Podin-PhD thesis - CDU eSpace

Transcription

The epidemiology and molecular
characterization of
Burkholderia pseudomallei
in
Malaysian Borneo
By
Yuwana Podin
(B.Sc, M.Sc.)
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Menzies School of Health Research
Institute of Advanced Studies
Charles Darwin University
December 2014
DECLARATION
I hereby declare that the work herein, now submitted as a thesis for the degree of
Doctor of Philosophy of the Charles Darwin University, is the result of my own
investigations, and all references to ideas and work of other researchers have been
specifically acknowledged. I hereby certify that the work embodied in this thesis
has not already been accepted in substance for any degree, and is not being
currently submitted in candidature for any other degree.
Yuwana Podin
December 2014
ACKNOWLEDGMENTS
I would like to thank those who have contributed directly or indirectly in making
my PhD journey more pleasant and at times, more bearable.
My deepest gratitude to my supervisors: Prof. Bart Currie for introducing me to
the world of melioidosis as well as for his wisdoms and insights; Prof. Phil
Giffard for his critical inputs which often pushed me to aim higher and for his
musical guidance in our ukulele band which kept me sane through tough times;
Dr. Mirjam Kaestli for her patience and for her help in connecting the dots,
especially when it comes to the statistical analyses for my thesis.
My heartfelt thanks to the Melioid Mob: Mark Mayo the Team Manager, the pillar
of strength for the team, who was always looking out for me and everyone else;
Dr. Derek Sarovich and Dr. Erin Price for their mentoring as well as for their help
especially with the whole genome sequence analysis of the samples used for this
study; Vanessa Theobald (Ness) for being ever so helpful and cheerful; Leisha
Richardson for showing me a lot of the laboratory techniques when I first started
at Menzies and for the shared random laughter in the laboratory; Glenda
Harrington for being the ever so caring colleague; Audrey Hill for the insightful
conversations and ideas; Evan for being a sport.
Thank you to the collaborators from the hospitals and health offices in Belaga,
Bintulu, Kapit, Kuching, Miri, Sibu and Kota Kinabalu who helped in the sample
collection especially Dr. MongHow Ooi, Dr. Anand Mohan, Dr. SeeChang Wong,
Dr. Timothy William, Dr. HengGee Lee, Beatrice, Dr. Jack Wong, Dr. TemLom
Fam, Mr. Charles, Mdm. SuLin Chien, Mdm. Irene Tan, Mr. Alexander Juing,
Ms. Suriya, Ms. Desiree Wong, to all the environmental health officers and nurses
of the Sarawak Health Department whom I cannot possibly mention, and to all the
patients, their guardians and the property owners for consenting to be part of the
study.
Thank you Prof. Paul Keim and Assist. Prof. Apichai Tuanyok for the technical
support in the whole genome sequencing of the isolates for my thesis; Jann
i
Hennesy, Nicole McMahon and Rob Baird for their assistance with the E-test and
Vitek 2 test of the Malaysian Borneo isolates at the Royal Darwin Hospital
pathology laboratory.
A huge thank you to the laboratory support team (Jo Bex, Bronwyn Kennard,
Rebecca Watson), Operations (Sue Hutton, Jo Bex, Julie Green, Julianne Giffard)
and the Menzies IT support team via Area 9.
The Spin Doctors of the Centrifugation Room (which was where we were seated):
Dr. Robin Marsh, Dr. Wajahat Mahmood, Dr. Tegan Harris, soon-to-be Dr.
Jacklyn Ng, Grennady, Zuly, Evan who shared the office space, laughters and
crazy moments. My other friends at Menzies; Ammar, Irene, Shez, Anna Stephen,
Kim, the Strum Pets (Brooke, Robyn, Anna Nicholson, Jutta, Julie, Tegan, Phil)
and everyone else at Menzies for the ever-so-entertaining tearoom conversations.
My friends in Darwin; the Jollys (Peter, Teresa, Ione, Sarah, ) who took me in as
one of theirs, Ian Crawshaw for allowing me to escape to your tropical haven on
the weekends, Sarah (Hobgen) for being my kitchen buddy at the student
accommodation during the early days of our PhD, Sharon for the conversations on
the morning bus rides and other friends whom I cannot possibly mention all of
your names here.
A big thank you to M&M, my neighbours during the final 2 years of my PhD;
Marlene for the Twin Peak DVDs that got me through my writer’s block moments
and for letting me conquer the gardens; and Mark for your music which kept me
entertained. Thank you Bruno the cat who at times was my only form of
interaction with another life form for up to 2 weeks straight! (RIP Bruno).
And because you are so special, you get mentioned twice, Jacklyn Ng and
husband HG Lim; for being with me through thick and thin, and for those yummy
Malaysian cuisines.
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My friends in UNIMAS; Andrew for being a very good friend and confidante;
Kathy for being the sister I never had; my colleagues at IHCM for their support in
whatever way.
Finally, the biggest thank you to my family for their undying love and support
despite my constant absence in many of our family gatherings; my older brother
Irwan, my sister-in-law Ervina, my younger brother Nikman, my nieces Ira and
Mira. Love you all to bits.
Dedication
This thesis is dedicated to both my departed parents. I will not be what or where I
am without your love and sacrifices. Thank you Papa and Mummy for being the
wind beneath my wings.
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DECLARATION OF AUTHOR’S CONTRIBUTION IN THE
PUBLICATIONS ARISEN FROM THIS THESIS
This thesis is substantially my own work including developing the research
questions, designing the methodology, applications of research permits, collecting
and analysing data and interpreting the results, performed under the guidance of
my supervisors Prof. Phil Giffard, Prof. Bart Currie and Dr. Mirjam Kaestli. I
have written all the chapters in this thesis and the papers where I am the first
author arisen from this thesis with contribution of wordings by my supervisors.
I hereby acknowledge the following contributions of co-authors in the
publications arisen from this thesis:
Chapter 3: Automated biochemical characterization of Burkholderia pseudomallei
from Malaysian Borneo
I applied for research permits and ethics approval from the various government
agencies in Malaysia and Sarawak state for the collection of B. pseudomallei
isolates used in this study, visited the hospitals that provided the clinical isolates,
prepared and coordinated for the isolates to be transported from the various
hospitals to the collaborative centre at Universiti Malaysia Sarawak and
eventually to Menzies School of Health Research, conducted all the laboratory
tests on the isolates, performed the data analysis and wrote the manuscript for
publication and the chapter for this thesis.
Dr. Mirjam Kaestli assisted in
statistical analysis of the work in this chapter and provided critical input in the
writing process of the manuscript. Ms. Nicole McMahon, Ms. Jann Hennessy and
Mr. Robert Baird assisted in performing tests on the isolates using the Vitek 2
machine at different times. Dr. HieUng Ngian, Dr. JinShyan Wong, Dr. Anand
Mohana, Dr. SeeChang Wong and Dr. Timothy William recruited the patients and
provided clinical data of the patients whose isolates were used in this study. Mr.
Mark Mayo assisted in re-culturing of the isolates at Menzies School of Health
Research PC3 laboratory. Prof. Bart Currie conceived the idea and contributed in
providing critical input as well as wording of some parts of the manuscript.
iv
Chapter 4: Characterization of aminoglycoside and macrolide susceptible B.
pseudomallei from Malaysian Borneo
I applied for research permits and ethics approval from the various government
agencies in Malaysia and Sarawak state for the collection of B. pseudomallei
isolates used in this study, visited the hospitals that provided the clinical isolates,
prepared and coordinated for the isolates to be transported from the various
hospitals to the collaborative centre at Universiti Malaysia Sarawak and
eventually to Menzies School of Health Research, designed the reversion assay
experiments, conducted all the laboratory tests and experiments on the isolates,
performed the data analysis and wrote the manuscript for publication and the
chapter for this thesis. Dr. Derek Sarovich assisted in the analysis of the whole
genome sequence, provided intellectual input into the design of the experiments
and provided critical input into the manuscript and chapter arisen from this work.
Dr. Erin Price assisted in the analysis of the whole genome sequence of the
isolates used in the study and provided critical input during the preparation of the
manuscript and the chapter arisen from this work. Dr. Mirjam Kaestli assisted in
the molecular testing of the isolates and provided critical input during the
preparation of the manuscript and the chapter arisen from this work. Mr. Mark
Mayo assisted in the culture of the isolates and provided critical input during the
preparation of the manuscript and the chapter arisen from this work. Dr.
KingChing Hii, Dr. HieUng Ngian, Dr. SeeChang Wong, Dr. IngTien Wong, Dr.
JinShyan Wong, Dr. Anand Mohan, Dr. MongHow Ooi, Dr. TemLom Fam and
Dr. Jack Wong recruited the patients and provided clinical data of the patients
whose isolates were used in this study. Assist. Prof. Apichai Tuanyok and Prof.
Paul Keim provided support in the whole genome sequencing of the isolates used
in this study. Prof. Phil Giffard and Prof. Bart Currie conceived the idea and
contributed in providing critical input and wordings into the manuscript
preparation as well as the chapter arisen from this work.
v
TABLE OF CONTENTS
Acknowledgments…………………………………………………………………i
Declaration of author’s contributions…………………………………………….iv
Table of contents………………………………………………………………….vi
Table of figures…………………………………………………………………...xi
Table of tables……………………………………………………………………xii
Abstract………………………………………………………………………….xiii
Abbreviations…………………………………………………………………….xv
Chapter 1: General introduction and literature review……………………….1
1.1 General introduction: Burkholderia pseudomallei and melioidosis……...……1
1.1.1 Burkholderia genus……………………………………………………..1
1.1.2 Burkholderia pseudomallei……………………………………………..1
1.1.3 Melioidosis……………………………………………………………...3
1.1.4 Melioidosis in animals………………………………………………….6
1.2 Literature review: Melioidosis in Malaysia…………………………………...9
1.2.1 Epidemiology of melioidosis in Malaysia……………………………...9
1.2.2 Melioidosis in animals in Malaysia…………………………………...15
1.2.3 Scientific interests on melioidosis in Malaysia………………………..16
1.3 Summary and implications of literature review……………………………...27
1.4 B. pseudomallei and melioidosis in Malaysian Borneo……………………...28
1.5 Study aims……………………………………………………………………32
1.6 Thesis outline………………………………………………………………...33
Chapter 2: Phenotypic characterization and molecular epidemiology of
clinical B. pseudomallei isolates from Malaysian Borneo…………………….35
2.1 Introduction…………………………………………………………………..35
2.1.1 Culture methods……………………………………………………….35
2.1.2 Confirmation methods…………………………………………………37
2.1.3 Methods of characterizing B. pseudomallei…………………………...39
2.1.4 Study hypothesis………………………………………………………44
2.1.5 Study objectives……………………………………………………….44
2.2 Materials and methods……………………………………………………….45
2.2.1 Ethics…………………………………………………………………..45
2.2.2 Collection of B. pseudomallei clinical isolates from hospitals………..45
2.2.3 Movement of B. pseudomallei clinical isolates……………………….45
2.2.4 Revival of B. pseudomallei clinical isolates transport from UNIMAS to
Menzies School of Health Research………………………………………...47
2.2.5 Morphological identification of B. pseudomallei from culture……….47
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2.2.6 B. pseudomallei culture confirmation by antigen detection using latex
agglutination test…………………………………………………………….47
2.2.7 Antibiotic susceptibility testing (E-test)………………………………49
2.2.8 Testing of colistin susceptibility of GEN sensitive B. pseudomallei
isolates and modification of Ashdown’s agar…………………………….....50
2.2.9 DNA extraction using QIAamp DNA mini kit………………………..51
2.2.10 Type III secretion system real-time PCR…………………………….51
2.2.11 Detection of the B. thailandensis-like flagellum and chemotaxis
biosynthesis (BTFC) and the Yersinia-like fimbrial (YLF) gene clusters…..52
2.2.12 bimABm and bimABp allele real-time PCR methods…………………..52
2.2.13 Multilocus sequencing typing …………………...…………………..53
2.2.14 Multiple-locus VNTR analysis methods ……………………………54
2.2.15 Analysis of MLVA-4 results ………………………………………..55
2.2.16 Whole genome sequencing…………………………………………..55
2.3 Results and discussions………………………………………………………57
2.3.1 Morphotypes…………………………………………………………..57
2.3.2 Latex agglutination test……………………………………………….57
2.3.3 Antibiotic susceptibility testing (E-test)…………………..…………..59
2.3.4 Colistin susceptibility experiment……………………………………..59
2.3.5 TTS1, BTFC/YLF gene cluster, bimABm/bimABp alleles real-time PCR
assays………………………………………………………………………..65
2.3.6 MLST results………………………………………………………….66
2.3.7 MLVA-4 results……………………………………………………….75
2.3.8 Whole genome sequencing……………………………………………80
2.4 Conclusions…………………………………………………………………..84
Chapter 3: Automated biochemical characterization of B. pseudomallei from
Malaysian Borneo………………………………………………………………86
3.1 Introduction………………………………………………………………….87
3.2.1 Clinical isolates used for the study……………………………………88
3.2.2 VITEK 2 system……………………………………………………….88
3.2.3 Confirmation of B. pseudomallei isolates by PCR……………………89
3.2.4 Confirmation of B. pseudomallei isolates by latex agglutination test…89
3.2.5 Statistical analysis……………………………………………………..89
3.3 Results and discussions………………………………………………………90
3.4 Conclusions…………………………………………………………………..94
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Chapter 4: Characterization of aminoglycoside and macrolide susceptible
B. pseudomallei from Malaysian Borneo………………………………………95
4.1 Introduction…………………………………………………………………..96
4.2.1 Ethics…………………………………………………………………..97
4.2.2 Bacterial strains………………………………………………………..97
4.2.3 MLST………………………………………………………………….97
4.2.4 Antibiotic susceptibility testing……………………………………….99
4.2.5 WGS and analysis……………………………………………………..99
4.2.6 Allele-specific PCR……………………………………………………99
4.2.7 Restoration of aminoglycoside resistance……………………………..99
4.2.8 Di-deoxy sequencing…………………………………………………100
4.2.9 Primer design………………………………………………………...100
4.3 Results and discussions……………………………………………………..103
4.3.1 MLST of Sarawak isolates indicates restricted geographical
distribution…………………………………………………………………103
4.3.2 ST881 and ST997 isolates are susceptible to the aminoglycosides and
macrolides………………………………………………………………….104
4.3.3 Reversion of the C1102G mutation restored aminoglycoside and
macrolide resistance………………………………………………………..105
4.3.4 Clinical and environmental significance of aminoglycoside and
macrolide sensitive B. pseudomallei in Sarawak…………………………..105
4.4 Conclusions…………………………………………………………………106
Chapter 5: Environmental investigation of B. pseudomallei and other
Burkholderia spp. in Sarawak………………………………………………...111
5.1 Introduction…………………………………………………………………111
5.1.1 Burkholderia cepacia complex………………………………………111
5.1.2 Ecology of B. pseudomallei………………………………………….112
5.1.3 Environmental surveys of B. pseudomallei…………………………..114
5.1.4 Unchlorinated water supply harbouring B. pseudomallei……………115
5.1.5 Landscape use in central Sarawak …………………………………..116
5.1.6 Statement of working hypothesis…...………………………………..116
5.2 Isolation and epidemiology of environmental B. pseudomallei and
Burkholderia spp. in Sarawak…………………………………………………..119
5.2.1 Materials and methods……………………………………………….119
5.2.1.1 Environmental sampling strategies……………….…….……119
5.2.1.2 Sample size power calculation………………………………119
5.2.1.3 First environmental sampling………………………………..120
5.2.1.4 Second environmental sampling……………………………..120
5.2.1.5 Permission and consent……………………………………...122
5.2.1.6 Soil sample collection………………………………………..122
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5.2.1.7 Water sample collection……………………………………..122
5.2.1.8 Recovery of B. pseudomallei from soil samples…………….123
5.2.1.9 Recovery of B. pseudomallei from water samples…………..123
5.2.1.10 Use of Ashdown’s agar with gentamicin and with
colistin……………………………………………………………….124
5.2.1.11 Morphological identification of B. pseudomallei from
culture…………………………………………..……………………126
5.2.1.12 Transport of isolates from UNIMAS laboratory to Menzies
School of Health Research…………………………………………..126
5.2.1.13 Revival of sweeps and isolates transported from UNIMAS to
Menzies School of Health Research…………………………………126
5.2.1.14 Storage of bacterial isolates………………………………...127
5.2.1.15 DNA extraction of sweeps of bacterial culture using
Chelex ® 100……………………………………………..…..……...127
5.2.1.16 Type III secretion system real-time PCR…………………..127
5.2.1.17 recA PCR………………………..………………………….128
5.2.1.18 recA sequencing…………………………………………….128
5.2.1.19 16S ribosomal DNA sequencing…………………………...129
5.2.1.20 Analysis of DNA sequences………………………………..129
5.2.2 Results and discussions………………………………………………130
5.3 Soil inoculation experiment………………………………………………...140
5.3.1 Materials and methods……………………………………………….140
5.3.1.1 Modified Ashdown’s agar with 50 μg/ml colistin…………...140
5.3.1.2 Sterilization of soil and quality control……………………...140
5.3.1.3 Preparation of B. pseudomallei isolates……………………...140
5.3.1.4 Soil inoculation with selective B. pseudomallei isolates…….141
5.3.1.5 Soil DNA extraction…………………………………………141
5.3.1.6 PCR of B. pseudomallei inoculums into soil………………...141
5.3.1.7 Recovery of viable B. pseudomallei from inoculated soil
samples………………………………………………………………142
5.3.1.8 DNA extraction and TTS1 real-time of sweeps from plates A, B
and C…………………………………………………………………142
5.3.1.9 Experimental limitations……………………………………..142
5.4 Antibiosis activities of Burkholderia spp. towards aminoglycoside susceptible
B. pseudomallei from Sarawak………………………………………..……..…147
5.4.1 Materials and methods………………………………………….……147
5.4.1.1 Antagonism screening……………………………………….147
5.4.1.2 Bacterial isolates used……………………………………….147
5.4.1.3 Preparation of bacterial culture………………………………149
5.5 Summarized discussions and conclusions for Chapter 5…………………...156
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Chapter 6: Concluding chapter………………………………………………161
6.1 General discussions…………………………………………………………161
6.2 Future directions…………………………………………………………….167
References……………………………………………………………………...174
Appendix A: Summary of research permits and ethics approval for
melioidosis study in Malaysian Borneo……………………………………....205
Appendix B: Soil sampling form for B. pseudomallei……………..………...206
Appendix C: Published papers from this thesis……………………………..208
x
TABLE OF FIGURES
Figure 1.1 Map of the states in Malaysia………………………………………………..14
Figure 1.2 Melioidosis in average annual rate per 100 000 population in Sarawak
from 2007-2010 ………………………………………………………………………….31
Figure 2.1 Latex agglutination test for B. pseudomallei…………………………….…..48
Figure 2.2 B. pseudomallei isolates from Malaysian Borneo regrown on Ashdown’s
agar showing different morphotypes………………………………………….………….58
Figure 2.3 Illustration of colistin susceptibility test on GENs B. pseudomallei from
Sarawak (MSHR5097&MSHR5104) with controls P. aeruginosa and MSHR668
from the NT………………………………………………………………………………64
Figure 2.4 Agar plate layout of colistin susceptibility test showing the positions of discs
impregnated with different concentrations of colistin…………………………………...64
Figure 2.5 Map of Malaysian Borneo showing the different STs from hospitals in Kota
Kinabalu, Bintulu, Kapit and Sibu ..……………………………………………………..68
Figure 2.6 eBURST of all Malaysian isolates….…………………………………….….71
Figure 2.7 eBURST of Malaysian and other Asian strains .…………………………….72
Figure 2.8 eBURST of Australian and Asian isolates…………………………………...74
Figure 2.9 Morphotype variants in isolates from patient SBU-1 having the same MLVA4 type 374 ……………………………………………………………………...………...78
Figure 2.10 Different morphotypes from isolates of patient SBH-5 with the same
MLVA-type 360………………………………………………………………………….79
Figure 2.11 Radial genetic relatedness tree constructed using ~91,000 SNPs across 23 B.
