ISOLATION AND IDENTIFICATION OF BURKHOLDERIA SPECIES FROM WATER SAMPLES

Mirna Binti Nasir

QR Bachelor of Science with Honours84 (Resource Biotechnology)

M675 2015

2015

Pus~n hldma M I lul'11a Aka ( n ik U IVE ' iTI MALAYSIA SARAWA

ISOLATION AND IDENTIFICATION OF BURKHOLDERIA SPECIES FROM

WATER SAMPLES

MIRNA BINTI NASIR (36920)

A thesis submitted in partial fulfillment of the Final Year Project (STF 3015)

Supervisor: Dr. Yuwana Podin

Co-supervisor: Dr. Micky Vincent

•.

Resource Biotechnology Programme Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

2014/2015

•]

ACKNOWLEDGEMENTS

In the Name of Allah, the Most Beneficent, the Most Merciful.

First of all, I would like to express my highest gratitude to Allah s.w.t for the ease that He

gave to complete my thesis, for the patience, strength and blessing.

I would like to express my sincere appreciation to my supportive supervisor, Dr

Yuwana Podin for constantly guiding and encouraging me throughout this project. Thanks

a lot for giving me a professional training, useful advices and suggestions to bring this

project to its final fonn. Appreciation is extended to the Universiti Malaysia Sarawak

(UNIMAS) especially the Institute of Health Community Medicine (IHCM) for providing

good facilities in the campus, and to all the staff, a very special thank you to all of you.

Next, a special thanks to my beloved parents, Nasir Bin Rahman, Asiah Binti

Labanda and my siblings; Namria, Irwan, Ani, 10han and Wahyudi for their constant

encouragement and love while completing this project.

Nevertheless, I want to thank to all the authors that I have made references in

completing the thesis work. Your works are very useful and much appreciated. In

particular, my sincere thankful is also extends to all my colleagues, lab mates and others

for their views and tips which are very useful indeed.

Alhamdulillah. Above all, I am grateful to ALLAH for the journey so far and for what lies

ahead.

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DECLARATION

I hereby declare that this thesis entitled "Isolation and Identification of Burkholderia

Species from Water Samples" is my own work and all sources have been quoted and

referred to have been acknowledged by means of complete references. It has been

submitted and shall not be submitted to other university or institute of higher learning.

(MIRNA BINTI NASIR)

RESOURCE BIOTECHNOLOGY

DEPARTMENT OF MOLECULAR BIOLOGY

UNIVERSITI MALAYSIA SARA W AK

iii

Pusat Khidmat Maklumat Akacf~,...; ': . li VERS TI MALAYSI A SARAWAK

TABLE OF CONTENTS

PAGECONTENT

Title (Front Cover)

