DETECTION AND MOLECULAR CHARACTERIZATION OF SHIGA-LIKE TOXIN PRODUCING Escherichia coli AND Escherichia coli

0157: H7 ISOLATED FROM RAW BEEF MARKETED IN EAST MALAYSIA

Chang Pheh Ping

Master of Science (Molecular Microbiology)

2003

Pusat Khidmat Maklumat Akademik UNtVERSCI'1 MALAYSIA SARAWAK

P. KMIDMAT MAKLUMAT AKADEMIK MAR

III1I ýiiiIiT 11ýý I11ý IIII 1000246210 DETECTION AND MOLECULAR

CHARACTERIZATION OF SHIGA-LIKE TOXIN PRODUCING Escherichia coli AND Escherichia coli

0157: H7 ISOLATED FROM RAW BEEF MARKETED IN EAST MALAYSIA

Chang Pheh Ping

A thesis submitted

in fulfillment of the requirements for the degree of

Master of Science

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2003

ACKNOWLEDGEMENTS

I wish to express my deepest gratitude and appreciation to my supervisor, Associate

Professor Dr. Kasing Apun for her support, guidance, advice and encouragement

throughout the Master project and thesis preparation. My sincere thanks to Professor

Dr. Mohd. Azib Salleh for his support and some advice during the initial part of the

work. My gratitude also goes to the Ministry of Science, Technology and

Environment (MOSTE) for providing the National Science Fellowship (NSF) to

enable me to further my studies at University Malaysia Sarawak. I wish to thank the

University Malaysia Sarawak for providing the funding for this project with the grant

No. 243/2001 (2).

I would like to extend my sincere thanks to Associate Professor Dr. Son Radu,

University Putra Malaysia for providing the reference culture E. coli 0157: H7 strain

EDL933 and Dr. Abdul Karim Russ Hassan, Faculty of Medicine and Health Science,

University Malaysia Sarawak for the clinical isolate of E. coli 0157: H7 strain SGH1.

My deepest appreciation to the lecturers of Faculty of Resource Science and

Technology, especially Dr. Edmund Sim for his invaluable suggestions and opinions

on the polymerase chain reaction (PCR) and sequencing part. My deepest gratitude

goes to Miss Ooi Wai Ling, University Putra Malaysia, for her invaluable opinions

and advice on some aspects of the experimental procedures. I am pleased to thank

Miss Ivyna Bong, Limjatai, Aziz, Haji Karni Taha, Puan Dayang, Puan Siti Fatimah,

Puan Roki, and Cik Jennifer for their friendship and excellence technical assistance.

ii

My thanks also to Goh Soon Hian for some guidance on the use of the software

RAPDistance and to the postgraduate office staffs for their excellent assistance.

Finally, I would like to thank my parents, family members, my friends and

those who have given their moral support as well as encouragement so that I can

complete my thesis.

Ill

ABSTRACT

A total of 88 raw beef samples marketed in Sarawak and Sabah, East Malaysia, were

investigated for the presence of Shiga-like toxin producing coli, . coli serogroup

0157: H7 and E. coli 0157. Identification of the bacterial species were based on

morphological and biochemical tests, followed by immunological test. The presence

off. coli 0157. -H7 and its virulence properties, Shiga-like toxins, Stxl and Stx2 were

confirmed by multiplex polymerase chain reaction (PCR) using four PCR primer

pairs that simultaneously amplified segments of sal, stx2, rE and iC 7 genes in a

single reaction. Five isolates of Shiga-like toxin producing E. coli 0157: H7, four non

Shiga-like toxin producing E. coli 0157 and two non-0157 Shiga-like toxin

producing E. coli were isolated from 1.1%, 2.3%, and 2.3% of raw beef samples

respectively. The prevalence of E. coli 0157: H7 and STEC in East Malaysia were

found to have a link with the location, with 54.5% (6/11) isolated from locations

situated in the central region of Sarawak. The STEC 0157: H7 isolates were detected

only in frozen imported beef whereas non-0157 STEC in local beef samples. In an

attempt to improve PCR-based assay to allow rapid detection, multiplex PCR

fl- iC K genes were conducted directly in primary targeting on stxl, stx2, rE and

enriched beef samples. The multiplex PCR could detect 51 organisms in one gram of

beef sample, demonstrating a higher sensitivity of the assay as compared to the

conventional culture method with cefixime-tellurite Sorbitol MacConkey agar, CT-

SMAC. The use of multiplex PCR was shown to provide rapid and sensitive

identification of E. coli 0157. H7 in raw beef. Analysis of 33 beef samples marketed in