pseudomallei including selected isolates from Sarawak…………………………………82
Figure 2.12 Map showing the demarcation of the Wallace’s Line…………...…………83
Figure 3.1 Nonmetric multidimensional scaling (nMDS) ordination on the Euclidean
distance resemblance matrix of the Vitek-2 biochemical profile of 235 B. pseudomallei
and B. cepacia isolates from Australia and Malaysian Borneo……………………...…..93
Figure 4.1 Map of study sites ………………………………………………………......98
Figure 4.2 Amplification plot of GENs allelic-specific real-time PCR………………..108
Figure 4.3 Alignment of the amrB hyperconserved membrane spanning domain in
selected B. pseudomallei isolates………..……………………………………………...108
Figure 5.1 Mean annual rainfall for the years 1995-2009 in Sarawak districts selected
for the environmental survey for B. pseudomallei……………………………..……….121
Figure 5.2 Map showing the environmental sampling sites …………………………...121
Figure 5.3 ‘Sandak’, a locally available tool that was used to dig holes………………124
Figure 5.4 Procedures for culturing B. pseudomallei from soil samples……………....125
Figure 5.5 Procedures for culturing B. pseudomallei from water samples…………….125
Figure 5.6 Map of Sarawak overlaid with human activities…………………………...132
Figure 5.7 Comparison of different morphologies of selected non-B. pseudomallei
environmental and B. pseudomallei isolates……………………………………………138
Figure 5.8 Set-up of plates for antagonism experiment………………………………..149
Figure 5.9 Zones of inhibition around the producers (arrows)………………………...154
Figure 5.10 Lysis of inoculated producers …………………………………………….154
Figure 5.11 Other forms of antibiosis. Panels B and C show no antibiosis activities by
either the target cells or the challengers. Panels A and D show that the producers were
taken over by the indicator……………………………………………………………...155
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TABLE OF TABLES
Table 1.1 Animal melioidosis cases reported thus far………..………………..….8
Table 1.2 Studies on melioidosis conducted in Malaysia………………………..18
Table 1.3 Summary of clinical presentations in melioidosis cases reported in
Malaysia………………………………………………………………………….22
Table 1.4 Clinical risk factors for melioidosis cases in Malaysia……………….24
Table 1.5 Occupations of melioidosis patients in Sarawak from years 2007 to
2010………………………………………………………………………………30
Table 2.1 Number of B. pseudomallei Malaysian Borneo isolates collected and
information on the hospitals……………………………………………………...46
Table 2.2 B. pseudomallei MLST loci…………………………………………...53
Table 2.3 B. pseudomallei isolates from Sarawak subjected to WGS…………..56
Table 2.4 Minimum inhibitory concentrations results for B. pseudomallei isolates
used in this study………………………………………………………..………..60
Table 2.5 STs found in Malaysian Borneo …………………………………...…70
Table 2.6 MLVA-4 types and VNTR allelic profiles of selected patients
……………………………………………………………………………………77
Table 3.1 Number of isolates tested with VITEK 2 system……………………..92
Table 4.1 Accession numbers of partial amrB gene sequences deposited in
GenBank………………………………………………………………………...101
Table 4.2 B. pseudomallei isolates used in this study………………………….101
Table 4.3 Minimum inhibitory concentrations for B. pseudomallei isolates
obtained in this study…………………………………………………………...109
Table 5.1 Number of soil samples collected from the different districts in
Sarawak…………………………………………………………………………130
Table 5.2 Comparison of culture yields using Ashdown’s agar with gentamicin
and colistin……………………………………………………………………...135
Table 5.3 Soil samples positive for Burkholderia spp. by culture……………..135
Table 5.4 Type of Burkholderia spp. isolated from the different types of sampling
sites……………………………………………………………………………..136
Table 5.5 Ct values of TTS1 real-time PCR of soil extracts and plates A, B and
C………………………………………………………………………………...146
Table 5.6 Isolates used for antagonistic screening……………………………..148
Table 5.7 Overall results of antagonistic experiment…………………………..151
xii
ABSTRACT
Melioidosis is a potentially fatal disease caused by Burkholderia pseudomallei
which is endemic in Malaysian Borneo. The general aim of this study is to
elucidate the molecular epidemiology of B. pseudomallei in Malaysian Borneo.
Consistent with the Wallace line theory of separation, genotyping showed
Malaysian Borneo clinical B. pseudomallei isolates were more related to
Southeast Asian strains than to Australian strains. Whole genome sequencing
demonstrated that B. pseudomallei from Sarawak were very closely related to
each other. Biochemical testing using VITEK 2 revealed that 25% of B.
pseudomallei from Malaysian Borneo were misidentified as B. cepacia,
suggesting that specificity of that identification system is regionally dependent. A
major and unexpected finding was that 88% of Sarawak B. pseudomallei were
gentamicin susceptible, with these B. pseudomallei being restricted to multilocus
sequence type ST881 and its single locus variant ST997. A novel nonsynonymous mutation was identified within amrB, an essential component of the
AmrAB-OprA multi-drug efflux pump. Reversion of the mutation to the wild-type
sequence confirmed the role of this mutation in conferring aminoglycoside and
macrolide sensitivity. No environmental B. pseudomallei were isolated from
Sarawak but other Burkholderia species were isolated, prompting the formulation
of hypotheses to explain the lack of environmental B. pseudomallei. Although
inconclusive, experiments showed antagonistic activities by other environmental
Burkholderia spp. recovered from environmental sampling studies towards B.
pseudomallei and also that gentamicin susceptible B. pseudomallei were slightly
xiii
less robust than gentamicin resistant strains in competing with other soil
microorganisms. This thesis contributed to the understanding of the population
structure of B. pseudomallei in Malaysian Borneo, Southeast Asia and globally.
The discovery of gentamicin susceptibility in Sarawak B. pseudomallei has
significant implications for laboratory diagnosis and environmental sampling of B.
pseudomallei in Malaysian Borneo and potentially in other melioidosis endemic
regions.
Although
the
exact
distributions,
quantification and
potential
environmental hazards and implications of B. pseudomallei in Malaysian Borneo
remain uncertain, these studies have led to important research questions now to be
explored. Most immediate is further searching for the proposed existence of an as
yet unidentified localized niche of B. pseudomallei in Malaysian Borneo.
xiv
ABBREVIATIONS
amrB-C1102G
AMX
ANOSIM
AQIS
AS-PCR
AZM
Bcc
bimABm
bimABp
BNAG
bp
BPSA
BTFC
C
C→G
CAZ
CF
CLSI
Ct
dNTP
DOX
ELISA
EPS
FAM
G
GEN
GENr
GENs
HBA
IFAT
IgG
IgM
IHA
KAN
kb
LGT
MALDI-TOF
MS
Mbp
MEM
MH
MIC
MLST
MLVA
Cytosine to guanine transition at 1102 nucleotide
position of the amrB gene
Amoxicillin-clavulanic acid
Analysis of similarities
Australian Quarantine and Inspection Service
Allelic-specific PCR
Azithromycin
Burkholderia cepacia complex
B. mallei-like BimA allele
B. pseudomallei BimA allele
β-N-acetyl-glucosaminidase
Base-pairs
B. pseudomallei selective agar
B. thailandensis-like flagellum and chemotaxis
biosynthesis gene cluster
Cytosine
Cytosine to guanine transition
Ceftazidime
Cystic fibrosis
Clinical and Laboratory Standard Institute, USA.
Threshold cycle
Deoxynucleoside triphosphates
Doxycycline
Enzyme-linked immunosorbent assays
Exopolysaccharide
FAM blue reporter dye
Guanine
Gentamicin
Gentamicin resistance
Gentamicin sensitive
Horse blood agar
Immunofluorescent assay test
Immunoglobulin G
Immunoglobulin M
Indirect hemagglutination assay
Kanamycin
Kilo base-pairs
Lateral gene transfer
Matrix-assisted laser desorption ionization-time of
flight mass spectrometry
Mega base-pairs
Meropenem
Mueller- Hinton’s agar
Minimal inhibition concentration
Multilocus sequence typing
Multilocus VNTR analysis
xv
MGBNFQ
NAGA
NED
NGS
nMDS
NT
OD
orf2
PC2
PET
PFGE
PNAG
R
RAPD
RDH
rDNA
RFLP
RND
rpm
SIMPER
SLV
SNP
spp.
ST
SXT
T
T367R
TSA
TTS1
UMMC
UNIMAS
US CDC
USAMRU
VNTR
WGS
YLF
∆Ct
Molecular-groove binding non-fluorescence quencher
β-N-acetyl-galactosaminidase
NED yellow reporter dye
Next-generation sequencing
Nonmetric multidimensional scaling
Northern Territory
Optical density
Open reading frame 2
Physical Containment Level 2 (equivalent of BSL-2)
PET red reporter dye
Pulsed-field gel electrophoresis
Poly-β-(1-6)-N-acetyl-glucosamine
Arginine
Randomly amplified polymorphic DNA
Royal Darwin Hospital
Ribosomal DNA
Restriction fragment length polymorphism
Resistance-nodulation-division
Revolutions per minute
Similarity percentages
Single locus variant
Single nucleotide polymorphism
Species
Sequence type
Trimethoprim-sulfamethoxazole
Threonine
Threonine to arginine substitution at amino acid
position 367
Tryptic soy agar
Type III secretion system
University Malaya Medical Centre
Universiti Malaysia Sarawak
United States Centers for Disease Control and
Prevention
United States of America Medical Research Unit
Variable numbers tandem repeat
Whole genome sequencing
Yersinia-like fimbrial gene cluster
Threshold cycle difference
xvi
Chapter 1
General Introduction
and
Literature Review
Chapter 1: General introduction and literature
review
____________________________________________
1.1 General introduction: Burkholderia pseudomallei and melioidosis
1.1.1 Burkholderia genus
The Burkholderia genus was formed in 1992 when seven members of the
Pseudomonas species were transferred over to the newly formed Burkholderia
genus (Yabuuchi et al., 1992). The name “Burkholderia” was coined to honour
Walter Burkholder who discovered Pseudomonas cepacia which caused onion
diseases in the 1940s and 1950s (Yabuuchi et al., 1992, Coenye and Vandamme,
2007). To date, the Burkholderia genus has over 60 species residing in diverse
ecologies
including
environmental
and
(http://www.bacterio.net/burkholderia.html)(Coenye
biological
and
Vandamme,
niches
2003,
Vandamme and Dawyndt, 2011). Most Burkholderia species (spp.) are considered
plant pathogens, plant commensals and soil bacteria, but some have the ability to
cause infections in humans and animals (Coenye et al., 2001, Coenye and
Vandamme, 2003, Coenye and Vandamme, 2007, Mahenthiralingam et al., 2005).
There are 17 closely related Burkholderia spp. that are categorised as
Burkholderia cepacia complex (Bcc). While some Bcc members are considered
plant pathogens, others are plant commensals and also bioremediation agents
which degrade environmental pollutants (Mahenthiralingam et al., 2005). In
addition to their roles in plants and environments, some Bcc have been reported to
cause opportunistic infection in both immunocompromised and cystic fibrosis
(CF) patients (Coenye and Vandamme, 2003, Coenye and Vandamme, 2007,
Vandamme and Dawyndt, 2011).
1.1.2 Burkholderia pseudomallei
Burkholderia pseudomallei is the bacterium that causes melioidosis which was
first described by Whitmore and Krishnaswami in Rangoon, Burma in 1911
(Whitmore and Krishnaswami, 1912). Due to the resemblance of the bacterium to
Bacillus mallei which causes glanders disease, it was named Bacillus
pseudomallei. Over the years, the bacterium was also known by different names
such as Bacillus whitmorii (Bacille de Whitmore in French), Malleomyces
1
pseudomallei, Pseudomonas pseudomallei and finally B. pseudomallei in 1992
(Yabuuchi et al., 1992). It is a non-spore forming motile gram-negative bacillus
with bipolar staining, often described as resembling a safety pin in a Gram stain
(Cheng and Currie, 2005). The genome of B. pseudomallei is comprised of two
circular chromosomes with a combined length of 7.2 mega base-pairs (Mbp),
encompassing approximately 5800 genes (Holden et al., 2004, Nandi et al., 2010).
Chromosome 1 contains housekeeping genes for macromolecular biosynthesis,
amino acid metabolism, cofactor and carrier synthesis, nucleotide and protein
biosynthesis, chemotaxis, and mobility. Chromosome 2 contains genes for
accessory functions such as adaptations to atypical conditions, iron homeostasis,
secondary metabolism, regulation and horizontal gene transfer (Holden et al.,
2004). Due in part to the high virulence of this organism and increased concerns
for transmission by aerosolization, B. pseudomallei was classified as a Tier 1
select agent by the U.S. Centres for Disease Control and Prevention (US CDC) in
2012 (http://www.selectagents.gov/).
B. pseudomallei is closely related to two other Burkholderia spp. namely
Burkholderia thailandensis and Burkholderia mallei (Brett et al., 1998, Nierman
et al., 2004). Despite its high genomic similarities to B. pseudomallei, B.
thailandensis is an avirulent soil bacterium that is able to utilize arabinose as a
sole energy source (Brett et al., 1998, Trakulsomboon et al., 1999). B. mallei is a
zoonotic host-adapted pathogen affecting mainly equines and causes glanders
disease (Nierman et al., 2004). Studies showed that B. mallei evolved from a
single strain of B. pseudomallei ancestor through erosion of nonessential genomes
responsible for environmental survival and became completely host-adapted. It
was also shown that the genome of B. mallei is approximately 1.5 Mbp smaller
than that of its ancestor (Godoy et al., 2003, Nierman et al., 2004, Losada et al.,
2010).
B. pseudomallei is commonly found in the soil and water of melioidosis endemic
regions, which include Southeast Asian countries, northern Australia and other
tropical regions (White, 2003, Cheng and Currie, 2005). As more melioidosis
areas are discovered, the knowledge on global distribution will keep expanding
over time (Currie et al., 2008, Wiersinga et al., 2012).
2
1.1.3 Melioidosis
Coined by Stanton and Fletcher in 1921, melioidosis was derived from Greek
which means ‘distemper of asses’ (Stanton and Fletcher, 1921). Patients with
melioidosis can present with a spectrum of clinical presentations including
localized skin abscess, acute or chronic pneumonia, genitourinary, bone, and joint
infections and severe systemic sepsis (with or without foci of multiple abscesses
in internal organs). Patients with septic shock may have a mortality of >90%
(White, 2003, Cheng and Currie, 2005). Asymptomatic and chronic infections
have been reported where the organism has been shown to evolve within the host
(Price et al., 2010, Price et al., 2013a).
Mode of acquisition
The mode of acquisition of melioidosis is via percutaneous inoculation, inhalation
or ingestion of B. pseudomallei contaminated wet soil or surface water or aerosols
(Puthucheary and Vadivelu, 2002, Cheng and Currie, 2005, Wiersinga et al.,
2012).
Inoculation has been suggested to be the main mode of acquisition. Individuals
with occupational risks such as farmers and construction workers were reportedly
exposed to B. pseudomallei through open wound or penetrating injuries
(Chaowagul et al., 1989, Cheng and Currie, 2005, Kaestli et al., 2009, Wiersinga
et al., 2012). In a study in the Top End, Australia, 25% of melioidosis patients
were shown to have some history of inoculation via skin breakage prior to disease.
Despite being acquired through skin inoculation, the range of disease in the
patients in this study was not confined to mere subcutaneous symptoms as many
patients presented with more severe illnesses (Currie et al., 2000b).
Inhalation was implicated as the primary mode of acquisition for melioidosis
cases amongst helicopter crews of the United States armies in Vietnam in the
1960s (Howe et al., 1971). Since then, inhalation has been considered to be an
important mode of acquisition especially during increased heavy rainfall and
during extreme weather events such as strong winds, cyclones or typhoons
(Puthucheary and Vadivelu, 2002, Currie and Jacups, 2003, Ko et al., 2007, Lo et
al., 2009, Su et al., 2011) .
3
The first implication of ingestion as a mode of acquisition was by Stanton and
Fletcher in 1925 (Stanton and Fletcher, 1925). Although this route has not been
described much in detail, reports of melioidosis outbreaks due to contaminated
water supply have implicated ingestion as a mode of acquisition (Inglis et al.,
1999, Currie et al., 2001). The high incidence of suppurative parotitis amongst
paediatric patients mainly in South East Asia has been suggested to be associated
with ingestion (Dance et al., 1989a) and a recent case control study from Thailand
has supported this, together with the high rates of recovery of B. pseudomallei
from domestic water supplies in that study (Limmathurotsakul et al., 2013b).
B. pseudomallei acquisition has also been associated with tsunami where
survivors of the disaster in affected countries were presented with various
symptoms including cutaneous infection, pulmonary involvement and septicaemia
(Allworth, 2005, Athan et al., 2005, Chierakul et al., 2005, Nieminen and Vaara,
2005, Svensson et al., 2006, Othman et al., 2007, Currie et al., 2008, Arzola et al.,
2007).
Other modes of acquisition are via laboratory-acquired infection (Green and
Tuffnell, 1968, Schlech et al., 1981), contaminated detergent and other medical
supplies (Punyagupta, 1989, Gal et al., 2004), breast milk (Ralph et al., 2004),
person-to-person transmission (McCormick et al., 1975, Holland et al., 2002),
possible sexual transmission (McCormick et al., 1975, Webling, 1980) and intrauterine transmission (Abbink et al., 2001). Although rare, zoonotic human
infections have also been reported (Low Choy et al., 2000).
Treatment of melioidosis
Due to both the intracellular nature of the organism after infection and its
potential for latency, there are two phases in melioidosis treatment; a short-term
intensive acute phase and a long-term oral eradication phase (Wiersinga et al.,
2012). In general, the intensive acute phase treatment involves bactericidal drugs
with or without post-antibiotic effect, while the eradication phase treatment
involves bacteriostatic drugs (Estes et al., 2010, Wiersinga et al., 2012). The
current recommended treatment of melioidosis consists of an intensive phase of at
least 10 to 14 days of intravenous ceftazidime, meropenem, or imipenem,
4
followed by oral eradication therapy of trimethoprim–sulfamethoxazole for three
to six months (Wiersinga et al., 2012).
Drug susceptibility and resistance
B. pseudomallei is intrinsically resistant to numerous antibiotics including firstgeneration
and
cephalosporins,
second-generation
all
macrolides,
most
quinolones,
all
penicillins,
all
narrow-spectrum
polymyxins,
and
aminoglycosides (Livermore et al., 1987, Dance et al., 1989c, Dance et al., 1989b,
Jenney et al., 2001, Estes et al., 2010, Wuthiekanun et al., 2011), which limits
treatment options for melioidosis. Although rare, aminoglycosides and macrolides
sensitivity has been reported (Simpson et al., 1999, Trunck et al., 2009, Podin et
al., 2014). B. pseudomallei is generally susceptible to carbapenems, some
fluoroquinolones,
and
amoxicillin-β-lactamase
inhibitor
combinations,
chloramphenicol, tetracyclines and trimethoprim-sulfonamides in vitro (Ashdown,
1988, Dance et al., 1989c, Wuthiekanun et al., 2011). However, clinical studies
have shown that fluoroquinolones are associated with higher treatment failure
rates (Chaowagul et al., 1997, Chetchotisakd et al., 2001, Steward et al., 2005,
Wuthiekanun et al., 2011). Although uncommon, acquired resistance to
ceftazidime during therapy has been documented and genetically characterised
(Jenney et al., 2001, Chantratita et al., 2011, Sarovich et al., 2012a, Sarovich et
al., 2012b).
Relapse of melioidosis
Relapse in melioidosis upon completion of treatment has been well documented
(Puthucheary and Vadivelu, 2002, White, 2003, Cheng and Currie, 2005, Sarovich
et al., 2014b). There have also been reports of reactivation of melioidosis for as
long as 62 years after primary exposure to the causative agent (Ngauy et al.,
2005). The propensity for relapse is caused by factors such as poor adherence to
treatment, severe diseases, inadequate use of drug type during the intensive and
eradication phase, and eradication phase of less than eight weeks (Chaowagul et
al., 1993, Currie et al., 2000a). While most of the relapse cases were associated
with reactivation of the original infecting strain, re-infection with a different strain
has also been reported (Desmarchelier et al., 1993, Vadivelu et al., 1998, Currie et
al., 2000a, Sarovich et al., 2014b).
5
Predisposing factors
Predisposing factors such as diabetes mellitus, chronic renal disease, chronic lung
disease and excessive alcohol consumption have been associated with higher risk
of melioidosis (Merianos et al., 1993, Suputtamongkol et al., 1994,
Suputtamongkol et al., 1999, Currie et al., 2000b, Puthucheary and Vadivelu,
2002, White, 2003, Cheng and Currie, 2005). Although uncommon, thalassemia
has been documented to be associated with increased risk of melioidosis in
Thailand and Malaysia (Hoc et al., 1993, Suputtamongkol et al., 1994)(A. Mohan,
personal communication). In Thailand, the use of a steroidal herbal remedy called
“yaa chud” has been also associated with increased risk of melioidosis
(Suputtamongkol et al., 1994). Similar risk was observed in Australia with the
usage of kava amongst Australian Aboriginals (Currie et al., 2000b). Interestingly,
HIV patients have not been reported to be necessarily more at risk of melioidosis
(Chierakul et al., 2004).
