UNIVERSITI PUTRA MALAYSIA

MOLECULAR CHARACTERISATION OF ESCHERICHIA COLI SEROGROUP 0 157:H7

AHMAD ZAINURI MOHD DZOMIR

FSMB 1998 1

MOLECULAR CHARACTERISATION OF ESCHERICHIA COLI SEROGROUP 0 1 57:H7

By

AHMAD ZAINURI MOHD DZOMIR

Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science in the Faculty

of Food Science and Biotechnology University Putra Malaysia

September 98

ACKNOWLEDGEMENTS

My deepest gratitude to my supervisor, Professor Dr. Gulam Rusul

Rahmat Ali for his continuous support, patience and advice given to me

throughout the course of this project. I also wish to thank my co­

supervisors, Dr. Son Radu, for his valuable guidance and constant

encouragement, and Dr. Zaiton Hassan, for her suggestions and support.

Thanks also to all of the members and ex-members of the Microbiology

Laboratory; Ibu Endang, Liew Wang Shiang, Loke Chui Fung, Kak

Dayang, Kak Mai, Kak Sahilah, Sharmila, Sudha, Nimita, Rozila, Kamil,

Razali, Sam, Reddy, Senthil, Nasseer, Yuherman and Bushtari for their

kindness, friendship, helpful discussions and comments.

Lastly, I would like to thank my family, Pak Su, Mak Su and nenek for

their love and support.

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . ii

LIST OF TABLES. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

LIST OF PLATES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . V111

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

ABSTRAK. . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . .. . xi

CHAPTER

I �ENE� INTR()J)l1CTI()N... ..... .. . ........ . . .. . . . . . . .. . . . .. .. . . .. 1

II LITE�T� �E�. . . .. . . . . ... .. . . . . . . ... . . . . .. . . . .. . . . . . . . . .. . ... !5 Microbiology of Escherichia coli 01!57:H7........................ !5

Biochemical Characteristics... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !5 Growth and Survival Properties. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 7

Pathogenicity . . . . . . . . . . . . . .. . . . . . . .. . . . . . , . . . . . . . . . . . . , .. . . . . . , . . . . . . . . . . . . , . 8 Verocytotoxins . .. . . . . . . .. . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . , . . . .. . 9 Adherence and Attachment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0

Complication of E. coli 0 1!57:H7 Infections.. . . . . . . . . . . . . . . . . . . . . 1 2 Hemorrhagic Colitis.. . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . 1 3 Hemolytic Uremic Syndrome (HUS).. . .. . . . .. . .. . . . . . .. . . . . .. . 14 Thrombotic Thrombocytopenic Purpura (TIP}.. . . . . . .. . . . 14

Epidemiology. . . .. . . . . . . . .. . . . . . .. . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1!5 Outbreak Investigations. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1!5 Studies of Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 1 9 Animal Reservoir. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . 20

Detection and Characterisation of E. coli 0 1!57:H7 in Foods. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 23

Conventional Methods.. . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . .. 24 Alternative Methods... . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . 27 Barriers to Implementation. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

Typing of A New Clonally Derived Pathogen - Escherichia coli 0 1!57:H7. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. 32

Biochemical Promes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 33 Antibiotic Susceptibility Pattern and Plasmid Prome. . . 36 Pulsed-Field Gel Electrophoresis (PFGE} . . . .. . . . . . . . . . . . . '" 38 Arbitrarily Primed Polymerase Chain Reaction (AP-PCR).. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 40

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Page

III ANTIBIOTIC SUSCEPTIBILITY PATTERN AND 43 PLASMID PROFILES . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . Introduction......... .. .. .. . . . . ............. .... ........ .... ...... ..... . ..... 43 Methods.............. ... . ............ . . . ... . . . . . ..... . . .......... . . ... .... . . .. 44