Acknow ledgements 11

IIIDeclaration

IVTable of Contents

viList of Abbreviations

VlllList of Tables

List of Figures IX

List of Appendices X

XlAbstract

11.0 INTRODUCTION

42.0 LITERATURE REVIEWS

2.1 Burkholderia species Characteristics 4

2.1.1 Burkholderia pseudomallei 4

42.2 Melioidosis

2.3 Identification of Burkholderia spp. 5

2.3.1 Phenotypic Identification (Bacterial Culture) 5

2.3.2 Genotypic Identification by PCR 5

2.4 Previous studies of water samples collected related to B. pseudomallei 7

3.0 MATERIALS & METHODOLOGY 8

3.1 Materials and Apparatus 8

3.2 Moore'$ Swab Preparation 8

3.3 Preparation of Reagents 8

3.3.1 Crystal violet, 0.1 % w/v 8

3.3.2 Neutral red, 0.1 % w/v 8

3.3.3 Chelex 100, 10% w/v 9

3.4 Preparation of Culture Media 9

3.4.1 Ashdown's Selective Broth 9

3.4.2 Ashdown's Selective Agar (ASH) 9

3.5 General Methodology & Experimental Design 10

3.6 Water Sampling 11

iv

3.7 Primary Isolation of Burkholderia spp. from Water Samples 12

3.8 DNA Extraction 13

3.9 Genotypic Identification ... 13

3.9.1 Detection of B. pseudomallei DNA by TIS 1 real-time PCR 13

3.9.2 Confirmation of Burkholderia spp. by PCR 14

3.9.3 Gel Analysis ofPCR Amplicons 14

4.0 RESULTS 15

4.1 Culture Characteristics of Bacterial Isolates 15

4.1.1 Colony Morphology on Selective Media 15

4.2 Genotypic Identification of Bacterial Isolates 16

4.2.1 Detection of B. pseudomallei DNA using TTS1 real-time PCR 16

4.2.2 Burkholderia recA gene PCR Assay 21

5.0 DISCUSSION 25

5.1 Culture Characteristics of Bacterial Isolates 25

5.1.1 Colony Morphology on Selective Media 25

5.2 Genotypic Identification of Bacterial Isolates 26

5.2.1 Detection of B. pseudomallei DNA using TTS 1 Real-Time PCR 26

5.2.2 Burkholderia recA gene PCR i\.ssay 27

5.3 Assessment of Health Impacts ofIsolated Burkholderia spp. 27

6.0 CONCLUSION 29

REFERENCES 30

APPENDICES 33

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., v

LIST OF ABBREVIATIONS

spp. species

PCR polymerase chain reaction

DNA deoxyribonucleic acid

UV Ultraviolet

UHQ Ultra-High Quality

°c degree Celsius

mm minute/minutes

w/v weight per volume

~M micromolar

mg milligram

MgCh magnesium chloride

dNTPs deoxynucleoside triphosphate

GPS Global Positioning System

RO reverse osmosis

vol volume

V volt

.' s second/second

rpm revolution per minute

h hourlhours

g Gram

L Liter

cm centimeter

ml milliliter \..

1 .. vi

,....

microliter

CF cystic fibriosis

GR GeneRuler

CDC Center for Disease Control and Prevention

BPSA Burkholderia pseudomallei Selective Agar

III

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1 vii

...~-------------------------------------------~--~

LIST OF TABLES

Tables Caption Page

Table 2.1 A summary of previous studies related to water samples 7 conducted in Australia and Laos

Table 3.1 Sampling sites for the collection of water samples 12

Table 4.1 Results of the occurrence of B. pseudomallei 18

Table 4.2 Occurrence ofBurkholderia spp. from different sampling site 21

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•, viii

LIST OF FIGURES

Figures Caption Page

Figure 3.1 Overview of the experimental design of the project 10

Figure 4.1 Colony morphologies of different bacteria isolates on the ASH 16 agar.

Figure 4.2 The amplification plots for TTS 1 real-time PCR 17

Figure 4.3 Estimation of the prevalence of B. pseudomallei from different 19 sampling site

Figure 4.4 Percentage from the total bacterial isolates for the prevalence of 20 B. pseudomallei

Figure 4.5 Estimation of the prevalence of Burkholderia spp. at different 22 sampling sites

Figure 4.6 Gel image of recA gene PCR products of selected isolates from 23 site 2

Figure 4.7 Percentage from the total isolates for prevalence of 24 Burkholderia spp .

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1 ix

LIST OF APPENDICES

Appendix Caption Page

Appendix A List of materials and apparatus used 33

Appendix B The colony morphology of the bacterial isolates 36

Appendix C Results of TIS 1 real-time peR 52

Appendix D Gel Image of bacterial isolates from Site 1- 8 55

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x

ISOLATION AND IDENTIFICATION OF BURKHOLDERIA SPECIES FROM WATER SAMPLES

MIRNA BINTI NASIR

Resource Biotechnology Programme Department of Molecular Biology

Faculty of Resource Science and Technology Universiti Malaysia Sarawak

ABSTRACT

Water has a strong association with soil, the high microbial presence in soil directly affects the microbial presence in water within the same vicinity. Water is an important element in our daily lives and hence the presence of any microbes in the water may potentially cause severe infections in humans and animals. Burkholderia pseudomallei currently denoted as Tier I Select Agent and it can caused disease known as melioidosis. The aim of this project is to isolate and identify Burkholderia species from the water samples, which water sources are much related to human activities. There were 8 total sites included in this project and originally water samples were collected from Tawau, Sabah and Kota Samarahan, Sarawak. Moore's swabs were used for the water samples collection, and their sources were mainly from lake, pond, construction sites and orchard. The swabs were used because it is more cost effective compared to conventional method. All water samples collected were cultured on Ashdown's agar. Fifty-six percent of the water samples collected around Tawau and Kota Samarahan, were culture positive for Burkholderia spp. and less than 2% of them were culture positive for B. pseudomallei.

