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ISOLATION, ANTIMICROBIAL RESISTANCE AND GENOTYPING OF Escherichia coli FROM SELECTED WILDLIFE IN SARAWAK

Kho Kai Ling

Master of Science 2011

Nusat khidmat Makluruat Akademik UNIVEILSITI MALAYSIA SAIta\V"AK

ISOLATION, ANTIMICROBIAL RESISTANCE AND GENOTYPING OF Escherichia coli FROM SELECTED WILDLIFE

IN SARAWAK P. KMIDMAT MAKLUMAT AKADIMIK

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KHO KAI LING

A thesis submitted in fulfillment of the requirement for the Degree of

Master of Science (Biotechnology)

Faculty of Resources Sciences and Technology UNIVERSITI MALAYSIA SARAWAK

2011,

ACKNOWLEDGEMENT

I would like to express my greatest appreciation to my supervisor, Prof. Dr. Kasing Apun, co-

supervisor Dr. Lesley Maurice Bilung for their advice, guidance and supervision throughout

this lengthy project.

A special acknowledgement is attributed to the Eco-zoonosis project leader, Prof. Dr.

Mohd. Tajuddin Abdullah. I would like to express my special gratitude to Mr. Wahap Marni,

Mr. Besar Ketol, Mr. Azis Ajim, Mr. Dahlan Rambli, and all the staff, lab assistants and

students from Department of Zoology and IBEC in the Faculty of Resource Science and

Technology, UNIMAS who assisted in the sampling trips. Samples collected were approved

by the Sarawak Forestry Department under the permit of 37/2008 for Bukit Lima Forest Park

Entrance and NCCD. 907.4 (IV) - 29 for sampling permit in Bukit Lima Forest Park.

Sampling in Bukit Aup Recreational Park was approved by Majlis Perbandaran Sibu and the

sampling in Naman Oil Palm Plantation was approved Ta Ann Plywood Sdn Bhd with the

permit number TAP-OP/GM/0508/11. Sampling in Kapit was approved by Sarawak Planning

Unit with permit reference number (13) UPN/S/G1/I/10.1 Vol. 26. This project was funded by

UNIMAS grant E14006/F07/06/ZRC/03/2007(03). I also would like to thank UNIMAS

ZAMALAH scholarship.

I would like to thank Dr. Samuel Lihan and Dr. Mickey Vincent for their advice in

genotyping in this study. My sincere thanks to, zoology and molecular biology department

tutors, Miss Chong Yee Ling and Miss Hashimatul Fatma Hashim for their advice and

assistance throughout the field work. Special thanks to the lab assistants, Mr. Azis Ajim, and

1

Miss Limjatai for their technical assistance. Not forgetting my fellow partners for this project

and in the biotechnology labs, for the companionship and knowledge exchange. My sincere

appreciation to all my friends, especially Mr. Adorn Benjamin, Miss Anita Tahir, Miss Chen

Yik Ming, Miss Harttini Neeni Hatta, Miss Lee Jong Jen, Miss Ng Lee Tze, and Miss Sarina

Niyup.

Lastly, I would like to express my gratitude to my family for their continuous care,

patience, and support throughout the years of this study. Last but not least, I would like to

thank all the lecturers in Faculty of Resource Science and Technology for their guidance.

ii

ABSTRACT

(The potential for dissemination of pathogenic enteric bacteria by wild animal to

human is of concern due to their ability to cause endemic and epidemic human diseases.

Among the enteric bacteria, Shiga toxin-producing Escherichia coli (E. coli 0157: H7) are

recognized as an important cause of diarrhea and emerging food borne zoonoses outbreaks.

Destruction or disturbances of habitat have been identified as a factor that leads to the

emergence of these zoonoses outbreaks. Therefore, this study was performed to compare the

occurrence of E. coli and detection of E. coli 01 57: H7 in selected wildlife comprising of birds,

bats and rodents from a natural habitat and disturbed habitat)The disturbed habitats

comprised of two urban forests, an oil palm plantation habitat located in Sibu and a human

settlement area. A forest area located in Nanga Merit, Kapit represented a natural habitat.