Bacterial Strains Used in this Study... ... . ...... .. ........ ... 44 Maintenance of Bacterial Strains....... .... . ....... . . .. . ....... 45 Antibiotic Discs......... ... . ............. .. . . .. . .... ........ . . . . . .. . . .. 45 Antibiotic Susceptibility Test. . . .... . . ...... . ....... . .... . . ..... .. 46 Plasmid DNA Isolation..... ................. .. . ..... . . . ... ..... .. ... 47 Agarose Gel Electrophoresis....... ... . . .. . . . . . . .. . . ........ . . . ... 48 Plasmid DNA Visualisation and Photography... ..... ... 49 Plasmid DNA Molecular Weight Determination......... .. 49

Results................... ........... ..... ...................................... 50 Susceptibilities of E. coli 0 1 57:H7 Isolates ........... . .... 50 Plasmid Promes of E. coli 0 157:H7 ........................... 54 Combination of Antibiotype and Plasmid Prome to Differentiate Isolates. . . .............................................. 57

Discussion ............. ................ , ... ...... ... ...... ............. ...... 60

IV. MACRORESTRICTION DIGESTION PATTERN BY PULSED-FIELD GEL ELECTROPHORESIS (PFGE). . . . . .. .. . .. . ... . . . .. . . . ... . . .... . . . .. . .. . . . . . . ... . . . .. .. . ... ..... ....... 65 Introduction......... ........... . . .... . . .. .. ............................ ..... 65 Methods....................... .................. .... ......................... . 66

Preparation of Intact Genomic DNA........................... 66 Digestion of the Agarose Embedded DNA............... .... 67 Electrophoresis................ ........................................ 67 DNA Bands Visualisation and Photography............. . .. 69 Genomic DNA Fragments Molecular Weight Determination ..... ...... ............... , ..... . ... ... ...... ............ . 69 Genetic Analysis... ... ... ...... ... ...... ... .. . ... . . . ... . . . . .. . .. ... ... 69

Results... ... ............... ....... . . . ..... ............ ........ ................. 70 Examining the Integrity of DNA Samples...... .. . ..... .. . . .. 70 Macrorestriction Digestion Pattern by PFGE............... 74 Genetic Distance between Different Macrorestriction Digestion Pattern..................................................... 80 Restriction Polymorphisms Resolved by PFGE Distinguished between Isolates............. .............. . . . ... 83

Discussion.. . ... . ............. ........... .. ... . ... ... . ........... ....... . . ... 87

iv

V DETECTION OF POLYMORPHISMS AMPLIFIED BY ARBITRARILY PRIMED POLYMERASE CHAIN

Page

REACTION (AP-PCR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Introduction . .. . . . ... .. . . . ..... . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Methods....................................................................... 9 1

Bacterial Growth and DNA Extraction........................ 9 1 Spectrophotometric Quantitation of DNA................... 92 Standardisation of Primers Unit......... ....................... 92 AP-PCR Amplifications.............................................. 93 Electrophoresis.............................. ... . . . . . . . . . . . . . . . . . . . . . . . 94 Visualisation of DNA Bands and Photography............ 94

Results......................................................................... 95 Optimisation of Concentration of Arbitrary Primer, DNA template and Mg2+ Ion for Discriminatory Power...................................................................... 95 Identification of Useful Primers................................. 97 Strain Discrimination with Five 1 0-nt Primers with G+C Content of 50%. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Discussion .............. , . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04

VI . GENERAL DISCUSSION AND CONCLUSION......... . . . . . . . . . 1 08

REFERENCES........................................................................ 1 12

VITAE.................................................................................... 1 23

v

LIST OF TABLES

Table

1 Outbreaks of Escherichia coli 0 1 57:H7 infection in the United States, and selected outbreaks in Canada and

Page

the United Kingdom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 18

2 Summary of the biochemical reactions of E. coli 0 1 57:H7 and E. coli other than serotype 0 1 57:H7. . . . . . . . . . 35