1.1.4 Melioidosis in animals
The first ever recorded cases of animal melioidosis was an outbreak amongst
laboratory guinea-pigs and rabbits in the animal facility at the Institute if Medical
Research, Kuala Lumpur in what was then known as the Federated States of
Malaya (Malaya) in 1913. The causative agent of the outbreak was only identified
in 1917 (Stanton and Fletcher, 1925). Animal melioidosis have been documented
globally involving a wide range of animal species including sheep (Cottew, 1950,
Dance, 1991, Mustaffa Babjee and Nor Aidah, 1994), goats (Stanton and Fletcher,
1925, Dance, 1991, Ouadah et al., 2007, Lu et al., 2013), pigs (Ketterer et al.,
1986, Dance, 1991, Mustaffa Babjee and Nor Aidah, 1994), horses (Stanton and
Fletcher, 1925, Stanton et al., 1927, Dance, 1991, Mustaffa Babjee and Nor
Aidah, 1994, White, 2003, Ladds et al., 1981), panda (Dance, 1991, White, 2003),
camels (Low Choy et al., 2000, Dance, 2000b), fallow deer (Mustaffa Babjee and
Nor Aidah, 1994), alpaca (Low Choy et al., 2000), crocodile (Low Choy et al.,
2000), koalas (Ladds et al., 1990), kangaroos (Low Choy et al., 2000, Egerton,
1963), cattle (Egerton, 1964, Ketterer et al., 1975, Mustaffa Babjee and Nor
Aidah, 1994, Low Choy et al., 2000), dogs (Mustaffa Babjee and Nor Aidah,
1994, Low Choy et al., 2000), cats (Stanton and Fletcher, 1925, Low Choy et al.,
2000), serows (Vellayan, 1994), primates (de Silva, 1971, Peters et al., 1976,
6
Vellayan, 1994, Ouadah et al., 2007, Sommanustweechai et al., 2013, Ritter et al.,
2013), iguanas (Zehnder et al., 2014), birds (Thomas et al., 1980, Vellayan, 1994,
Hampton et al., 2011, Ritter et al., 2013) and captive marine mammals (Hicks et
al., 2000, Kinoshita, 2008). The severity and manifestation of the disease varied
from one animal species to another where organs that were most commonly
affected were the lungs, spleen, liver and associated lymph nodes (Low Choy et
al., 2000, Sprague and Neubauer, 2004). A summary of types of animals affected
by melioidosis reported thus far is shown in Table 1.1.
7
Table 1.1 Animal melioidosis cases reported thus far
Animal type
Reference(s)
Alpaca
(Low Choy et al., 2000)
Birds
(Thomas et al., 1980, Vellayan, 1994, Hampton et al.,
2011, Ritter et al., 2013)
Camels
(Low Choy et al., 2000, Dance, 2000b)
Captive marine
mammals
Cats
(Hicks et al., 2000, Kinoshita, 2008)
Cattle
(Egerton, 1964, Ketterer et al., 1975, Mustaffa Babjee
and Nor Aidah, 1994, Low Choy et al., 2000)
Crocodile
(Low Choy et al., 2000)
Dogs
(Mustaffa Babjee and Nor Aidah, 1994, Low Choy et
al., 2000)
Fallow deers
(Mustaffa Babjee and Nor Aidah, 1994)
Guinea-pig
(Stanton and Fletcher, 1925)
Goats
(Stanton and Fletcher, 1925, Dance, 1991, Ouadah et
al., 2007, Lu et al., 2013)
Horses
(Stanton and Fletcher, 1925, Stanton et al., 1927, Dance,
1991, Mustaffa Babjee and Nor Aidah, 1994, White,
2003, Ladds et al., 1981)
Iguanas
(Zehnder et al., 2014)
Kangaroos
(Low Choy et al., 2000, Egerton, 1963)
Koalas
(Ladds et al., 1990)
Panda
(Dance, 1991, White, 2003)
Pigs
(Ketterer et al., 1986, Dance, 1991, Mustaffa Babjee
and Nor Aidah, 1994)
Primates
(de Silva, 1971, Peters et al., 1976, Vellayan, 1994,
Ouadah et al., 2007, Sommanustweechai et al., 2013,
Ritter et al., 2013)
Rabbits
(Stanton and Fletcher, 1925)
Serows
(Vellayan, 1994)
Sheep
(Cottew, 1950, Dance, 1991, Mustaffa Babjee and Nor
Aidah, 1994)
(Stanton and Fletcher, 1925, Low Choy et al., 2000)
8
1.2 Literature review: Melioidosis in Malaysia
1.2.1 Epidemiology of melioidosis in Malaysia
Melioidosis has been recognized as an endemic disease in Malaysia since it was
first documented in Kuala Lumpur in Malaya in 1913 by Stanton and Fletcher
(Stanton and Fletcher, 1925). There have been melioidosis cases documented in
different parts of Malaysia namely Kuala Lumpur, Pahang, Kedah, Kelantan,
Johor, Perak, Sarawak and Sabah (map in Figure 1.1). Interestingly, the first
documentation of melioidosis in the country involved animals and not humans
(Stanton and Fletcher, 1925).
Kuala Lumpur Federal Territory
Due to its location as the capital of the country with better infrastructure, there
were more reports of melioidosis cases than in other parts of Malaysia. In
addition, a surge of interest on melioidosis was created with the establishment of
the Department of Medical Microbiology at the Faculty of Medicine, University
of Malaya (UM) which led to publications on baseline data of the disease in cases
referred to University Malaya Medical Centre (UMMC) (Puthucheary et al., 1981,
Yee et al., 1988, Puthucheary et al., 1992, Puthucheary, 2009). In one of the
earliest and most comprehensive study on melioidosis in the country and indeed at
the time anywhere globally, Puthucheary et al. reviewed 50 septicaemic
melioidosis cases that were presented to UMMC. The study revealed that 58% of
the cases had pulmonary involvement with 24% having soft tissue involvement.
Diabetes mellitus was the most common predisposing factor at 38% while 34%
had more than one predisposing factors. The 65% mortality rate of the cases
reviewed suggested that the diagnosis and therapy of the disease then was
inadequate (Puthucheary et al., 1992). Interest in melioidosis eventually
developed in institutes in the rest of the country especially after the first
international symposium on melioidosis was held in Kuala Lumpur in April 1994.
Pahang
Pahang is a state located in the central part of Peninsular Malaysia. A study
conducted from year 2000 to 2003 indicated that the annual incidence of adult
melioidosis in the state was 6.07 per 100,000 population per year with a mortality
rate of 54%. The same study showed that 91% of the adult cases had underlying
9
predisposing factors with 89% suffering from diabetes mellitus and that
pulmonary involvement contributed to 58% of the total cases and 25% had
multiple organ involvement (How et al., 2005b). In a similar study, the incidence
rate for paediatric cases was 0.68 per 100,000 population per year, with localized
infection being the most common clinical presentation, followed by septic shock
(How et al., 2005a). Due to the importance of the disease in the state, the first
melioidosis registry in the country was established in Pahang in 2005 (How et al.,
2009). In 2010, an outbreak of melioidosis and leptospirosis co-infection occurred
where 22 out of 153 personnel of a rescue operation fell ill. Investigations
confirmed that 10 cases were associated with melioidosis which resulted in seven
deaths (Sapian et al., 2012).
Kedah
Kedah is a major rice growing state located in the northern part of Peninsular
Malaysia bordering Thailand. The first record suggesting the presence of
melioidosis in Kedah was from a nationwide serosurveillance in the 1960s where
the highest sero-prevalence against B. pseudomallei was among study participants
from Kedah (Strauss et al., 1969a). A more recent study reported that the
incidence of melioidosis in Alor Setar, the capital of Kedah, was 16.35 per
100,000 population per year with an overall mortality rate of 34% (Hassan et al.,
2010b). The study also found that consistent with previous studies, diabetes
mellitus is a major risk factor (57%) for morbidity and mortality. Clinically,
pneumonia was the most common primary clinical presentation followed by soft
tissue abscess (Hassan et al., 2010b).
Kelantan
Kelantan is a state located in the northeast of Peninsular Malaysia bordering
southern Thailand where rice farming is common. In a study where B.
pseudomallei culture-positive cases from 2001 through 2005 were reviewed
retrospectively, lung involvement was shown to be the highest presentation with
67%, followed by liver abscess, prostatic infection and brain involvement. In this
study series, the mortality rate was 65% where 70% cases had diabetes mellitus,
14% cases had chronic renal diseases and 20% of the surviving patients suffered
relapse (Deris et al., 2010).
10
Johor
Johor is a state in southern Peninsular Malaysia bordering Singapore. The earliest
record of melioidosis in Johor was in 1955 involving goats at the Central Animal
Husbandry Station (CAHS) (Mustaffa Babjee and Nor Aidah, 1994). In a study at
a hospital in Johor, 44 culture-positive human melioidosis cases presented
between January 1999 and December 2003 were reviewed retrospectively. Out of
the 32 cases with sufficient records, 75% were shown to have suffered from
diabetes mellitus as risk factor and 63% presented with pneumonia as their
primary clinical presentation with a large number also having multi-organ
involvement (Pagalavan, 2005).
Sarawak
Sarawak, located in the southwest coast of the Borneo Island, is the largest state in
Malaysia. Due to its large area and challenging terrains, some districts in the
central region of Sarawak are only accessible by long boat rides and helicopter
flights. The last 20 years has seen increased land-clearing activities associated
with logging, oil-palm plantations and dam constructions in the state.
Despite Sarawak being classified as hyper-endemic for melioidosis in Malaysia
(Puthucheary and Vadivelu, 2002), there have not been publications that
document the actual incidence of melioidosis in the state. A serosurvey carried out
by Strauss et al. in the 1960s suggested that the seropositivity of participants in
Sarawak were not significant (Strauss et al., 1969a). Interestingly, almost 30 years
later, another serosurvey carried out amongst army recruits in Sabah and Sarawak
in the early 1990s suggested otherwise with >50% of the 420 army recruits having
antibodies against either the whole-cell antigen or the exotoxin of B. pseudomallei
(Embi et al., 1992). However the specificity of the assays used in that study
remains uncertain.
Since 2003, melioidosis has been an administratively notifiable disease in the state
of Sarawak. Cases that were clinically suspected melioidosis with either serology
or culture positive results are required to be reported to the Sarawak Health
Department (Sarawak Health Department, unpublished data).
11
Based on data from 2002 to 2009, there were 541 melioidosis-related admissions
into government hospitals throughout Sarawak. However, this number is likely a
considerable underestimate of melioidosis incidence as there have been many late
hospital presentations where B. pseudomallei was not laboratory-diagnosed at the
time of death. Hence, such cases would not have been reported as melioidosis due
to absence of clinical and laboratory diagnoses. Despite the high disease burden,
the detection rate of melioidosis is still very poor with low suspicion level of
melioidosis amongst primary healthcare providers. In addition, there is also a very
low public awareness of the disease which results in delayed health-seeking
behaviour.
Several clusters of melioidosis cases have been reported in Sarawak. In 2003, an
outbreak of melioidosis involving high-school students was described. Out of 57
students involved in a sports match, 10 presented with multiple skin abscesses and
samples collected from seven cases tested positive for B. pseudomallei.
Environmental samples collected from the sports field also cultured B.
pseudomallei (Sarawak Health Department, unpublished data). Between 2006 and
2007, there was a cluster of melioidosis cases of unknown risk factors involving
workers at the construction site of the Bakun Hydroelectric Power Plant which
resulted in five deaths (Sarawak Health Department, unpublished data). The
construction site is located at an area that had previously been recognised as a
highly endemic site in the Kapit division. In general, a high proportion of Sarawak
melioidosis cases involved farmers, logging camp workers, students and
construction workers from the Kapit and Bintulu districts located in the central
region of Sarawak (Sarawak Health Department, unpublished data).
Sabah
Sabah is a state located on the northern part of the island of Borneo. Similar to
Sarawak, logging, oil palm plantation and rural lifestyle are common in Sabah.
However, unlike Sarawak, due to its close proximity to the islands of southern
Philippines and northeast Indonesia, there are a lot more human movements in
Sabah.
12
The earliest indication of the presence of melioidosis in Sabah is from a
serosurvey carried out by Strauss et al. in the 1960s suggesting that Sabah is one
of the two states in the country with the highest seropositivity against B.
pseudomallei (Strauss et al., 1969a). Another serosurvey conducted among army
recruits in the 1990s implicated high seropositivity among Sabah-based armies
(Embi et al., 1992). Based on these results, Sabah was considered one of the
hyper-endemic areas for melioidosis in Malaysia (Puthucheary and Vadivelu,
2002). In addition to serosurveillance, a melioidosis case with prostatitis was
reported (Kan and Kay, 1978). Other than that, published records of human
melioidosis in Sabah have been rare. The earliest cases of animal melioidosis
reported in Sabah involved imported pigs from Australia in 1963. There was no
indication of whether the pigs originated from the southern or the northern regions
of Australia. Neither was there any indication whether the pigs were either
exposed to the organism upon arriving in Sabah or were already infected if they
originated from northern Australia (Ouadah et al., 2007). Melioidosis cases
involving orang-utans with high mortality have also been reported (de Silva,
1971, Peters et al., 1976, Ouadah et al., 2007).
Imported melioidosis cases from Malaysia
In addition to reports of local cases, there have also been reports of melioidosis
cases involving returning travellers or imported cases from Malaysia to United
Kingdom (Spencer and Robert, 1978), Germany (Steinmetz et al., 1996), Nepal
(Shrestha et al., 2005) and China (Zong et al., 2012). Imported animal cases from
Malaysia have also been reported involving felines (O'Brien et al., 2003), and
primates (Britt et al., 1981).
13
Figure 1.1 Map of the states in Malaysia (Adapted from http://www.ecoi.net/file_upload/470_1283265713_malaysia-adm98.jpg)
14
1.2.2 Melioidosis in animals in Malaysia
Melioidosis was first reported in 1913 among laboratory guinea pigs and rabbits at
the Institute of Medical Research, Kuala Lumpur in what was then known as
Malaya (Stanton and Fletcher, 1925). Subsequent animal cases were mostly
reported around Kuala Lumpur involving a cat in 1918 (Stanton and Fletcher,
1925), an imported Australian racehorse which may have been infected in
Malaysia in 1925 (Stanton et al., 1927), a dog in 1926 (Mustaffa Babjee and Nor
Aidah, 1994) and birds in captivity in 1932 (Vellayan, 1994).
There were several melioidosis outbreaks involving livestock such as goats, pigs,
sheep, cattle and fallow deer imported from New Zealand. (Mustaffa Babjee and
Nor Aidah, 1994, Ouadah et al., 2007). An outbreak of melioidosis in breeding
pigs imported from Australia was also reported in Sabah in 1963 (Ouadah et al.,
2007). There was no clear evidence indicating whether the animals may have
already been infected with B. pseudomallei before their arrival to Malaysia or that
they were infected in Malaysia.
Apart from livestock, melioidosis cases involving zoo animals have also been
reported in Malaysia. Zoo animal melioidosis cases include serow (Capricornis
sumatrensis) in Perak in 1932, pig-tailed macaques (Macaca nemestrina) in Johor
in 1963 and in Kuala Lumpur in 1965, spider monkey (Brachytelis arachnoides)
and a gibbon (Hybolates lar) in Johor in 1968 (Vellayan, 1994). These cases were
attributed to contaminated water and surface soil especially in zoos that adopt the
open parks concept. Due to low suspicion level of melioidosis, most of the cases
were only detected at the necropsy stage with >95% mortality (Vellayan, 1994).
Melioidosis cases among orang-utans have also been reported to occur in forest
reserve areas in Sabah and Sarawak (de Silva, 1971, Peters et al., 1976, Ouadah et
al., 2007)(Sarawak Veterinary Department, unpublished data). In a study, de Silva
reported of fatal melioidosis cases among some orang-utans at the Sepilok
rehabilitation centre in Sandakan in 1965 and 1968 (de Silva, 1971). Despite
failed initial attempt in isolating B. pseudomallei from surface water and soil from
the centre in 1965 (Strauss et al., 1967), B. pseudomallei was eventually isolated
from environmental samples in the area in 1968 (de Silva, 1971). A serology test
15
conducted on 13 orang-utans at the centre showed very low titre (all 1:10);
suggesting that despite being successfully isolated in the area, B. pseudomallei
may be present in low level. It was postulated too that this low titre of antibody
against B. pseudomallei among the orang-utans tested was due to the animal
having natural immunity against B. pseudomallei (de Silva, 1971).
Records at the Animal Disease Research Centre (ADRC) showed that melioidosis
has also been reported in goats, cattle, pigs, deer, monkey, orang-utans, cat, dogs,
horse, buffalo and wild birds in Sabah from 1994 and 2003 (Ouadah et al., 2007).
A nationwide melioidosis seroprevalence conducted on livestock such as
buffaloes, pigs, goats, cattle and sheep indicated that seropositivity was highest in
buffaloes (48%) and the lowest was in goats (3%). The study also suggested that
low seropositivity in goats is due to the animals being kept in intensive systems
with minimal contact with soil (Musa et al., 2012).
1.2.3 Scientific interests on melioidosis in Malaysia
The scientific interest in melioidosis in Malaysia began when the disease was first
reported at the Institute of Medical Research in Kuala Lumpur, Malaya in the
1920s (Stanton and Fletcher, 1921, Stanton et al., 1924). There was a long hiatus
in documented melioidosis studies in the country possibly due to the World War
II and the post-World War II insurgencies that plagued the nation through the
1950s and 1960s. Malaya gained independence from Great Britain on 31st August
1957 and the Malaysian Federation was formed with the union of Malaya (now
known as Peninsular Malaysia), Singapore, Sabah and Sarawak on 16th September
1963. Interest on melioidosis in the country was reignited with environmental
surveillances and serosurveillances conducted in several states in the country by
the United States of America Medical Research Unit (USAMRU) in the 1960s
(Strauss et al., 1967, Strauss et al., 1969c, Strauss et al., 1969a, Strauss et al.,
1969b, Ellison et al., 1969).
Publications on melioidosis led by local Malaysian researches only started gaining
momentum in the late 1980s especially in clinical reports, reviews and
immunology. Evidence of research interests on microbiology, molecular and
genomics, and pathogenesis was only apparent in the 2000s, possibly due to better
16
facilities and funding. Table 1.2 provides an overview of studies on melioidosis
published thus far in Malaysia including historical publications.
