UNIVERSITI PUTRA MALAYSIA

CHARACTERISATION OF SALMONELLA ENTERITIDIS ISOLATES FROM HUMANS, ANIMALS AND ENVIRONMENTAL SOURCES.

MAIMUNAH BT. MUSTAKIM

FSMB 1998 2

CHARACTERISATION OF SALMONELLA ENTERITIDIS ISOLATES FROM HUMANS, ANIMALS AND ENVIRONMENTAL SOURCES.

By

MAIMUNAH BT. MUSTAKIM

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

Food Science and Biotechnology, Universiti Putra Malaysia.

August 1 998

With special dedication to my loving children:

Hanis Syafiqah

Arinah

Naim

Nazrin

ACKNOWLEDGEMENTS

The magnitude and scope of work involved in this research

required the assistance of numerous people, to whom the author

indebted, however great or small their respective contribution.

F irst, the author expresses her great appreciation to Dr. Son

Radu, the Committee Chairman, for his kindness, wi l l ingness to help,

and encouraging advise throughout the research work.

Sincere appreciation was extended to Professor Dr. Gulam Rusul

Rahmat Ali and Dr. Zaiton Hassan, the Committee Members,

especially for their suggestions and critical reviews of the thesis.

Very special thanks dedicated to al l her friends in Microbiology

Laboratory : Sahi lah, Kak Endang, Zainuri , Zunita, Dayang Fredal ina,

Loke Chui Fung, Rozila, Wong Wooi Koon, Yuherman, Samuel, Kami l

and Razal i .

The family : beloved mother, husband and chi ldren, their never

ending devotions and moral support throughout her education were

gratefully acknowledged.

i i i

TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . i i i L IST OF TABLES . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . v i i L IST OF F IGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . ix L IST OF PLATES . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . x ABSTRACT . . . . . . . . , . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i i i

CHAPTER

I I

I I I

GENERAL INTRODUCTION . . . . . . . . . . . . . . , . . . . . . ... . . . . 1

LITERATURE REViEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Genus Salmonella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Salmonella in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Salmonella in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella i n Natural Environment . . . . . . . . . . .

Worldwide Problem of Salmonel losis . . . . . . . . .

The Systematics of Salmonella . . . . . . . . . . . . . . .. . . . . . . . . . . . . . D ivisions of Salmonella into Serotype . . .

Subdivisions of Salmonella Serovars . . . . . .

Appl ication of Molecular Techniques in Subdivision of Salmonella Serovars . . . . . , . . . . . . . . . . . . . .

Plasmid Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conjugal Transfer of R Plasmids . . . . . . . . .

Chromosomal DNA Analysis by Pulse-field Gel Electrophoresis . . . . . . . . . . . . . . . . , . . . . . . . . . . .

Pulsed-field Gel Electrophoresis . . . . . . . . . . . .

Restriction Endonuclease for Chromosomal DNA Analysis . . . . . . . . . . . . . . . . . . . .

BIOTYPING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . .

Materials and Methods . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . .

Collection and Maintenance . . . . . . . . . . . . . . . . . .

Biotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .

Results . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . .

Biotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iv

7 7 8

1 0 1 4 1 6 1 7 1 8 24 27

33 34 38 39

42 43

45

47 47 50 50 51 55 55

Biotyping for S. enteritidis Phage-typed Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

IV PLASMID PROFILING AND ANTIMICROBIAL RESISTANCE OF S. ENTERITIDIS ISOLATES . . . 58 Introduction . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Bacterial Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Isolation of Plasmid DNA . . . . . . . . , . . . . . . . . . . . . 60 Detecting Plasmid DNA by Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Gel Visual isation and Photography . . . . . . . . . . . . 62 Plasmid DNA Size Reference . . . . . . . ; . . . . . . . 63