East Malaysia with multiplex PCR and conventional culture method has indicated

that the detection of the simultaneous presence of two target genes, rE and iC 7

iv

genes was sufficient for a 0157: H7 positive diagnosis in a beef sample. The inclusion

of primer sets for stxl and stx2 genes in the same reaction provided additional

information on the toxin profiles of a sample and were useful for detecting STEC

0157: H7 and other STEC. Pulsed-field gel electrophoresis (PFGE) with restriction

enzyme, Xbal was used to assess the genetic relatedness of the isolated E. coli

0157: H7, E. coli 0157, non-0157 STEC and other E. coli strains. The PFGE profiles

indicated that five E. coli 0157: H7 and three E. coli 0157 isolates presumably

represented a single strain of E. toll 0157: H7 and E. coli 0157 respectively.

Identical PFGE profiles found in the same E. coli serotype of the same beef sample

indicated that each individual beef marketed in East Malaysia harboured only one

type of STEC 0157: H7 or E. coli 0157. A large variety of PFGE patterns (45

patterns) were found among E. coli isolates demonstrating a high E. coli diversity in

the beef marketed in East Malaysia. From the dendrogram generated, there was no

close relation observed between STEC 0157: H7 and non-0157 STEC to the non-

pathogenic E. coli strains. In this study, PFGE typing method was shown to possess

high discriminatory power and proved its usefulness in differentiating among E. coli

0157: H7 and STEC. The PFGE profiles obtained from the E. coli beef isolates could

provide a database that would aid in the epidemiological investigation of the study

area in future.

V

ABSTRAK

Sebanyak 88 sampel daging lembu yang dipasarkan di Sarawak dan Sabah, Malaysia

Timur, telah dikaji untuk mengesan kehadiran bakteria E. coli yang berupaya

menghasilkan toksin berupa Shiga, E. coli 0157: H7 dan E. coli 0157.

Pengenalpastian dibuat berdasarkan ciri morfologi dan ujian biokimia, diikuti dengan

ujian immunologi. Ujian pengesahan untuk E. coli 0157: H7 dan kehadiran faktor

virulens, iaitu toksin berupa Shiga, Stxl dan Stx2 dibuat dengan kaedah tindak balas

berantai polimeras multipleks (multiplex polymerase chain reaction, PCR). Dalam

kaedah ini, empat pasangan primer PCR akan mengamplifikasi segmen gen stxl, stx2,

rfbE, dan fliCh7 dalam satu tindak balas tunggal. Sebanyak lima pencilan E. coli

0157: H7, empat pencilan E. coli 0157 yang tidak berupaya menghasilkan toksin

berupa Shiga, dan dua pencilan E. coli bukan 0157 yang berupaya menghasilkan

toksin berupa Shiga telah dipencilkan daripada 1.1%, 2.3% dan 2.3% daging lembu

masing-masing. Kehadiran E. coli 0157: H7 dan E. coli yang menghasilkan toksin

berupa Shiga di Malaysia Timur didapati ada hubungan dengan faktor lokasi, iaitu

54.5% (6/11) daripadanya dipencilkan daripada sampel yang didapati di bahagian

tengah Sarawak. Pencilan-pencilan E. coli 0157: H7 yang berupaya menghasilkan

toksin berupa Shiga didapati dalam daging lembu yang beku sahaja manakala E. coli

bukan 0157 yang berupaya menghasilkan toksin berupa Shiga hanya didapati dalam

daging lembu tempatan. Bagi mengecam dan mengenalpasti E. coli 0157: H7 dan gen

virulens pada daging lembu dengan lebih pantas , kaedah tindak balas berantai

polimeras multipleks, iaitu PCR multipleks yang mengamplifikasi segmen gen stxl,

stx2, rfbE, dan fliCh7 dilakukan secara terus dalam sampel asas yang telah

dikonsentrasikan E. coli. Kaedah PCR multipleks dapat mengecam 51 organisma

V1

dalam satu gram daging lembu dan kebolehan ini juga menunjukkan bahawa kaedah

PCR multipleks mempunyai sensitiviti yang lebih tinggi berbanding dengan kaedah

kultur tradisional yang menggunakan agar cefixime-tellurite Sorbitol MacConkey, CT-

