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

ii

Dedicated to my loving husband, Ir. Adly Havendri, MSc and my children Aditya Rahmat, Adrian Faisal, Arief Darmawan and Putri Nadhira Adelina

iii

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

iv

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,

v

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

vi

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

vii

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

viii

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,

ix

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

x

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,

xi

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

xii

Syarifah and mother in law mama Hajjah Mahyar for their love and support,

encouragement who share much of my joy and sorrow.

MARLINA

xiii

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:

xiv

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

xv

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

xvi

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

xvii

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

64

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)

77

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

111

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

xviii

5.5 Random Amplified Polymorphic DNA Fingerprinting 139 5.6 Enterobacterial Repetitive Intragenic Concensus (ERIC) PCR for Characterization of V. parahaemolyticus Isolates

145

VI. CONCLUSION 151 REFERENCES 156 APPENDICES 183 BIODATA OF THE AUTHOR 184  

                             

LIST OF TABLES  Table    Page

xix

  

 

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

90 

   

4.4  Characterization of V. parahaemolyticus from human stools samples

92 

   

4.5  Distribution of antimicrobial resistance of V. parahaemolyticus isolated from shellfish, hospital wastewater and human stools

96   

  

 

4.6  Characterization of V. parahaemolyticus clustered defined in hierarchic analysis performed with antibiotic resistance data

97 

   

xx

 4.7  Plasmid profile patterns of V. parahaemolyticus isolated from

shellfish, hospital waste water and human stools 104 

                                     

LIST OF FIGURES  

Figure  Page

xxi

  

2.1 Polymerase Chain Reaction (PCR) technique (Adopted from Kimball, 2005)

38

2.2 Map of Indonesia Country and West Sumatera Province (Adopted from Archipelago Indonesia).

50

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

83

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)

84

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

85

4.7 The partial sequence of toxR gene producing significant alignments compared with in database through BLASTQ2 Program

93

xxii

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

94

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

101

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

106

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).

110

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).

109

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

xxiii

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

110

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).

112

 

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.

113

                  

                     

LIST OF ABBREVIATIONS  

xxiv

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