UNIVERSITI PUTRA MALAYSIA

SELECTION OF POTENTIAL BACTERIAL PROBIOTICS FOR TIGER GROUPER (Epinephelus fuscoguttatus Forsskal) LARVICULTURE

NURHIDAYU BINTI AL-SAARI

FP 2012 31

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SELECTION OF POTENTIAL BACTERIAL PROBIOTICS FOR TIGER GROUPER (Epinephelus fuscoguttatus Forsskal) LARVICULTURE

By

NURHIDAYU BINTI AL-SAARI

Thesis Submitted to the School of Graduate Studies, Universiti Putra

Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science

June 2012

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Specially dedicated to my lovely parents to whom I indebted my life.

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

SELECTION OF POTENTIAL BACTERIAL PROBIOTICS FOR TIGER GROUPER (Epinephelus fuscoguttatus Forsskal) LARVICULTURE

By

Nurhidayu Binti Al-saari

June 2012 Chairman: Ina Salwany Md Yasin, PhD Faculty: Agriculture

Rapid development in today’s aquaculture industries have expanded to the level

of commercial scale and has indirectly led to environmental degradation and

catastrophic losses due to bacterial diseases. Thus, the application of probiotics

was proposed as an environmental-friendly preventive measure. This study was

carried out specifically to search for probiotic candidates for use in the

larviculture of Epinephelus fuscoguttatus. Hypothetically, selected intestinal

microfloras of E. fuscoguttatus are able to confer protection on larvae by

exhibiting antagonistic activity against pathogenic bacteria.

A set of criteria for bacterial probiotics was used in this study, focusing on

phenotypic and genotypic characterizations of the candidate strains, in vitro

capacity to inhibit the growth of targeted competitive strains, the haemolytic

activity and antibiotic susceptibility of selected candidates and virulence

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assessment to the host. Initially, the intestines of healthy individual grouper

which were randomly collected from three geographically distant farms in

Malaysia were processed and bacterial isolation was performed. A preliminary

identification and grouping method successfully identified 31 species, while 22

isolates as unidentified from a total of 123 isolated aerobes and facultative

anaerobes.

The first screening process was undertaken to assess the antagonistic activity

exhibited by the isolates against four common fish pathogens. Of the 123

isolates, 40 (32.5%) displayed strong antagonistic activity, mostly Gram-positive

bacteria (n=32; 80%). The haemolytic nature of the short listed probionts was

then assessed where only nine (22.5%) showed no breakdown of red blood cells

and they were grouped into five genera based on 16S rRNA gene sequence

analysis. This genotypic strategy was used to eliminate another four isolates

considering their potential pathogenic nature to human and sequences similarity

from which they were considered identical. Screening process was ended with

the antibiotic susceptibility assay of five selected isolates which resulted in the

elimination of V. harveyi i.e. JAQ01 and JAQ02 due to their opportunistic nature

and the availability of incomprehensible evidence that these strains may

potentially carry resistance genes for β-lactamase in a transferrable genetic

material.

Considering the needs for an advanced study to measure the antagonism

capacity of three selected candidates, bacteriocin-like inhibitory substances

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(BLIS) and co-culture assays were performed. The former assay nominated

Bacillus cereus JAQ04 as the most promising candidates with initial addition at

108 and/or 109 cfu/mL with 48 and/or 72 h pre-incubation time. Alternatively, for

co-culture method, the growth of pathogenic V. alginolyticus can also be

inhibited at 105 cfu/mL of all probionts tested. Both methods revealed that the

highest inhibition of pathogens has always associated with the introduction of

significantly higher densities of probionts.

Lastly, virulence assessment was made by determining the relationship

observed between the probiont concentrations with larval survival. Conclusively,

in relation to the previous antagonistic assay, 105 cfu/mL of B. cereus (JAQ04)

and Micrococcus luteus (JAQ07) were recommended as the safest and

adequate initial amount for use in field trial. Both bacteria needed at least 48 h to

proliferate before being able to counteract the pathogens. Thorough studies on

these bacteria including in-depth assessment on their effect in vivo are

recommended.

