UNIVERSITI PUTRA MALAYSIA

ISOLATION AND MOLECULAR CHARACTERISATION OF LISTERIA MONOCYTOGENES AND LISTERIA INNOCUA FROM POULTRY

MEAT

LESLEY MAURICE BILUNG

FSMB 2002 15

ISOLATION AND MOLECULAR CHARACTERISATION OF LISTERIA MONOCYTOGENES AND LISTERIA INNOCUA FROM POULTRY MEAT

By

LESLEY MAURICE BILUNG

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, In Fulfilment of the Requirement for the Degree of Master of

Science

April 2002

DEDICATION

Dedicated to Father of Glory, Lord of Glory and Spirit of Glory

"Blessed are You, Lord God of Israel, our Father, forever and ever.

Yours, 0 Lord, is the greatness, The power and the glory,

The victory and the majesty; for all that is in heaven and in earth is Yours;

Yours is the kingdom, 0 Lord, and You are exalted as head over aIL

Both riches and honor come from You, And You reign over all.

In Your hand is power and might; In Your hand it is to make great

and to give strength to aIL Now therefore, our God, we thank You

And praise Your glorious name. But who am I, and who are my people,

That we should be able to offer so willingly as this? .

For all things come from You, And of Your own we have given You."

1 Chronicles 29:10-14

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

ISOLATION AND MOLECULAR CHARACTERISATION OF LISTERIA MONOCI'TOGENES AND LISTERIA lNNOCUA FROM POULTRY MEAT

By

LESLEY MAURICE BILUNG

Apri12002

Chairman: Associate Professor Dr. Son Radu

Faculty: Food Science and Biotechnology

Thirty isolates of Listeria monocytogenes (12) and Listeria innocua (18) were isolated

from poultry meat. All isolates were confirmed by Microbact (Medvet, Australia)

identification kits. All the isolates were subjected to chromosomal and plasmid

DNA screening and antibiotic resistance test. Based on the antibiotic resistance

profiles, Listeria monocytogenes and Listeria innocua were differentiated into 10 and

9 profiles respectively. The antibiotyping procedure discriminated the Listeria

monocytogenes and Listeria innocua into 10 and 3 different groups respectively.

Most of the isolates were resistant to nalidixic acid (100%), c1indamycin (97%),

spectinomycin (97%), cefuroxime (93%), cefriaxone (80%), cephalothin (73%),

cefotaxime (67%), novobiocin (37%), chloramphenicol (27%), kanamycin (20%),

rifampicin (20%), tobramycin (17%), norfloxacin (13%), netilmicin (10%) and

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imipenem (3%). The results of the plasmid profiles and antibiotyping show that

there is no correlation between them. RAPD-PCR has been used to generate

polymorphic genomic fingerprints to discriminate the Listeria isolates. Primer

GEN15009 was chosen whereby it produced reproducible and typeable results in

all isolates examined with the bands ranging from 0.25 to 3.0 kilobase pairs.

From the dendrogram generated L. monocytogenes were separated from L. innocua

and the strains in each species were differentiated as well. The data indicate that

RAPD-PCR based approaches is a valid means of discriminating strain

differences among isolates of L. monocytogenes and L. innocua and as an adjunct to

differentiate among Listeria spp.

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

PEMENCILAN DAN PENCIRIAN MOLEKULAR LISTERIA MONOCYTOGENES DAN LISTERIA INNOCUA DARI DAGING A YAM

Oleh

LESLEY MAURICE BILUNG

April 2002

Pengerusi: Profesor Madya Dr. Son Radu

Fakulti: Fakulti Sains Makanan dan Bioteknologi

Tiga puluh pencilan Listeria mOllocytogenes (12 pencilan) dan Listeria imlOcua (18 pencilan)

telah dipencilkan dari daging ayam. Semua pencilan telah dianalisis menggunakan

Microbact (Medvet, Australia) identification kit. Semua pencilan telah dikaji untuk

kerintangan kepada antibiotik dan polimorfik menggunakan analisis random

amplification of polymorphic DNA (RAPD). Berdasarkan kepada profil kerintangan

antibiotik, Listeria mOllocytogenes dan Listeria illnocua dibezakan kepada 10 dan 9 profil

masing-masing. Kaedah antibiotip mengasingkan Listeria mOllocytogelles dan Listeria

illl10ata kepada 10 dan 3 kumpulan-kumpulan yang berbeza-beza. Kebanyakan pencilan

rintang terhadap nalidixic acid (100%), clindamycin (97%), spectinomycin (97%),

cefuroxime (93 % ), cefriaxone (80 % ), cephalothin (73 % ), cefotaxime (67% ),

novobiocin (37%), chloramphenicol (27%), kanamycin (20%), rifampicin (20%),

tobramycin (17%), norfloxacin (13%), netilmicin (10%) dan impenem (3%).