Both of the districts are located along the Rejang Basin, in the state of Sarawak, which is one

of the world's richest and most diverse ecosystems. Anal and cloacal swab collected from a

total of 682 wildlife hosts were screened for E. coli and E. coli 0157: H7 using standard

microbiological methods and molecular approach. The occurrence of E. coli was consistently

higher in rodents regardless of the habitats. Sit-I, sit-II and rfbE genes were not detected in

any of the 129 isolates during the detection of E. coli 0157: H7 by using multiplex PCR.

However, fliCh7 gene was detected in 27 E. coli isolates. Sequence analysis of E. coli

amplicon positive withliCh7 gene showed that four E. coli isolates from Bukit Aup Jubilee

Park were aligned to extraintestinal pathogenic E. coli strains harbouring f1iCh7 gene. Another

23 E. coli isolates positive with fiiCh7 gene were aligned to E. coli reference strain 14097

harbouring fliCh7 gene which is also not related to E. coli 0157: H7. Absence of pathogenic

genes indicated that wildlife in the studied area of Sarawak does not serve as important

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reservoirs of STEC, E. coli 0157 or E. coli 0157: H7. The frequency of antimicrobial agent

resistance was higher in the E. coli isolated from the disturbed habitats (town) in Sibu

compared to the rural area (Nanga Merit human settlement) and natural habitats (Nanga Merit

forest area). Isolates from the natural habitat displayed 100% of susceptibility to the 12

antimicrobial agent tested. E. coli isolates from the sampling habitats in Sibu showed

multidrug resistance. However, multiple antibiotic resistance indices (0.0283 and 0.0020 in

Sibu and Nanga Merit, respectively) indicated that the E. coli isolates were not recovered

from samples originated from high-risk source. Comparison of the genetic relatedness among

the 83 representative E. coli isolates based on the ERIC genetic fingerprinting patterns

showed that E. coli isolates from all the wildlife hosts and habitats were genetically

heterogeneous. PFGE genotyping for 35 representative isolates also indicated high degree of

diversity of E. coli isolated from selected wildlife in the studied habitats of Sarawak. All of

the isolates were arbitrarily grouped within the dendrogram regardless of the wildlife hosts

and habitats. Simpson's index of diversity for ERIC-PCR and PFGE are 99.89% and 99.84%,

respectively. Considering the great variety of restriction endonuclease digestion profiles found

among the E. coli isolated within the same sampling habitat, our results suggested limited

application of ERIC-PCR and PFGE for the purpose of general genotyping for E. coli in bats,

birds and rodents in the studied habitats. The findings of the overall study provide baseline

data for the comparison with any E. coli future study, surveillance and development of the

prevention steps in the state of Sarawak, Malaysia.

iv

ABSTRAK

Pemencilan, Rintangan Antimikrob dan Genotyping untuk Escherichia coli daripada

Hidupan Liar yang Tertentu dalam Sarawak.

Potensi penyebaran bakteria enterik oleh hidupan liar kepada manusia menjadi perhatian

kerana kemampuan mereka untuk menyebabkan penyakit endemik dan epidemik manusia. Di

antara bakteria enterik, Escherichia coli (E. coli 0157: H7) yang menghasilkan toxin Shiga

diakui sebagai penyebab utama cirit-birit dan wabak zoonosis bawaan makanan.

Pencerobohan atau gangguan habitat telah dikenalpasti sebagai faktor yang menyebabkan

munculnya wabak zoonosis. Oleh itu, kajian ini dilakukan untuk membandingkan kadar

pemulihan E. coli dan penemuan E. coli 0157: H7 daripada tiga jenis hidupan liar iaitu

burung, kelawar dan tikus dalam habitat semulajadi dan habitat terganggu. Habitat

terganggu terdiri daripada dua hutan bandar yang terletak tidak berjauhan dengan bandar,

satu ladang kelapa sawit yang terletak di Sibu dan penempatan manusia. Kawasan hutan di

Nanga Merit, Kapit mewakili habitat semulajadi. Kedua-dua daerah ini terletak di sepanjang