17
Table 1.2 Studies on melioidosis conducted in Malaysia
Topic
Study location (References)
Antimicrobial activities against
B. pseudomallei
Kuala Lumpur (Cheong et al., 1987, Koay et al., 1997,
Karunakaran and Puthucheary, 2007, Sam et al., 2009,
Liew et al., 2011, Ahmad et al., 2013, Lee et al., 2013)
Biochemical
Kuala Lumpur (Liew et al., 2012)
Sarawak (Wong et al., 2013b)
Current study (Podin et al., 2013a)
Clinical
(case reports and review)
Kelantan (Elango and Sivakumaran, 1991, Halder et al.,
1993, Halder et al., 1998)
Kuala Lumpur (Lin et al., 1980, Puthucheary et al.,
1981, Yee et al., 1988, Majid, 1990, Puthucheary et al.,
1992, Hoc et al., 1993, Koh, 1995, Puthucheary et al.,
2001, Ganesan et al., 2003, Raja, 2003, Azizi et al., 2005,
Joazlina et al., 2006, Asiah et al., 2006, Ng et al., 2006,
Sam and Puthucheary, 2006, Raja, 2007, Zainal Abidin et
al., 2007, Raja, 2008, Wong, 2013, Ding et al., 2013)
Melaka (Azali et al., 2007)
Pahang (Ang, 2005, How et al., 2005a, How and Liam,
2006, How et al., 2005b, Kandasamy and Somasundaram,
2007, How et al., 2009, Ng et al., 2009, Kuan et al., 2010,
Yew et al., 2011, Hin et al., 2012, How et al., 2013a)
Selangor (Pit et al., 1988, Noordin et al., 1995, Ng et al.,
2006, Othman et al., 2007, Ariza, 2008)
Sarawak (Chan et al., 2013, Wong et al., 2013a, Yew,
2013)
Sabah (Kan and Kay, 1978)
Perak (Lim et al., 2001)
Environmental surveillance
Sabah (Strauss et al., 1967)
Kuala Lumpur (Ellison et al., 1969)
Whole of Peninsular Malaysia (Strauss et al., 1969c,
Hassan et al., 2013)
Selangor (Strauss et al., 1969b)
Sarawak (Podin et al., 2013b)
Epidemiology
Kuala Lumpur (Sam and Puthucheary, 2007)
Pahang (How et al., 2005b, How et al., 2009, Sapian et
al., 2012)
Johor (Pagalavan, 2005)
Kedah (Hassan et al., 2010b)
Kelantan (Deris et al., 2010)
Sarawak (Fazlina et al., 2012)
Histopathology
Kuala Lumpur (Wong et al., 1996)
Immunology
Kuala Lumpur (Vadivelu et al., 1995, Wong et al., 1996,
Vadivelu and Puthucheary, 2000, Chenthamarakshan et
al., 2001a, Chenthamarakshan et al., 2001b, Nathan et al.,
2005, Nathan and Puthucheary, 2005, Puthucheary and
Nathan, 2006, Puthucheary and Nathan, 2008,
Puthucheary et al., 2010, Hara et al., 2013, Puah et al.,
2013)
Pahang (How et al., 2013b)
Selangor (Su et al., 2003, Tay et al., 2012, Chin et al.,
2012b)
18
Table 1.2 (continued)
Microbiology
Pahang (Francis et al., 2006) Selangor (Lee et al., 2007)
Penang (Yuen et al., 2012)
Kuala Lumpur (Ramli et al., 2012, Roesnita et al., 2012,
Koh et al., 2013, Lee et al., 2013)
Molecular and genomics
Kuala Lumpur (Vadivelu et al., 1997, Vadivelu et al.,
1998, Tay et al., 2010, Chua et al., 2010, Azura et al.,
2011, Chua et al., 2011, Khoo et al., 2012, Koh et al.,
2012, Eu et al., 2013)
Selangor (Radu et al., 1999, Radua et al., 2000, Su et al.,
2008, Chin et al., 2010, Sulaiman et al., 2013, Wong et al.,
2013d)
Current study (Podin et al., 2014)
Pathogenesis
Sabah (Ismail et al., 1988)
Kuala Lumpur (Nathan and Puthucheary, 2005,
Puthucheary and Nathan, 2006, Puthucheary et al., 2012,
Kang et al., 2013, Vellasamy et al., 2013)
Selangor (Su et al., 2008, Lee et al., 2011, Chin et al.,
2012a, Chin et al., 2013, Eng and Nathan, 2013, Wong et
al., 2013c, Pramila et al., 2013)
Kuala Lumpur (Al-Maleki et al., 2013, Mariappan et al.,
2013, Kalaiselvam et al., 2013)
Sabah (Jayaram, 2005)
Kuala Lumpur (Raja et al., 2005, Puthucheary, 2009,
Choh et al., 2013)
Proteomics
Review
Serosurveillance
Malaysia (Strauss et al., 1969a, Thin, 1976)
Malaysian Borneo (Embi et al., 1992, Hassan et al.,
2004, Hassan et al., 2010a)
Kuala Lumpur (Norazah et al., 1996)
Pahang (Aniza et al., 2013)
Vaccine
Selangor (Hara et al., 2009, Su et al., 2010)
Veterinary
Penang (Saroja, 1979)
Kuala Lumpur (Stanton and Fletcher, 1925, Stanton et
al., 1927, Mustaffa Babjee and Nor Aidah, 1994)
Selangor (Mustaffa Babjee and Nor Aidah, 1994, Musa et
al., 2012)
Johor (Mustaffa Babjee and Nor Aidah, 1994, Vellayan,
1994)
Perak (Mustaffa Babjee and Nor Aidah, 1994, Vellayan,
1994)
Sabah (de Silva, 1971, Peters et al., 1976, Ismail et al.,
1991, Ouadah et al., 2007)
Sarawak (Sarawak Veterinary Department, unpublished
data)
19
Melioidosis serosurveys in Malaysia
In the 1960s, a survey carried out in Malaysia using the indirect hemagglutination
assay (IHA) method indicated 7.3% positivity amongst survey recruits with the
highest from Kedah, Peninsular Malaysia and Sabah (Strauss et al., 1969a, Ismail
et al., 1987). Further surveys conducted in Malaysia indicated that 17 to 22%
farmers and 26% blood donors were shown to have antibodies against B.
pseudomallei, suggesting previous exposure to the bacteria (Strauss et al., 1969a,
Thin, 1976, Vadivelu et al., 1995, Ahmad et al., 1996). In 1992, a nationwide
serosurveillance study conducted involving military personnel suggested that
seropositivity against B. pseudomallei was higher among armies serving in Sabah
and Sarawak (Embi et al., 1992). In a more recent serosurveillance study, Hassan
et al. tested indigenous communities in the Loagan Bunut National Park located in
the northern region of Sarawak (Hassan et al., 2004) and in Perak, and showed
that seropositivity is higher in communities that live in rural areas (Hassan et al.,
2010a).
Despite the relatively extensive serosurveillance studies conducted in Malaysia,
antibody detection in melioidosis-endemic areas has been shown to be
problematic and at times possibly unreliable due to previous exposure to B.
thailandensis or other related species, which potentially results in cross-reactivity
(Anuntagool et al., 1998, Cheng and Currie, 2005). Since it has already been
established that melioidosis is endemic in Malaysia, serosurveillance will only
add value to the current knowledge of melioidosis if statistically significant
difference in the seropositivity of various communities against B. pseudomallei is
shown upon elimination of background cross-reactivity with other Burkholderia
spp. In such cases, the information on the higher seropositivity of a particular
population can be used to inform public health on the possibility of higher
prevalence of B. pseudomallei in that particular region.
Melioidosis clinical case reports from Malaysia
There have been relatively substantial case reports and clinical reviews on
melioidosis in hospitals throughout Malaysia with most reports focussing on
hospitals in Peninsular Malaysia, particularly Kuala Lumpur being the capital of
the country (Table 1.3). The mortality rates of melioidosis reported in several
20
states thus far range from 45% to 63% (Pagalavan, 2005, How et al., 2005b,
Hassan et al., 2010b, Deris et al., 2010, Wong et al., 2013a). The most common
clinical presentation in melioidosis patients in Malaysia is septicaemia (Lin et al.,
1980, Puthucheary et al., 1981, Yee et al., 1988, Puthucheary et al., 1992, How et
al., 2005b, Pagalavan, 2005, Sam and Puthucheary, 2006, Kandasamy and
Somasundaram, 2007, Ng et al., 2009, Hassan et al., 2010b, Chan et al., 2013),
followed by pulmonary involvement (Puthucheary et al., 1992, Liam, 1993,
Puthucheary et al., 2001, Pagalavan, 2005, Asiah et al., 2006, Sam and
Puthucheary, 2006, Ariza, 2008, Yee et al., 1988, Hassan et al., 2010b, Deris et
al., 2010, Yew et al., 2011, Chan et al., 2013, Wong et al., 2013a). Table 1.3
provides a summary of clinical presentations and organs involved in melioidosis
cases reported in Malaysia thus far. Although rare, cases with vascular
involvement (Noordin et al., 1995, Azizi et al., 2005) have also been reported.
Paediatric cases were common with several neonatal melioidosis cases reported
(Halder et al., 1993, Halder et al., 1998, Sam and Puthucheary, 2006, Ang, 2005,
How et al., 2005a, Deris et al., 2010) where most paediatric cases involved
localised infection (How et al., 2005a).
As shown in Table 1.4, the most common underlying risk factor among
melioidosis patients in Malaysia is diabetes mellitus (Yee et al., 1988, Liam,
1993, How et al., 2005b, Pagalavan, 2005, Sam and Puthucheary, 2006, Ng et al.,
2009, Hassan et al., 2010b, Deris et al., 2010, Ding et al., 2013, Chan et al., 2013,
Wong, 2013, Wong et al., 2013a), followed by chronic renal diseases (Yee et al.,
1988, How et al., 2005b, Pagalavan, 2005, Hassan et al., 2010b, Wong, 2013).
Chronic lung disease has also been reported as a risk factor where several cases
had a history of tuberculosis (Yee et al., 1988, Pagalavan, 2005, Azali et al., 2007,
Hassan et al., 2010b). History of alcohol abuse is only common in populations
that are predominantly non-Muslim such as Sarawak (How et al., 2005b, Chan et
al., 2013)(Sarawak Health Department, unpublished data). Although it has been
reported that HIV positive individuals may not have necessarily higher risk for
melioidosis (Chierakul et al., 2004), cases have been reported among
immunocompromised individuals including those with primary immunodeficiency
and HIV/AIDS (How et al., 2005b, Pagalavan, 2005, Sam and Puthucheary, 2006,
Hassan et al., 2010b).
21
Table 1.3 Summary of clinical presentations in melioidosis cases reported in Malaysia
Clinical presentation
Pulmonary
(Pneumonia, lung abscess)
Kuala Lumpur (Yee et al., 1988, Majid, 1990, Puthucheary et
al., 1992, Liam, 1993, Puthucheary and Vadivelu, 2002, Asiah
et al., 2006, Sam and Puthucheary, 2006)
Selangor (Ariza, 2008)
Pahang (How et al., 2005b, Yew et al., 2011, Hin et al., 2012,
How et al., 2013a)
Sarawak (Fazlina et al., 2012, Chan et al., 2013, Wong et al.,
2013a)
Septicaemia
Kuala Lumpur (Lin et al., 1980, Puthucheary et al., 1981, Yee
et al., 1988, Puthucheary et al., 1992, Sam and Puthucheary,
2006)
Pahang (How et al., 2005b, Kandasamy and Somasundaram,
2007, Ng et al., 2009)
Sarawak (Chan et al., 2013)
Musculoskeletal
Kuala Lumpur (Yee et al., 1988, Puthucheary et al., 1992,
Sam and Puthucheary, 2006, Ng et al., 2006)
Brain or CNS involvement
Kuala Lumpur (Yee et al., 1988, Puthucheary et al., 1992,
Sam and Puthucheary, 2006, Zulkiflee et al., 2008)
Selangor (Pit et al., 1988)
Kelantan (Halder et al., 1998, Deris et al., 2010)
Skin or soft tissue abscess
Kuala Lumpur (Puthucheary et al., 1992, Yee et al., 1988,
Sam and Puthucheary, 2006)
Pahang (How et al., 2005a)
Sarawak (Sarawak Health Department, unpublished data)
Prostatic abscess
Kuala Lumpur (Koh, 1995)
Pahang (Ng et al., 2009)
Parotid abscess
Sarawak (A. Mohan, personal communication)
Septic arthritis
Kuala Lumpur (Puthucheary et al., 1992, Raja, 2007)
Cervical/neck abscess
Kuala Lumpur (Zulkiflee et al., 2008)
22
Lymph abscess
Kuala Lumpur (Yee et al., 1988, Sam and Puthucheary, 2006)
Scalp abscess
Pahang (Kuan et al., 2010)
Suppurative otitis media
Parapharyngeal space
Kelantan (Elango and Sivakumaran, 1991)
Spinal epidural abscess
Kuala Lumpur (Ganesan et al., 2003)
Heart
Kuala Lumpur (Majid, 1990, Puthucheary et al., 1992, Zainal
Abidin et al., 2007)
Pahang (Yew et al., 2011)
Liver
Kuala Lumpur (Yee et al., 1988, Puthucheary et al., 1992)
Sarawak (Chan et al., 2013, Wong et al., 2013a)
Spleen
Kuala Lumpur (Puthucheary et al., 1992, Hoc et al., 1993,
Joazlina et al., 2006, Sam and Puthucheary, 2006)
Pahang (How et al., 2005b, Yew et al., 2011)
Sarawak (Chan et al., 2013)
Renal
Kuala Lumpur (Puthucheary et al., 1992, Ding et al., 2013)
Gall bladder
Kuala Lumpur (Puthucheary et al., 1992)
Pancreas
Kuala Lumpur (Puthucheary et al., 1992)
Vascular
Selangor (Noordin et al., 1995)
Kuala Lumpur (Azizi et al., 2005)
Other intra-abdominal
Kuala Lumpur (Wong, 2013)
23
Table 1.4 Clinical risk factors for melioidosis cases in Malaysia
Risk factor
Diabetes mellitus
Kuala Lumpur (Yee et al., 1988, Liam,
1993, Sam and Puthucheary, 2006, Ding et
al., 2013, Wong, 2013)
Pahang (How et al., 2005b, Ng et al., 2009,
Sapian et al., 2012)
Sarawak (Chan et al., 2013, Wong et al.,
2013a)
Chronic renal disease
Kuala Lumpur (Yee et al., 1988, Wong,
2013)
Chronic lung disease
Kuala Lumpur (Yee et al., 1988)
Pahang (Sapian et al., 2012)
Alcohol abuse
Sarawak (Chan et al., 2013) (Sarawak
Health Department, unpublished data)
Immunocompromised individuals
(including primary immunodeficiency,
HIV/AIDS, patients undergoing
corticosteroid therapy)
Kuala Lumpur (Yee et al., 1988)
Kuala Lumpur (Sam and Puthucheary,
2006)
Others
(Thalassemia, cystic fibrosis,
leukaemia, malnutrition, cancer)
Kuala Lumpur (Yee et al., 1988, Hoc et
al., 1993, Asiah et al., 2006, Sam and
Puthucheary, 2006)
Sarawak
(J.S.
Wong,
personal
communication)
24
Drug susceptibility of Malaysian B. pseudomallei
Studies on drug susceptibility of B. pseudomallei in vitro had also been carried out
where findings are consistent with those found elsewhere (Cheong et al., 1987,
Koay et al., 1997, Karunakaran and Puthucheary, 2007, Sam et al., 2009, Ahmad
et al., 2013) except for the rare gentamicin sensitivity isolates in Sarawak
discussed in Chapter 4 of this thesis (Podin et al., 2014).
Development of immunoassays in Malaysia
A large number of researchers in Malaysia have been interested in the
identification of immunogenic proteins of B. pseudomallei and the development
of diagnostic methods, particularly immunoassays. Several studies on enzymelinked immunosorbent assays (ELISAs) using various antigen targets have been
described (Ismail et al., 1987, Ismail et al., 1991, Embi et al., 1992, Vadivelu et
al., 1995, Vadivelu and Puthucheary, 2000, Puthucheary et al., 2010, Hara et al.,
2013, Puah et al., 2013). Immunofluorescent assay test (IFAT) assays were also
described and this method is widely used in some hospitals within Malaysia
(Chenthamarakshan et al., 2001a, Chenthamarakshan et al., 2001b, Vadivelu and
Puthucheary, 2000, Puthucheary et al., 2010). In addition to ELISA and IFAT, an
immunohistochemical test was described as a method for diagnosing melioidosis
(Wong et al., 1996). A rapid diagnostic test in the form of dot enzyme
immunoassay has been explored, and the test is currently in the final stage of
being commercialized (Zainoodin and Asma, 2010). This surge in the
development of diagnostic kits is possibly due to incentives offered by the
Ministry of Science, Technology and Innovation of Malaysia for researches that
have commercialisation potentials. Nevertheless confirmation of melioidosis
requires culture of B. pseudomallei, so improved serology assays detecting
antibodies to various B. pseudomallei antigens only have limited potential for
improving diagnosis of individual cases. Indeed where a laboratory focuses on
serology rather than bacterial culture, both false positive and false negative
diagnoses will occur.
25
Molecular characterization of Malaysian B. pseudomallei
Initially, there was a slow start in molecular-based researches in Malaysia. Pulsedfield gel electrophoresis (PFGE) appeared to be one of the favourite methods
where comparison of genetic clusters was done (Vadivelu et al., 1997, Vadivelu et
al., 1998, Chua et al., 2010, Hassan et al., 2013). Other restriction fragment length
polymorphisms (RFLP), randomly amplified polymorphic DNA (RAPD) analysis
and 16S ribosomal DNA sequencing were also employed in some studies in
characterizing B. pseudomallei (Radu et al., 1999, Radua et al., 2000, Lee et al.,
2007, Tay et al., 2010). Apart from McCombie et al. who analysed some
Malaysian historical B. pseudomallei isolates collected by the USAMRU in the
1960s using multilocus sequence typing (MLST), no other researchers in
Malaysia have systematically utilized this method thus far (McCombie et al.,
2006). In the recent years, there has been a shift into the genomics realm with the
acquisition of state-of-the-art facilities among better resourced groups (Su et al.,
2008, Khoo et al., 2012, Wong et al., 2013d, Eu et al., 2013)
Microbiology of melioidosis in Malaysia
In microbiological methods, Francis et al. proposed a selective media called
Francis media which has since been utilized in some government hospitals in
Peninsular Malaysia (Francis et al., 2006). While Francis media has been found to
be useful in some clinical laboratories, the use of gentamicin as the selective in
this medium may be problematic in regions with high number of gentamicin
sensitive B. pseudomallei isolates (Podin et al., 2014). In addition to selective
media development, other microbiological studies include biofilm formation,
morphotype variants, and comparison of various selective media for B.
pseudomallei (Ramli and Vadivelu, 2008, Roesnita et al., 2012, Ramli et al., 2012,
Koh et al., 2013).
Virulence, pathogenesis and host response studies in Malaysia
There have also been interests among the local research groups in Malaysia in the
virulence, pathogenesis and host response of B. pseudomallei, with the eventual
aim of vaccine development. Nathan et al. thus far, has been the leading group on
B. pseudomallei pathogenesis research in the country with their various
publications specifically on immunogenic bacterial protein as potential vaccine
26
candidate (Su et al., 2003, Nathan et al., 2005, Nathan and Puthucheary, 2005,
Puthucheary and Nathan, 2006, Su et al., 2008, Hara et al., 2009, Su et al., 2010,
Lee et al., 2011). Nathan et al. had also investigated the host-pathogen
relationship in a mouse model by using DNA microarray technology (Chin et al.,
2010). The intracellular adaptations of B. pseudomallei and cellular responses
during an infection have also been described (Mohamed et al., 2002, Nathan and
Puthucheary, 2005, Puthucheary and Nathan, 2006, Kumutha et al., 2008,
Thimma et al., 2008). Exotoxin of B. pseudomallei is another research focus by
some groups where results have been applied in serosurveys in humans and sheep
(Ismail et al., 1987, Ismail et al., 1991, Embi et al., 1992, Su et al., 2003,
Vellasamy et al., 2009). In addition, B. pseudomallei was shown to be resistant to
serum bactericidal properties in an older study (Ismail et al., 1988).
Environmental surveys and ecology of B. pseudomallei in Malaysia
Since B. pseudomallei is naturally found in the soil of endemic areas,
environmental investigations have been carried out. In the 1960s, Strauss et al.
embarked on an extensive study by sampling all the states in Peninsular Malaysia
except for Penang, with B. pseudomallei found in the soil and water (Ellison et al.,
1969, Strauss et al., 1969c, Strauss et al., 1969b). An environmental survey of B.
pseudomallei was also conducted in Sabah in 1967 (Strauss et al., 1967). In more
recent studies, B. pseudomallei was isolated in various locales in Perak, Johor,
Kedah and Perlis (Hassan et al., 2013). Environmental surveys conducted in
northern and central Sarawak suggested that the environmental prevalence of B.
pseudomallei was geographically variable (Inglis, 2002, Hassan et al., 2004).
1.3 Summary and implications of literature review
The substantive work albeit in different aspects of melioidosis and B.
pseudomallei, indicated the importance of the disease in Malaysia. Despite this
work, the epidemiology of melioidosis in the country remains uncertain with
sporadic reports available from Kuala Lumpur (Puthucheary and Vadivelu, 2002),
Pahang (How et al., 2005b, How et al., 2009), Kedah (Hassan et al., 2010b),
Kelantan (Deris et al., 2010), Johor (Pagalavan, 2005), Sarawak (Puthucheary,
2009)(Sarawak Health Department, unpublished) and Sabah (Puthucheary and
Vadivelu, 2002, Puthucheary, 2009).
27
In addition, there is limited knowledge about the molecular epidemiology of B.
pseudomallei in Malaysia in comparison with strains found elsewhere.
Characterization of Malaysian B. pseudomallei using previous molecular methods
such as PFGE (Chua et al., 2010), other RFLP (Radu et al., 1999) and RAPD
(Radua et al., 2000) was informative but not robust enough for comparison of
strains between laboratories (Godoy et al., 2003). Hence, characterization of
Malaysian B. pseudomallei using more current molecular methods will be useful
in providing better understanding of the population structure of the organism in
relation to the overall worldwide B. pseudomallei population. Currently, the data
available are the MLST of the historical isolates collected by the USAMRU in the
1960s (McCombie et al., 2006). On a broader scope, molecular epidemiology B.
pseudomallei will also provide insight into the biogeography and the factors that
influence the distribution of the organism within a particular ecology where such
knowledge can be used to inform public health (Baker et al., 2013).
Despite the extensive serosurveillance conducted nationwide by several groups at
different time points involving different communities (Strauss et al., 1969a, Thin,
1976, Ismail et al., 1987, Embi et al., 1992, Vadivelu et al., 1995, Ahmad et al.,
1996, Hassan et al., 2004, Hassan et al., 2010a), antibody detection in
melioidosis-endemic areas has been shown to be problematic and unreliable
(Anuntagool et al., 1998, Cheng and Currie, 2005).