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Keputusan profil plasmid dan anbbiotip menunjukkan tidak ada perkaitan di antara

mereka. RAPD-PCR telah digunakan untuk menghasilkan corak genomic polimorfik

untuk membezakan penci1an - penci1an Listeria. Primer GEN15009 dipilih selepas

menyaring 10 primer. Primer tersebut menghasilkan keputusan yang pada keseluruhan

penci1an yang dikaji dengan saiz jalur di antara 0.25 kb ke 3.0 kb. Daripada

dendrogram yang diperolehi L. monocyrogenes dipisahkan dari L. innocua dan

pencilan setiap spesis juga dibezakan. Data menunjukkan RAPD-PCR dapat

digunakan sebagai satu cara untuk mendiskriminasikan pencilan - penci1an L.

monocytogenes dan L. innocua, dan untuk membezakan spesis Listeria.

Have you not known?

Have you not heard?

ACKNO�EDGEMENTS

The everlasting God, the Lord,

The Creator of the ends of the earth,

Neither faints nor is weary.

His understanding is unsearchable.

He gives power to the weak,

And to those who have no might He increases strength.

He gives the power to the weak,

And to those who have no might He increases strength.

Even the youths shall faints and be weary,

And the young men shall utterly fall,

But those who wait on the Lord

Shall renew their strength;

They shall mount up with wings like eagles,

They shall run and not be weary,

They shall walk and not faint (Isaiah 40: 28-31)

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I've been constantly reminded by God that He is my strength and only in Him I

can be fruitful. I do believe that where I am now is because of His grace, love and

beautiful plan for me. He's always been my ability, my wisdom, my peace, my

joy, my strength, my confidence ......... and He is my everything, He is my all.

My deepest gratitude goes to my supervisor, Associate Professor Dr. Son Radu

for trusting me and giving me the opportunity to do my MSc. I treasured and

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appreciate his patience, guidance, advices and encouragement through out my

study years (since I was doing my BSc). I learn a lot from him.

My appreciation also goes to Dr Kasing and Dr Raha for their support and co­

operation. They have been very helpful.

Not forgetting my family (dad, mum, Gadreal, Timothy and Rachel) for their

unending love, prayers and encouragement. Words could not express my

gratitude to all of you.

Special thanks goes to all my cousins (Bhie, Emma, Daniel & team), aunts, uncles,

and grandparents (tepu' Liang, the late of tepu' Sheila, tepu' Siren and tepu'

Bulan) for their love and prayers.

Also, I thank my lab friends (Mild, Yousr, Sam, Liha, Zainuri, Kqueen, Wai Ling,

Nizam, Yin Sze, Yock Ann, Apitnya, Wu, Yanti, Alis, Yuherman, !bu Endang,

Chee Keong, Kak Zaleha, Kak Zunita, Kak Sahilah, Naseer, Sushil, Lawrence,

Lim, Wan, Gwen, Priyan, Ziana, Ain, Mai, Jurin and Vicky) for helping me in my

work and patiently teaching me so many things. Thank you for your friendship,

it means a lot to me.

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Last but not least, I thank my housemates (Zain, Abit, Bees, Mag, Win, Sera,

Chris, Thy, Irene, Ang), our pastor Sisto Annie, deacons (Navies, Sibez, Kie, Mim,

Elis, Alan, Bhie, Roger, Jiw and Pilai), teachers in S.R. Sri Sempurna, our cell

group members and brothers and sisters in Christ SIB Serdang for their support,

help and prayers. I am so bless to have all of you by my side ...... All glory, honor,

praise and thanksgiving be unto the Lord.