SMAC. Penggunaan kaedah PCR multipleks tertunjuk membolehkan pengenalpastian

E. coli 0157: H7 dalam daging lembu dilakukan dengan cepat dan sensitif. Analisis

menggunakan kaedah PCR multipleks dan kaedah kultur tradisional yang

menglibatkan 33 sampel daging lembu yang dipasarkan di Malaysia Timur ini telah

menunjukkan bahawa sistem PCR dengan kombinasi dua gen, rfbE, danfliCh7 sudah

memadai dalam pengecaman E. coli 0157: H7 dalam sampel. Penambahan gen

stxldan stx2 ke dalam sistem PCR ini bertujuan memberi informasi tambahan tentang

profil toksin sesuatu sampel dan adalah berguna untuk mengecam E. coli 0157: H7

dan E. coli yang berupaya menghasilkan toksin berupa Shiga. Dalam usaha untuk

mengenalpasti perhubungan genetik di antara pencilan, kaedah elektroforesis "gel

pulse field" (Pulsed-field gel electrophoresis, PFGE) yang menglibatkan enzim

pemotong, Xbal telah digunakan untuk mendapatkan kesamaan dan perbezaan. bagi E.

coli 0157: H7, E. coli 0157, E. coli bukan 0157 yang berupaya menghasilkan toksin

berupa Shiga dan E. coli lain yang telah dipencilkan. Profil PFGE yang diperolehi

menunjukkan bahawa lima pencilan E. coli 0157: H7 dan tiga pencilan E. coli 0157

masing-masing mewakili stren E. coli 0157: H7 dan E. coli 0157 yang tunggal. Profil

PFGE yang serupa hanya didapati pada serotaip E. coli yang sama yang dipencilkan

daripada sampel daging lembu yang sama. Ini menunjukkan bahawa daging lembu

yang dipasarkan di Malaysia Timur secara individunya merupakan habitat bagi satu

jenis E. coli 0157: H7 atau E. coil 0157. Pelbagai jenis profil PFGE (45 jenis) telah

diperolehi daripada pencilan-pencilan E. coli dan ini menunjukkan bahawa daging

lembu yang dipasarkan di Malaysia Timur mempunyai diversiti E. coli yang tinggi.

vii

Daripada dendrogram yang dihasilkan, tiada perhubungan rapat didapati di antara E.

coli 0157: H7 yang berupaya menghasilkan toksin berupa Shiga dan E. coli bukan

0157 yang menghasilkan toksin berupa Shiga dengan stren E. coli yang bukan

patogenik. Kaedah PFGE telah tertunjuk mempunyai kuasa diskriminasi yang tinggi

dan berguna dalam pembezaan di antara E. coli 0157: H7 dan E. coli yang

menghasilkan toksin berupa Shiga. Butiran data daripada profil PFGE yang diperolehi

daripada E. coli yang dipencil daripada daging lembu turut akan membantu dalam

kajian epidemiologi berkenaan kelak.

Pusat Khidmat Maklumat Akademik UNIVERSITI MALAYSIA SARAWAK

TABLE OF CONTENTS

Acknowledgements

Abstract

Abstrak

Table of Contents

List of Tables

List of Figures

List of Abbreviations

1. General Introduction

2. Literature Review

2.1 Escherichia coli

2.1.1 Characteristic

2.1.2 Taxonomy

2.1.3 Habitat

2.1.4 Growth

2.2 Shiga-like toxin producing Escherichia coli (STEC)

2.3 Escherichia coli 0157 and 0157: H7

2.3.1 Biochemical characteristic

2.3.2 The disease

2.3.3 Prevalence and incidence

2.3.4 Mode of transmission

2.3.5 Pathogenicity

2.3.5.1 Shiga-like toxins (Stx)

2.3.5.2 Attaching and effacing adherence

2.3.5.3 Pathogenesis

11

iv

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ix

xiv

xv

xvi

I

8

8

9

11

12

13

13

14

14

16

22

24

25

27

28

ix

2.4 Isolation of Escherichia coli 0157 and Escherichia coli 0157: H7 29

2.4.1 Enrichment media 30

2.4.2 Plating media 31

2.4.3 Incubation condition 34

2.5 Identification of Escherichia coli 0157 and Escherichia coli 0157: H7 34

2.6 Detection of Escherichia coli 0157 and Escherichia coli O157: H7 35

2.6.1 Immunological detection systems 35

2.6.2 Polymerase chain reaction (PCR)

2.6.2.1 Multiplex PCR

2.6.2.2 Direct multiplex PCR

2.7 Nucleic acid sequencing

2.8 Molecular typing: Pulsed-field gel electrophoresis

3. The occurrence of Shiga-like toxin producing Escherichia coli, Escherichia coli 0157: H7, and Escherichia coli 0157 in raw beef marketed in East Malaysia