Keywords: Burkholderia spp., disease, microbe, Moore's swab, water sample

ABSTRAK

Air mempunyai hubungan yang kuat dengan tanah, kehadiran mikrob yang tinggi di dalam tanah secara langsung memberi kesan kehadiran mikrob di dalam air di dalam kawasan yang sarna. Air adalah e1emen penting dalam kehidupan seharian kita dan dengan itu kehadiran mana-mana mikrob di dalam air yang berpotensi boleh menyebabkan jangkitan teruk pad a manusia dan haiwan. Burkholderia pseudomallei kini ditandakan sebagai Tier I Agen Pilihan dan ia boleh menyebabkan penyakit iaitu melioidosis. Tujuan projek ini adalah untuk mengasingkan dan mengenal pasti spesies Burkholderia dari sampel air, yang sumber air yang banyak berkaitan dengan aktiviti manusia. Terdapat 8 jumlah tapak yang termasuk dalam projek ini dan asalnya sampel air telah dikumpulkan dari Tawau, Sabah dan Kota Samarahan, Sarawak. Kapas kesat Moore telah digunakan untuk pengumpulan sam pel air, dan sumber mereka adalah dari tasik, kolam, tapak pembinaan dan dusun. Kapas kesat telah digunakan kerana ia lebih kos efektif berbanding kaedah konvensional. Semua sampel air yang dikumpul dikulturkan pada agar Ashdown. Lima puluh enam peratus daripada sampel air yang dikumpul di sekitar Tawau dan Kota Samarahan, adalah positif kultur species Burkholderia dan kurang daripada 2% daripadanya adalah positifkultur B. pseudomallei.

Kata kunci: species Burkholderia, penyakit, mikrob, kapas kesat Moore, sampel air

xi

1.0 INTRODUCTION

The Burkholderia species has been known to be naturally found in environmental water

and soil where some members of the species have been known to cause disease in humans

and animals (Mahenthiralingam et al., 2005). For example, Burkholderia pseudomallei

may cause a potentially fatal disease called melioidosis due to exposure to contaminated

environmental water or soil through skin abrasions, ingestion or inhalation

(Mayo et al., 2011).

B. pseudomallei is also known as biothreat agent (White, 2003). Dance (1991) reported

that melioidosis is endemic to Southeast Asia, Northern Australia including the temperate

regions that border the equator. It had been gradually reported from various countries

across South and East Asia as well as parts of South America, Papua New Guinea and the

Caribbean (Limmathurotsaku1 et aI., 2013).

Coenye and Vandamme (2003) stated that the majority of Burkholderia spp. are

categorized as soil bacteria. Thus, the prevalence of Burkh0 lderia spp. in the soil especially

B. pseudomallei is much linked with increased rainfalls as reported by Currie and Jacups

(2003).

.' Besides B. pseudomallei, other members of the Burkholderia spp. that potentially cause

disease are known as Burkholderia cepacia complex (Bcc), which have been reported to

cause infection to humans and animals also lead to plant disease (Mahenthiralingam et aI.,

2005). Besides, Bcc also known as plant pathogen pseudomonad, which can caused

variable lung infections in cystic fibrosis (CF) patient s which result in asymptomatic

carriage, chronic infection or 'cepacia syndrome' (Coenye & Vandamme, 2003;

Mahenthiralingam et al., 2005; Mahenthiralingam et al., 2008).

1

Mahenthiralingam et al. (2005) stated Bcc bacteria plays are environmental agent,

where helps to produces antimicrobial compounds, promotes colonizes plant root and fixes

nitrogen as well as utilize a wide variety of carbon compounds. However, due to some

human activities like agriculture and industry may post potential risk of being exposed to

the member of the Burkholderia spp. Frequent occupational exposure that have direct

contact to soils and ground water gives to an increased possibility of contracting

melioidosis, placing rice farmers and laborers at high risk (Inglis et al., 2006).

There is still limited information on the Burkholderia spp. in terms of their existence

and diversity in the environment (Stopnisek et al., 2014). In Malaysia, only a few studies

have been conducted on the prevalence of Burkholderia spp. in the environment.

According to the guidelines from Jabatan Kesihatan Negeri Pahang (n.d.), melioidosis is

endemic in that state where a study in 2003 showed that the rate of incidence was 6.1 per

100,000 population. Besides, in July 2010, there was an outbreak of melioidosis and

leptospirosis co-infection was reported among publics who involved for the search and

rescue operation of a drowning victim at Lubuk Yu, Maran recreational area that resulted

in eight fatalities. The latest issue about melioidosis was reported by Musa et al. (2015)

where it involves the epidemiology of melioidosis and investigation on the risk factors for

exposure to B. pseudomallei in small ruminant farms in Peninsular Malaysia .

.'