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I certify that an Examination Committee has met on 17th April 2002 to conduct the final examination of Lesley Maurice Bilung on her Master Science thesis entitled IIIsolation and Molecular Characterisation of Listeria monocytogenes and Listeria innocua from Poultry Meat" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regu1ations 1981. The Committee recommends that the candidate be awarded the relevant degree. The Committee Members for the candidate are as

follows:

Fatimah Abu Bakar, Ph.D, Lecturer, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Chairperson)

Son Radu, Ph.D, Associate Professor, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Member)

Raha Abdul Rahim, Ph.D, Associate Professor, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Member)

Kasing Ap� Ph.D, Lecturer, Faculty of Resource Science and Technology, Universiti Sarawak Malaysia (Member)

L---J � MOHAMAD RAMADILI, Ph.D, Professor/Deputy DeaI\ School of Graduate Studies, Universiti Putra Malaysia

Date: 1 e JUN 2002

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfillment of the requirement for degree of Master of Science.

AINI !DERIS, Ph.D, Professor jDean, School of Graduate Studies, Universiti Putra Malaysia

Date: 9 JAN 2003

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DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

Lesley Maunce Bilung

Date: 11( b / {)l-

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TABLE OF CONTENTS

Page DEDICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 2 ABSTRACT . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 3 ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ACKNOVVLEDGEMENTS . . . . . . ... . . . . . . . . . . . . . . . .. . ... . . . .. . . .... . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ....... 7 APPROVAL . . . . . . . . ... . . . . . . . . . . ... . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . ... . . . . . . ... 10 DECLARATION . . ... . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . ... . .. . . . . ... . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . 12 LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 15 LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 16 LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . .... . . . . . . . . . . . . . . . . . . . . . 17

CHAPTER

1 INTRODUCTION . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . ... . . .. . . . . . . . . . . . . . . . . .. . . . . .... . . 19

2 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . .. . . .. . ... ... . .. 25 2.1 Taxonomy of Listeria . . . ... . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 25 2.2 Habitat. . . . . . ... . . .... . . . . . ... . . . . ... . . . . . . . . . . . . . . . . . . ... . . . . . . ... . . .. . . . . . .... 28 2.3 Epidemiology ... . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . .... .28 2.4 Listeriosis . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . 30 2.5 Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . ....... 32 2.6 Pathogenicity . . . . . . . . . . . . . . . . . . . . . . ... . . . . . .... . . . . . . . . . . . . . . . ....... ........ .. 34 2.7 Occurrence in Foods . . .. . . . . . . . . ..... . . . . . . . . . . . . . . ... . . . . . . . .. . . .. ......... 35

2.7.1 Listeria in poultry meat. . . . ... . . . . . . . . . . . . . . . . ... . . . ..... . . . . .... . .37 2.8 Growth . . . . . . . . . . . . . . . . . ... . . . . . ... .. . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . .... 38 2.9 Control of listeriosis . . . . . ... . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . ... . . . . . . . . . . .. 39 2.10 Isolation of Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . ...... .41 2.11 Identification of Listeria species . . . . ........ .. . . ... . . . .. . . . . . . ... . . . ...... 42 2.12 Definition of drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 44 2.13 Antimicrobial Susceptibility . . . .. . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . ... 44

2.13.1 The Action of Antimicrobial Drugs ..... . . . . . . . . . . . . . . , ...... ..46 2.13.1.1 The Inhibition of Cell Wall

Synthesis . . .. . . . . .. . . . ..... . . . . ... . . . . . . . . . . . . . . . . 46 2.13.1.2 The Inhibition of Protein Synthesis ......... 47 2.13.1.3 Injury to the Plasma Membrane . . . . .. . ..... .47 2.13.1.4 The Inhibition of Nucleic Acid

Synthesis . . . . . . . . . . . .. . . . .. . . . . ... . . . . . . . . . . . . ..... 48 2.13.1.5 Inhibiting the Synthesis of Essential

Metabolites . . . . . . ... . . . . . . . . . . . . . . . . . ... . . . . . .... . 48 2.13.2 Drug Resistance ... . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . .49 2.13.3 Antibiotic Resistance in Listeria species . . . . . . . . . . .. . . . . . . .. .. 51

