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)
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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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