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Plasmid Profiles Analysis of Salmonella enteritidis Isolates . . . . . . . . . . . . . . . . . . . 65 Antimicrobial Resistance in S. enteritidis. . . . . 73 Relationships between Antibiotic Resistance and Possession of P lasmid . . . . . 75 Subdivision of S. enteritidis Phage-typed Strains by Plasmid Profi l ing and Antibiotic Resistance Patterns . . . . . . . . . . . . . . . . . . . 75

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

V CONJUGATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . . . . . 82 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Bacterial Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Conjugation of Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Preparation of Selective Media . . . . . . . . . . . . . . . 85 Determination of Plasmid(s) in Transconjugant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Discussion ... . . .. " ... ... ... ... ... ... ... ... ... ................ 90

VI RESTRICTION ENDONUCLEASE DIGESTION POLYMORPHISM OF S. ENTERITIDIS BY PULSED-FIELD GEL ELECTROPHORESIS . . . . . . 93 Introduction . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" . . . . . . 93 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Preparation of H igh Molecular Weight DNA by Agarose Embedding Bacterial Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Restriction Enzymes Digestion of DNA Plugs . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

v

Detecting Restriction Endonuclease Digestion by Pulsed-field Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Restriction Endonucleases . . . ... . .. . .. ... ... .... 98 Genome S ize Measurement . . . ... ... ... ......... 99 Data Analysis . . . ... ... ... ... .. . ... ... ... ... . .. . .. . . . . 99

Result . . . . . . . . . . . . . . , ... ...... ......... ... '" ... ... ... ... ... 1 02 Restriction Polymorphism of Chromosomal DNA of the S. enteritidis Isolates . . . . . . . . . . . . . . . . . 1 02 Distribution of Restriction Polymorph isms in S. enteritidis Isolates. . . . . . ... ... ... ... .......... 1 1 0 Variations of PFGE Patterns in Humans, Animals and Environmental Isolates . . . . . . . . . 1 1 2 Subdivision of S . enteritidis Phage-typed Strains by REDP-PFGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1"1'9 Characterisation of S . enteritidis Isolates by Restriction Polymorphism of the Chromosomal DNA. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . . . . . 1 1 9 Factors Influencing the Separation of DNA Fragments by Pulsed-field Gel Electrophoresis . . . . . . . . . . . . . . . .................... ... ... 1 20 Comparison of the Restriction Endonuclease Patterns in Humans, Animals and Environmental Sources . . . ... ... 1 2 1 Genomic Size of S . enteritidis. . . . . . . . . . . . . . . . . . . . . 1 23 Characterisation of S . enteritidis Phage-typed Strains by REDP-PFGE . . . . . . . . . . . . . . . . . . . 1 23

VI I GENERAL DISCUSS ION AND CONCLUSION . . . . . 1 26

REFERENCES . . . ... ... . .. .. . . . . . .. .. . ... ... ... ... . . . ... ... ... ... ... .... 1 31

APPEND IX . . . .... . . . . . . . . . . . ....... ....... . . ... . . . . . ....... ......... . . ... ............. 1 39

VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" ... ... . . . ... ... . . . ... ... ... ... ... . . . . . 1 49

vi

LIST OF TABLES

Table Page

1 Source of Food Contamination . . . . . . . . . . . . . . . . . . . . . . . . 1 2

2 Differentiation of Salmonella with other genus of the Enterobactericeae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

3 Abbreviated Kaufmann-White Scheme of Salmonella Serovar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

4 Determination of S. typhimurium Phage Type . . . . . . 30

5 Biotyping Scheme for Salmonella enteritidis . . . . . . . . 49

6 The 1 05 S. enteritidis Isolates . . . . . . . . . . . . . . . . . . . . . . . . 50

7 Biotyping of S. enteritidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

8 Biotyping of S. enteritidis Phage-typed Strains . . . . . 56

9 Plasmid Profi les in Salmonella enteritidis . . . . . . . . . . . 66

1 0 Plasmid Classes of S. enteritidis Isolates . . . . , . . . . . . . 66

1 1 Antimicrobial Resistance Patterns of S . enteritidis from Various Sources . . , . . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . 74