2.14 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . ... . . . . . .. . .. . . . . . . .. . . . . . . . . . . ... 52 2.14.1 Plasmids in Listeria species .. . . . . . . . . . . . . . . . .... . . . . . . . . . . ...... . .53

2.15 Polymerase Chain Reaction (PeR) . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . 54

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2.16 . Random Amplified Polymorphic DNA (RAPD) ....... .... .......... 58 216. 1 RAPD in listeria species . . .. . ............. ... ............. . ........ 59

3 ISOLATION AND IDENTIFICATION OF LISTERIA SPP. FROM roULTRY MEAT . . . . ............. ....... ...................................... ..... ... 61 3.1 Introduction ................. . ... . ....... ....... ................................ 61

3.1.1 Isolation .... ..................... . . . ..... . .. .... . . .... ................. 6 2 3.1.2 Biochemical tests and Identification of listeria

spp ......... .............. .............. ..... ...... . ............ ...... ... 65 3.2 Materials and Method .. . ....... ................. ......... . .................. 65

3.2.1 Samples Collection ............ ......... ........... .................. 65 . 3.2.2 Isolation and Identification of listeria spp. from Poultry

Meat Samples ........................................................ 66 3.23 Biochemical tests .................................................... 69

3.3 Results ... ........ ... .......... . ............ .... . ... . . .............. .... : ... .... .. 69 3.4 Discussion ......... ... � ............ .................. ...................... . ..... 72

4 ANTIBIOTIC SUSCEPTIBILITY TESTING OF LISTERIA SPP. . ... ........ 76 4.1 Introduction .... ........ .... ....... ..... .... . .................. ... ............... 76 4.2 Materials and Method .... ........... ......... . ............................... 78 4.3 Results ... . ..... ....................... .......................... .... ............. 79 4.4 Discussion ............................ ................................... ........ 84

5 PLASMID PROFILING OF USTERIA SPP . ...... .......................... ...... 91 5.1 Introduction .................. .............................. ... . .......... . ..... 91 5.2 Materials and Method ..... . ....... ............................ ..... .......... 92

5.2.1 Plasmid DNA Extraction Technique ............................ 92 5.2.2 Agarose Gel Electrophoresis ............ . ..... ................... 94 5.2.3 Molecular Weight Determination of Plasmids ............... 96

5.3 Results ...................... .... ......................... .......... .............. 96 5.4 Discussion .......................... . ... ....................................... 101

6 RAPD FINGERPRINTING OF LISTERIA SPP . ... ............................ 106 6.1 IIttroduction ...................... ..................... ......... ....... ......... 106 6.2 Materials and Method .............................................. , . .. . . . .. 107

6.2.1 Genomic DNA Isolation .. ....... ................................. 107 6.2.2 Primer of RAPD-PCR Reaction ........................ ...... .... 108 6.2.3 RAPD-PCR Cocktail Mixture ......... ..... ..... .. ............... 109 6.24 PCR Thermal Cycler ............................ ................... 109 6.25 Gel Electrophoresis for PCR .............. ...... ..... ......... ... II0

6.3 Results ...... . .............. ........ ..... ......... ....... . ........ ............ ... 111 6.4 Discussion . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . .. . ... . . . . . . . . . .. 112

7 CONQ.USION . ...... ................................................. ......... ... ...... 122 REFERENCE ....................... ... .... " .. . . . . . . . . . . . . .. .. . . . . . . . . . . , .... . . . . . . . . . . . . 126 APPENDIX ............................................................................... 146 BIODATA OF TIIE AUfHOR. ............. ....... ........................ ......... 152

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LIST OF TABLES

Table Page

Symptoms and clinical manifestation of listeriosis ......... . ..... . . . .. . . .... ... .... .24

U.S Meat and Poultry Recalls (1994-1998) ............................................ .24

Biochemical characteristic of the genus Listeria ...... . . ... . .. . . . . ..... . . . . ........... 43

Total Usage of Different Types of Antibiotic for Humans and Animals . . . .. . . . . . .. . ......... . . .. . . . ...... . . . . . . . .. . . . . ..... . . ..... ... .. .... .. . . . . . . . . ..... . . .. 50