38

39

43

43

46

3.1 Introduction 49

3.2 Materials and methods 51

3.2.1 Meat samples 51

3.2.2 Isolation of Shiga-like toxin producing E. coli (STEC) and E. 52 coli 0157: H7

3.2.3 DNA isolation 55

3.2.4 Primers

3.2.5 Multiplex polymerase chain reaction (PCR)

3.2.6 PCR optimization

3.2.7 Gel electrophoresis

3.2.8 Automated DNA sequencing

56

57

57

58

58

X

3.2.9 Data analysis 60

3.3 Results 60

3.3.1 Comparison of the methods used in the isolation of Shiga-like 60 toxin producing E. coli and E. coli 0 157: H7

3.3.2 The prevalence of E. coli based on location 61

3.3.3 Prevalence of Shiga-like toxin producing E. coli 0 157: H7, E. 67

coli 0157 and Shiga-like toxin E. coli (STEC)

3.3.4 Presence of E. coli 0157: H7,0157 and STEC based on the 69

country source of beef samples

3.3.5 Presence of E. coli 0 157: H7,0157 and STEC based on the 69

storage condition

3.3.6 Presence of E. coli 0157: H7,0157 and STEC based on the 70 different beef part

3.3.7 The specificity of the multiplex PCR assay 70

3.3.8 DNA sequencing 74

3.4 Discussion 80

3.5 Conclusion 91

4. Rapid detection and identification of Escherichia coli 0157: H7 and the virulence genes in raw beef marketed in East Malaysia by direct

multiplex PCR

4.1 Introduction 92

4.2 Materials and methods 94

4.2.1 Meat samples 94

4.2.2 Bacterial strain and culture condition 94

4.2.3 Seeding experiment 95

4.2.4 Culture preparation for detection of E. coli 0157: H7 in raw 96 beef samples

4.2.5 Conventional culture method used for comparison 96 4.2.6 Preparation of DNA template for multiplex PCR assay 97

4,23 Primers 99

X1

4.2.8 Multiplex PCR

4.2.9 Automated DNA sequencing

4.3 Results

99

100

102

4.3.1 The sensitivity of the direct multiplex PCR 102

4.3.2 Detection of virulence genes and identification of E. coli 104 0 157: H7 in raw beef

4.3.3 The effectiveness of boiling cell extraction method in the 106

preparation of template DNA

4.3.4 The specificity of direct multiplex PCR and the generated 110

products

4.4 Discussion 114

4.5 Conclusion 121

5. Molecular characterization of Shiga-like toxin producing Escherichla

coli and serogroup 0157: H7, Escherichia coli 0157, and other Escherichia coli strains isolated from raw beef by pulsed-field gel electrophoresis

5.1 Introduction

5.2 Materials and methods

5.2.1 Bacterial strains

5.2.2 Bacterial culture conditions

5.2.3 DNA preparation

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124

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125

5.2.4 Restriction endonuclease digestion 126

5.2.5 Running the pulsed-field gel electrophoresis (PFGE) 127

5.2.6 Data analysis 128

5.3 Result 130

5.3.1 Optimization of running condition and gel resolution 130

5.3.2 PFGE profiles 133

xu

5.4 Discussion

5.5 Conclusion

6. General Conclusion

7. References

8. Appendix

143

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177

PUBLICATION/ CONFERENCE PROCEEDINGS

Apun, K., Chang, P. P., and Sim, E. U. H. (2003) Survey of raw beef marketed in East Malaysia for Escherichia coli 0157 and Escherichia coli 0157: H7. Journal of Bioscience (accepted for publication).

Apun, K. and Chang, P. P. (2003) Application of pulsed-field gel electrophoresis to characterize pathogenic and non-pathogenic Escherichia coli isolates obtained from beef marketed in East Malaysia. Presented at 3rd Federation of Asia Pacific Microbiology Societies Conference, Kuala Lumpur, October 2003.