Basin Rejang, Sarawak, yang merupakan salah satu ekosistem terkaya di dunia dan

mempunyai kepelbagaian ekosistem. Swab dubur dikumpulkan daripada 682 hidupan liar

untuk pemencilan E. coli dan E. coli 0157. " H7 dengan menggunakan kaedah mikrobiologi

piawai dan pendekatan molekul. Kadar pemulihan E. coli adalah sentiasa tertinggi dalam

tikus di semua habitat. Gen slt-I, sit-II and rýbE tidak dapat dikesan dalam kesemua 129 wakil

pencilan dengan menggunakan_kaedah tindak balas berantai polimeras multipleks E. coil

0157: H7. Namun begitu, gen f1iCh7 hadir dalam 27 pemencilan E. coli. Analisa proses

penjujukan DNA menunjukan bahawa empat pencilan E. coli dari Bukit Aup, Sibu

V

dikumpulkan sebagai `extraintestinal' E. coli yang mempunyai fliCh7 gene. Dua puluh tiga

pencilan E. coli lain yang positif dengan fliCh7 gene merupakan strain rujukan E. coli 14097

yang tidak berkaitan dengan E. coli 0157: H7. Ketidakhadiran gen berpatogen menunjukkan

bahawa hidupan liar di kawasan yang dikaji dalam Sarawak bukan merupakan pembawa

penting untuk STEC, E. coli 0157 atau E. coli 0157: H7. Frekuensi rintangan agen

antimikrob adalah lebih tinggi dalam pencilan E. coli dari bandar (Sibu) berbanding dengan

kawasan luar bandar (penempatan manusia Nanga Merit) dan habitat asli (kawasan hutan

Nanga Merit). Pencilan daripada hutan semulajadi menunjukkan 100% kerentanan terhadap

12 agen antimikrob yang diuji. E. coli dari habitat yang dikaji di Sibu menunjukkan rintangan

pelbagai antibiotik. Namun demikian, indeks multiple-antibiotic-resistance (MAR) (0.00206

and 0.00017 untuk Sibu dan Nanga Merit, masing-masing) menunjukkan bahawa pencilan E.

coli bukan terdiri daripada sampel yang berasal dari sumber yang berisiko tinggi.

Perbandingan hubungan genetik antara 83 wakil pencilan E. coli berdasarkan corak cap-

jarian genetik menunjukkan bahawa pencilan E. coli dari semua jenis hidupan liar dan

habitatnya adalah pelbagai. Kaedah PFGE untuk 35 wakil pencilan juga menunjukkan

kepelbagaian diversiti antara E. coli yang dipencil daripada hidupan liar di habitat yang

dikaji dalam Sarawak. Semua pencilan telah dikumpulkan secara rawak di dalam

dendrogram tanpa mengira jenis hidupan liar dan habitat. Nilai Simpson 's index of diversity

untuk ERIC-PCR dan PFGE ialah 99.89% dan 99.84%, masing-masing. Dengan

mempertimbangkan kepelbagaian profil pencernaan endonuklease pembatas antara E. coli

yang dipencil daripada habitat pensampelan yang sama, hasil kajian ini menunjukan bahawa

ERIC-PCR dan PFGE mempunyai aplikasi yang terhad sebagai kaedah genotip am untuk

strain E. coli yang dipencil daripada kelawar, burung dan tikus dalam habitat yang dikaji.

Hasil kajian ini dapat memberikan panduan dalam perbandingan kepada kajian E. coli,

vi

pengawasan dan pembangunan langkah-langkah pencegahan dalam daerah di negeri

Sarawakpada masa hadapan.

vii

Pusat Khidinat Müklumat Akatlemiº. UMVERSITI MALAYSIA SARAWAK

TABLE OF CONTENTS

ACKNOWLEDGEMENT

ABSTRACT

ABSTRAK

TABLE OF CONTENTS

PUBLICATION/CONFERENCE PROCEEDINGS

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER 1 GENERAL INTRODUCTION

CHAPTER 2 LITERATURE REVIEW

2.1 Zoonoses

2.2 Escherichia coli and Shiga toxin producing E. coli

2.2.1 Escherichia coli

2.2.2 Shiga-toxin producing Escherichia coli 0157: H7

2.2.3 Virulence factor and pathogenesis of E. coli 0157: H7

2.2.4 Mode of transmission for E. coli o 157: H7

2.2.5 Clinical symptoms of E. coli 0157: H7 infection

2.2.6 Diagnosis and treatment of E. coli 0157: H7 infection

Animal and wildlife reservoir of E. coli, E. coli 0157: 117 and 2' 2' 7 STEC E. coli