As suggested in previous studies, Malaysian Borneo is the ‘hyper-endemic’ foci
for melioidosis (Puthucheary and Vadivelu, 2002). Despite this recognition, the
magnitude of the epidemiology of melioidosis and B. pseudomallei in the region
remains unclear. As evident in the summary in Table 1.2, there has been limited
information on melioidosis and B. pseudomallei in Malaysian Borneo.
1.4 B. pseudomallei and melioidosis in Malaysian Borneo
Malaysian Borneo consists of two states, Sabah and Sarawak; divided by Brunei
Darussalam in between. Sarawak is located in the southwest coast of the Borneo
Island and is the largest state in Malaysia. The central region of Sarawak shares a
long and porous border with West Kalimantan of Indonesia. Sabah is a state
28
located on the northern part of the island of Borneo where it is in close proximity
to the islands of southern Philippines and northeast Indonesia, and also shares a
border with East Kalimantan of Indonesia.
In Malaysian Borneo, the last 20 years have seen extensive logging, dam
constructions and proliferation of oil-palm plantations. While these activities
bring about major changes to the ecosystem and human movements, little is
known about how such changes affect the ecology of B. pseudomallei and cases of
melioidosis in the areas involved. Previous studies have shown land use such as
constructions or excavation (Inglis et al., 2001, Cheng and Currie, 2005)(M.
Kaestli, manuscript in preparation) and agricultural activities, and use of fertilizers
(M. Kaestli, manuscript in preparation) have direct and indirect impacts on the
ecology and distribution of B. pseudomallei. Despite being suggested as ‘hyperendemic’ for melioidosis, there is limited knowledge of the epidemiology of
melioidosis and the molecular epidemiology of B. pseudomallei in Malaysian
Borneo.
As shown in Figure 1.2, the central region which includes Kapit and Belaga
districts has the highest cumulative number of melioidosis cases in Sarawak.
Preliminary data on melioidosis patients in Sarawak from the year 2007 to 2010
revealed that 27% of the total number of cases reported were farmers or workers
in agriculture sector (Table 1.5). Interestingly, 35% of melioidosis cases involve
students and children younger than 15 years. In addition, Sarawak Health
Department had reported that 16% of melioidosis cases in the state have had
history of heavy alcohol consumption. The population in the areas with the
highest number of melioidosis (Figure 1.2) is the Iban ethnic group where alcohol
drinking culture is deeply woven into the fabric of this community (Sarawak
Health Department, unpublished data). Although Type 2 diabetes had been
reported as the most important risk factor for melioidosis (Cheng and Currie,
2005), there had not been any data from Sarawak Health Department associating
diabetes with melioidosis. Despite the high disease burden, the detection rate of
melioidosis is still very poor and there is low suspicion level of melioidosis
amongst primary healthcare providers. In addition, there is a low public awareness
29
of the disease in the region. As a result, the extent of melioidosis in Malaysian
Borneo remains uncertain despite having relatively high endemicity.
Hence, a comprehensive study on melioidosis in Malaysian Borneo is necessary to
understand the extent of the disease in the region including how the changes in
land use affect the ecology of B. pseudomallei, and to elucidate the relatedness of
B. pseudomallei from Malaysian Borneo compared with strains found elsewhere.
Table 1.5 Occupations of melioidosis patients in Sarawak from years 2007 to
2010
Occupation
Cases (%)
Land surveyor
5 (1)
Mechanic
5 (1)
Office job
6 (1)
Logging
14 (3)
Pensioner/elderly
15 (3)
Unemployed
23 (5)
Housewife
28 (6)
Self-employed
34 (7)
Underage of 12
57 (11)
Construction
62 (12)
Student (up to 15 years old)
122 (24)
Farmer/Agriculture
134 (27)
Total
505
(Source: Sarawak Health Department)
30
Figure 1.2 Melioidosis in average annual incidence rate per 100,000 population in Sarawak from 2007-2010 (Source: Sarawak Health Department)
31
1.5 Study aims
The major goal of this study is to investigate the epidemiology of melioidosis and
B. pseudomallei in Malaysian Borneo. In addition to being a human (and animal)
pathogen, B. pseudomallei is also a soil bacterium. Building on this premise,
characterizing B. pseudomallei strains that have caused human diseases is equally
as important as investigating the ecology of B. pseudomallei in communities that
have been affected by melioidosis. To achieve the major goal of this study, five
aims have been outlined as below. The first three aims are relevant to clinical B.
pseudomallei isolates from Malaysian Borneo while the last two aims are relevant
to environmental investigation of B. pseudomallei in the region:

To characterize the phenotypic and molecular attributes of clinical B.
pseudomallei from Malaysian Borneo.

To characterize Malaysian Borneo B. pseudomallei isolates using the
VITEK 2 biochemical automated system.

To determine the prevalence and mechanism of gentamicin and macrolide
susceptibility in B. pseudomallei isolates from Sarawak.

To determine if B. pseudomallei could be isolated from central Sarawak
soil samples.

To assess the relatedness between environmental B. pseudomallei isolates
and clinical isolates from the same region.
Knowledge gained from answering the research questions based on the aims listed
above will provide insight into the population structure of B. pseudomallei in
Malaysian Borneo, Malaysia and also contribute to current knowledge of the
worldwide population structure. This study will also provide insight into the
extent of gentamicin and macrolide susceptibility in B. pseudomallei isolates in
Sarawak and the mechanism behind such occurrence. Furthermore, knowledge
gained from investigating the different facets of melioidosis will also inform
public health on the magnitude of melioidosis and environmental hazards of B.
pseudomallei in the region.
32
1.6 Thesis outline
Chapter 2 describes the B. pseudomallei clinical isolates collected from
Malaysian Borneo and their attributes defined by the different characterization
methods. This chapter also provides insight into the population structure of
clinical B. pseudomallei from Malaysian Borneo in comparison with isolates
found elsewhere.
Chapter 3 describes the characterization of B. pseudomallei from Malaysian
Borneo using the automated biochemical VITEK 2 system.
Chapter 4 provides an insight into the aminoglycoside and macrolide susceptible
clinical B. pseudomallei isolates from Sarawak. The chapter includes results of
experiments that explained the mechanism which conferred aminoglycoside and
macrolide susceptibility to these isolates.
Chapter 5 describes the environmental investigation of B. pseudomallei in
Sarawak. Although no B. pseudomallei was isolated from the environmental
samples collected, other Burkholderia spp. were isolated. The rest of the chapter
explored possible reasons and theories which may have led to the lack of B.
pseudomallei from the environmental samples in Sarawak with further
investigations based on the secondary hypotheses.
Chapter 6 discusses the implications and the significance of the study findings.
Future works in relation to the findings from this study are also proposed in this
chapter.
33
Chapter 2
Phenotypic characterization and molecular
epidemiology of clinical B. pseudomallei
isolates
from Malaysian Borneo
Chapter 2: Phenotypic characterization and molecular
epidemiology of clinical B. pseudomallei isolates from
Malaysian Borneo
____________________________________________
Study aims
To characterize the phenotypic and molecular attributes of clinical B. pseudomallei
from Malaysian Borneo.
2.1 Introduction
B. pseudomallei has the potential to cause severe and sometimes fatal diseases
(Wiersinga et al., 2012). Due to the potential severity of disease caused by B.
pseudomallei, laboratory investigations are important to shed light on the nature of
this organism. Various laboratory methods have been developed that can be useful in
both routine clinical laboratory diagnoses and in scientific research of B. pseudomallei
while other methods are more applicable solely in scientific research settings. These
laboratory methods are discussed in detail in the following sections.
2.1.1 Culture methods
Culture method remains the “gold standard” as the first step in identifying B.
pseudomallei recovered from either clinical or environmental samples despite the
availability of newly developed technologies. In the early days of melioidosis
discovery, Whitmore used different media to culture B. pseudomallei such as nutrient
agar, broth, agar with salt, gelatine, glycerine agar and potato slopes (Whitmore and
Krishnaswami, 1912). Since then, culture methods of B. pseudomallei have evolved
with the development of different selective and differential media.
In 1968, Farkas-Himsley described the addition of colistin-S in MacConkey for the
growth selection of what was then known as Pseudomonas pseudomallei (Farkas
Himsley, 1968). More than a decade later, Ashdown described an agar medium with
higher selectivity for B. pseudomallei by replacing colistin with gentamicin and by
adding crystal violet (Ashdown, 1979a). Despite the introduction of gentamicin into
selective medium for B. pseudomallei, colistin was still used in other broths such as
crystal violet-colistin broth (CVCB) containing trypticase soy broth 5 mg/L crystal
35
violet and 20 mg/L colistin (Ashdown, 1979b) and threonine-basal salt solution
(TBSS) (Galimand and Dodin, 1982). CVC50 was later described with increased
concentration of colistin (50 mg/L) for better recovery rate of B. pseudomallei from
environmental samples (Wuthiekanun et al., 1995). All these selective broth media
were recommended to be used in combination with Ashdown’s agar for the isolation
of B. pseudomallei. Particularly, samples from clinical and non-clinical non-sterile
sites (e.g. sputum, throat swabs, environmental samples) are initially inoculated into
these broths before being subcultured onto Ashdown’s agar (Ashdown, 1979b,
Galimand and Dodin, 1982, Wuthiekanun et al., 1995).
In addition to selective broth, several other selective agars have also been developed
such as B. pseudomallei selective agar (BPSA) which uses Nile blue as an indicator
dye, 1% glycerol, maltose and 20 mg/L gentamicin (Howard and Inglis, 2003), and
Francis media which uses glycerol, 0.1% crystal violet and 0.2% bromocresol purple
(Francis et al., 2006). In one study, a comparison was done between Ashdown’s
medium, BPSA and B. cepacia medium, which showed that Ashdown’s medium has
superior selectivity than BPSA despite equal sensitivity (Peacock et al., 2005). Hence,
to date, Ashdown’s agar remains the most widely used medium for the recovery of B.
pseudomallei from both clinical and environmental samples.
In his first report of what is now known as B. pseudomallei, Whitmore described that
some colonies possessed the “wrinkled appearance” while others had the
“liquefaction” morphology, which is now known as the smooth and mucoid
appearance (Whitmore and Krishnaswami, 1912). Morphological identification of
suspected positive colonies is still an important practice in identifying B.
pseudomallei. The bacterium has been reported to have the ability to switch from the
classical dry, wrinkled morphotype to the smooth mucoid morphotype depending on
various factors such as growth temperature at either 37 °C or 42 °C, starvation, pH
and iron content of media (Chantratita et al., 2007). In addition, a recent study
suggested that morphology variants in a particular B. pseudomallei strain (NCTC
13392) were not due to genomic sequence difference but rather, due to epigenetic
factors, gene expression programs, and/or proteome variations (Vipond et al., 2013).
36
Nevertheless, while the current selective media used may be the best option thus far,
the selectivity of these media is not 100%. This is due to the complex ecology in the
environment and in some clinical specimens where some Burkholderia spp. co-exist
with B. pseudomallei, and confounded by similarities in the morphologies of some
Burkholderia spp. and B. pseudomallei (Dance, 2000a, Chantratita et al., 2007, Levy
et al., 2008, Price et al., 2013b). Hence, B. pseudomallei cannot be identified based
solely on culture method alone.
2.1.2 Confirmation methods
Positive cultures of B. pseudomallei can be confirmed using methods such as
immunoassays (Samosornsuk et al., 1999, Wuthiekanun et al., 2002), manual or
automated biochemical tests (bioMérieux, France), molecular assays (Novak et al.,
2006, Price et al., 2012) and mass spectrometry (Emonet et al., 2010, Sauer and
Kliem, 2010, Dubois et al., 2012).
Immunoassays have been developed to detect presence of either B. pseudomallei
antigen or antibodies. Assays that use antibodies to detect the specific proteins or
antigens of B. pseudomallei include latex agglutination and immunofluorescence
tests, which have been shown to be useful in both routine clinical diagnostic and
research settings (Naigowit et al., 1993, Walsh et al., 1994, Samosornsuk et al., 1999,
Anuntagool et al., 2000, Wuthiekanun et al., 2002). Thus far, several latex
agglutination assays which employed monoclonal antibodies targeting either the
lipopolysaccharide or exopolysaccharide of B. pseudomallei, have been shown to
possess sensitivity ranging between 95% to 100%, as well as specificity of up to
100% in confirming B. pseudomallei positive culture (Dharakul et al., 1999,
Samosornsuk et al., 1999, Anuntagool et al., 2000, Wuthiekanun et al., 2002). Due to
their rapidity (<2 minutes), and no requirement for specialised equipment or skills,
latex agglutination assays are useful for routine culture confirmation tests in lower
resourced laboratories in melioidosis endemic areas.
Immunofluorescence tests that have been developed thus far allow detection of B.
pseudomallei in culture (Naigowit et al., 1993) and also direct detection of the
organism in suitable clinical specimens with specificity of up to 99% and sensitivity
37
of up to 73% (Walsh et al., 1994). This method however is not widely utilized and it
requires specialised microscopy facilities (Wuthiekanun et al., 2004).
Examples of immunoassays that have been developed to detect B. pseudomalleispecific
immunoglobulin
M
(IgM)
and
IgG
antibodies
include
indirect
hemagglutination (IHA) for (Ashdown, 1987), enzyme-linked immunosorbent assay
(ELISA) (Chenthamarakshan et al., 2001b), immunofluorescent assay (IFAT)
(Vadivelu
and
Puthucheary,
2000),
and
a
commercially
available
rapid
immunochromogenic test (Cuzzubbo et al., 2000)(PanBio Ltd., Australia) that was
shown to have poor specificity and is no longer available (O'Brien et al., 2004). With
their sensitivity ranging from 67% to 79%, with specificity of 68% to 90% (Ashdown,
1987, Wuthiekanun et al., 2004), these tests are generally not useful in endemic
regions due to high background seropositivity. However, serology assays may be
useful in melioidosis non-endemic regions or in diagnosing non-local patients who are
suspected of having melioidosis in an endemic region (Sirisinha et al., 2000,
Wuthiekanun et al., 2004).
Manual and automated biochemical test panels such as API 20E or 20NE
(bioMérieux), Vitek 2 (bioMérieux, France), BBL Crystal (Bectin Dickinson, USA),
Phoenix (Bectin Dickinson, USA) provide rapid and high throughput diagnosis but
misidentification of B. pseudomallei as either another Burkholderia spp. or another
non-related bacterium has been widely reported (Dance et al., 1989d, Inglis et al.,
1998, Lowe et al., 2002, Koh et al., 2003, Lowe et al., 2006, Zong et al., 2012, Podin
et al., 2013a). Because of this, laboratory scientists in melioidosis endemic regions
now practise caution when interpreting results using these commercial biochemical
assays.
Most of the molecular confirmation methods for B. pseudomallei are polymerase
chain reaction (PCR)-based assays, which detect specific genetic markers (Thibault et
al., 2004, Tomaso et al., 2005, Merritt et al., 2006, Novak et al., 2006, Neubauer et al.,
2007, Suppiah et al., 2010, Bowers et al., 2010, Ho et al., 2011, Price et al., 2012).
Depending on the availability of resources and their applicability, molecular tests
have been shown to be favourable over other methods due to their abilities to detect
specific genes, to allow for quantification of bacterial load with accuracy of up to
38
100%. For instance, the BurkDiff assay by Bowers and colleagues has been shown to
remain 100% specific for B. pseudomallei and another assay developed by Price et al.
was also shown to have 100% specificity when a single false negative was excluded
based on a genetic mutation in the probe-binding region (Bowers et al., 2010, Price et
al., 2012). However, not all molecular confirmation assays described above have been
proven useful in routine diagnostic laboratories. Thus far, a PCR assay targeting the
open reading frame 2 (orf2) of the type III secretion system (TTS1) of B.
pseudomallei has been shown to be the most reliable assay and has been considered
the gold standard for molecular confirmation for B. pseudomallei in both routine
diagnostic and research settings (Novak et al., 2006, Meumann et al., 2006, Kaestli et
al., 2007, Kaestli et al., 2012a).
In recent years, matrix-assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF) has been recognised as the latest identification tool for
clinical microbiology which has been speculated to replace automated biochemical
test systems (Emonet et al., 2010, Sauer and Kliem, 2010, Dubois et al., 2012).
2.1.3 Methods of characterizing B. pseudomallei
Upon confirmation of species identification, there are several methods commonly
employed for strain characterization of B. pseudomallei. Depending on circumstances,
some methods can only be useful for solely scientific explorations purposes while
other methods can be of scientific research, clinical as well as public health relevance.
Characterization methods for B. pseudomallei are discussed in detail in the following
sections.
Drug susceptibility
In addition to being useful in determining treatment dosage in routine clinical settings
(Cheng and Currie, 2005, Wiersinga et al., 2012), drug susceptibility tests have also
been shown useful in identifying potentially drug resistant or sensitive strains and in
understanding implications of such occurrences in scientific researches (Moore et al.,
1999, Jenney et al., 2001, Chan et al., 2004, Kumar et al., 2008, Trunck et al., 2009,
Mima and Schweizer, 2010). In addition to the conventional antimicrobial
susceptibility tests (bioMérieux, France), antibiotic resistance can also be
characterised using molecular methods. For example, molecular assays that detect the
39
presence of mutations in the penA gene which confers ceftazidime resistance (the
first-line treatment for melioidosis) have been described (Tribuddharat et al., 2003,
Sam et al., 2009, Sarovich et al., 2012b).
Geographic attribution
There have been several significant and important studies published in recent years
which improved scientists’ knowledge on the molecular epidemiology of B.
pseudomallei. For instance, Tuanyok et al. described the B. thailandensis-like
flagellum and chemotaxis biosynthesis (BTFC) and the Yersinia-like fimbrial (YLF)
gene clusters, which are useful (although not 100% specific) markers for determining
the geographical origin of B. pseudomallei strains (Tuanyok et al., 2007). Multilocus
sequence typing (MLST), a method which interrogates seven housekeeping genes of
B. pseudomallei has also been used to provide insights into the molecular
epidemiology of the organism (Godoy et al., 2003). Using MLST, studies have shown
geographical distinctions between B. pseudomallei isolates from Australia and
Southeast Asia (Cheng et al., 2004, McCombie et al., 2006, Vesaratchavest et al.,
2006, Currie et al., 2007b, Pearson et al., 2009). In an extensive study, Pearson and
colleagues demonstrated clear geographical distinctions between B. pseudomallei
isolates from Australia with those from Southeast Asia as well as the rest of the world
(Pearson et al., 2009). In addition, MLST was also used in understanding the
molecular epidemiology of B. pseudomallei in Papua New Guinea (PNG) (Warner et
al., 2008, Baker et al., 2011a) and the Torres Straits islands (Baker et al., 2013).
Current initiatives to analyse whole genome sequences from diverse B. pseudomallei
strains collected globally will help unravel the global phylogeny of B. pseudomallei
and provide important new insights into the global spread and timelines, and the
nature of dissemination of the organism.
Virulence factors
B. pseudomallei encodes for a suite of diverse virulence factors such as
lipopolysaccharide (DeShazer et al., 1998, Atkins et al., 2002, Tuanyok et al., 2012),
two capsular polysaccharide clusters (DeShazer et al., 2001, Reckseidler et al., 2001,
Reckseidler-Zenteno et al., 2005, Sarkar-Tyson et al., 2007, Reckseidler-Zenteno et
al., 2010), three type III secretion system clusters (Attree and Attree, 2001, Rainbow
et al., 2002) and six type VI secretion system clusters (Shalom et al., 2007). In
40
addition to these variably present virulence factors, Sitthidet described a real-time
PCR assay that determined whether a particular strain possesses either one of the two
alleles of actin-based motility gene bimA; B. mallei-like BimA (bimABm) and B.
pseudomallei BimA (bimABp) (Sitthidet et al., 2008). bimA gene is essential in actinbased motility of B. pseudomallei which helps the bacteria to evade host immune
responses and has been suggested to be associated with virulence (Stevens et al.,
2005). Another study reported that B. pseudomallei isolates possessing the bimABm
allele are 14 times more prevalent in neurological melioidosis cases. The same study
also showed that fhaB3, another virulence factor for B. pseudomallei, is positively
correlated with patients presented with systemic infection of the bacteria (Sarovich et
al., 2014a).
In characterizing immunogenic proteins in B. pseudomallei, Tay et al. described
analysis methods of the flagellin gene for a possible vaccine candidate (Tay et al.,
2010) while Tuanyok et al. reported two different types of lipopolysaccharide (LPS)
in B. pseudomallei which correlated with strains of different geographical origins and
virulence levels (Tuanyok et al., 2012).