3 Information regarding the E. coli 0 1 57:H7 studied. . . . . . . . . 44

4 Distribution of antimicrobial resistance of E. coli 0 1 57:H7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1

5 Predominant patterns of antimicrobial resistance of beef isolates of E. coli 0 157:H7. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

6 Susceptibility testing of resistance to multiple antimicrobial agents among E. coli 0 1 57:H7 beef isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 52

7 Plasmids of Escherichia coli 0 1 57:H7 . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 56

8 Number of plasmid DNA harbored by E. coli 0 1 57:H7 isolates . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . , . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

9 Plasmid pattern of E. coli 0 1 57:H7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 56

1 0 Combination of antibiotype and plasmid pattern to differentiate isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

1 1 Optimal electrophoretic conditions for each restriction endonuclease used. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

1 2 F values for each PFGE pattern for E. coli 0 1 57:H7 isolates . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. 8 1

1 3 Restriction digestion pattern by PFGE distinguished between isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 86

14 The sequence of each oligonucleotide tested for AP-PCR analysis of E. coli 0 1 57:H7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 93

1 5 Purity index and quantity of DNA extracted in E. coli 0 1 57:H7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

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16 AP-PCR profile pattern and genetic types of E. coli

Page

0157:H7. .......... . ... ................. . ......... ........................... . 103

vii

LIST OF PLATES

Plate Page

1 Plasmid profiles of E. coli 0 1 57:H7 isolates . . . . . . . . . . . . . . . . . . . . 55

2 Screening of the 14 chromosomal DNAs of E. coli 0 1 57:H7 for integrity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

3 PFGE of the 10 E. coli 0 1 57:H7 genomic samples after performing a mock digestion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4 Comparison of Xbal digests of genomic DNA of E. coli 0 1 57:H7 beef isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

5 Comparison of SpeI digests of genomic DNA of E. coli 0 1 57:H7 beef isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

6 Comparison of HindIII digests of genomic DNA of E. coli 0 1 57:H7 beef isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

7 Results of tests to identify useful primers for AP-PCR analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

8 Representative arrays of fragments generated by arbitrary primer PCR with primer GEN 1 5002.

9 Random amplified polymorphic DNA patterns of 1 5 E.

1 00

coli 0 1 57:H7 beef isolates using primer GEN 1 5003. . . . . . . . 1 0 1

1 0 Representative arrays of fragments generated by arbitrary primer PCR with primer GEN 1 5009.

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102

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

MOLECULAR CHARACTERISATION OF ESCHERICHIA COLI

SEROGROUP 0 157:H7

BY

AHMAD ZAINURI MOHD DZOMIR

SEPTEMBER 1998

Chairman: Professor Dr. Gulam Rusul Rahmat Ali

Faculty: Food Science and Biotechnology

Fourteen E. coli 0 1 57:H7 beef isolates were characterised by using

four specific epidemiological markers: combination of antibiogram and

plasmid profiling, pulsed-field gel electrophoresis (PFGE) and arbitrary

primed polymerase chain reaction (AP-PCR). These markers were

assessed for their reliability, typability, rapidity and discriminatory

power in differentiating beef E. coli 0 1 57:H7 strains from different

locations, namely Bangsar, Kajang, Petaling Jaya and Serdang. The

majority of the isolates were resistant to penicillin G ( 1 00%), vancomycin

( 100%) , trimethoprim/ sulphametoxazole ( 100%) , bacitracin ( 100%) and

erythromycin (92 .8%) . Only 2 1.4% were resistant to carbenicillin. 1 4.3%

and 7. 1% were resistant to ampicillin and cephalotin, respectively.