2.3 Isolation and Identification of E. coli and E. coli 0157: H7

2.3.1 Isolation and identification of E. coli

2.3.2 Isolation and identification of E. coli 0157: H7

2.3.3 Multiplex PCR for the identification of E. coli 0157: H7 virulence gene

2.3.4 DNA sequencing

2.4 Antimicrobial susceptibility

2.4.1 Emergence and spreading of resistant bacteria

2.4.2 Mechanisms of antibiotic resistance

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2.5

CHAPTER 3

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

2.4.3 Susceptibility test methods

Genetic fingerprinting

2.5.1 Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR)

2.5.2 Pulsed field gel electrophoresis (PFGE)

2.5.3 Other genotyping methods

MATERIALS AND METHODS

Sampling sites

Samples collection Bacterial Isolation and Identification

Identification of E. coli 0157: H7

Multiplex Polymerase Chain Reaction (PCR)

3.5.1 Bacterial strains

3.5.2 DNA extraction

3.5.3 Multiplex PCR

3.5.4 Gel Electrophoresis

3.5.5 DNA sequencing of thefliCh7 gene

3.5.6 Sequence alignment of partialfliCh7 gene

Antimicrobial Susceptibility Test

3.6.1 Bacterial strains and preparation of inoculum

3.6.2 Antimicrobial susceptibility test

3.6.3 Data analysis Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR)

3.7.1 Bacterial strains

3.7.2 DNA isolation

3.7.3 ERIC primers

3.7.4 ERIC-PCR

3.7.5 Gel electrophoresis

3.7.6 Fingerprint data analysis

Pulsed- field gel electrophoresis (PFGE)

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3.8.1 Standard method

3.8.1.1 Pulsed Field Gel Electrophoresis Plug Preparation

3.8.1.2 Restriction Endonuclease Digestion

3.8.1.3 Pulsed-field Gel Electrophoresis

3.8.2 Rapid Method

3.8.2.1 PFGE plugs preparation

3.8.2.2 Lysis of cells in agarose plugs

3.8.2.3 Restriction Endonuclease (RE) Digestion

3.8.2.4 Pulsed-field Gel Electrophoresis

3.8.2.5 Data analysis

CHAPTER 4 RESULTS

4.1 The occurrence of E. coli in bats, birds and rodents in Sarawak.

4.2

4.3

4.4

Identification of pathogenic strain of E. coli isolated from wildlife by multiplex PCR and DNA sequencing. Antimicrobial susceptibilities of E. coli in bats, birds and rodents in Sarawak. Genotyping of E. coli isolated from different wildlife species by ERIC- PCR.

4.5 Genotyping of E. coli isolated from different wildlife species by PFGE.

4.5.1 Standard method

CHAPTER 5

5.1

5.2

5.3

5.4

CHAPTER 6

4.. 5.2 Rapid method

DISCUSSION

The occurrence of E. coli in bats, birds and rodents in Sarawak.

Identification of pathogenic strain of E. coli isolated from wildlife by multiplex PCR and DNA sequencing. Antimicrobial susceptibilities of E. coli in bats, birds and Sarawak.

Genotyping of E. coli isolated from different wildlife.

GENERAL CONCLUSION

REFFERENCES

APPENDIX A

APPENDIX B

APPENDIX C

rodents in

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PUBLICATION/CONFERENCE PROCEEDINGS

Kasing Apun, Kho Kai Ling, Chong Yee Ling, Hashimatul Fatma Hashim, M. T. Abdullah & Mustafa Abdul Rahman. (2008). Enterobacteria isolated from birds and rodents from the

urban forest in Sibu, Sarawak. Proceedings of the 10th MSAB Symposium, Kuching 2008,

pg693-696. ISBN: 978-983-44335-1-2.