1 2 Relationships between Antibiotic Resistance Patterns and Plasmid Profi les . . . . . . . . . . . . . . . . . . . . . . . . 76

1 3 Relationships Among Phage-type, P lasmid Profi les and Antibiotic Resistance Patterns . . . . . . . . . 77

1 4 The Abi l ity of Gene Transfer Among S . enteritidis Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

1 5 Characterization of Transconjugants . . . . . . . . . . . . . . . 89

1 6 REDP-PFGE of S . enteritidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1

1 7 Interpretation of Pulse-field Gel Electrophoresis Patterns . . , ' " . . . . . , . . . ' " . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3

vii

1 8 Simi larity Index ( F Values) for S . enteritidis Strains from Xbal Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 1 1 5

1 9 Simi larity Index (F Values) for S . enteritidis Strains from Spel Digestion . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . , . . 1 1 6

20 Subdivision of S. enteritidis Phage-typed Strains by REDP-PFGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8

viii

LIST OF FIGURES

Figure Page

1 Comparison of Reported Cases of Salmonellosis Excluding Typhoid Fever between the Year 1 941 to 1 985 and 1 988 to 1 995 . . . . . . . . . . . . ... . . . . . . . . . . . . . . . 3

2 Polysaccharide Structure of the B Antigenic Group of Salmonella . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . . . 25

3 Physical Forms of Plasm ids . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

4 Graph of E lectrophoretic Mobi l ity for DNA S ize Standard of V51 7 ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

5 Graph of Electrophoretic Mobi l ity for DNA S ize Standard of A, Concatamer . . . . . . . . . . . . . . . . . . . . . ' " . . . . . . 1 00

6 A Schematic Representative of Xbal-PFGE F ingerprints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03

7 A Schematic Representative of Spel-PFGE Fingerprints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . 1 07

ix

LIST OF PLATES

Plate Page

1 E lectron micrograph of P lasmid DNA . . . . . . . . . . . . . . . 36

2 Electron micrograph of Bacterial Conjugation . . . . 40

3 Plasmid Patterns of S. enteritidis Isolates from Several Animal Sources . . . . . . . , . . . . . . . . . . . , . . . . . . . . . . . , . 69

4 Plasmid Patterns of S. enteritidis Isolates from Several Animal Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 70

5 Plasmid Patterns of S. enteritidis Isolates from Human Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1

6 Plasmid Patterns of S. enteritidis Isolates from Human Sources . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . 72

7 Plasmids in Transconjugant of S . enteritidis Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

8 REDP-Xbal of S . enteritidis Isolates . . . . . . . . . . . . . . . . . . 1 05

9 REDP-Xbal of S . enteritidis Isolates . . . . . . . . . . . . ' " . . . 1 06

1 0 REDP-Spel of S. enteritidis Isolates . . . . . , . . . . . . . . . . . , 108

1 1 RED P-Spel of S. enteritidis Isolates . . . . . , . . . . . . . . . . . , 1 09

x

Abstract of thesis presented to the Senate of Universit i Putra Malaysia in fulfi lment of the requirements for the degree of Master of Science.