The selection of location for water sampling sites mainly involved random sites where

people could have a direct contact with sources of water which may be contaminated with

Burkholderia spp. The main intent of this present study is to assess the prevalence of

Burkholderia spp. in the water samples, B. pseudo mallei. Therefore, the isolation and the

identification of Burkholderia spp. from water samples were carried out.

2

The objectives of this project are:

1. To identify the identity of Burkholderia spp. from the water samples collected.

2. To compare the level of prevalence of Burkholderia spp. in the different water

sources sampled .

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3

2.0 LITERATURE REVIEWS

2.1 Burkholderia species Characteristics

Choh et al. (2013) reported that the Burkholderia genus comprises of a diverse group of

gram-negative bacteria, motile bacilli which usually found in the soil and water. The size

of the bacteria is 1~5 ~m in length and 0.5-1.0 ~m in width. Burkholderial infections are

naturally hard to treat due to their multiple antibiotic resistance properties, their ability to

form biofilms and their tendencies to cause chronic infection in the hosts. Hence,

infections of the bacteria lead to a diversity of acute, sub-acute, and chronic clinical

manifestations.

2.1.1 Burkholderia pseudomal/ei

According to Vongphayloth et al. (2012), B. ps.eudomallei is broadly scattered in Southeast

Asia as well as Northern Australia. As mentioned by McRobb et al. (2013), B.

pseudomallei has been listed as a Tier 1 Select Agent by the Center for Disease Control

and Prevention (CDC) in October 2012. This due to the B. pseudomallei itself where they

can contribute and have potential for bioweaponization, lack of vaccine availability and

also high rate of mortality.

2.2 Melioidosis

Melioidosis is a disease caused by B. pseudomallei, a saprophytic bacterium (Brook et aI.,

1997) and it has lethal possibility (Podin et al., 2014). Schweizer (2012) also mentioned

that melioidosis as the multifaceted disease and it is difficult to treat this disease therefore,

causing high rate of morbidity and mortality. Due to the slow growth of the bacteria and '.

4

PuS t Khidmat Maklumal AkndemHt J \iERSITI MALAYSIA SARAWAK

their resistance of multiple drugs, the treatment of melioidosis involves few phases namely

the intensive phase, the maintenance phase and the eradication phase for up to 3 months to

avoid relapse and recurrence (Puthucheary, 2009; Schweiger, 2012). The culture-based

isolation method in clinical laboratory remains as the "gold standard" for the infection of

B. pseudomallei as mentioned by Puthucheary (2009).

2.3 Identification of Burkholderia spp.

2.3.1 Phenotypic Identification (Bacterial Culture)

Gentamicin resistance is basically a trademark of B. pseudomallei (Podin et al.. 2014).

Ashdown's medium is an effective to be used as they mainly have the components of

crystal violet and gentamicin which acts as selective agents to isolate B. pseudomallei. The

appearance of dry wrinkled colonies is often produced by this microbe. However, Howard

and Inglis (2003) had established another selective medium called B. pseudomallei

selective agar (BPSA). The medium enhanced the isolation of culture where it increases

the colony size, improved recovery of some strains of B. pseudo mallei and also superior

selection against Burkholderia cepacia and Pseudomanas aeruginosa. However,

Ashdown's medium is concluded as more selective than BPSA by Peacock et al. (2005) in

a study where a comparison of both media was done .

.'

2.3.2 Genotypic Identification by PCR

Bacterial chromosomal DNA of B. pseudomallei can be detected using polymerase chain

reaction (PCR) assays targeting various genes. For instance, a real-time PCR targeting a

115 base pair region within orj2 of the type III secretion system (TISl) as was described

by Novak et al. (2006) and the assay has previously been determined to be significantly

more sensitive than cultivation techniques (Baker et al., 2011). In addition, a scheme for

'.. 5

the identification of all Burkholderia spp. based on the recA gene has been developed

where isolates that are tested positive by peR targeting this gene is further subjected to

DNA sequencing (Payne et al., 2005).

TTS 1 real-time peR is used to detect the presence of B. pseudomallei for the

isolates is a powerful method for rapid identification and can potentially complement to the

current confirmatory diagnostic procedures for melioidosis as described by Novak et al. in

2006.

The application of TIS 1 real-time peR is beneficial in terms of time saving. This

assay minimized the time from days to just a couple of hours to complete for the diagnosis.

Novak et al. (2006) added the adjusted assay reduces the time allowed for primary

definitive antibiotic intervention and lessen laboratorian contact time.