Chang, P. P., Apun, K., and Sim, E. U. H. (2002) A multiplex polymerase chain reaction assay for identification of Escherichia coli 0157: H7. Proceedings NSF Seminar 2002, Kuala Lumpur, December 2002, pg. 46-49.

Chang, P. P. (2002) The occurrence of Escherichia coli 0157 in raw beef from local

and imported sources marketed in Sarawak. Proceedings of the Postgraduate Colloquium in Science and Technology 2002, Kuching, February 2002, pg. 9- 12.

xiii

LIST OF TABLES

TABLE 2.1 Clinical features of E. coli 0157: H7 infection 16

TABLE 3.1 Oligonucleotide primers used in this study 56

TABLE 3.2 Bacterial count (aerobic plate counts, total coliform counts and 62

presumptive E. coli counts) in fresh, frozen, and thawed samples purchased according to two bacterial isolation methods used

TABLE 3.3 Biochemical tests for the identification of E. coli 64

TABLE 3.4 E. coli 0157: H7, E. coli 0157 and non-0157 STEC isolated 68 from 88 raw beef samples marketed in East Malaysia

TABLE 3.5 The nucleotide sequence homology and the total nucleotides 76

sequenced (in percentage) of the PCR products obtained with primer pairs SLTIF-SLTIR, SLTIIF-SLTIIR, RfbF-RfbR, and FLICh7F-FLICh7R from the E. coli strains

TABLE 4.1 The evaluation of the sensitivity of multiplex PCR and culture 103

method with seeding experiments

TABLE 4.2 Detection of stxl, stx2, rfbE and/ orliCh, genes with multiplex 107 PCR in 33 raw beef samples collected in Sarawak and Sabah

TABLE 4.3 The detection of E. coli 0 157: H7 directly from enrichment 107

cultures with multiplex PCR assay and conventional culture method, and the identification of the specific genes according to location of beef purchased

TABLE 4.4 The detection of E. coli O157: H7 directly from enrichment 109

cultures with multiplex PCR assay and conventional culture method, and the identification of the specific genes according to the country source of beef samples

TABLE 4.5 The detection of E. coli O157: H7 directly from enrichment 109

cultures with multiplex PCR assay and conventional culture method, and the identification of the specific genes according to the types of retailers

TABLE 4.6 The nucleotide sequence homology and the total nucleotides 112

sequenced (in percentage) of the PCR products obtained with primer pairs SLTIF-SLTIR, SLTIIF-SLTIIR, RfbF-RfbR, and FLICh7F-FLICh7R

TABLE 5.1 The source and characteristics of E. coli isolates 135

TABLE 5.2 Distance matrix of 51 E. coli isolates, including 49 isolates from 139 beef samples marketed in East Malaysia

xiv

LIST OF FIGURES

FIG 2.1 The illustration of the polymerase chain reaction (PCR) process 41

FIG 2.2 The illustration of automated DNA sequencing using the 45 dideoxynucleotide triphosphate chain termination method

FIG 3.1 The biochemical tests for the identification of E. coli 64

FIG 3.2 The phenotypic characteristics on different selective agar of E. coli 65

FIG 3.3 The geographic distribution of typical and atypical E. coli isolates 66

which were categorized according to the ability to ferment sorbitol (SF) and exhibit ß-glucuronidase (GUD) activity

FIG 3.4 Amplicons obtained by multiplex PCR of the respective 12 E. coli 71

strains carrying virulence gene(s) were analyzed by agarose (1.2%)

gel electrophoresis

FIG 3.5 The amplified products of the independent rfbE- andliCh7-specific 73 PCR assay and duplex PCR of both for KKW1,3,5 and KCD8 isolates

FIG 3.6 Multiple sequence alignment of fliCh7 gene of beef isolates BTC 11,77 BTC20, and KKA7 with respect to reference strain EDL933

FIG 3.7 Multiple sequence alignment of stx2 gene of beef isolates BTC 11, BTC20, and KKA7 with respect to reference strain EDL933

78

FIG 3.8 PHYLIP rooted tree-a Phenogram of the stx2 genes as determined by DRAWGRAM