CHARACTERISATION OF SALMONELLA ENTERITIDIS ISOLATES FROM H UMANS, ANIMALS AND ENVIRONMENTAL SOU RCES

By

MAIMUNAH MUSTAKIM

August 1 998

Chairman Dr. Son Radu

Faculty : Food Science and Biotechnology

One hundred and five strains of Salmonella enteritidis isolated

from five different sources including humans, food animals, avian, wi ld

animals and envifOnments were studied. All isolates were

characterised for their biotype, plasmid profi les and resistance to

antibiotics. Biotyping of the isolates subdivided them into two biotypes

i .e., A and E . Subdivision of the isolates by plasmid profi l ing identified

fifteen plasmid patterns i . e. , PPO to PP1 4. Plasmid pattern P P 1 4 was

the most common pattern encountered in 47 (44%) of the total isolates

from various sources. Resistance to antibiotics was not uncommon

with 1 04 (99%) of the isolates were resistant to more than one

antibiotics. Subdivision by antibiotic resistance pattern exhibited ten

antibiograms designated as R1 to R1 0. A total of 87 (82%) of

xi

the isolates fel l into resistant pattern R 1 which showed resistant

towards erythromycin, penici l l i n G and triple sulpha. In conjug�tions

studies, the abi l ity of transferring antibiotic resistance was

demonstrated in the transconjugants with the concomitant transfer of

the respective p lasmid and resistant to tetracycl ine. S ixty seven

representative strains of S. enteritidis studied for restriction

endonuclease digestion polymorphism by - pulsed-field gel

electrophoresis (REDP-PFGE) exhibited different PFGE fragment

patterns. Digestion with low-frequency-cleavage restriction

endonucleases, i . e . , Xbal ( 5'- TCTAGA-3' )and Spel (5'-ACTAGT-3')

produced five and three restriction patterns respectively. Inter­

relationships of the PFGE patterns among the isolates could be

determined by the calculation of simi larity index, F. The F values

grouped the strains as closely related, unrelated or ind istinguishable.

The phage-typed strains of S. enteritidis were subdivided into one

biotypes, eleven p lasmid profi les, six resistance patterns and seven

restriction polymorph isms. In general , characterisation by DNA-based

method of plasmid profil ing gave more discrimination compared to

other methods. These methods also provide effective adjunct to

antibiotic resistance pattern and phage typing for subdivision of S.

enteritidis.

xii

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

PENCIRIAN PENCILAN SALMONELLA ENTERITIDIS DARI SUMBER­SUMBER MANUSIA, HAIWAN DAN PERSEKITARAN

Oleh

MAIMUNAH MUSTAKIM

Ogos 1 998

Pengerusi : Dr. Son Radu

Fakulti : Sains Makanan dan Bioteknologi

Seratus l ima penci lan Salmonella enteritidis diperolehi dari

pelbagai sumber termasuk manusia, haiwan ternakan, beburung,

haiwan l iar dan persekitaran telah dikaji . Kesemua penci lan itu

dicirikan secara biotip , pemprofi lan plasmid dan sifat kerintangan

kepada antibiotik. Secara b iotip , pencilan itu telah dibahagikan kepada

dua iaitu biotip A dan E. Pembahagian secara plasmid pemprofi lan

menunjukkan penci lan itu boleh dibezakan dengan l ima belas corak

plasmid iaitu PPO - PP1 4. Corak PP1 4 tersebar secara meluas di

kalangan sumber-sumber yang dikaj i . Kerintangan kepada antibiotik

adalah perkara biasa bagi penci lan dengan 1 04 (99%) penci lan

adalah rintang kepada sekurang-kurangnya satu jenis antibiotik.

Pembahagian secara corak kerintangan menghasi lkan sepuluh corak

iaitu R1 h ingga R1 0. Sebanyak 87 (82.9%) penci lan itu termasuk di

xiii

dalam corak R 1 yang menunjukkan kerintangan kepada eritromicin,

penici l l in G dan 'triple-sulpha' .Di dalam kajian konjugasi, kebo.lehan

memindahkan s ifat-sifat kerintangan telah ditunjukkan di dalam

transkonjugan di mana sifat kerintangan telah dipindahkan bersama­

sama dengan plasmid. Kajian ke atas enampuluh tujuh waki l isolat­

isolat dari pelbagai sumber secara pol imorfisma penghadaman enzim

restriksi yang dihasi lkan melalui e lectrophoresis gel "pulse-field"