ix

Plasmid analysis revealed three basic plasmid patterns among E. coli

0 1 57:H7 strains, profile 1 characterised by plasmid DNA of 60 and 2.5

MDa, profile 2 characterised by plasmid of 60 MDa, and profile 3

characterised by the absence of any plasmid in the strains. Grouping

according to combination of antibiogram and plasmid analysis indicated

eight different groups as two strains with similar antibiotype could be

distinguished into two different strains by their dissimilar plasmid

profile. However, the reliability of antibiogram and plasmid analysis in

typing E. coli 0 1 57:H7 can be questioned. Thus, other reliable methods

such as PFGE and AP-PCR were then applied. In the present study,

macrorestriction of genomic DNA of E. coli 0 1 57:H7 using Xbal, SpeI and

HindIII and analysed by PFGE successfully grouped ten out of fourteen

isolates into five groups and provided evidence of epidemiologically

related strains between strains of different and same locations. However,

AP-PCR using three short primers grouped the isolates into fourteen

distinct groups and differentiates isolates that were not differentiated by

PFGE. The overall analysis of the present study revealed AP-PCR as the

most suitable method to differentiate E. coli 0 1 57:H7 because it was

more discriminatory, less labor intensive and applicable to all isolates.

Using this method, it was clearly shown that all fourteen E. coli 0 1 57:H7

existed as independent isolates.

x

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Master Sains

PENCIRIAN MOLEKULAR ESCHERICHIA COLl SEROGROUP 0157:H7

OLEH

AHMAD ZAINURI MOHD DZOMIR

SEPTEMBER 1998

Pengerusi: Professor Dr. Gulam Rusul Rahmat Ali

Fakulti: Sains Makanan dan Bioteknologi

Empat belas isolat E. coli 0 1 57:H7 diciri menggunakan empat metod

epidemiologi iaitu gabungan ujian ketahanan terhadap antibiotik dan

analisis plasmid, PFGE dan AP-PCR. Marker diuji dari segi kemantapan,

kebolehan pencirian, kepantasan dan nilai pemisahan untuk

membezakan E. coli 0 1 57:H7 yang diisolat dari pelbagai tempat seperti

Bangsar, Kajang, Petaling Jaya dan Serdang. Kebanyakan isolat adalah

resistan kepada penicillin G ( 100%), vancomycin ( 100%), trimethoprim/

sulphametoxazole ( 1 00%) , bacitracin ( 100%) dan erythromycin (92 . 8%) .

Hanya 2 1 .4% resistan kepada carbenicillin. 1 4 .3% dan 7 . 1% resistant

kepada ampicillin dan cephalotin masing-masing. Analisis plasmid

menunjukkan tiga corak asas di antara E. coli 0 1 57:H7, profil 1 diciri

oleh kehadiran dua plasmid bersaiz saiz 60 MDa dan 2 .5 MDa, profil 2

xi

diciri oleh kehadiran satu plasmid bersaiz 60 MDa dan profil 3 diciri

oleh ketidakhadiran plasmid. Pengkelasan mengikut penggabungan

antibiograrn dan analisis plasmid menunjukkan lapan kumpulan

bardasarkan pada pendapat yang mengatakan dua isolat yang

mempunyai antibiotaip yang serupa boleh dibezakan dengan profil

plasmid yang berlainan. Walaubagaimanapun, kebolehan antibiograrn

dan analisis plasmid untuk menciri E. coli 0157:H7 boleh dipersoalkan.

Jadi, metod lain yang lebih mantap seperti PFGE dan AP-PCR telah

dicuba. Dalarn kajian ini, pemotongan DNA genomik E. coli 0157:H7

menggunakan enzim Xbal, SpeJ dan HindIII, dan dianalisis

menggunakan PFGE mengkelaskan sepuluh dari empatbelas isolat

kepada lima kumpulan dan memberi bukti perkaitan epidemiologi

antara isolat yang dipencil dari temp at yang sarna atau pun berlainan.

