UNIVERSITI PUTRA MALAYSIA

PREVALENCE AND RISK ASSESSMENT OF SALMONELLA SPP. IN SLICED FRUIT

PUI CHAI FUNG

FSTM 2011 2

PREVALENCE AND RISK ASSESSMENT OF SALMONELLA SPP. IN SLICED FRUIT

PUI CHAI FUNG

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2011

PREVALENCE AND RISK ASSESSMENT OF SALMONELLA SPP. IN SLICED FRUIT

By

PUI CHAI FUNG

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Master of Science

March 2011

Dedicated to my beloved mum (Chu Su Chey), family and friends for their eternity love and endless support

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

PREVALENCE AND RISK ASSESSMENT OF SALMONELLA SPP. IN SLICED FRUIT

By

PUI CHAI FUNG

March 2011

Chairman: Professor Son Radu, PhD

Faculty: Food Science and Technology

Fresh fruits and vegetables are increasingly recognized as a source of

salmonellosis outbreaks in many parts of the world. Such products are

always eaten raw or with minimal processing and if contaminated with

Salmonella, they may represent a health hazard to the consumers. The public

health importance of Salmonella in man led the present study to determine

the prevalence and quantity of Salmonella spp., Salmonella Typhi and

Salmonella Typhimurium in sliced fruits. In domestic kitchen, the food

contact surfaces are sometimes improperly cleaned which ease the

formation of biofilm by Salmonella. Subsequent detachment from the biofilm

layer can be at risk for contamination of fresh fruits and vegetables. Hence,

the quantification of biofilm formed by Salmonella Typhi and Salmonella

Typhimurium on plastic cutting board used in domestic kitchen was

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determined in this study prior to analyzing the transfer rate of these two

pathogens to the fruit model.

The most probable number (MPN)-multiplex polymerase chain reaction

(PCR) was used for the prevalence and quantification studies. A multiplex

PCR was optimized for the simultaneous detection of Salmonella spp.,

Salmonella Typhi and Salmonella Typhimurium in sliced papaya, watermelon,

mango, sapodilla, jackfruit, dragon fruit and honeydew. The prevalence of

Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in 210

samples of sliced fruits examined were 23.3%, 7.6% and 3.8%, respectively

with estimated quantity varied from 0 to 19 MPN/g. On the other hand, the

prevalence of Salmonella spp. discovered from the total sliced fruits of

hawker stalls were three times higher than those from hypermarkets at 30%

and 10% respectively at p<0.05. Salmonella spp. were detected in 23.3% fruit

samples using MPN-multiplex PCR and at 9.5% using MPN-plating method.

Out of the seven types of fruits examined, dragon fruit from hawker stalls

showed the highest prevalence of contamination with 75% Salmonella spp.,

40% Salmonella Typhi and 25% Salmonella Typhimurium. Hence, it was used

as the fruit model to study the biofilm formation by Salmonella Typhi and

Salmonella Typhimurium on plastic cutting board and their subsequent

transfer to dragon fruit. By using crystal violet assay, it was found that

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biofilm formation of Salmonella Typhi and Salmonella Typhimurium on

plastic cutting board was the highest at 12 h when they are incubated in

physiological saline solution at 28°C. On the transfer of these two pathogens

from plastic cutting board to dragon fruit, results showed their mean

transfer percentages were 92.38 and 91.30% respectively for Salmonella

Typhi and Salmonella Typhimurium. These highlighted that biofilm can

cross-contaminate dragon fruit and contribute to cross-infection. As a

conclusion, there is a need for the health authority to look into the risk

assessment of sliced fruits in Malaysia. The risk of microbial transfer from

food contact surface to fresh fruits should also be of concern to prevent

foodborne disease outbreaks.

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

PREVALEN DAN PENILAIAN RISIKO SALMONELLA SPP. DALAM BUAH HIRISAN

Oleh

PUI CHAI FUNG

Mac 2011

Chairman: Profesor Son Radu, PhD

Faculty: Sains dan Teknologi Makanan

Buah-buahan dan sayur-sayuran segar semakin diakui sebagai sumber

wabak salmonelosis di kebanyakan bahagian dunia. Produk-produk ini

selalu dimakan mentah atau melalui pemprosesan minima dan jika

dikontaminasi oleh Salmonella, ini membahayakan kesihatan pelanggan.

Kepentingan kesihatan masyarakat Salmonella ke atas manusia telah

mendorong kajian ini dilaksanakan supaya prevalen dan kuantiti Salmonella

spp., Salmonella Typhi and Salmonella Typhimurium dalam buah-buahan

hirisan boleh ditentukan. Di dapur rumah tangga, permukaan sentuh

makanan kadang-kadang tidak dibersihkan dengan betul dan ini

memudahkan pembentukan biofilem oleh Salmonella. Penyingkiran

selanjutnya lapisan biofilem berisiko ini dapat mengkontaminasi buah-

buahan dan sayur-sayuran segar. Oleh itu, kuantiti biofilem yang

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dihasilkan oleh Salmonella Typhi dan Salmonella Typhimurium pada

permukaan papan potong plastik yang digunakan di dapur ditentukan

terlebih dahulu, sebelum menganalisa kadar pemindahan kedua-dua

patogen ini kepada model buah.