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

PEMILIHAN BAKTERIA BERPOTENSI PROBIOTIK BAGI KULTUR LARVA KERAPU HARIMAU (Epinephelus fuscoguttatus Forsskal)

Oleh

Nurhidayu Binti Al-saari

Jun 2012

Pengerusi: Ina Salwany Md Yasin, PhD Fakulti: Pertanian

Pembangunan pesat sektor akuakultur pada hari ini telah berkembang kepada

skala komersial dan secara tidak langsung telah mengakibatkan pencemaran

alam sekitar dan kerugian besar yang disebabkan oleh penyakit bakteria. Oleh

itu, penggunaan probiotik telah dicadangkan sebagai langkah pencegahan yang

mesra alam. Kajian ini telah dijalankan khusus untuk mencari calon-calon

probiotik untuk digunakan dalam ternakan larva Epinephelus fuscoguttatus.

Diandaikan, microflora terpilih dari usus E. fuscoguttatus dapat memberi

perlindungan kepada larva dengan cara menentang bakteria pathogen.

Satu set kriteria pemilihan bakteria probiotik telah digunakan dalam kajian ini

memberi tumpuan kepada pencirian fenotip dan genotip terikan calon, kapasiti

in vitro untuk menghalang pertumbuhan strain sasaran, aktiviti hemolitik dan

kerentanan antibiotik calon-calon yang dipilih dan penilaian kebisaan tuan

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rumah. Pada mulanya, usus kerapu dari individu sihat yang dikumpulkan secara

rawak dari tiga ladang berasingan di Malaysia telah diproses dan diikuti dengan

pemencilan bakteria. Kaedah pengenalan dan pengkelasan awal berjaya

mengenal pasti 31 spesies, manakala 22 isolat dikelaskan sebagai tidak dikenali

daripada jumlah keseluruhan 123 aerobes dan anaerobes fakultatif terpencil.

Proses saringan pertama telah dijalankan untuk menilai aktiviti antagonistik

yang dipamerkan oleh isolat-isolat yang dikaji terhadap empat patogen yang

biasa bagi ikan. Daripada 123 isolat, 40 (32.5%) memaparkan aktiviti

antagonistik yang kuat, yang mana kebanyakannya adalah bakteria Gram-positif

(n = 32; 80%). Sifat hemolitik probionts yang telah disenarai pendek

kemudiannya dinilai di mana hanya sembilan (22.5%) tidak menunjukkan

pecahan sel darah merah dan mereka dikumpulkan kepada lima genus

berdasarkan analisis jujukan gen 16S rRNA. Strategi genotip ini telah digunakan

untuk menghapus kira empat lagi isolat disebabkan mereka berpotensi menjadi

patogen terhadap manusia dan mempunyai persamaan jujukan gen di mana

mereka dianggap serupa. Proses saringan diakhiri oleh penilaian kerentanan

antibiotik oleh lima isolat terpilih yang menyebabkan penghapusan V. harveyi

JAQ01 dan JAQ02 kerana sifat oportunis mereka dan kewujudan bukti sedia

ada yang masih belum jelas yang menerangkan bahawa terikan ini mungkin

membawa gen rintangan bagi β-lactamase dalam bahan genetik boleh pindah.

Melihat kepada keperluan kajian yang lebih dalam untuk mengukur kapasiti

antagonisma tiga calon yang dipilih, bahan-bahan rencatan menyerupai

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bacteriocin (BLIS) dan ujian kultur bersama telah dilakukan. Asai pertama telah

menamakan Bacillus cereus JAQ04 sebagai calon yang paling berpotensi

digunakan pada kepekatan 108 dan/atau 109 cfu/mL dengan 48 dan/atau 72 jam

masa pengeraman. Sebagai alternatif, bagi kaedah kultur bersama,

pertumbuhan patogen V. alginolyticus juga boleh direncatkan dengan

penambahan 105 cfu/mL probionts yang diuji. Kedua-dua kaedah mendedahkan

bahawa perencatan tertinggi patogen sentiasa dikaitkan dengan penambahan

probionts pada ketumpatan yang ketara lebih tinggi.