Dengan menggunakan AP-PCR dan tiga primer pendek, semua isolat

berjaya dibezakan ke dalarn empat belas kumpulan yang berlainan dan

ia juga dapat memisahkan isolat yang tidak boleh dibezakan dengan

PFGE. Secara keseluruhan, kajian ini mendapati bahawa AP-PCR

merupakan metod yang paling sesuai untuk membezakan E. coli

0157:H7 kerana ianya lebih cepat, mempunyai nilai pemisahan yg lebih

tinggi, kurang memerlukan tenaga kerja dan boleh diaplikasikan keatas

semua isolat. Dengan metod ini, arnat jelas ditunjukkan bahawa

empatbelas isolat yang dikaji tidak mempunyai perkaitan di antara

sarna satu lain.

xii

CHAPTER I

GENERAL INTRODUCTION

The bacteria constituting the species Escherichia coli were first

discovered by a German microbiologist, Theobald Escherich in 1 885 and

were commonly thought as normal flora of man and animals (Sojka,

1 965) . However, until late 1950's, certain strains were found to be

capable of inducing disease, and E. coli were therefore regarded as a

potential pathogen. These strains are classified into several groups based

on their distinct clinical manifestations and virulence determinants, for

example, the enterotoxigenic E. coli (ETEC) , enteropathogenic E. coli

(EPEC) and enteroinvasive E. coli (EIEC) (Olsvik et al., 1 99 1 ).

In 1 982, following two outbreaks of a distinctive bloody diarrheal

syndrome and a sporadic case of bloody diarrhea, a new bacterial

pathogen, E. coli 0 1 57:H7 was identified (Riley et al. , 1 983). E. coli

0 1 57:H7 comprises a fourth group of E. coli associated with human

diarrhea, i .e., enterohemorrhagic E. coli (EHEC). The first two outbreaks

that occurred in Michigan and Oregon, involved a national fast-food

chain distributing hamburgers. Since then, several additional outbreaks

have been reported in other parts of United States (Ryan et al. , 1 986,

2

Griffin et al. , 1988), Canada (Borczyk et al. , 1987) and with increased

surveillance, E. coli 0157:H7 outbreaks have also being reported in other

parts of the world including Mexico (Cravioto et aI. , 1990), China (Xu et

al. , 1990), Argentina (Lopez et al. , 1989), Belgium (Pierrard et al. , 1990)

and Malaysia (Son et al. , 1996).

As the incidence of by E. coli 0157:H7 is in the increase in many

parts of the world, specific surveillance of this pathogen is essential. For

identifying the sources and monitoring the spread of E. coli 0157:H7, a

number of epidemiologic markers have been use, including antibiotic

susceptibility pattern (antibiotype) , plasmid profile, pulsed field gel

electrophoresis (PFGE) , biotype, phage typing and restriction analyses of

chromosomal DNA by classical electrophoresis. The use of epidemiologic

markers enables the bacterial stains to be typed and establish degree of

relatedness between the strains.

As phenotypic systems have limitations in typing and stability, and

most typing systems on their own have a low discriminatory ability,

therefore, in the present study, we combined antibiotic susceptibility

pattern and plasmid profile for subtyping E. coli 0157:H7 beyond their

serotype. Among genotypic analysis techniques, PFGE of

macrorestriction fragments appears to be a highly sensitive method that

may detect subtle genetic variations among phylogenetically and

epidemiology related isolates of E. coli OI57:H7.

3

Recently, a polymerase chain reaction (PCR)-based method of

genotypic analyses that is sensitive and yet efficient for typing pathogenic

microbe has become very popular because of its convenience and

simplicity. Arbitrarily primed-PCR has been reported to be useful for

tracing route of infection and also for understanding the spread of

pathogen, especially 0157:H7 serotype. In the present study, we attempt

to type strains of E. coli 0157:H7 isolated from different samples and

locations according to three methods, namely the combination of plasmid

proflle and antibiotype, PFGE and arbitrarily primed (AP)-PCR. The study

here is motivated by the view that E. coli 0157:H7, though members of

one clone, can often be distinguished by methods that have been clarified

by previous workers.