Kho Kai Ling, Kasing Apun, Chong Yee Ling, Hashimatul Fatma Hashim, Mohd. Tajuddin Abdullah, Lesley Maurice Bilung'and Mustafa Abdul Rahman (2009). Occurrence of Escherichia coli in Wildlife at Urban Forests and Oil Palm Plantation in Sibu, Sarawak. Proceedings of the 2 "d UNIMAS Colloquium, 2009, pg 60-64. ISBN 978-983-9257-99-1.

Kho Kai Ling, Kasing Apun, Lesley Maurice Bilung, Hashimatul Fatma Hashim, Chong Yee Ling, Mohd. Tajuddin Abdullah, and Mustafa Abdul Rahman (2009). Antibiotic Resistance of Escherichia coli isolated from wildlife in two different habitats in Sarawak, Malaysia.

Proceedings of the International Congress of Malaysian Society for Microbiology 2009, Penang, pg 259. ISBN 978-983-41487-4-4.

Kho Kai Ling, Kasing Apun, Lesley Maurice Bilung, Hashimatul Fatma Hashim, Chong Yee Ling, Mohd. Tajuddin Abdullah, and Mustafa Abdul Rahman (2010). Detection and characterization of Escherichia coli isolates by multiplex PCR and ERIC-PCR. Proceedings

of the 3rd UNIMAS Colloquium, 2010, pg176-184. ISBN 978-967-5418-08-2.

Kasing Apun, Kho Kai Ling, Chong Yee Ling, Hashimatul Fatma Hashim, Mohd. Tajuddin Abdullah, Mustafa Abdul Rahman, Lesley Maurice Bilung and Samuel Lihan (2011). Detection of Escherichia coli 0157: H7 in wildlife from disturbed habitats in Sarawak, Malaysia. Research Journal of Microbiology, 6,132-139. ISSN 1816-4935.

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LIST OF TABLES

Table Page Table 2.1 Comparison of the characteristics of several microbial typing methods. 34

Table 3.1 Biochemical characteristic of E. coli. 48

Table 3.2 Wildlife hosts and number of E. coli isolates use for multiplex PCR. 49

Table 3.3 Oligonucleotide sequences of primers used for multiplex PCR reaction. 51

Table 3.4 Cycle profiles of multiplex PCR. 52

Table 3.5 Classification of antimicrobial agents used for this study. 54

Table 3.6 Oligonucleotide sequences of primers used in ERIC-PCR. 57

Table 3.7 Cycle profiles of ERIC PCR. 58

Table 3.8 Parameter for PFGE standard method. 62

Table 3.9 Parameter for PFGE rapid method. 65

Table 4.1 Number of wildlife hosts tested for the occurrence of E. coli in Sibu. 68

Table 4.2 Number of wildlife hosts tested for the occurrence of E. coli in Nanga Merit, 68 Kapit.

Table 4.3 Number of animal hosts with E. coli isolated and total number of E. coli 69 isolates of each sampling sites in Sibu.

Table 4.4 Number of animal hosts with E. coli isolated and total number of E. coli 69 isolates of each sampling sites in Nanga Merit, Kapit.

Table 4.5 Occurrence of E. coli in wildlife of Sibu, Sarawak. 70

Table 4.6 Occurrence of E. coli in wildlife of Nanga Merit, Kapit, Sarawak. 71

Table 4.7 Nucleotide sequence homology identities and sequence with significant 76 alignments from Genbank for the sequenced PCR products of flich7 amplicon-positive E. coli isolates.

Table 4.8 The frequency of E. coli isolates resistant towards each antimicrobial agent 79 tested.

Table 4.9 MAR indices for the E. coli isolates from wildlife in Sibu. 80

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Table Page Table 4.10 MAR indices for the E. coli isolates from wildlife in Nanga Merit, Kapit. 80

Table 4.11 Calculation of the Simpson Index of Diversity of E. coli isolates for ERICIR 84 and ERIC2.

X111

LIST OF FIGURES

Figure Page Figure 2.1 The route of transmission for resistant bacteria between the environment, 29

animal, wildlife and humans.

Figure 2.2 The core inverted repeats in ERIC sequence. 35

Figure 2.3 Complete ERIC sequence shown as a hairpin where lines and colons 36 connect bases in the two arms complementary in DNA and RNA.