79

FIG 4.1 A representative gel for seven combinations of genetic profiles 108

obtained from 33 beef samples

FIG 4.2 An example of a sequencing result analyzed with the blastn 113

program

FIG 5.1 PFGE of chromosomal DNA digested with XbaI of E. coli isolates 131

with 1 program block and initial parameters

FIG 5.2 PFGE of chromosomal DNA digested with Xbal of E. coli isolates 132

with two program blocks: Comparison of the gel resolution before

and after optimization

FIG 5.3 PFGE of chromosomal DNA digested with XbaI of 51 E. coli 134 isolates with two program blocks

FIG 5.4 Dendrogram of E. coli 0157: H7, E. coli 0157, STEC, and E. coli 138

strains isolated in beef marketed in East Malaysia

xv

LIST OF ABBREVIATIONS

A BLAST BCIG bp C cm CDC cfu CTAB CT-SMAC DNA EHEC dNTPs ELISA EMB ERIC-PCR

g G GUD HC HUS IMS IMViC kb LB LPS mEC+n MgC12 M Mda Mb mg min ml mm µm µg µl mol sec

NaCI Na-EDTA NCBI PATS PCI PCR PFGE

adenine Basic Local Alignment Search Tool chromogen 5-bromo-4-chloro-3-indolyl-b-D-glucuronide base pairs cytosine centimeter Centers for Disease Control and Prevention colony forming unit cetyltrimethylammonium bromide cefixime-tellurite Sorbitol MacConkey agar deoxyribonucleic acid Enterohemorrhagic Escherichia coli deoxynucleotide triphosphates enzyme-linked immunosorbent assay Eosin Methylene Blue agar Enterobacterial repetitive intergenic consensus sequence-based polymerase chain reaction gram guanine 13-glucuronidase activity Hemorrhagic colitis Hemolytic-uremic syndrome Immunomagnetic separation Indole-Methyl red-Voges-Proskauer- Citrate tests kilobase pairs Luria Bertani lipopolyssaccharide modified Escherichia coli medium with novobiocin magnesium chloride Molar or molarity (moles of solute per liter of solution) megadalton megabase pairs milligram minute(s) milliliter milliMolar micrometer microgram microliter mole second(s) sodium chloride sodium ethylenediamine tetra-acetic acid National Center for Biotechnology Information Polymorphic amplified typing sequences phenol/chloroform/isoamyl alcohol Polymerase Chain Reaction Pulsed-Field Gel Electrophoresis

xvi

psi pound(s) per square inch (Ib/in2) RAPD Random Amplification of Polymorphic DNA rpm revolution per minute SDS sodium dodecyl sulfate SF sorbitol-fermenting/fermenter SNF non-sorbitol-fermenting/fermenter SMAC Sorbitol MacConkey agar SMAC-BCIG Sorbitol MacConkey agar supplemented with chromogen 5-bromo-4-

chloro-3-indolyl-b-D-glucuronide sp. species STEC Shiga-like toxin producing Escherichia coli Stx Shiga-like toxin T thymidine Taq Thermus aquaticus DNA polymerase TAE Tris-acetate electrophoresis buffer TBE Tris-Borate EDTA electrophoresis buffer TE Tris-EDTA buffer Tris Tris (hydroxymethyl) methylamine TTP Thrombotic thrombocytopenic purpura USA United States of America USDA United States Department of Agriculture USFDA United States Food and Drug Administration VT Verotoxin V volts % percent > more than < less and the same as °C degree Celsius

xvii

CHAPTER 1

GENERAL INTRODUCTION

Escherichia coli is a common commensal of the human gastrointestinal tract.

However, under certain conditions strains of E. coli can cause disease. Shiga-like

toxin-producing E. coli (STEC) has been implicated as the causative agent in several

human diseases (Nataro and Kaper 1998, Paton and Paton 1998a), with E. coli

0157: H7 to be the most well known among them. These diseases range from mild

diarrhea to severe and life threatening conditions, such as hemolytic-uremic syndrome

(HUS) and thrombotic thrombocytopenic purpura (TTP). Cattle are generally

considered to be the major reservoir of both E. coli 0157 and non-0157 STEC

(Bettelheim 2000). The organisms can be transmitted efficiently via contaminated,

undercooked food of animal origin, cross-contaminated food (Griffin 1995; Smith

1997), water (Olsen et al. 2002) and from human to human (Griffin 1995). The first

recorded outbreak associated with HUS in 1982 was attributed to E. coli O157: H7

(Riley et al. 1983), a serotype that still accounts for the greatest proportion of STEC

disease in North America, Europe and Japan. However, non-0157 STEC strains may

account for 20 to 70 percent of STEC infections overall in different countries (World

Health Organization 1999).