menunjukkan corak-corak P FGE yang berbeza. Penghadaman dengan

enzim restriksi pemotongan-frekuensi-rendah iaitu Xbal ( 5'-TCTAGA-

3') dan Spe l (5'-ACTAGT-3') masing-masing menghasi lkan l ima dan

tiga corak restriksi. Hubung-kait strain-strain di dalam sumber masing­

masing boleh dikira dari indeks persamaan, F. Dari ni lai F itu, strain­

strain dikumpulkan kepada berhubungrapat, tiada perhubungan atau

sama di antara satu sama lain. Pembahagian strain-strain fajtip

menghasi lkan satu corak biotip, sebelas corak plasmid dan tujuh

polimorfisma penghadaman enzim restriksi . Pada amnya, pencirian

dengan kaedah-kaedah berasaskan DNA menunjukkan lebih banyak

perbezaan di kalangan strain-strain. Kaedah tersebut juga sangat

berfaedah untuk digunakan bersama-sama kaedah antibiogram dan

fajtip di dalam pembahagian strain-strain S. enteritidis.

xiv

CHAPTER I

GENERAL INTRODUCTION

Typhoid, paratyphoid and salmonellosis are col lective terms

associated to infection and fevers caused by Salmonella species.

Salmonella typhi, the etiologic agent of typhoid fever has long been

threatened and led serious i l lness to human population in both

developed and third-world countries. The World Health Organisation

(WHO) has estimated that, annually, there are 1 6.6 mi l l ion cases of

typhoid fever with nearly 600,000 deaths, and 1 .3 bi l l ion cases of

acute gastroenteritis due to non-typhoidal salmonellosis with 3 mi l l ion

deaths. In developing countries, contaminated water supply due to

improper disposal of human and animal wastes is the usual mode for

transmission of the disease.

Typhoid fever was recognised by cl inical signs and symptoms

before the advent of bacteriological era. Beginning with the isolation

of Bacillus cholerae-suis from pigs with hog cholera in 1 885 by Salmon

and Smith, fol lowed by isolation of simi lar bacteria from foodborne

intoxication by Gaffky and Paak in 1 885 and Gartner in1 888 (Le Minor,

1

2

1 992) or animal disease by Loeffler in 1 892, those baci l lus were then

created a genus and named Salmonella by Lignieres in 1 900.

Salmonel la infection has been notified as a g lobal problem

ranking first or second as a cause of food and waterborne diseases

(Gangarosa, 1 980). In the United States for example, incidence of

sal monel losis increased gradually from below 1 000 cases in 1 941 to

70,000 cases in 1 985. The number has remained between 40,000 to

50,000 cases from 1 988 to 1 995 [Centre for Disease Contro l , 1 996;

Figure 1 (8)]. The epidemiological pattern of salmonel la infections has

changed with time, place and environmental conditions. These

changes have resulted due to the changes in food habits in the

community as wel l as changes in food production technology. Also, the

emergence of drug resistant salmonel la made the situation to be more

compl icated.

As Salmonel la problems have caused unnecessary suffering,

death and great economic losses which have led the public health

authorities in several countries to institute survei l lance programmes in

an attempt to control the rapidly increasing incidence of the disease. In

the United States, Salmonella survei l lance was instituted by Centre for

Disease Control (CDC) Atlanta, Georgia in 1 966 as a co-operative

undertaking by 50 states. In England and Wales, a systematic

col lection of information on salmonella infections was started in 1 949

70000

60000

50000

40000

30000

20000

10000

(A)

O+-���--�-r--��--'-��� 1941 1945 1951 1955 1961 1965 1971 1975 1981 1985

50,000 45,000 40,000 35,000 30,000 25,000 20,000 15,000 10,000

(8)

5,000 O+-�---r--���-r--'-� 1988 1989 1990 1991 1992 1993 1994 1995

Figure 1 : Comparison of Reported Cases of Salmonellosis excluding Typhoid Fever between the Year 1 941 to 1 985 (A) and 1 988 to 1 995 (8).

3

( Source : A - Salmonella, 1 992. 8 - Centre for Disease Control and Prevention, 1 996)

4

by Salmonella Subcommittee of the Publ ic Health Laboratory

Services. Survei l lance program for typhoid in Malaysia is managed by

the Epidemiology Division, Ministry of Health. General ly,

salmonel la survei l lance programme implemented in many countries

over the world are important in identifying outbreaks, high risk

foods and the mechanism by which transmission occurs.