Akhir sekali, penilaian kebisaan telah dilakukan dengan cara menentukan

hubungan diantara kepekatan probiont dengan kemandirian larva.

Kesimpulannya, dengan merujuk kepada penilaian antagonistik terdahulu, 105

cfu/mL B. cereus (JAQ04) dan Micrococcus luteus (JAQ07) adalah jumlah yang

disyorkan paling selamat dan mencukupi bagi kegunaan ladang. Kedua-dua

bakteria memerlukan sekurang-kurangnya 48 jam untuk berkembang sebelum

dapat mengatasi patogen. Kajian yang menyeluruh ke atas bakteria ini termasuk

penilaian terperinci mengenai kesan mereka secara in vivo adalah disyorkan.

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ACKNOWLEDGEMENTS

First and foremost, greatest praises and gratitude were dedicated to Allah, the

Almighty, Beneficial and the most Merciful, who has enabled me to finish this

thesis.

I would like to express my gratitude to everybody who helped me and gave me

the courage and possibility to complete this project. This work would not have

been possible without the support and encouragement of my supervisors,

colleagues and family and it is a great honor that I now have the opportunity to

express my gratitude to them.

The first person I would like to thank is my direct supervisor Dr. Ina Salwany Md.

Yasin whose help, stimulating suggestions and encouragement which eventually

helped me a lot in all the time of doing this project and writing this thesis. She is

the one who kept an eye on the progress of my work and always available when

I need her advices. Thanks to you for your untiring support, motivation and

encouragement in both morally and financially.

Many thanks go to my co-supervisors, Assoc. Professor Dr. Hassan Mohd Daud

and Professor Dr. Sharr Azni Harmin who has also been abundantly helpful, and

have assisted me in numerous ways. I owe them lots of appreciation in return to

their comments, ideas, critical suggestion and constant encouragement.

I furthermore want to acknowledge my fellow labmates and dearest friends,

Shikin, Kak Hidayah, Kak Zalina, Wanie and Edi for their unconditional support

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and warm friendship. Million thanks to Professor Dr. Mohd Zamri Saad and staff

from Faculty of Veterinary Medicine, En. Fadhil Syukri, Pn. Zaiton, En. Azmi, En.

Jasni, Pn. Shafika, Cik Azlina and En. Abdullah for their technical assistance,

critical reviews and guidance. I am deeply indebted to all of you and my prayer

and love will always be with you.

Finally, my special thanks were dedicated to my family (Abah, Mak, Isyam, Awi

and Ashraf), who I owed the most for their constant encouragement, love and

patience. I am grateful to be blessed by having them beside me. My parents’

courage and conviction will always inspire me. It is to them that I dedicate my

work.

I also wished to thank all farms involved in specimen collection and financial

support from Agro-Biotechnology Institute (ABI), Ministry of Science, Technology

and Innovation (MOSTI) Malaysia

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I certify that a Thesis Examination Committee has met on 12 June 2012 to conduct the final examination of Nurhidayu binti Al-saari on her thesis entitled " Selection of Potential Bacterial Probiotics for Tiger Grouper, (Epinephelus fuscoguttatus Forsskal) Larviculture " 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 Salleh Kamarudin, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Kamaruzaman Sijam, PhD

Associate Professor Name of Faculty Universiti Putra Malaysia (Internal Examiner) Che Roos Saad, PhD

Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Najiah Musa, PhD

Associate Professor Faculty of Fisheries and Aqua-Industry Universiti Malaysia Terengganu Malaysia (External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Ina Salwany Md Yasin, PhD Senior Lecturer Faculty of Agriculture Universiti Putra Malaysia (Chairman) Hassan Mohd Daud, PhD

Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Sharr Azni Harmin, PhD

Professor Faculty of Science and Biotechnology Universiti Selangor (Member)

BUJANG BIN KIM HUAT, 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 not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

NURHIDAYU BINTI AL-SAARI

Date: 12 June 2012

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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 INTRODUCTION 1

2 LITERATURE REVIEW 2.1 Aquaculture for Food Security 7

2.2 Background of Epinephelus fuscoguttatus (Forsskål,1775)

8

2.3 Larval Rearing and Bacterial Diseases of Grouper 10 2.4 Common Practices to Overcome Bacterial Diseases in

Fish Culture and Probiotics as an Alternative 13

2.5 Probiotics in Marine Larviculture 2.5.1 Probiotics in General 16

2.5.2 Current Status of Probiotics in Larviculture and Relevent Researches

17

2.6 Selection Protocols of Bacterial Probiotics for MarineFish Larvae

2.6.1 Acquisition of Bacterial Strains from Healthy Animals

19

2.6.2 In vitro Screening 21 2.6.3 Organism Identification 24 2.6.4 Safety Consideration of Potential Candidates 25

3 ISOLATION AND CHARACTERIZATION OF INTESTINAL MICROFLORA OF FARMED TIGER GROUPER (Epinephelus fuscoguttatus)

3.1 Introduction 29 3.2 Materials and Methods

3.2.1 3.2.1 Fish Samples 30 3.2.2 Bacterial Isolation 32 3.2.3 Bacterial Identification and Grouping 33

3.3 Results and Discussion 3.3.1 Bacterial Isolation 34

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3.3.2 Bacterial Identification and Grouping 37 3.4 Conclusion 43 4 IN VITRO SCREENING OF BACTERIAL PROBIONTS AND

16S rRNA SEQUENCE-BASED IDENTIFICATION

4.1 Introduction 44 4.2 Materials and Methods

4.2.1 Preparation of Bacterial Strains 45 4.2.2 Preliminary Screening based on the Antagonism

to Pathogens 46

4.2.3 Haemolysis Assay 47 4.2.4 DNA Extraction and 16S rRNA Gene Sequence

Analysis 47

4.2.5 Antibiotics Susceptibility Assay 48 4.2.6 Further Assesment on the Antagonistic Activity of

Probionts Against Pathogenic Bacteria 49

4.2.6.1 Preparation of selected probionts and target strains

49

4.2.6.2 Modified cross-streak method for BLIS assay

50

4.2.6.3 Co-culture of Vibrio alginolyticus with probionts

51

4.3 Results and Discussion 4.3.1 Screening of Antagonistic Activity and Haemolytic

Property 52

4.3.2 16S rRNA Gene Sequence Analysis 57 4.3.3 Antibiotics Susceptibility Assay 60 4.3.4 Bacteriocin-Like Inhibitory Substances (BLIS)

Assays 61

4.3.5 Co-culture Method 70 4.3.6 Relation between BLIS Assay and Co-culture

Method 74

4.4 Conclusion 74

5 TOLERANCE LEVEL ASSESSMENT OF PROBIOTIC CANDIDATES

5.1 Introduction 78 5.2 Materials and Methods 5.2.1 Experimental Animals 80 5.2.2 Preparation of Bacterial Suspension 80 5.2.3 Treatments and Experimental Design 80 5.2.4 Statistical Analysis 81 5.3 Results and Discussion 5.3.1 Effect of Bacterial Probionts on the Hatching

Rate of E. fuscoguttatus Eggs 82

5.3.2 Tolerance Level Assessment of Bacterial Probionts Against E. fuscoguttatus Larvae

84

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5.4 Conclusion 93 6 GENERAL DISCUSSION AND CONCLUSION 94 REFERENCES 102 APPENDICES 116 BIODATA OF STUDENT 122 LIST OF PUBLICATIONS 123