E. coli 0157: H7 and non-0157 STEC produce toxins, verotoxin 1 or Shiga-like

toxin (Stxl) and verotoxin 2 or Stx2. According to O'Brien and Holmes (1987), the

Stxs are cytotoxic for some cell lines, enterotoxic and paralytic-lethal when injected

I

intravenously in mice and rabbits. In addition, these Shiga-like toxins are reported to

be responsible in part for hemorrhagic colitis (HC) and hemolytic-uremic syndrome,

HUS (Griffin 1995).

STEC and E. coli 0157: H7 possess very low infectious dose, as few as 10

viable bacteria can initiate the pathogenesis of disease (Willshaw et al. 1994). Despite

the very low infectious dose, as well as the high pathogenicity of E. coli 0 15 7: H7 and

STEC, there is no current available specific treatment for the disease HUS. Hence,

there is an urgent need for effective preventive measures based on a detailed

understanding of the epidemiology of STEC infections. Such measures will also

depend on the availability of rapid, sensitive, simple and reproducible procedures for

the detection of these pathogens and for the characterization among the pathogens and

those of non-pathogenic E. coli strains. Consequently, attempts to develop suitable

and sensitive methods are initiated and the development has progressed rapidly for

better detection. Initial and traditional methods for the detection and identification of

STEC 0157 and other serogroups in samples involve enrichment cultures, selection of

bacterial colonies, biochemical analysis of the isolates and determination of the main

virulence markers, such as Shiga-like toxins (Tarr 1995, Clifton-Hadley 2000). This

approach is laborious, time-consuming and insufficiently sensitive in identifying

STEC organisms. To improve the sensitivity and reduce the total time involved in

detection, commercial rapid ELISA procedures have been introduced (Padhye and

Doyle 1991). With the advent of molecular biology techniques, genetically based

assay has been developed for the detection improvement of pathogenic E. coli and E.

2

coli 0157: H7. The available methods include DNA hybridization (Feng 1993) and

PCR-based assay (Hu et al. 1999).

A number of PCR-based assays have been developed for the detection of E. coli

0157 and other serogroups (Meng et al. 1997, Kumar et al. 2001, Toma et al. 2003).

Although significant developments have been made in the molecular diagnosis of

STEC bacteria, there is still a need to improve PCR-based assays so that specific and

direct identification of the important virulence factors and genotypic identification

factors can be made in a single reaction. Multiplex PCR is a PCR approach that uses

two or more primer sets to simultaneously amplify multiple target sequences in a

single reaction. Hence, the use of multiplex PCR has been widely studied with a

varying combination of target gene sequences in order to improve the sensitivity of

the method (Meng et al. 1997, Nagano et al. 1998). Most of the tests are based on the

identification of stx (Shiga-like toxin) and eaeA (intimin) gene sequences (Meng et al.

1997, Fagan et al. 1999). Currently, the use of multiplex PCR to amplify the presence

of the target genes in the primary sample, rather than in the broth culture of pure

colony has introduced a direct approach for detection and identification STEC (Osek

2002, Hu et al. 1999). This direct multiplex PCR approach not only reduces the time

consumed, it also eliminates the necessity for strain isolation, thereby negating the

potential biases during selection of bacterial colonies, in particular the non-0157

STEC.

The detection and identification of pathogenic E. coli and E. coli 0157: H7 are

very much of importance. Subserovar characterization of these pathogenic E. coli and

3

E. coli 0157: H7 is no less essential in order to investigate the genetic relationship

among pathogenic E. coli and E. coli 0157: H7, as well as to differentiate them from

non-pathogenic E. coli. Initial typing methods routinely used during outbreak

investigations include phage typing (Khakhria et al. 1990) and Shiga-like toxin typing

(Paros et al. 1993). In addition to that, plasmid profiling (Meng et al. 1995),

ribotyping (Apun et al. 1995), enterobacterial repetitive intergenic consensus (ERIC)

sequence-based PCR (ERIC-PCR) (Giammanco et al. 2002), randomly amplified

polymorphic DNA (RAPD) (Hopkins and Hilton 2000), pulsed-field gel

electrophoresis (Gautom 1997) and, recently, in a novel approach for differentiating

E. coli 0157: H7, polymorphic amplified typing sequences (PATS) (Kudva et al.

2002) have been used to characterize STEC E. coli and E. coli 0157: H7. Plasmid

profiling is a relatively simple and economical technique for subtyping, yet they are

not universally applicable to all bacteria as many bacterial species harbour plasmids

infrequently. Phage typing, Shiga-like toxin typing, and ribotyping have been reported

to be relatively lacking of discriminatory power (Saari et al. 2001, Martin et al. 1996).