4

Objectives of the Study

This study is carried out to determine the relatedness of a group of

14 E. coli 0157:H7 beef isolates using molecular techniques. The isolates

are from Bangsar, Kajang, Serdang and Petaling Jaya. Three methods are

used; combination of plasmid profiling and antibiotic susceptiblity

pattern, macrorestriction digestion pattern by PFGE and arbitrarily

primed polymerase chain reaction (AP-peR) to detect polymorphisms in

their genome. The results from each method are then discussed in order

to determine which is the most discriminative for the purpose of strain

differentiation.

CHAPTER II

LITERATURE REVIEW

Microbiology of Escherichia coli 0 157:H7

The strains of E. coli 0157:H7 that were isolated during the outbreak

in the Northwest Pacific, United States of America are typical of most E.

coli, with some exceptions. E. coli serotype 0157:H7 possess biochemical

markers, growth and survival characteristics that are significantly

different from those of other E. coli strains.

Biochemical Characteristics.

The inability to ferment sorbitol and the absence of f3-glucuronidase

activity are believed to be a specific phenotypic feature of E. coli

0157:H7. More than 90 % of E. coli isolates of human origin ferment

sorbitol within 24 hours; howeyer, E. coli 0157:H7 does not (Johnson et

al. , 1983; Ratnam et al., 1988). Furthermore, other workers reported that

E. coli 0157:H7 failed to ferment sorbitol as late as seven days (Wells et

al., 1983). Workers exploited this biochemical feature and developed a

modified MacConkey agar containing D-sorbitol (SMAC) instead of

5

6

lactose to detect E. coli 0 1 57:H7 (March and Ratnam, 1 986; Farmer and

Davis, 1 985) . The strain is similar to other E. coli on the lactose­

containing agar such as Eosin Methylene Blue Agar (EMBA) making it

very difficult to differentiate between them. However, on SMAC agar E.

coli 0 1 57:H7 could be recognised as colorless colonies showing that they

failed to ferment sorbitol. Ratnam et al. ( 1988) described supplementary

biochemical markers, such as lysine and ornithine decarboxylation to

increase the specificity of the sorbitol-only screened E. coli 0 1 57:H7. The

inclusion of these tests reduced the number of organism to be serotyped

and improved the specificity of the biochemical screen to 33.6% (Haldene

et ai., 1 986) .

In addition, E. coli 0 1 57:H7 strains are different from other E.

coli strains, as they do not posses �-glucuronidase activity. More than

90% of E. coli produce the enzyme �-glucuronidase, which is the basis

for a rapid flu orogenic assay for E. coli (Feng and Hartman, 1 982) . The

indicator, 4-methyl-umbelliferone glucuronide (MUG) used in this assay

is hydrolysed by �-glucuronidase enzyme possess by normal E. coli and

converted into 4-methylumbelliferone that fluoresces under radiation

with ultra-violet (UV) light (366 nm). In contrast to most E. coli strains, E.

coli 0 1 57:H7 is not capable of producing �-glucuronidase. When

irradiated with long wave UV light, no fluorescence is formed, hence E.

coli 0 1 57:H7 could be differentiated from other E. coli.

7

Growth and Survival Properties

Doyle and Shoeni ( 1984) showed that E. coli 0 1 57:H7 can survive

well up to 9 months at -20°C and at -80°C in ground beef. They also

observed another important characteristic of E. coli 0 1 57:H7 i .e . , they

grew poorly at 44-44.5°C, which is the temperature generally used for the

isolation of E. coli 0 1 57:H7. Hence, traditional procedures for detecting

E. coli in foods would not likely detect E. coli 0 1 57:H7. Temperature

range of 16.4 to 42 .5°C and 48 hours incubation has been suggested by

Raghukeer and Matches ( 1990) for detecting E. coli 0 157:H7 in foods.