Figure 3.1 Location of the sampling district Sibu and Nanga Merit along the Batang 43 Rajang, Sarawak.

Figure 3.2 Map showing sampling sites for the wildlife at Bukit Lima, Bukit Aup 44 and Naman Plantation, Sibu, Sarawak.

Figure 3.3 Map showing sampling sites in Nanga Merit 45

Figure 4.1 Occurrence of E. coli isolated from the five sampling sites in Sibu and 72 Nanga Merit.

Figure 4.2 Amplicon obtained by multiplex PCR for the 14 E. coli isolates isolated 74 from Sibu with flich7 gene with expected size of 625bp fragmented by 2% agarose gel electrophoresis.

Figure 4.3 Amplicon obtained by multiplex PCR for the nine E. coli isolates from 75 Nanga Merit with flich7 gene with expected size of 625bp fragmented by 2% agarose gel electrophoresis.

Figure 4.4 ERIC-PCR fingerprints of E. coli isolates from bats, birds and rodents. 82

Figure 4.5 Dendrogram of the E. coil isolates generated by ERIC-PCR. 83

Figure 4.6 Dendrogram of E. coli isolates from bat host generated by ERIC-PCR. 86

Figure 4.7 Dendrogram of E. coli isolates from rodent host generated by ERIC- 88 PCR.

Figure 4,8 Dendrogram of E. coli isolates from bird host generated by ERIC-PCR 89

Figure 4.9 PFGE chromosomal DNA digested with Xbal of representative E. coli 91 isolates.

Figure 4.10 PFGE chromosomal DNA digested with XbaI of representative E. coli 93 isolates by method 2.

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Figure Page Figure 4.11 PFGE chromosomal DNA digested with Xbal of 35 representative E. 96

coli isolates.

Figure 4.12 Dendrogram of 35 representative E. coli isolates generated based on 97 PFGE fingerprint by using XbaI.

Figure 4.13 Dendrogram of E. coli isolates from bat hosts generated based on PFGE 99 fingerprint by using XbaI.

Figure 4.14 Dendrogram of E. coli isolates from bird hosts generated based on PFGE 100 fmgerprint by using XbaI.

Figure 4.15 Dendrogram of E. coli isolates from rodent hosts generated based on 101 PFGE fingerprint by using XbaI.

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LIST OF ABBREVIATIONS

AE AFLP ATCC BAJP BaC12 BaC12"2H20 BaSOa BLASTN BLFP bp CLSI cm CT-SMAC ddH2O dNTPs DNA EAEC E. coli EDTA EIDs EIEC EMB ERIC EPEC ETEC

Jýl Ch7 fliCh7

g h HC HCI H2S H2SO4 HUS kb KIA L LEE LB LMP M MAC MAR Mb

Attaching and effacing Amplified fragment length polymorphisms American Type Culture Collection Bukit Aup Jubilee Park Barium chloride Barium chloride dihydrate Barium sulfate Basic Local Alignment Search Tool Bukit Lima Forest Park base pairs Clinical and Laboratory Standards Institute

centimeter Cefaxime-tellurite supplemented Sorbitol MacConkey Distilled water deoxynucleotide triphosphates Deoxyribonucleic Acid

enteroaggregative E. coli Escherichia coli Ethylenediamine Tetra-Acetic Acid emerging infectious disease enteroinvasive E. coli Eosin methylene blue Enterobacterial Repetitive Intergenic Consensus

enteropathogenic E. coli enterotoxigenic E. coli extraintestinal pathogenic E. coli Gene coded for antigen H7

gram hours hemorrhagic colitis Hydrochloric acid Hydrogen Sulphide Sulfuric acid hemolytic uremic syndrome Kilo base pair Kligler Iron Agar Liter locus of enterocyte effacement Luria Bertani Low melting point Molar MacConkey Multiple antibiotic resistances Mega base pair

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MIC ml MLC mm mm MgC12 NA NaOH NCBI nm PAls PBS PCR PFGE rfbE RAPD Rep-PCR RFLP RNA rRNA sec SMAC STEC Stx Stx 1 Stx2 TANP Taq TAE TBE TE tRNAs Tris TSI TTP U Uv WHO v v/v w/v µg/ml µg/U µl µM %