Strain variation using loci not under positive selection
In addition to the characterization methods mentioned above, B. pseudomallei have
also been genotyped using methods such as pulsed-field gel electrophoresis (PFGE)
using different restriction enzymes (Vadivelu et al., 1997, Currie et al., 2001, Gal et
al., 2004, Chua et al., 2010), BOX-PCR fingerprinting (Currie et al., 2007a), variable
number tandem repeat (VNTR) analysis (Pearson et al., 2007), multilocus variablenumber tandem repeat (VNTR) analysis (MLVA) (U'Ren et al., 2007, Currie et al.,
2009), multilocus sequence typing (MLST) (Godoy et al., 2003) and whole genome
sequencing (WGS). These methods have different resolutions that are useful for
answering different research questions, which can be of public health significance.
For instance, BOX-PCR fingerprinting, PFGE, VTNR and MLVA, which have better
fine-scale resolution compared with MLST and WGS, are useful in determining
clonal outbreaks. On the other hand, MLST and WGS provide insights into the
population structure of B. pseudomallei when comparing strains of diverse
geographical origins.
41
Although PFGE has now largely been superseded by less laborious genotyping
techniques, it was once a common method for characterising this bacterium and is still
used in some laboratories. There are two different restriction enzymes SpeI and XbaI
being used in PFGE for B. pseudomallei (Vadivelu et al., 1997, Currie et al., 2001,
Gal et al., 2004, Chua et al., 2010) where SpeI had been suggested to have higher
discriminatory power over XbaI (Chua et al., 2011). This method was reportedly used
in successfully identifying a common source of infection in a cluster of melioidosis
cases in the Northern Territory, Australia (Gal et al., 2004). Apart from PFGE, BOXPCR fingerprinting for B. pseudomallei, was shown to provide a rapid method for
detecting clonal melioidosis outbreaks where the results were comparable to MLST
and PFGE (Currie et al., 2007a).
In 2003, MLST for B. pseudomallei was developed to provide insight into the
population structure of the bacterium. This method involved the interrogation of seven
housekeeping genes ace, gltB, gmhD, lepA, lipA, narK and ndh located in
chromosome I (Godoy et al., 2003). Information on the sequence type (ST) of each
strain is uploaded to an online database where special software is made available to
analyse
the
relatedness
of
the
seven
loci
of
each
isolate
uploaded
(http://bpseudomallei.mlst.net/). The establishment of the website which provides free
access to information has encouraged researchers to share information of their B.
pseudomallei isolates. Subsequently, comparisons between isolates of different
geographical origins can be achieved at the website, resulting in a greater
understanding of the molecular epidemiology of B. pseudomallei worldwide (Cheng
et al., 2004, McCombie et al., 2006, Vesaratchavest et al., 2006, Currie et al., 2007b,
Warner et al., 2008, Pearson et al., 2009, Baker et al., 2011a, Baker et al., 2013).
MLVA has been shown to be a useful molecular typing method for various pathogens
(Keim et al., 2000, Johansson et al., 2004, Liu et al., 2006, Johansson et al., 2006,
Svraka et al., 2006), particularly as an adjunct to MLST. Pearson et al. employed
MLVA in analysing selected outbreaks of B. pseudomallei in Australia where the
method was found to be robust (Pearson et al., 2007). Based on the VNTR concept,
U’Ren developed a multiple-locus VNTR analysis (MLVA) genotyping system which
interrogated 23 polymorphic loci in B. pseudomallei (U'Ren et al., 2007).
Subsequently, Currie et al. described the use of a simpler and less laborious MLVA
42
system with four VNTR loci, which was able to reveal clonal outbreaks (Currie et al.,
2009). In addition, MLVA has also been utilized in investigating genetic variation in
acute melioidosis infections and in determining the likely point source of B.
pseudomallei infection (Price et al., 2010).
Whole genome sequencing (WGS) of B. pseudomallei
Since the first whole genome sequence of B. pseudomallei (K96243) was published in
2004 (Holden et al., 2004), researchers studying this bacterium have gained greater
insights into the pathogenesis of melioidosis, the virulence, the environmental
survival, the evolution, the antimicrobial resistance and the protein function of this
complex organism (Holden et al., 2004, Yu et al., 2006, Sim et al., 2008, Nandi et al.,
2010, Price et al., 2013a). WGS has also provided a greater insight into the population
structure and the evolution of B. pseudomallei (Pearson et al., 2009). WGS enables
characterisation of every nucleotide in the genome, providing unprecedented strain
resolution. Having a complex genome with two chromosomes and the tendencies for
horizontal gene transfer, there is a compelling need in defining core and accessory B.
pseudomallei genomes using WGS (Holden et al., 2004, Sim et al., 2008, Nandi et al.,
2010). Whole genome assembly of B. pseudomallei has proven a nontrivial task due
to the large number of long repetitive regions in the B. pseudomallei genome, which
require expensive and targeted sequencing for gap closure. To date there are seven
closed B. pseudomallei genomes with over 60 draft genome assemblies that are
publicly available (NCBI Microbes database). The introduction of massively parallel
next-generation sequencing (NGS) platforms such as 454, Illumina and Ion Torrent
has resulted in the explosion of NGS data being generated for B. pseudomallei, with
over 500 B. pseudomallei genomes generated from the isolate collection at Menzies
School of Health Research alone (E. P. Price, personal communication). With the
continuing decrease in WGS sequencing costs, this technology will soon become the
more favourable option for performing MLST since both methods require similar
resources.
Despite previous description of B. pseudomallei strains from Peninsular Malaysia
using various methods such as PFGE (Vadivelu et al., 1997, Chua et al., 2010, Chua
et al., 2011), drug susceptibility (Karunakaran and Puthucheary, 2007, Sam et al.,
43
2009, Ahmad et al., 2013) and MLST (McCombie et al., 2006), the information on B.
pseudomallei from Malaysian Borneo remains limited.
In this current study, 68 clinical B. pseudomallei isolates were collected from five
hospitals in Malaysian Borneo; namely, Queen Elizabeth Hospital in Sabah; and
Bintulu Hospital, Kapit Hospital, Miri Hospital and Sibu Hospital in Sarawak.
2.1.4 Study Hypothesis

B. pseudomallei strains from Malaysian Borneo are more closely related to
each other in comparison with Australian strains, and although ecologically
established, have narrow genetic diversity.
2.1.5 Study Objectives
1. To determine the population structure and infer dissemination of Malaysian
Borneo B. pseudomallei isolates using various well-described genotyping
methods.
2. To understand the evolution of the Malaysian Borneo B. pseudomallei in
relation to isolates from other parts of the world using WGS and MLST
analyses.
To achieve the objectives of this study, the clinical B. pseudomallei isolates from
Malaysian Borneo were subjected to various characterization methods as discussed in
the next section of this chapter. The significance and the implications of the results
are discussed in the subsequent sections of this chapter.
44
2.2 Materials and methods
2.2.1 Ethics
B. pseudomallei strain collection and research were approved by the Medical
Research Ethics Committee and registered with the National Medical Research
Registrar and the Clinical Research Centre, Ministry of Health of Malaysia
(Appendix A). All clinical isolates were from routine melioidosis laboratory
diagnosis and hence no written consent was obtained from patients.
2.2.2 Collection of B. pseudomallei clinical isolates from hospitals
A total of 68 B. pseudomallei isolates were collected from melioidosis cases from five
hospitals located in Kota Kinabalu in Sabah, and Bintulu, Kapit, Miri and Sibu in
Sarawak. Isolates from Sabah were collected between 1994 and 2012 whereas isolates
from Sarawak were collected from between 2010 and 2012. Table 2.1 provides
further details on number of isolates collected and information on each hospital.
2.2.3 Movement of B. pseudomallei clinical isolates
All B. pseudomallei isolates collected from the different hospitals were transported to
the laboratory at the Institute of Health and Community Medicine, Universiti
Malaysia Sarawak (UNIMAS) in trypticase soy broth (TSB) (Oxoid, Australia)
containing 20% glycerol with the cold-chain maintained as much as possible. Upon
arrival at the laboratory in UNIMAS, the isolates were revived by streaking on
trypticase soy agar (TSA) and Ashdown’s agar (Oxoid, Australia) before incubation at
37 °C. Plates were observed for up to seven days for growth and colonies selected for
regrowth on fresh agar plates. Pure bacterial cultures were inoculated into TSA slants
in screw cap tubes. Tubes containing inoculated agar slants were sealed tightly with
parafilm (Parafilm, USA) and transported from UNIMAS to Menzies School of
Health Research using authorized courier service at room temperature. The total
transportation time was up to 10 days. Export permits were obtained from the
Ministry of Health of Malaysia and the Australian Quarantine and import permit from
Inspection Service (AQIS) as shown in Appendix A.
45
Table 2.1 Number of B. pseudomallei Malaysian Borneo isolates collected and information on the hospitals
Hospital
Location
Queen Elizabeth
Hospital
Bintulu Hospital
Kapit Hospital
Miri Hospital
Kota Kinabalu,
Sabah
Bintulu, Sarawak
Kapit, Sarawak
Miri, Sarawak
Sibu Hospital
Sibu, Sarawak
No. isolates
collected
25
No. beds
Services
589
Catchment
population*
>1,000,000
10
17
1
284
134
333
<250,000
>100,000
>353,000
15
555
>355,000
General hospital with several specialist services
District Hospital
Referral hospital for northern region of Sarawak
with several specialist services
Main tertiary and referral hospital in central region
of Sarawak. Second largest hospital in Sarawak
(Sources: Sarawak Health Department and Department of Statistics of Malaysia)
46
The only tertiary and referral hospital in Sabah
2.2.4 Revival of B. pseudomallei clinical isolates transported from UNIMAS to
Menzies School of Health Research
Upon arrival at Menzies School of Health Research laboratory, bacterial culture in
agar slants were revived by streaking the cultures onto Chocolate agar (Oxoid,
Australia) and Ashdown’s agar (Oxoid, Australia) before incubating plates at 37 °C.
Plates were observed for growth and morphological identification of culture. Isolates
that did not grow on ordinary Ashdown’s agar containing 40 μg/ml of gentamicin
were grown on modified Ashdown’s agar with 50 μg/ml of colistin, based on colistin
susceptibility testing as discussed in Section 2.2.8.
2.2.5 Morphological identification of B. pseudomallei from culture
Inoculated Ashdown’s agar plates (with and without gentamicin) were observed every
day for growth of the classical purple dry, wrinkled colonies of B. pseudomallei for up
to 14 days (Chantratita et al., 2007). Suspected B. pseudomallei colonies were
subcultured onto fresh Ashdown’s agar plates. Pure cultures were subcultured onto
Chocolate agar for storage and for further characterization by latex agglutination,
antibiotic susceptible testing, and genetic analysis.
2.2.6 B. pseudomallei culture confirmation by antigen detection using latex
agglutination test
To confirm the identity of B. pseudomallei cultures, a latex agglutination test with
monoclonal antibodies targeting the exopolysaccharide was used (Samosornsuk et al.,
1999, Anuntagool et al., 2000). Two to three colonies of an overnight bacterial culture
grown on Chocolate agar were emulsified with 20 μl sterile water on a glass slide or
disposable agglutination test card by gently rubbing with a loop until a milky mixture
was formed. Then, 10 μl of the latex-coated monoclonal antibodies was added to the
cell suspension, and gently mixed with a loop or pipette for up to 10 seconds. Positive
agglutination was noted by clumping of the latex-coated monoclonal antibodies
(Figure 2.1).
47
POS
NEG
Figure 2.1 Latex agglutination test for B. pseudomallei. Left panel showing
agglutination reaction by a negative culture; Right panel showing positive
agglutination reaction by a B. pseudomallei culture.
48
2.2.7 Antibiotic susceptibility testing (E-test)
Minimum inhibitory concentrations (MICs) for amoxicillin-clavulanic acid,
azithromycin, ceftazidime, doxycycline, gentamicin, kanamycin, meropenem and
trimethoprim-sulfamethoxazole were determined using E-tests according to
manufacturer’s instructions (bioMérieux, France). Amoxicillin-clavulanic acid,
ceftazidime, doxycycline, meropenem and trimethoprim-sulfamethoxazole were
selected for MIC determination because these antibiotics are used in routine
melioidosis treatment. MICs for gentamicin were determined due to its use in
selective media for B. pseudomallei. Azithromycin, gentamicin and kanamycin were
tested due to their association with selective media for B. pseudomallei (Chapter 4).
B. pseudomallei strains were grown on Chocolate agar (Oxoid, Australia) 24 hours
before setting up plates for MIC determination. The next day, cell suspensions of all
isolates were prepared by emulsifying overnight culture with 0.85% saline (Pfizer,
USA) to optical density (OD) of ≈ 0.13 using WPA CO-7000 Medical Colorimeter
(Biochrom Ltd, Cambridge, UK) at 590ηm which is equivalent to 0.5 McFarland
Standard turbidity or approximately 108 to 109 cells/ml (bioMérieux, France). To
prepare the bacterial lawn, a sterile cotton swab was dipped into the inoculum cell
suspension and pressed against the inside of the tube wall to remove excess fluid. The
entire surface of Mueller-Hinton’s (MH) agar plate (Oxoid, Australia) was streaked
evenly in three directions. The MH plates were left in the biosafety cabinet until
excess liquid was fully absorbed, before E-test strips (bioMérieux, France) were
applied carefully onto the surface of the plate. The plates were incubated at 37 °C for
18 hours, then the zones for inhibition were observed.
The definition of MIC breakpoint values for susceptible (s), intermediate (i), and
resistant (r) for amoxicillin-clavulanic acid (s ≤ 8/4, i 16/8, r ≥ 32/16), ceftazidime (s
≤ 8, i 16, r ≥ 32), doxycycline (s ≤ 4, i 8, r ≥ 16), meropenem (s ≤ 4, i 8, r ≥ 16) and
trimethoprim-sulfamethoxazole (s ≤ 2/38, r ≥ 4/76) were based on recommendations
by the Clinical and Laboratory Standard Institute (CLSI) and the pathology laboratory
of the Royal Darwin Hospital approved guidelines (CLSI, USA). The MIC breakpoint
values of azithromycin (s ≤ 2, i 4, r ≥ 8), gentamicin (s ≤ 4, i 8, r ≥ 16) and kanamycin
(s ≤ 16, i 32, r ≥64) were based on manufacturer’s interpretive guidelines for aerobes
49
(bioMérieux, France). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa
ATCC 27853 were used as controls.
2.2.8 Testing of colistin susceptibility of gentamicin sensitive B. pseudomallei
isolates and modification of Ashdown’s agar
Colistin has been used as an additive in MacConkey agar for the selective growth of
what was then known as Pseudomonas pseudomallei since 1968 (Farkas Himsley,
1968). After the introduction of gentamicin as a more superior selective agent for B.
pseudomallei (Ashdown, 1979a), colistin was then used in broth media for isolating
B. pseudomallei from non-sterile sites in combination with MacConkey agar
(Ashdown, 1979b). Since then, various groups have shown that the use of broth media
in combination with Ashdown’s agar was able to increase the isolation rate of B.
pseudomallei from specimens of non-sterile clinical sites and environmental samples
(Ashdown, 1979b, Wuthiekanun et al., 1990).
Following the discovery that the majority of B. pseudomallei clinical isolates from
Sarawak were gentamicin sensitive (GENs) (Podin et al., 2014) (see Chapter 4),
colistin was considered as an alternative selective agent in replacement of gentamicin
(Ashdown, 1979b). This experiment was designed to determine whether colistin has
any inhibitory effect on GENs B. pseudomallei isolates from Sarawak.
A panel of bacterial strains consisting of 12 GENs B. pseudomallei isolates from
Sarawak and a gentamicin resistant (GENr) strain from the NT (MSHR668) was
selected. The control strain used for this experiment was P. aeruginosa ATCC 27853,
which is susceptible to colistin (bioMérieux, France). Based on previously published
work, the recommended concentrations for colistin in Ashdown’s broth for clinical
samples and environmental samples are 5 μg/ml and 50 μg/ml respectively (Ashdown,
1979b, Wuthiekanun et al., 1990, Kaestli et al., 2009). Serial dilutions of colistin with
the following concentrations were prepared: 500 μg/ml, 50 μg/ml, 12.5 μg/ml. Blank
discs (bioMérieux, France) were inoculated with 20 μl of different concentrations of
colistin and let dry before use. For negative control, blank discs were inoculated with
the same amount of sterile water and let dry.
50
A loopful of stored pure bacterial isolate from the -80 °C collection was streaked onto
Chocolate agar plates (Oxoid, Australia) and incubated overnight at 37 °C. A cell
suspension of the overnight culture was prepared by emulsifying half a loop of the
culture with 0.85% saline to OD≈0.13. Then, 1 ml of the bacterial cell suspension was
poured onto MH agar, excess liquid aspirated and plates were left in the biosafety
hood for the liquid to be fully absorbed. Each plate was then divided into four
quadrants where each quadrant was placed with one disc of the different
concentrations of colistin and one disc inoculated with sterile water as negative
control. In order to observe the difference of the effect of colistin with direct contact
on the bacterial lawn as compared with impregnated discs, an additional 10 μl of
colistin of the respective concentration was inoculated directly onto the bacterial lawn
next to the discs (Figure 2.4).
Upon confirming that all GENs B. pseudomallei from Sarawak were not susceptible to
colistin (<500 μg/ml tested in this study), 40 μg/ml of gentamicin in the Ashdown’s
agar recipe was substituted with 50 μg/ml of colistin. Hence, the bacterial cultures of
the clinical isolates from the first field trip were done using Ashdown’s agar with
gentamicin whereas the isolates from the second trip were cultured with colistin
instead of gentamicin.
2.2.9 DNA extraction using QIAamp DNA mini kit
Pure B. pseudomallei clinical isolates were grown on Chocolate agar (Oxoid,
Australia) overnight at 37 °C before being subjected to DNA extraction. Due to
inherent difficulties lysing B. pseudomallei compared with other Gram-negative
species, the “Gram-positive bacteria” protocol within the QIAamp DNA mini kit,
with the following modifications to maximise DNA yields; 1) 1 mg/ml lysozyme, 2)
45 minute incubation with Proteinase K at 55°C, 3) elution in 200 μl of the elution
buffer provided in the kit (Qiagen, Australia).
2.2.10 Type III secretion system real-time PCR
B. pseudomallei isolates were confirmed using primer sets targeting 115-base pair
(bp) in the open reading frame 2 (orf2) of the type III secretion system (TTS1) of B.
pseudomallei, with modifications (Novak et al., 2006, Meumann et al., 2006, Kaestli
et
al.,
2012a).
The
following
primers
51
were
used:
BpTT4176F;
5’-
BpTT4290R;
CGTCTCTATACTGTCGAGCAATCG-3’,
5’-
CGTGCACACCGGTCAGTATC-3’ and with a 6FAM-black hole quencher (BHQ)labelled probe BpTT4208P; 5’-6-FAM- CGGAATCTGGATCACCACCACTTTCCBHQ1-3’. Per 10 μl PCR reaction, 0.5 ng template DNA was added to 900nM
primers, 200nM probe and 1X Applied Biosystems Genotyping master mix (Life
Technologies, USA) with thermal cycling condition at 50 °C for 2 minutes, 95 °C for
10 minutes, and 40 cycles of 95 °C for 15 seconds and 58 °C for 1 minute on an ABI
PRISM 7900HT real-time PCR instrument (Life Technologies, USA) (Kaestli et al.,
2012a).
2.2.11 Detection of the B. thailandensis-like flagellum and chemotaxis
biosynthesis (BTFC) and the Yersinia-like fimbrial (YLF) gene clusters
The detection of BTFC and YLF gene clusters on all isolates was performed in the
multiplex real-time PCR platform, using primers and probes designed by Tuanyok et
al. (Tuanyok et al., 2007). The primers’ sequences were; BTFC_F; 5’BTFC_R;
GGCAGCGTCGAACTGTTCTAG-3’,
5’-
CGAATCAATTCGTTTCCCTTGT-3’, YLF_F; 5’- TGCTCGGCTTCCAGATCAG3’, YLF_R; 5’-CGGTCAGTTGCCCGCTATT-3’, with molecular-groove binding
non-fluorescence quencher (MGBNFQ)-labelled probes; BTFC_p; 5’- VICTTCGGCTGCGAAACA-MGBNFQ-3’, YLF_p; 5’-FAM-TCGGACCGCTTGCAMGBNFQ-3’ (Biosearch Technologies, Australia). In a 10 μl PCR reaction, 0.5 ng
template DNA was added to 900nM primers, 200nM probe and 1X Applied
Biosystems genotyping master mix (Life Technologies, USA) with thermal cycling
condition at 50 °C for 2 minutes, 95 °C for 10 minutes, and 40 cycles of 95 °C for 15
seconds and 58 °C for 1 minute on an ABI PRISM 7900HT real-time PCR instrument
(Life Technologies, USA) (Kaestli et al., 2012a).