E. coli 0 1 57:H7 has no unusual heat resistance. Most of the

organisms will be killed if the food was pasteurised or heated to some

extent. D' Aoust et al. ( 1 988) observed that more than 1 04 E. coli

0 1 57:H7 per ml were killed following pasteurisation of milk (72°C,

16 .2s). In addition, Kotula et al. ( 1977) observed ground beef patties that

were undercooked 'well done' (4 minutes per side at 1 49°F griddle

temperature) showed a reduction in the coliform count from four logs to

less than 1 / gram. Coliform count in retail raw ground beef are viable,

but may be expected to be 1 000/gram or more (Restaino and Lyon,

1 987) . This means that high initial coliform counts that results in a rare

hamburger are likely to permit the survival of coliforms after cooking.

8

Outbreaks of E. col i 0 1 57:H7 caused by drinking apple cider

indicates that E. col i 0 1 57:H7 is resistant to acidic pH and distinguished

0 1 57: H7 serotype from other E. col i (Miller and Kaspar, 1 994) . E. col i

0 1 57:H7 can grow at pH levels ranging from 4.0 to 9 .0 (Glass et al . ,

1 992) which is not significantly different from the pH range of 4 .4 to 9 .0

reported for other E. col i. However, the ability of E. col i 0 1 57:H7 to

survive at a pH of less than 4.0 was reported by Miller and Kaspar

( 1994) . Their study revealed that E. col i 0 1 57:H7 can survive in

unpasteurised apple cider at pH 3.6 to 4 .0 for as long as 3 1 days at BOC.

Similarly, Zhao and Doyle ( 1994) observed that E. col i 0 1 57:H7, when

initially present at 6 .5 x 1 03 CFU/g, can survive in mayonnaise (pH 3.6

to 3 .9) at 20°C for 2 1 days and at 5°C for 55 days.

Pathogenicity

The pathogenesis of infections with E. col i 0 1 57:H7 and other

enterohemorrhagic E. col i is not completely understood. E. col i 0 1 57:H7

do not elaborate the heat-labile and heat-stable enterotoxins that are

produced by enterotoxigenic E. col i and not enteroinvasive, as judged by

both Sereny test and the absence of invasion into epithelial cells in tissue

culture. However, E. col i 0 1 57:H7 do produce high levels of cytotoxin,

variously termed as verotoxin or Shiga-like toxin. In addition to toxin,

adherence to mucosal surfaces in the gastrointestinal tract is an

9

important primary step that results in bacterial colonisation of the

intestine necessary for delivery of elaborated toxins to enterocytes.

Verocytotoxins

Verocytotoxin-producing E. coli (VTEC) were first described by

Konowalchuk (1977), who identified a cytotoxin active on cultured Vero

cells (African green monkey kidney cells) and produced by certain strains

of E. coli; it was termed Vero cytotoxin (VT) . VT is clearly

indistinguishable, biologically and immunogically from the heat stabile

(ST) and heat-labile (LT) enterotoxins of E. coli, and reported to be closely

related to Shiga toxin which is produced by strains of Shigella dysentriae

type 1. Hence, these toxins were designated as Shiga-like toxins. Both

Shiga and Shiga-like toxin share the same receptor, and have similar

structure and modes of action (Robinson et al. , 1980). The gene coding

for Shiga-like toxin I is very similar to that of Shiga toxin, while the gene

for Shiga-like toxin II shows 58 percent overall homology with that for

Shiga-like toxin.

Shiga-like toxins from E. coli 0157:H7 were first reported in 1983

when OBrien and co-workers found that isolates from two outbreaks in

the United States produced high levels of a cell-associated cytotoxin for

HeLa and Vero cells. On further analysis, it is clear that this organism

produces two kind of cytotoxins, one of which can be neutralised by anti-

10

Shiga toxin. The neutralisable cytotoxin was designated as Shiga-like

toxin I (SLT-I), and the other Shiga-like toxin II (SLT-II). If both toxins are

produced by the same strains, SLT-I predominates in cell lysates while

SLT-II is more active toxin in culture filtrate. The two Shiga-like toxins

are antigenically distinct and differ in their biological effects: Shiga-like

toxin II is less toxic to Vero cells, but more toxic for mice, and causes

hemorrhagic colitis in the adult rabbit, while Shiga-like toxin I does not

(Evans et al., 1977) .