Prevalence of Listeria spp. in Poultry Meat Samples . . ...... . . . . .. . . . . . . ... . .... .... 71

Resistance percentage of Listeria species examined from poultry ... . . .. ........ 81

Resistance percentage of Listeria monocytogenes examined from poultry .... . . . . . .. . . . . . .... . . . . .. . . . ... . .. . . . . . . . .. . . . ....... . . . . .... . ....... . . .. . . ...... . . . . . . . 82

Resistance percentage of Listeria innocua examined from poultry . .. . .. . . . . .. . . . . . . . . . . . . . .. . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . .. ... ... ... . . . .. . . . .. . . . ....... 83

Plasmid profiling patterns of Listeria species isolated from poultry samples . . .................. . . . . . ... . . .. . . .. . . . . . . ... . . . ... . . . . . . .. . . . . . . . . . .... . . ... 98

Correlation between antibiotic resistance patterns and plasmid profiles among L. innocua . .. . . . . .. . . . . .. . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . .. 99

Correlation between antibiotic resistance patterns and plasmid profiles among L.monocytogenes . .. . . . . .. . . . . . . ... ..... .. .. ........ . . ... ......... . . ...... 100

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LIST OF FIGURES

Figure Page

Ways in which L. monocytogenes is disseminated in the environments, animals, foods and humans ............................................................... 31

A Canadian version of the U.S. FDA Listen'a isolation protocol for Listeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . 68

Growth of colony of Listeria spp. obtained on P ALCAM agar ................... 70

Plasmid profiling of L. monocytogenes obtained with 0.8% agarose gel electrophoresis ................................................................ 104

Plasmid profiling of L. innocua obtained with 0.8% agarose gel electrophoresis ................................ , ....... " ................ , ............... '" ... 105

Dendrogram based on Gel Compar showing relationships between 12 strains of Listeria monocytogenes (LI) from poultry meat.. ...................... 117

Dendrogram based on Gel Compar showing relationships between 12 strains of Listeria innocua (LM) from poultry meat.. . " ... '" .. , .. , .............. 118

Dendrogram based on Gel Compar showing relationships between 30 strains of Listeria monocytogenes (LI) and Listeria innocua (LM) from poultry meat ................................................................................... 119

RAPD fingerprinting of L monocytogenes from poultry isolates generated with primer GENl5009 .. , ......... " .. " ....... , ............ , . ............... 120

RAPD fingerprinting of L innocua from poultry isolates generated with primer GENl5009 ....................................................... 121

AOAC AP-PCR BLEB CAMP DNA dNlPs EC EDTA ELISAs EtBr FDA G HO I IDF ISO KAc KCl LEB L-PALCAMY

M MAR MBC MDa MgCb mg MIC min ml mm mM

f..t.g

f..t.ID

f..t.l mol

LIST OF ABBREVIATIONS

Association Official Analytical Chemistry arbitrary primered- polymerase chain reaction Buffered Listeria Enrichment Broth Christie-Atkins-Munch-Peterson deoxyribonucleic acid deoxynucleotide triphosphates European Community Ethylenediamine tetra-acetic acid enzyme-linked immunosorbent assays Ethidium bromide Food and Drug Administration gram hydrochloriC acid Intermediate International Dairy Federation International Standard Organisation potassium acetate potassium chloride Listeria Enrichment Broth

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Polymyxin Acriflavine lithium Chloride Ceftazidime Aesculin Mannitol Egg Yolk Broth Molar or molarity (moles of solute per liter of solution) Multiple Antibiotic Resistance Minimal Bactericidal Concentration Megadalton magnesium chloride milligram Minimal Inhibitory Concentration minute(s) milliliter millimeter miliMolar microgram micrometer microliter mole

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N Normal NaCI sodium chloride NaOH sodium hydroxide PO Phenol.chloroform�Isoamyl alcohol PCR Polymerase Chain Reaction PFGE pulsed-field gel electrophoresis psi pound(s) per square inch (Ib/in2) R Resistant RAPD Random Amplification of Polymorphic DNA RNA ribonucleic acid rpm revolution per minute rRNA ribosomal ribonucleic acid S susceptible subsp. subspecies SDS Sodium dodecyl sulphate spp. species Taq Thermus aquaticus DNA (polymerase) TBE Tri-Borate EDT A elctrophoresis buffer Tris Tris (hydroxymethyl) methylamine lSA Tryptone Soy Agar lSB Tryptic Soy Broth U.S United States USDA United States Department of Agriculture USFDA United States Food and Drug Administration UV ultraviolet DVM University of Vermont V volts WHO World Health Organisation w/v weight/volume v/v volume/volume > more than °C degree Celsius