2.2.12 bimABm and bimABp allele real-time PCR methods
All isolates were subjected to two separate real-time PCR assays that discriminated
two alleles of the actin-based motility gene bimA; B. mallei-like BimA (bimABm) and
B. pseudomallei BimA (bimABp) (Sitthidet et al., 2008). The sequences of the primers
and
probe
for
bimABm-specific
CGCTTCGCGCATCTACTATGT3’,
assay
are
bimA_Bm_F;
bimA_Bm_R;
5’5’-
GCGTTAAACGCCGTACTTTCA-3’. The primers and probe for bimABp-specific
52
assay
are
bimA_Bp_F;
5’-6FAM-ATGCGCTCGCTATC-MGBNFQ-3’,
bimA_Bm_p;
5’-GGAAGCTTTGGCGTGCATAT-3’,
bimA_Bp_R;
bimA_Bp_p6;
CCCATGCCTTCCTCGACTAAT-3’,
5’-
5’-FAM-
ACGTCACTGCCAATC-MGBNFQ-3’ (Biosearch Technologies, Australia). In a 10
μl PCR reaction, 0.5 ng template DNA was added to 900nM primers, 20nM probe and
1X Applied Biosystems genotyping master mix (Life Technologies, USA) with
thermal cycling condition at 50 °C for 2 minutes, 95 °C for 10 minutes, and 40 cycles
of 95 °C for 15 seconds and 58 °C for 1 minute on an ABI PRISM 7900HT real-time
PCR instrument (Life Technologies, USA).
2.2.13 Multilocus sequence typing (MLST)
MLST was performed on all isolates at Imperial College London using previously
published method (Godoy et al., 2003). The allele numbers of each of the seven loci
(Table 2.2) were assigned by comparing nucleotide sequences of the loci of isolates
to
sequences
available
at
the
MLST
database
for
B.
pseudomallei
(http://bpseudomallei.mlst.net/ - Accessed on 3 December 2013). The sequence type
(ST) of each isolate was then assigned based on the allelic profile with the
combination of the different seven allelic numbers of the loci. The relatedness of B.
pseudomallei isolates within a certain population was analysed using eBURST
version 3, an MLST algorithm available on the website (Feil et al., 2004).
Table 2.2 B. pseudomallei MLST loci
Locus name
Gene function
Genome location (kb)
ace
Acetyl coenzyme A reductase
1,780
gltB
Glutamate synthase
3,761
gmhD
ADP glycerol-mannoheptose epimerase
3,023
lepA
GTP-binding elongation factor
2,938
lipA
Lipoic acid synthetase
448
narK
Nitrite extrusion protein
2,784
ndh
NADH dehydrogenase
1,400
Adapted from (Godoy et al., 2003)
53
2.2.14 Multiple-locus VNTR analysis (MLVA) methods
Initially, a multiple-locus VNTR analysis (MLVA) genotyping system which
interrogated 23 polymorphic loci in B. pseudomallei was developed by U’Ren (U'Ren
et al., 2007). In this current study, a simpler and less laborious MLVA system was
utilized by interrogating four VNTR loci (389, 933, 1788, 2341) in four separate
PCRs as previously described (Currie et al., 2009).
DNA extracts of B. pseudomallei isolates were initially subjected to PCR for the four
different
loci
using
the
following
primers:
389
389
GTTACAAGCGCGGGTCGGCAAGAGGCTGAAA-3’,
933
ACGAGTGGGTGGCGTAAGC-3,
ATGGTGGCGGCCGTCGGCGAAAACC-3’,
forward;
5’-
VIC-
reverse;
5’-
forward;
933
5’-NED-
reverse;
5’-
GCTCGAATGGGTGTACGAAGGGCCACGCTGATTC-3’, 1788 forward; 5’-PETGCGCGGCGAGAACGGCAAGAACGAA-3’,
1788
GAGCATCGGGTGGGCGGCGCGTATTGAT-3’,
2341
GGCTTCGCACCCGCCCCATTTCAGC-3’,
2341
reverse;
forward;
reverse;
5’5’-FAM5’-
GCACCGGGCGCGGCGCACTCG-3’. Separate PCR reactions were conducted with
these different primers with each reaction containing 0.88 U HotStarTaq DNA
Polymerase (QIAGEN, Hilden, Germany), 1× PCR buffer, 1.2 M Betaine, 3 mM
MgCl2, 0.2 mM deoxynucleoside triphosphates, 0.2 μM fluorescently labeled forward
primer, 0.2 μM reverse primer, 0.5 ng/μl template DNA and double-distilled water to
give a volume of 11 μl per reaction. Thermo cycling was performed in a Palm Cycler
(Corbett Research Pty Ltd, Australia) with an initial denaturation of 95 °C for 5
minutes, followed by 34 cycles of 94 °C for 30 seconds, 68 °C for 30 seconds, and 72
°C for 30 seconds, and a final extension step of 72 °C for 5 minutes and 15 °C for 3
minutes.
Upon checking for PCR product quality by gel electrophoresis, PCR products of each
labelled primer were pooled before being diluted with 200 μl of double-distilled
water. Then, 1.2 μl of the pooled, diluted PCR product was added to a mixture of a
1:6 ratio of Hi-Di formamide (Applied Biosystems, USA) and GeneScan 1200 LIZ
(Applied Biosystems, USA) fluorescence-labelled size standard. PCR products were
then subjected to capillary electrophoresis using a 3100xl DNA Sequencer (Applied
54
Biosystems, USA) and analyzed by using the ABI software program GeneMapper
version 3.5 (Applied Biosystems, USA).
2.2.15 Analysis of MLVA-4 results
The analysis of the MLVA-4 loci was done as previously described (Currie et al.,
2009), where GeneMapper peak files were imported into BioNumerics version 4.6.1
(Applied Maths BVBA, Sint-Martens-Latem, Belgium). Each of the four loci had
different number of alleles; 389 (15 alleles), 933 (21 alleles), 1788 (16 alleles), 2341
(20 alleles) where each allele number was assigned based on the sizes of the tandem
repeats interrogated. MLVA-4 type of each isolate was then assigned based on the
combination of the four integers representing the allele numbers of the four loci
(Table 2.6).
2.2.16 Whole genome sequencing (WGS)
WGS was performed on 11 B. pseudomallei isolates (Table 2.3) from Sarawak using
the Illumina GAIIx platform according to standard protocols at the Center for
Microbial Genetics and Genomics, Northern Arizona University, USA. WGS data
were aligned to B. pseudomallei K96243 (Holden et al., 2004) using BurrowsWheeler Aligner (Li and Durbin, 2009) with subsequent conversion to binary
alignment map (bam) format using SAMtools (Li et al., 2009). Single nucleotide
polymorphisms (SNPs) and indels were called using the GATK (McKenna et al.,
2010) with parameters published previously (Price et al., 2010). Mutations were
assessed
for
change-of-function
using
55
PROTEAN
(Plasterer,
2000).
Table 2.3 B. pseudomallei isolates from Sarawak subjected to WGS
B. pseudomallei isolate
Patient code
GENr/GENs
Geographical origin
MSHR5080
KPT-1
GENr
Kapit
MSHR5085
BTU-1
GENr
Bintulu
MSHR5086
BTU-1
GENr
Bintulu
MSHR5087
KPT-2
GENs
Kapit
MSHR5093
KPT-3
GENs
Kapit
MSHR5095
BTU-2
GENs
Bintulu
MSHR5097
BTU-3
GENs
Bintulu
MSHR5100
BTU-4
GENr
Bintulu
MSHR5102
BTU-4
GENr
Bintulu
MSHR5105
BTU-5
GENs
Bintulu
MSHR5107
KPT-4
GENs
Kapit
56
2.3 Results and discussions
2.3.1 Morphotypes
A majority of the clinical isolates from Malaysian Borneo had the classical dry,
wrinkled colonies while others had either mucoid colonies or a mixture of dry,
wrinkled and mucoid colonies. As depicted in Figure 2.2, it appeared that some of the
clinical isolates possessed different morphotypes when grown on Ashdown’s agar.
This phenomenon is common as Chantratita et al. observed that mixed morphotypes
within a clinical isolate is common due to environmental stress, availability of
nutrient, iron limitation, and growth at a certain temperature. Particularly, it was
suggested that the smooth and mucoid morphotypes (Figure 2.2, Panels E-F) were
the bacteria’s responses towards a harsh environment such as starvation and dry
growth conditions (Chantratita et al., 2007).
A recent study concurred with
Chantratita et al.’s findings by suggesting that morphology variants in a particular B.
pseudomallei strain were not due to genomic sequence difference but rather, due to
epigenetic factors, gene expression programs, and/or proteome variations (Vipond et
al., 2013). The genotyping results discussed later provide more insight on this
phenomenon.
2.3.2 Latex agglutination test
All clinical isolates tested using agglutination test were positive, confirming that these
isolates were B. pseudomallei. The results also suggest that the exopolyssacharides of
B. pseudomallei from Malaysian Borneo are no different from those tested previously
(Samosornsuk et al., 1999, Anuntagool et al., 2000).
57
B
A
B
C
D
E
F
D
F
Figure 2.2 B. pseudomallei isolates from Malaysian Borneo regrown on Ashdown’s
agar showing different morphotypes. Panel A and B - Mixed colonies of dry-wrinkled
and mucoid; Panel C and D – Classic dry, wrinkled colonies; Panel E and F –
Mucoid colonies.
58
2.3.3 Antibiotic susceptibility testing (E-test)
Based on the results shown in Table 2.4, the MIC profiles of amoxicillin-clavulanic
acid, ceftazidime, doxycycline, meropenem and trimethoprim-sulfamethoxazole for
the Malaysian Borneo B. pseudomallei were all within the expected range, similar to
what was described earlier in isolates from elsewhere (Dance et al., 1989b, Jenney et
al., 2001, Chan et al., 2004, Ahmad et al., 2013). These results suggested that drug
resistance in Malaysian Borneo B. pseudomallei is not prevalent thus far. Intriguingly,
a majority of the isolates from Sarawak appeared to be GENs. Out of the total of 42
isolates tested from Sarawak, a staggering 88% appeared to be GENs. Isolates
possessing this particular attribute were found in all hospitals (Bintulu, Kapit, Sibu)
except for Miri. Although B. pseudomallei are intrinsically resistant to gentamicin,
sensitivity to gentamicin has been reported, being as rare as one in 1000 in Thailand
(Trunck et al., 2009). The characterization and the mechanism of this phenotype are
discussed in detail in Chapter 4.
2.3.4 Colistin susceptibility experiment
As expected, all 12 GENs B. pseudomallei from Sarawak selected for the experiment
were not susceptible to colistin irrespective of the concentration (<500 μg/ml tested in
this study). The control P. aeruginosa was shown to be susceptible to colistin and the
effect was more apparent when colistin was inoculated directly onto the bacterial lawn
(Figure 2.3). The results of this experiment confirmed that similar to GENr B.
pseudomallei, GENs are also resistant to colistin and hence, the use of colistin in
selective media will not inhibit the growth of GENs strains. These results also suggest
that the mechanism involved in conferring gentamicin susceptibility in the Malaysian
Borneo isolates (Chapter 4) is different from that of colistin.
59
Table 2.4 Minimum inhibitory concentrations results for B. pseudomallei isolates used in this study.
Strain
Source
Location
ST
AMX
MSHR313
MSHR314
MSHR316
MSHR317
MSHR318
MSHR319
MSHR320
MSHR323
MSHR324
MSHR325
MSHR326
MSHR327
MSHR330
MSHR331
MSHR4358
MSHR4363
MSHR4364
MSHR5079
MSHR5084
MSHR5087
MSHR5089
MSHR5091
MSHR5093
15 yo male
21 yo female. Blood culture.
64 yo, female. Blood culture.
1 yo, female.
5 yo, male. Blood culture.
24 yo, female. Blood culture (320)
63 yo, male. Blood culture. (321)
49 yo, female. Sinus (319)
50 yo, female. Pus (321)
49 yo, female. Pus culture(322)
45yo, male. Pus (323)
7 yo male, pleural fluid
(324)
10 yo, female. Abdominal abscess pus (325)
48 yo, male, Liver abscess (326)
2 yo, female. Blood culture (323)
3 yo, male. Right ear (341)
32 yo male. Blood
No clinical information.
67 yo female, osteomyelitis, blood culture.
3 yo female, abscess on scalp.
45 yo female.
3 yo female, abscess.
62 yo female, community acquired pneumonia,
blood culture.
MSHR5095 41 yo male, pneumonia, blood culture.
Minimum inhibitory concentration (μg/ml)
AZM
CAZ DOX
GEN
KAN
MEM
SXT
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Sabah
Kapit
Bintulu
Kapit
Kapit
Kapit
Kapit
50
58
50
273
272
271
250
272
250
250
50
84
232
50
50
875
232
50
658
881
881
881
881
6
3
4
3
4
6
3
3
ND
ND
3
3
3
3
3
ND
3
3
3
3
3
6
6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
16
ND
ND
ND
ND
ND
2
1.5
2
1.0
1.5
1.5
1.0
1.5
0.5
1.0
1.5
1.5
2
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
3
4
2
2
2
3
3
3
1.5
2
0.75
1.5
1.5
2
1.5
1.5
1.5
1.5
1.5
1.5
3
1.0
1.5
1.5
1.5
96
96
>256
64
>256
>256
128
96
48
48
48
24
>256
48
96
48
32
96
32
1.0
1.0
1.0
1.0
24
24
24
16
24
24
24
24
24
24
24
16
48
24
16
24
12
24
16
2
2
1.5
1.5
0.75
0.75
0.75
0.5
0.5
0.5
0.5
0.75
0.38
0.5
0.5
0.75
0.75
0.75
0.75
0.75
0.5
0.75
0.75
0.5
0.5
0.75
1.0
0.75
0.75
3
0.5
1.5
0.5
1.5
0.75
0.19
0.75
0.5
0.75
0.75
0.75
0.75
0.5
1.0
0.75
0.5
3
4
4
3
Bintulu
881
3
ND
1.0
1.5
1.5
2
0.5
0.75
60
MSHR5097 34 yo male, blood culture.
MSHR5102 17 yo male, neutropenic sepsis.
Bintulu
Bintulu
881
658
3
3
ND
24
1.0
2
1.5
3
1.0
≥256
2
128
0.5
2
MSHR5104 53 yo female, blood culture.
MSHR5105 2 yo female, pus from left cervical
lymphadenitis.
MSHR5107 18 yo male, pus from neck abscess.
MSHR6363 64 yo, female. Hypertension and chronic
kidney diseases, dry cough, deceased. Blood
culture (360)
MSHR6364 18 yo, male. Thalassemia with
cardiomyopathy. Left hip and kidney pain.
Swab culture (360)
MSHR6367 62 yo male. No clinical information (360)
MSHR6369 62 yo, male. No clinical information. (44)
MSHR6372 48 yo, male. Diabetes mellitus, hypertension,
Chronic kidney disease stage 5, peritonitis
(363)
MSHR6381 47 yo male, shoulder abscess.
MSHR6385 2 yo female, severe community acquired
pneumonia, blood culture.
MSHR6386 27 yo male, community acquired pneumonia,
blood culture.
blood culture.
MSHR6389 11 yo male, lower right eyelid and right
cervical abscess, pus.
Bintulu
Bintulu
881
881
2
3
ND
ND
0.75
1.5
1.0
1.5
1.5
1.5
2
1.5
0.19
0.5
0.5
0.12
5
0.75
4
Kapit
Sabah
881
84
3
2
ND
ND
1.0
1.0
1.5
1.0
1.0
24
1.5
12
0.5
1.0
1.0
1.5
Sabah
84
2
ND
1.5
1.0
16
12
0.75
0.5
Sabah
Sabah
Sabah
84
84
84
3
3
3
ND
24
ND
1.5
1.5
2
1.5
1.0
1
24
24
24
12
16
24
1.0
1.0
1.5
1.5
0.75
0.5
Kapit
Kapit
881
881
3
3
ND
ND
2
2
0.5
0.75
1.0
1.0
2
1.5
1.0
0.75
3
2
Kapit
881
3
ND
2
0.38
1.0
1.5
0.75
2
Kapit
881
3
ND
3
0.5
1.0
2
0.75
2
Sibu
881
3
ND
2
0.38
1.5
2
0.75
1.0
61
MSHR6390 30 yo male, community acquired pneumonia.
MSHR6392 18 yo male, ankle abscess.
blood culture.
MSHR6404 12 yo female, pus from lower eyelid abscess.
MSHR6408 No clinical information.
blood culture.
MSHR6410 45 yo male, knee septic abscess, blood culture.
blood culture.
MSHR6414 41 yo male, ankle septic arthritis.
MSHR6415 54 yo female, sepsis secondary to right leg
wound.
MSHR6418 15 months female, occipital abscess, culture
positive from abscess.
MSHR6761 32 yo, male. Blood
MSHR6762 49 yo, male. History of diabetes and gout.
Multiloculated liver abscess
MSHR6766 46 yo female. Liver abscess
MSHR6769 41 yo male, severe pneumonia with sepsis,
blood culture.
MSHR6774 14 yo male, pus from cervical abscess.
MSHR6778 11 yo female, neck abscess.
MSHR6780 31 yo male, pus.
MSHR6785 59 yo female, knee abscess.
Sibu
Sibu
Sibu
881
881
881
3
2
3
ND
ND
ND
1.5
1.5
4
0.5
0.25
0.75
1.0
0.75
1.5
2
2
2
0.75
0.75
1.0
2
1.5
1.5
Sibu
Sibu
Sibu
881
881
881
3
3
2
ND
ND
ND
2
2
1.5
0.5
0.5
0.5
1.5
1.0
0.5
2
1.5
2
1.0
0.75
0.5
2
2
2
Sibu
Sibu
881
881
3
3
1.0
ND
2
2
0.5
0.5
0.75
1.0
2
1.5
0.75
0.75
2
2
Sibu
Kapit
881
881
1.5
3
ND
ND
0.75
1.5
0.5
0.75
0.75
0.5
ND
2
0.5
0.75
0.5
3
Bintulu
881
3
ND
2
0.75
1.0
1.5
0.75
2
Bintulu
Sabah
Sabah
50
50
50
2
3
2
24
ND
ND
2
3
2
2
1.5
1
48
24
24
24
6
16
1.0
1.0
0.75
2
0.5
0.5
Sabah
Bintulu
50
881
3
2
ND
0.75
1.5
1.5
1.5
0.75
24
1.0
24
2
1.0
0.5
1.5
0.5
Kapit
Kapit
Kapit
Kapit
881
881
881
881
8
1.5
3
3
ND
ND
ND
ND
4
1.0
2
1.5
0.75
0.5
1.5
0.5
0.5
0.5
0.75
0.5
1.5
ND
2
1.5
1.0
0.5
0.75
0.75
2
2
1.5
3
62
MSHR6789
MSHR6793
MSHR6802
MSHR6803
MSHR6806
MSHR6808
12 yo male, blood culture.
Kapit
881
3
ND
1.5
0.5
0.5
1.5
0.75
1.5
37 yo female, blood culture.
Kapit
997
3
1.0
1.5 0.75 0.75
2
0.75
2
3 yo female, septic shock blood culture.
Sibu
881
3
ND
2
0.5
1.5
2
0.5
1.5
48 yo male.
Sibu
881
3
ND
1.5
0.5
0.75
2
0.75
4
37 yo female, blood culture.
Sibu
881
2
ND
3
0.75
1.0
2
1.0
3
61 yo, male, community acquired pneumonia, Sibu
881
3
1.0
3
0.5
0.75
1.5
1
1-2
blood culture.
Sibu
46
3
24
3
1.0
96
32
0.75
1.5
MSHR6824 50 yo female, abscess and sepsis, blood culture. Miri
271
2
ND
1.0
1.0
16
12
0.75
3
MSHR6828 Female, blood culture.
Bintulu
881
3
ND
1.0
1.0
0.75
2
1.0
2
Abbreviations: AMX, amoxicillin-clavulanic acid; AZM, azithromycin; CAZ, ceftazidime; DOX, doxycycline; GEN, gentamicin; KAN,
kanamycin; MEM, meropenem; SXT, trimethoprim-sulfamethoxazole; ND, Not determined.
63
P. aeruginosa
MSHR668
MSHR5104
MSHR5097
Figure 2.3 Illustration of colistin susceptibility test on GENs B. pseudomallei from
Sarawak (MSHR5097 & MSHR5104) with controls P. aeruginosa and MSHR668
from the NT. Arrows denote direct inoculation of colistin onto the bacterial lawn.