Phage conversion is responsible for controlling the production of

several important bacterial toxins, including diphtheria toxin,

streptococcal erythrogenic toxin, botulinum toxin and staphylococcal

enterotoxin (Betley et al. , 1986) . Recently, the production of Shiga-like

toxin was shown to be determined by specific phages in selected strains

of both EPEC and EHEC (0 1 57) serotype isolated from humans (Smith et

ai. , 1983) which indirectly suggests that E. coli 0 1 57:H7 may have

acquired these toxins through phage mediated transfer (Strockbine et ai. ,

1986) .

Adherence and Attachment

Another property that makes E. coli 0 1 57:H7 virulent is its ability to

adhere to intestinal cells. The mechanism of bacterial attachment to the

intestinal mucosal cell remains controversial because there is no direct

II

evidence from human cases to asses the nature of intestinal colonisation

by E. col i 0 1 57:H7. However, experiments to observe the phenomenon

have been conducted in animal and cell cultures.

Studies in gnotobiotic piglets and cell cultures showed no evidence

of the extensive invasion and intracellular multiplication as seen with the

invasive bacteria such as Shigella (Tzipori et al . , 1 986). E. col i 0 1 57:H7

produce a distinctive microscopic lesion characterised by intimate

attachment of the bacteria to the apical intestinal mucosal cell and

localised destruction of the microvilli (Tzipori et al., 1 988; Tzipori et al . ,

1 989). The bacteria also exhibit localised adherence to cells in culture,

with dense concentration of actin microfilaments in a cup-like structure

in the cytoplasm beneath the attached bacterium (Knutton et ai. , 1 989) .

This attaching-efficacing lesion resembles that produced by

enteropathogenic E. coli strains in piglets and cell culture (Knutton et al.,

1 989; Tzipori et al . , 1989) .

The mechanism may indicate an initial contact followed by more

intimate attachment, such as has been described by enteropathogenic E.

col i (Knutton et ai. , 1989) . The initial attachment may be mediated by the

60 MDa plasmid while the intimate attachment may be chromosomally

mediated (Toth et al., 1 990) . Most E. col i 0 1 57:H7 carry a 60 MDa

plasmid . There are several different opinions on the role of the plasmid.

Karch et ai. ( 1 987) determined that the plasmid was required for

1 2

expression of a fimbrial adhesin and adherence to Henle 407 intestinal

cells. The strains that were cured of the plasmid failed to express

fimbriae and lost the ability to adhere to intestinal cells. Recently, Toth et

al. ( 1 990) determined that the 60 MDa plasmid appear to modify the

eucaryotic cell adherence of E. coli 0 1 57:H7 and that adherence was

conferred to an E. coli transformant.

However, Junkin and Doyle ( 1 989) revealed that adherence to Henle

407 cells by E. coli 0 1 57:H7 strain 932 was not dependent on the 60

MDa plasmid mentioned. They showed that the test strain 932 adheres

to the human small intestine cell line INT407 at an average level of 7 . 1

bacteria per INT407 cell without the strain harboring the plasmid.

Complications of E. coli 0157:H7lnfections

Since the occurrence of the outbreaks in 1 982 , continued

epidemiologic, clinical, and laboratory investigations clearly established

E. coli 0 1 57:H7 to be an important etiologic agent of hemorrhagic colitis.

Typical hemorrhagic colitis can be distinguished clinically from bloody

diarrhea or dysentery seen in shigellosis, Campylobacter spp. and

enteroinvasive E. coli enteritis, amoebiasis, or other enteric illnesses such

as narcrotising enterocolitis or pseudomembraneous colitis by the lack of

prominent fever (Riley, 1987) . The illness caused by E. coli 0 1 57:H7

resolves in most patients with no sequel. Early outbreak studies in which