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CHAPTER 1

INTRODUCTION

The genus Listeria comprises six species: L. monocytogenes, L. innocua, L. ivanovii,

L. welshimeri, L. seeligeri and L. grayi. All species are ubiquitous and potential food

contaminants, but only L. monocytogenes is a human and animal pathogen,

although very rare cases of infection due to L. ivanovii and L. seeligeri have been

described (Boerlin et al., 1992; Rocourt et al., 1992; McLauchlin, 1997). L.

monocytogenes is gaining recognition as a severe human pathogen which can

cause listeriosis, a disease primarily affecting women and their neonates, patients

who are immunocompromised and the elderly as well. Listeriosis, mainly caused

by the consumption of foodstuffs, can occur as a sporadic disease or as part of an

outbreak (Hocking et al., 1997; Giovanacci et al., 1999). The symptoms and clinical

manifestation of listeriosis in human pathogens are summarized in Table 1.1

(Marth, 1988). Based on data collected on food consumption and association with

listeriosis, the most at risk foods are reported to be ready-to-eat foods such as

ready-cooked chicken, sliced ham, pate, processed meat pate, shellfish products,

soft and surface ripened cheese and foods held under refrigeration (Sutherland

and Poritt, 1997). From the data collected from USDA, 1999 (Table 1.2) reporting

the recalls of meat and poultry in the United States from 1994 to 1998, L.

monocytogenes caused the highest recalls of meat and poultry among the

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foodborne pathogens (Kuhn, 1998). Cells of Listeria are Gram positive, short,

regular rods with rounded ends that can occur singly, in parallel or in short

chains arranged to form a V shape. The cells are 0.4 - 0.5 J.lID in diameter and 0.5

- 2.0 f.1m in length. The organism is motile by means of few flagella when grown

at 20 - 25°C. Listeria spp. will grow in most bacterial culture media. Growth is

enhanced in the presence of glucose, serum and blood. Table 2.3 shows the

biochemical characteristics of the genus Listeria. The organisms are widespread

throughout nature, being commonly found in the environment and carried by

many species of both domestic and wild animals (Sutherland and Poritt, 1997).

The presence of any Listeria species in food maybe an indicator of poor hygiene.

Moreover the contamination of raw or processed food products by this organism

may result in drastic economic losses for the food industry. However, since L.

monocytogenes is the major human pathogen, it is important to be able to isolate

and correctly identify the different Listeria species, or at least differentiate L.

monocytogenes from the other genus (Mclauchlin, 1997; Giovanacci et al., 1999).

Methodology for the isolation of Listeria from food and environmental samples

relies on enrichment procedures, followed by selective plating. Traditional

isolation methods have partly been replaced by so-called rapid methods, of

which latex tests, enzyme-linked immunosorbent assays (ELISAs) and DNA

probes are commercially available (Hofstra et al., 1994). For identification on

21

species level, systems based on biochemical tests are available (Bannerman et ai.,

1992; Bille et al., 1992).

Listeriosis represents a public health problem since it is fatal in up to 30% of cases

(Farber and Peterkin, 1991; Jones and MacGowan, 1995). In general, isolates of

L. monocytogenes, as well as strains of other Listeria spp., are susceptible to a wide

range of antibiotics except cephalosporins and fosfomycin (Hof, 1991; Hof, et al.,

1997). The treatment of choice for listeriosis remains the administration of

ampicillin or penicillin G combined with an aminoglycoside, classically

gentamicin (Boisivon et al., 1990; MacGowan et a1., 1990; McLauchlin et al., 1991;

Franco et al., 1994; Jones and MacGowan, 1995; Lorber, 1997). The association of

trimethoprim with a sulfonamide, such as sulfamethoxazole in co-trimoxazole, is

considered to be a second--choice therapy. Other strains of Listeria spp. isolated

from food or the environment or in sporadic cases of human listeriosis resistant

to one or several antibiotics have been described (Quentin et al., 1990; MacGowan

et al., 1990; Slade and Collins-Thompson, 1990; Poyart - Salmeron et al., 1992;

Facinelli et aI., 1993; Hadorn et ai., 1993; Franco et al., 1994; Charpentier et al.,

1995).