Minimal Inhibition Concentration milliliter Minimal lethal concentration millimeter milimolar Magnesium chloride Nutrient agar Soduim hydroxide National Center for Biotechnology Information nanometer pathogenicity islands Phosphate Buffer Saline Polymerase Chain Reaction Pulsed Field Gel Electrophoresis Gene encoded for antigen 0157 Random amplified polymorphic DNA Repetitive sequence polymerase chain reaction Restriction Fragment Length Polymorphisms Ribonucleic acid Ribosomal ribonucleic acid seconds sorbitol MacConkey agar Shiga toxin- producing E. coli Shiga toxin Shiga toxin 1 Shiga toxin 2 Ta-Ann Naman Plantation Thermus aquaticus DNA polymerase Tris-acetate EDTA Tris-Borate EDTA Tris-EDTA buffer transfer ribonucleic acids Tris (hydroxymethyl) methylamine Triple Sugar Iron thrombotic thrombocytopenic purpura Unit Ultraviolet World Health Organization Volt Volume/volume Weight/volume Microgram per microlitre Microgram per unit Microlitre microMolar Percentage

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°C degree Celsius << More than or equal to + Positive

Negative

xviii

CHAPTER 1

GENERAL INTRODUCTION

Wildlife is well-known to be involved in most of the zoonotic diseases (Kruse et al., 2004).

Zoonotic diseases are defined as diseases and infections that are transmitted from vertebrate

hosts to human (Cleaveland et al., 2007). The zoonotic agents, typically various bacteria,

carried by wildlife serve as the major reservoirs for microbial transmission to both human and

domestic animals. According to Hart et al. (1999) there are over 500 different pathogens that

are transmitted from the animals to human. Wild animals are thought to be the source of more

than 70% of all emerging infections (Kuiken et al., 2005). Outbreaks of zoonoses are

receiving an increasing attention worldwide due to their major impact on human health,

agriculture production, wildlife-based economies and wildlife conservation (Chomel, 2007).

According to WHO (2010), millions of people are infected by zoonotic diseases every year.

Enterobacteriaceae are among the best-characterized group of microflora in the

gastro-intestinal tract of wildlife. Enterobacteria such as Escherichia coli (E. coli) have been

implicated as part of the normal microflora in the intestinal tract of mammals and birds

(Caprioli et al., 2005). However, certain strains of E. coli such as E. coli 0157, E. coli 018, E.

coli 026 and E. coli 0111 are most commonly involved with pathogenicity (Paton and Paton,

1998). E. coli 0157: H7, which is also known as enterohemorrhagic E. coli, has created the

global public health concern because it can cause human infection associated with a wide

range of clinical illness including asymptomatic shedding, non-bloody diarrhoea,

haemorrhagic colitis, haemolytic uremic syndrome and death (Griffin and Mead, 1998). E.

I

coli 0157: H7 was first recognized as a human pathogen in 1982 with the outbreaks of

hemorrhagic colitis occurred by consumption of undercooked meat in Oregon and Michigan

(Riley et al., 1983). E. coli 0157: H7 belongs to Shiga toxin-producing E. coli (STEC)

represents the only pathogenic group of the E. coli that has a definite zoonotic origin (Caprioli

et al., 2005). Ruminants, especially cattle, were the main reservoir for E. coli 0157: H7

(Caprioli et al., 2005). However, it has been reported to be isolated from wild birds (Wallace

et al., 1997; Foster et al., 2006) and rodents (Nielsen et al., 2004) which implicated wild

animals served as potentials vectors for the dissemination of E. coli 0 157: H7. Besides that, E.

coli 0157: H7 was also being isolated from other animals such as swine (Feder et al., 2003),

rabbit (Garcia and Fox, 2003) and sheep (Kudva et al., 1996).

