of the V. parahaemolyticus isolated from food, hospital wastewater and human stools. Lanes: M, λHindIII marker, lanes 2-17: isolates of V. parahaemolyticus
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4.11 RAPD-PCR fingerprints of the representative isolates of V. parahaemolyticus isolates from shellfish, hospital wastewater and human stools. Lane 1 – 18 with primer OPAR 3. Lane M contains DNA molecular weight marker in kilobase pairs (kb).
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4.12 RAPD-PCR fingerprints of the representative isolates of V. parahaemolyticus isolates from shellfish, hospital wastewater and human stools. Lane 1 – 18 with primer OPAR 4. Lane M contains DNA molecular weight marker in kilobase pairs (kb).
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4.13 RAPD-PCR fingerprints of the representative isolates of V. parahaemolyticus isolates from shellfish, hospital wastewater and human stools. Lane 1 – 16 with primer OPAR 8. Lane M contains DNA molecular weight marker in kilobase pairs (kb).
109
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4.14 Dendrogram showing the clustering of RAPD patterns for V. parahaemolyticus isolates from shellfish, hospital waste water and human stools using primer Gold Oligo PAR 3, Gold Oligo PAR 4 and Gold Oligo PAR 8 using the RAPD Distance Software Version 4.0
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4.15 ERIC-PCR fingerprints of the representative isolates of V. parahaemolyticus isolates from shellfish, hospital waste water and human stools. Lane 1 – 9 with primer R1 and R2, Lane M contains DNA molecular weight marker in kilobase pairs (kb).
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4.16 Dendrogram showing the clustering of ERIC patterns for V. parahaemolyticus isolates from shellfish, hospital waste water and human stools using the RAPD Distance Software Version 4. 0.
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LIST OF ABBREVIATIONS
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APW Alkaline Peptone Water
bp base pair
CDC Center of Disease Control and Prevention
CFU Colony-Forming Unit
CT Cholera Toxin
dATP Deoxyadenosine triphosphate
dCTP Deoxycytosine triphosphate
dGTP Deoxyguanine triphosphate
DNA Deoxyribo Nucleic Acid
dNTP Deoxynucleotide triphosphate
dTTP Deoxythymidine triphosphate
EDTA Ethylene Diamine Tetra Acetic
ERIC Enterobacterial Repetitive Intergenic Consensus
GET Glucose-EDTA-Tris base
KAc Kalium Acetate
kb kilobase
KP Kanagawa Phenomenon
LB Luria Bertani
MDR Multiple Drug Resistance
MBC Minimum Bactericidal Concentration
MIC Minimum Inhibitory Concentration
min minute(m)
NaCl Sodium chloride