, 6

2.4 Previous studies of water samples collected related to B. pseudomallei

Table 2.1 A summary of previous studies related to water samples conducted in Australia and Laos. The

presence of B. pselldomallei comes from water samples which are from different sources.

Seasonal changes also contributes to the occurrence of B. pseudomallei.

I 1"

• Dry season: Bore water collected from 47 properties (188 samples).

• 25 bores fed by carbonate rock aquifers and 22 by aquifers in fractured weathered rock.

• Wet season: 26 resampled (103 samples).

• In 55 blocks of the Darwin rural region, bore water collected.

• Water samples from the bore head, water storage tank, and other water exit points (taps, hoses).

I', '.

Draper et al.

pseudomallei in the dry season. • 12 of 47 bores (26%) tested positive for B.

(2010)

• These 12 were revisited together with 14 bores negative for B. pseudomallei in the wet season, and matched for aquifer type I and location.

• II of 12 initially positive bores were again positive, and 4 of 14 (29%) previously negative bores were newly positive in the wet season.

Mayo et al.

was cultured from 18 of 55 (33%) water • During the wet season, B. pseudomallei

(20 II)

samples. 16 of 45 (36%) blocks tested I

positive. • During the dry season, 2 of 10 (20%)

blocks tested positive. ~----~------------------+-----------~--------------------;-~-----------11

• Water were collected from • Sixty-five percent showed positive presence Baker et al. topsoil and water from seasonal ofB. pselldomallei in soil sample (2011)

groundwater seeps around the • Water samples collected showed the base ofCastle Hill. presence of viable B. pselldomallei up to

• After an intensive rainfall, water samples were collected from roads and gutters m nearby residential area.

• A collection of 200 samples. • 35 of 98 (36%) and 6 of 102 (6%), were Vongphayloth el

• 50 from each sites like lakes or positive for B. pseudomallei. al. (2012)

ponds, rice fields, and boreholes • River water samples were more frequently or domestic water tanks. contaminated. I

• 100 from the Sed one River, • I positive sample from lakes, ponds, downstream and up:ltream from boreholes and domestic tanks. each reference site.

McRobb et al.

rural area, unchlorinated bore B. pseudo mallei before UV irradiation • On a house block in the Darwin • All sampling points were culture positive for

(2013)

water collected. , • Following UV sterilizer installation, all

• 151 sample: 2 weeks before the installation of a UV sterilizer.

• 2nd sample: 3 weeks after UV irradiation.

• Water collected from multiple sites upstream and downstream of the sterilizer including the bore, storage tank, garden taps, shower, and laundry.

sampling points downstream of the UV sterilizer were B. pseudomallei culture negative, indicating successful decontamination of the house water supply.

• The sampling points upstream of the UV I

filter remained positive after installation of the filter, confirming that decontamination of the water supply was attributable to UV irradiation.

\.. r ..

7

3.0 MATERIALS AND METHODOLOGY

3.1 Materials and Apparatus

The materials and other apparatus used for this project are listed in Appendix A.

3.2 Moore's Swab Preparation

The swabs used were prepared according to methods described by Barrett et al. (1980). A

modification has been done for the size of the swab used where instead of using size of 120

x 16 cm, the swabs was prepared by cutting cotton gauze into an approximate size of 60 x

16 cm. Then, the cut cotton gauzes were folded longitudinally into long cylindrical shape.

Each of the swabs was tied in the middle with cotton string and sealed sterilizing bag

before being labeled and autoclaved. The aut?claved swabs were stored until further use.

3.3 Preparation of Reagents

3.3.1 Crystal violet, 0.1 % w/v

Crystal violet was prepared by dissolving 1 g of the crystal violet powder in UHQ water ~'

with a final volume of 100 ml to constitute a final concentration of 1% weight per volume

(w/v). Then, the mixture was stirred well and stored prior to use.

3.3.2 Neutral red, 1 % w/v

Similar to crystal violet, neutral red was prepared by dissolving 1 g of the neutral red

powder in UHQ water with the final volume of 100 ml to constitute a final concentration of

I% weight per volume. Then, the mixture was also stirred well and stored prior to use. \.•1

8

3.3.3 Chelex 100, 10% w/v

This solution which was used for DNA extraction, was prepared by adding 5 g the slurry of

the Chelex 100 Resin (Bio-Rad Laboratories, CA, USA) to 100 ml of UHQ water in a

sterile 100 ml Schott bottle.

3.4 Preparation of Culture Media

The culture media that were used in this project were sterilized by autoclaving at 121°C for

15 minutes.