The epidemiologic surveillance of diseases caused by several bacteria has been

made easier by the study of plasmid profiles (Mayer, 1988). The plasmids may

code for antibiotic resistance, toxins, adhesion fimbria, metabolic enzymes and

22

bacteriocin production (Mayer, 1988). The prevalence of plasmids and their

functions in Listeria is not well understood (Kolstad et al., 1990). Previous studies

had shown that Listeria harbour plasmid (Slade and Collins-Thompson.. 1990;

Kolstad et al., 1992; Peterkin et al., 1991; Lebrun et al., 1992; !som et al., 1995;

Harvey and Gilmour, 2001). The presence of plasmids in poultry bacteria is

indicative of a potential human health hazard, since these plasmid-containing

bacteria can be transferred to human through the consumption of contaminated

poultry meat (Son et al., 1999).

During the last decade, powerful bacterial molecular typing methods such as

pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis

(MLEE) and restriction enzyme analysis (REA) have been developed. These

methods usually exhibit a higher discriminatory power than phenotyping

methods and has proven useful to assess the distribution of Listeria strains

(Lawrence and Gilmour, 1995; Destro et al., 1996; Ojeniyi et al., 1996; Unnerstad et

al., 1996). Genomic fingerprinting via random amplification of polymorphic

DNA (RAPD) has been found to be of value with Listeria spp. and the usefulness

of RAPD analysis for the epidemiological study of Listeria isolates has been

confirmed by other groups (Mazurier and Wernars, 1992; Lawrence et al., 1993;

MacGowan et aI., 1993; Czajka and Batt, 1994; Farber and Addison, 1994;

Niederhauser et al., 1994; Boerlin et al., 1995).

23

The ability to identify Listeria quickly and reliably can be important in both

livestock industry and medicine for establishing the causes of undesirable

contaminations and precise determination of pathogenic strains. In Malaysia,

there is no report on the outbreak of listeriosis. However, as the isolation rate of

Listeria in foods is on the increased (Gulam et aI., 1991; Saleha et aI., 1998; Zaiton

et aI., 1998) there is an urgent need for precise determination and genetic

understanding of the organism. An understanding of genetic variability in

Listeria is important for the studies of the taxonomy, epidemiology and

pathogenicity of this species. The objectives of this study are to:

1. Isolate and identify Listeria spp. from poultry meat sample.

2. Determine the antimicrobial resistance patterns and plasmid profiles

among the Listeria isolates.

3. Detect the DNA sequence diversity of Listeria isolates by random

amplified polymorphic DNA (RAPD) analysis.

Table 1.1 : Symptoms and clinical manifestation of listeriosis

Symptoms Fever } Convulsions } Chills } LFlu' -like symptoms Backache } Headache } Diarrhoea Vomiting Discoloured urine

Oinical manifestations Pyrexia Meningitis / meningoencephalitis Septicaemia Spontaneous abortlon Granulomatosis infantiseptica (listeriosis of newborn) Conjunctivitis Oculoglandular listeriosis Cutaneous listeriosis Pneumonic listeriosis Cervicoglandular Mild, febrile gastroenteris

24

Sources: Marth, 1988. Disease characteristics of Listeria monocytogenes. Food Technology 42: 165-168.

Table 1.2 : US Meat and Poultry Recalls (1994-1998)

Cause 1994 1995 1996 1997 1998 Listeria monocytogenes 16 11 6 3 39 Extraneous material 13 9 2 7 37 E. coli 0157:H7 3 5 2 13 29 Processing failures 7 6 6 5 28 Misbranding 1 1 4 6 16 Drug 1 4 2 3 11

. Salmonella 0 2 1 3 7 All others 9 4 1 4 20 Total 50 42 42 44 18

Sources: US Department of Agriculture, Listeria monocytogenes, Kuhn, M.L. Food Processing, March 1998, p. 16-20.