The genus Salmonella belongs to the fam i ly Enterobactericeae

and comprises of more than 2,200 serotypes. Besides its medical

importance, this genus has attracted the interest of a great number of

scientists (bacteriologists, immunologists, etc. ) for its wide range of

variations in the classification systems. These organisms have been

subdivided in many ways especial ly for epidemiological purposes on

the basis of biochemical reactions, serological analysis or further

ampl ified by phage typing. Based on biochemical characteristics,

Kauffmann subdivided the genera into four major groups: I , I I , I I I and IV

(Le Minor, 1 992) . Then they were further differentiated into seven

subspecies i .e . , I , I I , ilia, I I Ib , IV, V and VI by studies of the thermal

stabi l ity of hybrids (Le Minor, 1 992) . Description of serological

identification based on the 0 (somatic), VI (capsular) and H (flagel lar)

antigens distinguished the strains into more than 2 ,200 serotypes

(Kauffmann and White scheme). In 1 938, Craigie and Yen introduced

phage typing for S. typhi which proved to be a valuable and sensitive

tool in the control of typhoid fever. The abil ity of phage to distinguished

5

varieties among identical serotypes led to the development and

acceptance of phage typing as a significant epidemiolog ical

procedure. With the advent of DNA technology era, several molecular

approaches for bacterial identification have been employed

in Salmonella. The DNA-related techniques such as plasmid analysis ,

restriction endonuclease digestion polymorphism (REDP) , ribotyping

and polymerase chain reaction (PCR) were able to identify individual

strains with remarkable precision.

Objectives

Nontyphoidal salmonellosis has been recognised as the most

frequent foodborne infection world-wide. Incidence of foodborne

outbreaks in recent years observed the emergence of Salmonella

enteritidis as the most commonly isolated serotype in many parts of the

world. For instance , the isolation frequency of S. enteritidis from

humans in Britain, Greece, Spain and the United States showed a

significant increase of more than 50% between 1 980 and 1 994 (Ridley

et a/. , 1 996 ; Vatopoulos et a/. , 1 994; Anon, 1 991 and Centre for

Disease Contro l , 1 993).

Due to the recent prominence of S. enteritidis, identification of the

strain is essential for an effective investigation of the outbreak cases.

Combination of conventional as wel l as molecular typing methods

6

have been employed for a precise differentiation of the individual

strain. In the present work, S. enteritidis strains which are multiply

resistant to antimicrobial agents isolated from animals, humans and

environmental sources were characterised by using conventional and

molecular typing methods.

The specific objectives of the present investigation were as

fol lows:

1 . To carry out biochemical tests for biotyping analysis.

2. To characterised the strains by plasmid content, antimicrobial

resistant patterns and their correlation.

3. To study the abi l ity to transfer resistance by conjugation studies.

4. To characterise the representative strains of S. enteritidis by

restriction endonuclease digestion polymorphism generated by pulsed­

field gel electrophoresis (REDP-PFGE).

5. To apply the REDP-PFGE technique in determining the genome

size.

6. To subdivide the S. enteritidis phage typed strains by biotyping,

plasmid profi l ing, antibiotic resistance patterns and REDP-PFGE

methods.

CHAPTER II

LITERATURE REVIEW

Introduction

The Salmonella was first coined by Lignieres in 1 900 as an

honour to D .E . Salmon, an American bacteriologist who reported on

"the hog cholera group of bacteria" in swine. These gram-negative rod­

shaped bacteria are confirmed to be the member of the fami ly

Enterobacteriaceae and were assigned to genus I I I in the fami ly (Le

Minor, 1 984). They were characterised as moti le with petrichous

flagel la, facultatively anaerobic and usual ly produced gas from g lucose

fermentation. They were also characterised as positive in hydrogen

sulphide production, citrate uti l isation and decarboxylation of lysine

and ornithine. In contrast, they were negative for indole, urease,

phenylalanine and tryptophan.