Most probable number (MPN)-multiplex polymerase chain reaction (PCR)

telah digunakan untuk kajian prevalen dan kuantifikasi. Multiplex-PCR

dioptimumkan untuk mengesan Salmonella spp., Salmonella Typhi dan

Salmonella Typhimurium secara serentak dalam hirisan betik, tembikai,

mangga, ciku, nangka, buah naga dan tembikai susu. Prevalen Salmonella

spp., Salmonella Typhi dan Salmonella Typhimurium pada 210 sampel buah-

buahan hirisan yang dikaji adalah masing-masing 23.3%, 7.6% dan 3.8%

dengan jangkaan kuantiti berbeza daripada 0 hingga 19 MPN/g. Selain

daripada itu, didapati bahawa prevalen Salmonella spp. pada buah-buahan

hirisan dari gerai penjaja adalah tiga kali ganda lebih tinggi berbanding

dengan buah-buahan dari pasar raya besar, masing-masing sebanyak 30%

dan 10% pada p<0.05. Dengan menggunakan kaedah MPN-multiplex PCR,

sebanyak 23.3% sampel telah dikesan mengandungi Salmonella spp. dan,

hanya 9.5% apabila menggunakan kaedah MPN-plating.

Daripada tujuh jenis buah-buahan yang dikaji, buah naga dari gerai penjaja

menunjukkan prevalen kontaminasi tertinggi dengan 75% Salmonella spp.,

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40% Salmonella Typhi dan 25% Salmonella Typhimurium. Oleh itu, buah

naga telah digunakan sebagai model buah untuk mengkaji pembentukan

biofilem oleh Salmonella Typhi dan Salmonella Typhimurium di atas papan

potong plastik dan juga kadar pemindahannya kepada buah naga. Dengan

menggunakan ujian crystal violet, didapati bahawa pembentukan biofilem

oleh Salmonella Typhi dan Salmonella Typhimurium atas papan potong

plastik adalah tertinggi pada 12 jam inkubasi dalam larutan garam fisiologis

pada suhu 28°C. Bagi pemindahan kedua-dua patogen dari papan potong

plastik kepada buah naga, keputusan menunjukkan bahawa purata peratus

pemindahan adalah 92.38 dan 91.30%, masing-masing untuk Salmonella

Typhi dan Salmonella Typhimurium. Ini menunjukkan bahawa biofilem

boleh mengkontaminasi buah naga secara bersilang dan menyumbang

kepada jangkitan silang. Kesimpulannya, pihak berkuasa kesihatan perlu

menilai risiko buah-buahan hirisan di Malaysia. Risiko pemindahan

mikroorganisma dari permukaan sentuh makanan kepada buah-buahan

segar seharusnya diberi perhatian untuk mencegah wabak penyakit yang

berpunca daripada makanan.

ACKNOWLEDGEMENTS

I would like to express my sincere and deepest appreciation to Professor Dr.

Son Radu, the chairman of my supervisory committee for the invaluable

guidance and support throughout my study. Thank you very much for the

advice and motivation which bring me a step closer to my dream.

A million thanks to my co-supervisors, Associate Professor Dr. Cheah Yoke

Kqueen from Faculty of Medicine and Health Sciences and Dr. Farinazleen

Mohamad Ghazali from Faculty of Food Science and Technology for the

advice and encouragement throughout my study. Thank you very much!

Sincere thanks to all my dearest laboratory colleagues (Dr. Chai Lay Ching,

Dr. John Tang Yew Huat, Jeyaletchumi Ponniah, Lee Hai Yen, Noorlis

Ahmad, Tuan Zainazor Tuan Chilek, Tunung Robin, Afriani Sandra, Chai

Li Fen, Elexson Nilian, Jeshveen Singh, Mohd. Zarrul Nizam Bin Zaaba,

Noor Hidayah bt. Mohd. Shahroni, Petrus Edmund, Ubong Anyi and Wong

Woan Chwen). All of you have enlightened my life. Thank you for the help

and assistance given to me. I will remember the laughter and sweet

memories while working with all of you.

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Not forgetting to acknowledge all my dearest friends especially Cindy

Thing, Kon Mee Jing, Suyi Yap, Victoria Lee and Wong Woan Chwen who

support me morally and accompany me throughout my study. Thank you

very much for the love and care given to me. My sincere gratitude is

extended to all staffs and colleagues of Faculty of Food Science and

Technology, UPM, who contributed one way or another towards the

completion of my study.