Pathogenic strains of E. coli which cause diseases are difficult to be distinguished

from those that are part of the normal microflora. Traditional microbiology methods for the

identification of E. coli 0157: H7, involving enrichment of cultures plating on sorbitol

MacConkey agar, analysed the colonies with biochemical test and latex agglutination kit. The

confirmed cultures are further typed for Shiga like toxin (SLT) by the use of cell culture and

neutralizing antibody to the toxin (Hu et al., 1999). Traditional methods are labour intensive

and time consuming which may provide false positive results due to atypical phenotypic

properties of some strains (Rahman, 2002). Polymerase Chain Reaction (PCR) assays

represents good alternatives to traditional typing methods for the diagnosis of pathogenic E.

coli due to their simplicity, rapidity and specificity (Olsvik and Strockbine, 1993). Multiplex

PCR allows simultaneous detection of two or more different virulence genes in a single

reaction, inevitably leads to a greater number of positive samples compared to conventional

methods (Gallian, 2003). Several articles have reported that multiplex PCR have been

2

successfully employed to detect E. coli 0157: H7 (Hu et al., 1999; Radu et al., 2001; Apun et

al., 2006; Bai et al., 2010).

Nowadays, antimicrobial agents are widely used in human diseases treatment and

animal husbandry to prevent infectious diseases and to promote growth. The antibiotics used

in animal husbandry are identical or closely resemble drugs used in humans (McEwen and

Fedorka-Cray, 2002). The wide applications of antibiotic have lead to emergence and

dissemination of resistant gene and bacteria among the animals and the environment.

Antibiotic resistant bacteria are disseminated through manure and liquid manure of animals

and human excretions (Reinthaler et al., 2003). As a consequence, wildlife may acquire the

antibiotic resistant strains from human and food animals when they are in close proximity.

Antimicrobial resistance has emerged in zoonotic enteropathogens for example E. coli,

Salmonella spp. and Campylobacter spp. In animals, antibiotic resistance in zoonotic

pathogens including E. coli 0157: H7 and commensal microorganisms for most E. coli strains

are of special concern to human health because these bacteria can be transferred to human and

animal via food chain. Besides, resistance genes from commensal bacteria may be transferred

to the zoonotic pathogens (McEwen and Fedorka-Cray, 2002). Therefore, to ensure the public

health, there is a need to examine and identify the antibiotic resistance profile of E. coli

isolated from wildlife as a result of apparent increased in antibiotic usage. In this research, the

antibiotic resistant patterns of E. coli in wildlife were compared between disturb habitats and

natural habitats with less perturbation.

Molecular typing methods based on the analysis of chromosomal DNA have broad

applications in public health bacteriology and epidemiological study. The process of

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molecular typing is epidemiologically crucial for recognizing the outbreaks of infection,

analysis of strain relatedness in order to identify transmission route, detecting the cross-

transmission of pathogens and determining the source of the infection (Olive and Bean, 1999).

Epidemiological typing of bacterial strains can be carried out by various methods. The

subtyping methods chosen must be typeable, reproducible, high discriminatory power and

practical (Thong et al., 2003). In recent year, the molecular typing methods such as pulsed-

field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP); PCR-

based and sequenced-based methods have been increasingly used for bacteria outbreak

investigation (Patel and Graham, 2007). Enterobacterial repetitive intergenic consensus (ERIC)

sequences are highly conserved sequence but their chromosomal locations differ between

species (Hulton et al., 1991). ERIC sequences were utilized as efficient primer binding sites in

the PCR to produce fingerprints of different bacterial genomes (Versalovic et al., 1991).

ERIC-PCR had been widely used for the genetic characterization of E. coli (Casarez et al.,

2007; Prabhu et al., 2010; Yuan et al., 2010). Another well known genotyping method, PFGE,

is known to be highly reproducible, having high discrimination power, excellent typeability

and high inter-laboratory reproducibility which fulfil most of the criteria for molecular typing

(vanBelkum et al., 2001). This technique has been frequently used for the characterization of

E. coli and E. coli 0157 isolates (Radu et al., 2001; Casarez et al., 2007; Bentancor et al.,

2010). Therefore, ERIC-PCR and PFGE were selected to study the genetic relatedness

between the E. colt strains isolated from different animal hosts and sampling habitats in

Sarawak.

The potential of wildlife involved in dissemination of pathogenic microorganisms is of

concern and vigilance worldwide. The surveillance of the epidemiology of pathogenic

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