Although this group of bacteria are ubiquitous in nature, some

are strictly host adapted. They have been known to be pathogenic to

humans causing enteric fever, gastroenteritis and septicaemia. These

pathogens also infect many animal species.

7

8

The systematics with respect to classification and nomenclature

of Salmonella have been in a state of flux for many years. The

classification authorities in different countries placed these bacteria in

different species or nomenclature that was inconsistent to the

international agreement. The existing taxonomic schemes based on

the biochemical characteristics and serology (Kaufmann, 1 966;

Edwards and Ewing 1 986; Le Minor, 1 992) have been traditional ly

attributed in the classification of this genus. In general practiceL these

bacteria were named either for the disease caused or animal involved.

For example, the human medical community embraced and used such

specific epithets as S. typhi, S. paratyphi etc. The veterinary medical

community were preferable to name the bacteria with the affected

animal species such as Salmonella choleraesuis, S. pollorum, S.

gallinarum, S. bovis morbificans etc. In addition, there were also

names associated to geographical areas such as S. arkansas, S.

birmingham, S. jurusalem, S. panama etc.

The Genus Salmonella

The Salmonella are natural inhabitants of the intestinal tract of

food animals including cattle, swine, sheep, chicken, turkeys and

ducks. This bacteria are also found in pet animals such as dogs, cats

and turtles. The wi ld animals l ike pigeon , repti les, m ice, rats and

insects are also natural reservoirs of the Salmonella. Usual ly,

9

disposals (faeces and urine) of the infected animals have been the

main sources of Salmonella spread to the environment and then

transmit the infection to other susceptible animals or human being.

This genus are classified into three main groups by World Health

Organisation (WHO) according to the host and mode of transmission

(Guthrie, 1 991 ) . The first group consisting of S. typhi, S. paratyphi A, B

and C , only associated to human infections. These serovars are

spread primarily either by food or water which have been d irectly or

indirectly contaminated by human waste. The second group is

considered to include serovars that are host-adapted in animals.

Included in this group are S. gallina rum adapted to poultry, S. dublin

adapted to catt le, S. abortus equi adapted to horse, S. abortus ovis

adapted to sheep, S. choleraesuis and S. typhisuis adapted to swine.

Some of this serovars particularly the dubl in and choleraesuis , are

pathogenic to human under certain condition. The third group are

serovars which show no particular host adaptation and are pathogenic

in either humans or animals. Including in this group are S. enteritidis

and S. typhimurium which cause the majority of salmonel los is

infection. Salmonel losis in humans caused by organisms from the

second and third group would be considered as zoonotic

salmonel losis.

1 0

Salmonella in Humans

Typhoid fever or enteric fever has plagued the human

populations for more than a century. Salmonella typhi, the etiologic

agent of typhoid fever was one of the earliest pathogenic bacteria to

be isolated and characterised (Le Minor, 1 992). This fever is an acute

i l lness causing bacteremic or septicemic infections. Paratyphoid fever

caused by S. paratyphi A, 8 or C, is pathological ly and clinical ly

s imi lar to typhoid, but i t forms mi lder i l lness. Typhoid infection in

humans is initiated by ingestion and survival of the bacteria in the

acidic pH of the stomach. With adequate numbers of infectious dose,

the bacteria penetrate the mucosa of the smal l intestine to the midlayer

where they are engulfed into the epithel ial cel ls . At this stage, the

bacteria wi l l cause inflammatory response in the small bowel and the

colon which then produced diarrhoea in the infected persons. A

systemic infection characterised by prolonged fever, headache,

enlargement of the spleen, rose spots on the abdominal surface,

constipation (50% of the cases) , diarrhoea (20% of cases) and muscle

soreness. The incubation period in such an infection may be as long

as 3 weeks fol lowing ingestion of the infectious dose of organisms. In

the past, the mortal ity rate in the untreated typhoid fever was as high

as 1 5%, but with antibiotic treatment this has been reduced to less

than 1 % (Guthrie, 1 991 ).