3.4.1 Ashdown's Selective Broth

Ashdown's selective broth was prepared by mixing 12 g of tryptone and 4 ml of 0.1 %

crystal violet with additional of UHQ water to a final volume of 1 L. The mixture was

stirred well and labelled before being autoclaved at 121°C for 15 minutes. Upon cooling

off to 50°C, 50 mglL of colistin was added aseptically.

3.4.2 Ashdown's Selective Agar (ASH)

Ashdown's selective agar was prepared by mixing 6 g oftryptone, 6 g agar powder, 16 ml

of glycerol, 2 ml of 0.1 % crystal violet and 2 ml of 1% neutral red with additional of UHQ .'

water to a final volume of 400 ml. The mixture was 'stirred well and labelled before being

autoclaved 121°Cfor 15 minutes. The mixture was allowed to cool at 55°C before 50 mglL

of colistin was added. The mixture was then poured into sterile petri dishes, allowed to

harden and cool before storing at 4°C.

9

3.5 General Methodology & Experimental Design

In general, the project was started after identification of sites were done. A survey was

carried out to identify suitable sampling site and confirmation whether there are water

sources within that site. After that, all the materials needed to collect water samples were

brought to the site.

SAMPLING (**RANDOM SITES) 8Sites (10 water samples/ site)

V · ENRICHMENT OF WATER SAMPLES

20 ml ofAshdown's Selective Broth

f­\JI

CULTURING Ashdown's Selective Agar (contains 50 mgIL Colistin)

V PHENOTYPIC IDENTIFICATION

Colony Morphology ~

1­'. tJI ~ .

GENOTYPIC IDENTIFICATION PCR(recA PCR& TIS! Real-Time PCR)

Figure 3.1 Overview of the experimental design of the project

.' Figure 3.1 shows the general experimental design of the project. The project was

initiated with a random sampling site; where water samples were collected. Later, each

water samples were enriched with Ashdown's selective broth before culturing process was

carried out. Culturing process was done by streaking the layer formed of the broth onto

Ashdown's selective agar. Phenotypic identification was carried out based on colony

, 10

morphology fonned by the bacterial isolates. Identification based on their genotype was

done through TTS 1 real-time PCR and Burkholderia-specific recA PCR.

3.6 Water Sampling

Water samples were collected from eight randomly selected sites between December 2014

and March 2015 within the UNIMAS campus and in some sites in Sabah. Table 3.1 shows

the sites where the collection of water samples have been done.

"Random sites" in this project is defined as sites selected not based on any specific

criteria. Water sampling sites were categorized into different sources which include lake,

pond, construction sites and orchard using Moore's swabs as described earlier.

GPS coordinates were recorded for every each site of each water sample collected.

The water samples were collected using Moore's swab and 10 swabs were prepared for

every each site. Swabs were tied with a fishing line and left overnight immersed in the

water. The following day, the swabs were collected and kept in sterilized plastic

containers, labelled and were prevented from exposure to direct sunlight. During

transportation to the laboratory, swabs were stored at ambient temperature until further

processing. .'

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Table 3.1 Sampling sites for the collection of water samples. Water collection have been done in two

different states involves Sabah and Sarawak.

Pond near UNIMAS stadium UNIMAS, Kota Samarahan, 1.461626; 110.435173 Sarawak

Small banana orchard lalan Kubota, Tawau, Sa bah 4.254991; 117.913996 2

Construction site I an Kubota, Tawau, Sabah 4.257265; 117.915233 3

Construction site 2 ' lalan Kubota, Tawau, Sabah 4.255847; 117.914314 4.

Walkway construction site along UNIMAS, Kota Samarahan, 1.463479; 110.431053 the CTF3 Sarawak

5

UNIMAS East Lake UNIMAS, Kota Samarahan, 1.459525; 110.431454 Sarawak

6

UNIMAS Lake UNIMAS, Samarahan, 1.468029; 110.431454 Sarawak

7

Residence area Kampung Sebayor, Kota 1.478685; 110.435472 Samarahan, Sarawak

8

3.7 Primary Isolation of Burkholderia spp. from Water Samples

The water samples were enriched with 20 ml of Ashdown's selective broth and incubated

for 2-4 days before subculturing onto Ashdown's selective agar. The tiny biofilm layer that

formed in each water sample container was streaked on the ASH agar and incubated at .'

39°C incubator. The culture plates were observed every day for colony fonnation. Each

isolate on the agar was also subcultured until pure cultures were obtained. All the colony

morphology of the isolates were recorded.

12