This thesis is especially dedicated to my dearest mother (Chu Su Chey) who

is the VIP in my life and also to my family members. Thank you very much

for everything, the sacrifices and endless love for me. Without your support,

I will never move this far in my life. I love you all!

I certify that a Thesis Examination Committee has met on 24 March 2011 to conduct the final examination of Pui Chai Fung on her thesis entitled “Prevalence and Risk Assessment of Salmonella Spp. in Sliced Fruit” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Master of Science. Members of the Thesis Examination Committee were as follows: Mohd Yazid bin Abd Manap, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Saleha binti Abdul Aziz, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) Shuhaimi bin Mustafa, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) I. Karunasagar, PhD Professor Karnataka Veterinary, Animal and Fisheries Science University India (External Examiner)

_____________________

NORITAH OMAR, PhD Associate Professor and Deputy Dean

School of Graduate Studies Universiti Putra Malaysia Date: 24 May 2011

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APPROVAL

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Son Radu, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Cheah Yoke Kqueen, PhD. Associate Professor Faculty of Medical and Health Sciences Universiti Putra Malaysia (Member) Farinazleen Mohamad Ghazali, PhD Senior Lecturer Faculty of Food Science and Technology Universiti Putra Malaysia (Member)

__________________________________ HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is nor concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

_______________ PUI CHAI FUNG Date: 24 March 2011

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TABLE OF CONTENTS Page DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xxi CHAPTER 1 GENERAL INTRODUCTION 1.1 Introduction 1 1.2 Objectives 5 2 LITERATURE REVIEW 2.1 Salmonella spp. 6 2.1.1 History background 6 2.1.2 Classification and nomenclature 8 2.1.3 Characteristic 11 2.1.4 Clinical manifestation 11 2.1.5 Epidemiology 17 2.1.6 Pathogenicity 21 2.1.7 Transmission vehicles 23 2.1.8 Antibiotic resistance 32 2.1.9 Biofilm formation 33 2.1.10 Quorum sensing 36 2.2 Isolation and identification of Salmonella spp. 38 2.2.1 Conventional methods 38 2.2.2 Rapid methods 40 2.2.3 Quantitative-qualitative methods 43 2.3 Molecular typing of Salmonella spp. 45 2.3.1 Nucleic acid amplification 45 2.3.2 Nucleotide sequencing technique 46 2.3.3 Restriction endonuclease digestion 47 2.4 Control and prevention 50 2.4.1 Guidelines for the control of Salmonella infection 50 2.4.2 Guidelines for the control of biofilm formation 54 by Salmonella

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3 OPTIMIZATION OF MULTIPLEX PCR FOR THE DETECTION OF SALMONELLA SPP., SALMONELLA TYPHI AND SALMONELLA TYPHIMURIUM IN SLICED FRUITS 3.1 Introduction 56 3.2 Materials and methods 58

3.2.1 Bacterial strains and culture conditions 58 3.2.2 DNA template preparation 59 3.2.3 Oligonucleotide primers 59 3.2.4 Multiplex PCR amplification 60 3.2.5 Agarose gel electrophoresis 62 3.2.6 Optimization of multiplex PCR 62 3.2.7 Specificity of multiplex PCR 62

3.3 Results 63 3.4 Discussion 67 3.5 Conclusion 71 4 PREVALENCE AND QUANTITY OF SALMONELLA SPP.,

SALMONELLA TYPHI AND SALMONELLA TYPHIMURIUM IN SLICED FRUITS IN MALAYSIA 4.1 Introduction 72 4.2 Materials and methods 74

4.2.1 Sample collection 74 4.2.2 Most probable number (MPN) method 75 4.2.3 Plating method 76 4.2.4 DNA extraction 76 4.2.5 Multiplex PCR 77 4.2.6 Statistical analysis 78

4.3 Results 80 4.4 Discussion 86 4.5 Conclusion 90 5 BIOFILM FORMATION BY SALMONELLA TYPHI AND

SALMONELLA TYPHIMURIUM ON PLASTIC CUTTING BOARD AND ITS TRANSFER TO DRAGON FRUIT 5.1 Introduction 91 5.2 Materials and methods 95

5.2.1 Bacterial strains and culture conditions 95 5.2.2 Test surfaces 95 5.2.3 Biofilm formation on plastic cutting board 96 5.2.4 Transfer of Salmonella Typhi and Salmonella 97 Typhimurium biofilm from plastic cutting board to dragon fruit 5.2.5 Statistical analysis 98

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5.3 Results 99 5.4 Discussion 102 5.5 Conclusion 106 6 GENERAL CONCLUSION AND DISCUSSION 107 REFERENCES 112 APPENDIX A 131 APPENDIX B 140 BIODATA OF STUDENT 147 LIST OF PUBLICATIONS 149

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