However, they can exhibit superior discriminatory performance when combined with

other typing technique. Preston et al. (2000) reported that combination of phage typing

and PFGE fingerprinting could provide optimal discrimination for E. coli 0157

subtyping. Likewise, combined use of PFGE and ribotyping in the characterization of

E. coli 0157 from animal origin food, retail meats and cases of human disease has

demonstrated superior discriminatory performance (Avery et al. 2002). The PCR-

based assay, ERIC-PCR has been successfully used in differentiating laboratory

strains of E. coli by Versalovic et al. (1991) and suggested its potential for subtyping

gram-negative enteric bacteria. In contrast, Giammanco et al. (2002) reported that

4

Pusat Kliidmat Maklumat Akademik UMVEFLSITI MALAYSIA SARAWAK

ERIC-PCR was unable to distinguish epidemiologically unrelated strains of E. coli

0157: H7.

Another subtyping method, PFGE, was widely used for bacterial

characterization. PFGE has been described as the "gold standard" of genetic

fingerprinting methods for E. coli 0157 with high discriminatory performance and

reproducible result (Preston et al. 2000). An alternative to PFGE for E. coli 0157

subtyping is RAPD analysis and it was shown to be more discriminatory than phage

typing (Grif et al. 1998), ribotying and serotyping (Kärkkitinen et al. 1996).

Nevertheless, unlike PFGE, it often presents intra- and inter-laboratory reproducibility

problems in particular when subtyping data are used to track strains over long periods

and in different laboratories. Polymorphic amplified typing sequences (PATS)

analysis is a very newly devised simple PCR-based strain typing technique for E. coli

0157. In a study of Kudve et al. (2002), PATS was able to type all the E. coli 0157

tested while PFGE was unable to type a few of the isolates. However, PFGE had

higher discriminatory power compared to PATS.

In recent years, PFGE has increasingly been used for the molecular subtyping of

a wide range of bacterial and fungal pathogens. For E. coli 0157: H7, the usefulness of

PFGE fingerprinting during outbreak investigations has been demonstrated by Barrett

et al. (1994) and Preston et al. (2000). In 1993, the Centers for Disease Control. and

Prevention (CDC) has applied PFGE to characterize clinical and food isolates of E.

coli 0157: H7 during a large outbreak of foodborne illness caused by E. coli 0157: H7

that occurred in the western United States of America. In addition, CDC has

5

standardized PFGE methods in order to allow isolates to be compared from different

parts of the country, enabling recognition of nationwide outbreaks attributable to a

common source of infection, particularly those in which cases are geographically

separated.

E. coli 0157: H7 is a newly emerged foodborne pathogen and has been

associated with human disease such as hemorrhagic colitis and hemolytic-uremic

syndrome (HUS). This important emerging pathogen, E. coli 0157, has been reported

in West Malaysia (Son et al. 1998a). However, there is no published report on the

occurrence of this pathogen in East Malaysia, either in food or in clinical samples. As

cattle is the main reservoir of E. coli 0157 and non-0157 STEC, it is necessary to

initiate a study on the occurrence of the E. coli 0157: H7 and STEC in beef marketed

in Sarawak and Sabah. The data generated would assist the relevant authorities

(Health and Agriculture sectors) in planning proper procedure for detection of these

bacteria in beef. Most laboratories in East Malaysia detect the pathogens with

conventional culture method, involving enrichment cultures, selection of bacterial

colonies, biochemical analysis of the isolates and determination of the main virulence

markers. This is laborious and time consuming. Therefore, a rapid and reproducible

method for the detection and identification of E. coli 0157: H7 is needed for the

diagnosis and detection of this pathogen in the laboratories in East Malaysia. The

characterization of E. coli 0157: H7 and STEC detected are as vital. The data on the

genetic relatedness or divergence among the E. coli 0157: H7 and STEC can be used

in studying possible clonal relationship among strains. Hence, under the condition

whereby any outbreak occurs, investigation and determination of foodborne illness

6