Figure 2.4 Agar plate layout of
colistin susceptibility test showing
500μg/ml
50μg/ml
the positions of discs impregnated
with different concentrations of
colistin. In addition to the discs,
extra 10 μl of colistin of the
12.5μg/ml
Water
respective
concentration
inoculated
directly
bacterial lawn.
64
onto
was
the
2.3.5 TTS1, BTFC/YLF gene cluster, bimABm/bimABp alleles real-time PCR
assays
Real-time PCR using TTS1 primers confirmed that all the isolates from Malaysian
Borneo collected for this study are indeed B. pseudomallei. That this well-validated
assay was able to detect B. pseudomallei from Malaysian Borneo is an indication that
there is nothing unusual with the TTS1 of the organisms from this region (Novak et
al., 2006, Meumann et al., 2006).
As for the BTFC/YLF gene cluster real-time PCR assay, similar to B. pseudomallei
from Thailand and other Southeast Asian strains, the isolates from Malaysian Borneo
were shown to possess the YLF allele. This is consistent with previous work, which
described that B. pseudomallei harbouring the YLF gene cluster were predominantly
found in Asian countries whereas the BTFC gene cluster is predominantly found in
Australia (Tuanyok et al., 2007). However, the geographical restrictions of B.
pseudomallei possessing either the YLF or the BTFC allele remain to be defined
(Tuanyok et al., 2007). Likewise, the paradox remains that while the Australian B.
pseudomallei strains mostly possess the BTFC allele, B. thailandensis has to date
been hard to find in the environment of Australia.
When tested with the bimABm/bimABp allelic real-time PCR assay, all isolates from
Malaysian Borneo appeared to possess the bimABp allele. Thus far, isolates shown to
possess the bimABm allele have been all from Australia (Sitthidet et al., 2008) or India
(Mukhopadhyay et al., 2011). The bimA gene of B. pseudomallei encodes for actinbased motility which mediates cell-to-cell spread and subsequently helps the organism
to evade host immune responses (Stevens et al., 2005, Sitthidet et al., 2011). The
significance of B. pseudomallei harbouring the bimABm allele has yet to be determined
although it had been suggested that this particular allele is associated with higher
virulence (Sitthidet et al., 2008). In Australia, B. pseudomallei isolates harbouring the
bimABm allele have been shown to possess 14 times more probability of being
associated with melioidosis of neurological involvement (Sarovich et al., 2014a).
65
2.3.6 MLST results
Higher diversity in B. pseudomallei from Sabah compared to Sarawak
As shown in Figure 2.5, B. pseudomallei isolates from Sabah had a higher diversity
of ST when compared with isolates from Sarawak. There were nine STs from a total
of 25 isolates collected from the hospital in Kota Kinabalu in Sabah whereas there
were only six STs from 42 Sarawak isolates. The genetic diversity index (D) of B.
pseudomallei STs from Sabah and Sarawak were calculated using previously
published method (Grundmann et al., 2001). The statistical analysis revealed that the
genetic diversity of STs in Sabah was 80.6% [CI 71.8% to 89.5%] whereas that of
Sarawak was 28.9% [CI 10.9% to 46.8%] (no overlap in values).
There is a possibility that this higher diversity of ST observed in Sabah may be due to
the Kota Kinabalu hospital being a tertiary hospital where it received referral cases
from other hospitals throughout the state. Sibu and Bintulu hospitals are also tertiary
hospitals and yet isolates from these hospitals still had lower ST diversity. The
difference in the diversity of ST from hospitals in Sabah and Sarawak may relate to
more human movements between the different regions of Sabah and the islands in
southern Philippines which may have facilitated the dispersal of B. pseudomallei in
comparison to the regions of Sarawak, which are more geographically isolated.
Currently, there are only four B. pseudomallei isolates from the Philippines on the
MLST database of which, three were from monkeys in 1990 and one was from a
clinical case in 1969 (http://bpseudomallei.mlst.net/ - Accessed on 12 February 2014).
An eBURST analysis of STs of these four B. pseudomallei isolates from the
Philippines and isolates from Malaysia indicated that one isolate from a monkey from
the Philippines (ST57) was a SLV of an isolate from Sabah (ST58). Another B.
pseudomallei isolate from the Philippines of human origin (ST99) is a double locus
variant (DLV) of an isolate from Sabah (ST232). The similarity in two of the STs
(ST57 and ST99) of B. pseudomallei from the Philippines with isolates from Sabah
(ST58 and ST232) may reflect that the patients in Sabah who had these STs isolated
were infected in the Philippines but were subsequently diagnosed in Sabah. Another
possibility is local spread of B. pseudomallei between Sabah and the Philippines
through importation of animals as previously reported (Dance et al., 1992, Ouadah et
al., 2007). MLST and WGS analyses of a broader range of isolates from different
geographical locations within the Philippines and comparison with isolates from
66
diverse locations in Malaysia and elsewhere in Southeast Asia are required to further
elucidate the diversity of B. pseudomallei within the region and to test the various
hypotheses of spread of B. pseudomallei.
Different STs between Sabah and Sarawak
Of the 13 STs found in Malaysian Borneo, only ST50 and ST271 were found in both
Sabah and Sarawak. These results suggest that despite being located on the same
island of Borneo, the B. pseudomallei strains found in Sabah are different from those
found in Sarawak. Human movements have been postulated to play an important role
in the diversity of B. pseudomallei in studies from Papua New Guinea (PNG) (Baker
et al., 2011a). Similarly, this phenomenon of different STs found in Sabah and
Sarawak might be due to limited human movements between these two states or due
to reasons mentioned above. Apart from flying, the only other mode of travels
between the two states involves crossing the international border of Brunei
Darussalam twice as shown in the map in Figure 2.5. While this may be a plausible
reason for the wide difference in the STs between the two states, more isolates need to
be collected from areas bordering the two states to confirm this hypothesis.
ST881, ST997 and GENs
In Sarawak, a majority of isolates collected from Kapit, Sibu and Bintulu hospitals
belonged to ST881, which appeared to be GENs when results were crosschecked with
antibiotics susceptibility test. The single locus variant (SLV) of ST881, ST997 was
also found to be GENs. Trunck and colleagues have previously reported that GENs in
Thai B. pseudomallei isolates occurs infrequently, being present in approximately one
out of every thousand strains (Trunck et al., 2009). This intriguing phenomenon
where a majority of B. pseudomallei isolates from these regions in Sarawak was GENs
is discussed further in Chapter 4.
67
Figure 2.5 Map of Malaysian Borneo showing the different STs from hospitals in Kota Kinabalu, Bintulu, Kapit and Sibu. Denoted by the red
dots. (Adapted from http://en.wikipedia.org/wiki/East_Malaysia#mediaviewer/File:Borneo2_map_english_names.svg).
68
Common STs in Malaysian Borneo and Peninsular Malaysia
Based on the database available on the MLST website, there were only three STs that
were common between Malaysian Borneo and Peninsular Malaysia, namely ST46,
ST50 and ST84 (http://bpseudomallei.mlst.net/ - Accessed 30 November 2013).
Table 2.5 showed that apart from the sole ST46 from the collection for the study from
Miri hospital in Sarawak, 22 other isolates belonging to this ST were found in
Peninsular Malaysia with a majority of them from the 1960s collection sequence
typed by the USAMRU (McCombie et al., 2006). ST46 is widespread in Southeast
Asia and Asia regions, appearing in Thailand, Indonesia, Vietnam, Cambodia and
Bangladesh. It was also found in a patient in the USA, possibly a returning traveller
from an endemic region. Importantly, ST46 had been isolated in Malaysia over a span
of almost 50 years from 1964 to 2012. A similar phenomenon was observed in ST109
which was found circulating in Australia over a span of more than 10 years (Cheng et
al., 2004)(E. McRobb, manuscript in preparation). That ST46 is the SLV of ST50
which is the group founder (Figure 2.6) and the fact that it is found in six other
countries
support previous observation that there is a
high degree of
interconnectedness in the Southeast Asian B. pseudomallei population compared with
the Australian population (Pearson et al., 2009).
Similar to ST46, ST84 was also described in the USAMRU 1960s collection
(McCombie et al., 2006). Intriguingly, ST84 isolates found in Sabah were collected
both in 1994 and 2012, suggesting that ST84 is common and widespread in both
Peninsular Malaysia and Sabah.
ST50 was one of the more widespread ST in Malaysian Borneo being found mostly in
Sabah but also in Kapit and Bintulu. This ST was also found in Thailand and China.
B. pseudomallei from Malaysian Borneo in relation to other Asian strains
As indicated in Figure 2.7 (ST of Malaysian and Asian strains) B. pseudomallei from
Peninsular Malaysia were more diverse compared with strains from Malaysian
Borneo. This probably reflects that Peninsular Malaysia is connected to the IndoChina mainland which results in it sharing many common STs with Thailand and
other countries in the Indo-China region. B. pseudomallei from Malaysian Borneo, on
69
the other hand, were less diverse, consistent to it being an island with harsher terrains,
rendering these areas geographically restricted. A similar phenomenon was observed
in PNG (Warner et al., 2008, Baker et al., 2011a) and the Torres Straits islands (Baker
et al., 2013), where certain STs were confined to a geographically restricted area
where there were limited human movements.
Table 2.5 STs found in Malaysian Borneo.
ST
Sabah
Sarawak
Peninsular
Other countries
Malaysia
46
No
Yes
Yes
Bangladesh, Cambodia, Indonesia,
Thailand, Vietnam, USA*
50
Yes
Yes
No
China, Thailand
58
Yes
No
No
Thailand
84
YesŦ
No
Yes
Thailand
232
Yes
No
No
Thailand
250
Yes
No
No
No
271
YesŦ
Yes
No
No
272
Yes
No
No
No
273
Yes
No
No
No
658
No
Yes
No
Laos, Thailand
875
Yes
No
No
No
881
No
Yes
No
China*
997
No
Yes
No
No
(*) – Returned traveller or infected while in endemic country
(Ŧ) – Isolated from samples in years 1994 and 2012
70
Figure 2.6 eBURST of all Malaysian isolates. Red dots - STs found only in Sarawak; Green dots – STs found only in Sabah; Blue dots –
STs found both in Sabah and Sarawak; Black dots - STs found only in Peninsular Malaysia. Size of the dot denotes the relative frequency of
the ST.
71
Figure 2.7 eBURST of Malaysian and other Asian strains. Red solid dots – Malaysia; Black solid dots - Other Asian strains, Blue – group
founder; Yellow – subgroup founder. The size of the dot is directly proportionate to the relative frequency of the ST.
72
B. pseudomallei from Malaysian Borneo in relation to Australia strains
The eBURST analysis of Thai, Malaysian and Australian strains indicated that the Malaysian and
Malaysian Borneo strains clustered together with the Thai strains rather than the Australian
strains (Figure 2.8). This observation is consistent with previous work by Pearson et al. which
described the distinction between B. pseudomallei from Southeast Asia and Australia (Pearson et
al., 2009). In their analysis, Pearson et al. suggested that B. pseudomallei strains in Southeast
Asia are a subset of the strains found in Australia where greater allelic diversity was observed in
the Australian B. pseudomallei population. That the Malaysian Borneo strains clustered with the
Thai strains strengthens previous observations by Pearson and colleagues.
Nonetheless, all these analyses were based on the snapshot of the current population available on
the MLST website where the majority of Malaysian isolates on the database consist of isolates
from historical collections by USAMRU (McCombie et al., 2006) and from this current study. To
date the majority of B. pseudomallei isolates collected from Peninsular Malaysia in more recent
times have not been subjected to MLST. The question remains whether the population structure
observed in this study will be different once more Malaysian isolates are included in the analysis.
73
Figure 2.8 eBURST of Australian and Asian isolates. Green circles - Thai; Black circles – Australian; Red – Other Malaysian strains; Pink
solid-Malaysian Borneo, Pink outer circles – Other Asians. Size of the dot denotes the relative frequency of the ST.
74
2.3.7 MLVA-4 results
A total of 115 isolates were subjected to MLVA-4 typing method where 40 isolates
were from Sabah and 75 isolates were from Sarawak. The 115 isolates tested were
collected from 65 melioidosis patients with 24 patients from Sabah and 41 patients
from Sarawak.
Same MLST type, same MLVA type within the same host, different samples
Patient SBH-1 had four B. pseudomallei isolates with two from blood sample
(MSHR4358, MSHR4359), and two from pleural fluid (MSHR4360, MSHR4361)
with all yielding ST50 and MLVA-4 type 323, indicating that they were clonal.
Similarly, patient SBH-9 had three B. pseudomallei isolates with one from blood
(MSHR0319) and two from urine samples (MSHR 4362, MSHR4370) belonging to
ST271 and MLVA-4 type 320.
Cluster of cases with same MLVA-4 type
Three patients (SBH-2, SBH-3, SBH-4) presented to Queen Elizabeth Hospital in
Sabah in 2012 had identical MLVA-4 type (360). Given that these patients had strong
epidemiological links being presented to the same hospital within 2012, these results
strongly suggested that these patients may had been infected by the same clone of B.
pseudomallei. Patients SBH-7 and SBH-8 also presented to the Sabah hospital and
had B. pseudomallei of identical MLST (ST250) and MLVA-type 321 isolated. A
similar instance was observed with patients BTU-1 and BTU-4 who presented to
Bintulu hospital in 2011, with isolates from both patients yielding ST658 and MLVA4 type 348. While further epidemiological investigation is required to surmise that this
is in fact a clonal outbreak, such occurrences have been investigated before using this
method in the Top End of Australia where the environmental source was identified
(Currie et al., 2009, McRobb et al., 2013).
Same MLST type, different MLVA-4 type
In some instances, variable MLVA-4 types within the same ST were also observed in
this study. Despite having identical ST881, isolates from two patients (SBU-1, SBU2) had different MLVA types with patient SBU-1 having MLVA-type 374, patient
SBU-2 MLTA-type 378. As shown in Table 2.6, isolates from both patients only
75
shared one similar locus (locus 1788) out of the four compared in MLVA-4. This is as
expected given the high mutation rate of the MLVA alleles in comparison to the
MLST loci and shows the utility of MLVA to support or refute epidemiological links
between two strains found to have the same ST (U'Ren et al., 2007, Currie et al.,
2009, Sarovich et al., 2012a). In contrast with patients SBH-2, SBH-3, SBH-4
discussed earlier, patients SBU-1, SBU-2 were less epidemiologically linked. Patient
SBU-1 was presented with melioidosis in 2010 and patient SBU-2 was presented
more than a year later in 2011. While there is little information on both patients, the
demographic of both patients seemed to also suggest both patients were unlikely to
have come from the same area; patient SBU-1 is an 18 year old student in potentially
a rural boarding school whereas patient SBU-2 is a 61 year old pensioner residing in
an urban area. MLVA is therefore useful in defining relationships among very closely
related isolates, but not broader resolution (Pearson et al., 2007, U'Ren et al., 2007).
Intra-host mutation
Apart from MLVA type variability within the same MLST sequence type between
patients, such variability within a single host was also observed in this study in patient
SWK-3. Patient SWK-3 was presented initially to Kapit hospital with community
acquired pneumonia and was subsequently referred to Sibu hospital the following day.
Blood samples collected from the patient in both hospitals were B. pseudomallei
positive and both were MLST ST881, but the isolate collected at Kapit hospital
(MSHR6386) was MLVA-4 type 371 whereas the isolate from Sibu hospital)
MSHR6411 was type 384. A closer look at the MLVA-4 allelic profiles of the four
loci revealed that there was a single locus variant at locus 933 (Table 2.6). In vivo
mutations of clonal B. pseudomallei isolates within a single host are not uncommon
but has been reported before (U'Ren et al., 2007, Price et al., 2010, Sarovich et al.,
2012a).
76
Table 2.6 MLVA-4 types and VNTR allelic profiles of selected patients
Isolate
Site
Year
Region
VNTR locus name
MLVA-4
Type
ST
Patient
Code
MSHR4358
Blood
1994
Sabah
389 933
10 7
1788
5
2341
9
323
50
SBH-1
MSHR4359
Blood
1994
Sabah
10
7
5
9
323
50
SBH-1
MSHR4360
Pleural
1994
Sabah
10
7
5
9
323
50
SBH-1
1994
Sabah
10
7
5
9
323
50
SBH-1
fluid
MSHR4361
Pleural
fluid
MSHR6363
Blood
2012
Sabah
3
12
6
9
360
84
SBH-2
MSHR6364
Swab
2012
Sabah
3
12
6
9
360
84
SBH-3
MSHR6367
NA
2012
Sabah
3
12
6
9
360
84
SBH-4
MSHR6369
NA
2012
Sabah
3
12
6
10
44
84
SBH-5
MSHR6370
NA
2012
Sabah
3
12
6
10
44
84
SBH-5
MSHR6371
NA
2012
Sabah
3
12
6
10
44
84
SBH-5
MSHR4364
Blood
1994
Sabah
4
5
8
3
342
232
SBH-6
MSHR4365
Blood
1994
Sabah
4
5
8
3
342
232
SBH-6
MSHR4366
Blood
1994
Sabah
4
5
8
3
342
232
SBH-6
MSHR4367
Blood
1994
Sabah
4
5
8
3
342
232
SBH-6
MSHR4368
Blood
1994
Sabah
4
5
8
3
342
232
SBH-6
MSHR4369
Blood
1994
Sabah
4
5
8
3
342
232
SBH-6
MSHR0320
Blood
1994
Sabah
2
8
4
4
321
250
SBH-7
MSHR0324
Pus
1994
Sabah
2
8
4
4
321
250
SBH-8
MSHR0319
Blood
1994
Sabah
7
5
3
4
320
271
SBH-9
MSHR4362
Urine
1994
Sabah
7
5
3
4
320
271
SBH-9
MSHR4370
Urine
1994
Sabah
7
5
3
4
320
271
SBH-9
MSHR5099
Blood
2011
3
9
11
5
348
658
BTU-4
MSHR5100
Blood
2011
3
9
11
5
348
658
BTU-4
MSHR5101
Blood
2011
3
9
11
5
348
658
BTU-4
MSHR5102
Blood
2011
3
9
11
5
348
658
BTU-4
MSHR5102
Blood
2011
3
9
11
5
348
658
BTU-4
MSHR5103
Blood
2011
3
9
11
5
348
658
BTU-4
MSHR5084
Blood
2011
Bintulu,
Sarawak
Bintulu,
Sarawak
Bintulu,
Sarawak
Bintulu,
Sarawak
Bintulu,
Sarawak
Bintulu,
Sarawak
Bintulu,
Sarawak
3
9
11
5
348
658
BTU-1
77
MSHR5085
Blood
2011
MSHR5086
Blood
2011
MSHR6386
Blood
2012
MSHR6411
Blood
2012
MSHR6392
Swab
2010
MSHR6393
Swab
2010
MSHR6391
Swab
2010
MSHR6394
Swab
2010
MSHR6400
Blood
2011
MSHR6401
Blood
2011
MSHR6402
Blood
2011
MSHR6403
Blood
2011
Bintulu,
Sarawak
Bintulu,
Sarawak
Kapit,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
Sibu,
Sarawak
3
9
11
5
348
658
BTU-1
3
9
11
5
348
658
BTU-1
6
9
4
14
371
881
SWK-3
6
10
4
14
384
881
SWK-3
7
16
4
11
374
881
SBU-1
7
16
4
11
374
881
SBU-1
7
16
4
11
374
881
SBU-1
7
16
4
11
374
881
SBU-1
4
5
4
10
378
881
SBU-2
4
5
4
10
378
881
SBU-2
4
5
4
10
378
881
SBU-2
4
5
4
10
378
881
SBU-2
* NA - Information not available
Different morphotypes but same MLVA-4 type
Despite having different morphotypes, isolates from patient SBU-1 (Figure 2.9) had
the same MLVA-4 type 374. Similarly, isolates from patient SBH-5 (Figure 2.10)
were both MLVA-4 type 360. This observation is not surprising as Chantratita and
colleagues had demonstrated that morphotype switching in B. pseudomallei isolates is
common as the strains respond to environmental stress, change in nutrient or
starvation, iron limitation, and temperature of growth (Chantratita et al., 2007).
78
MSHR6392
MSHR6394
Figure 2.9 Morphotype variants in isolates from patient SBU-1 having the same
MLVA-4 type 374
MSHR6369
MSHR6370
MSHR6371
Figure 2.10 Different morphotypes from isolates of patient SBH-5 with the same
MLVA-type 360. It is not possible to get single colonies for isolates possessing
mucoid morphotype as shown here.
79
2.3.8 Whole genome sequencing
Sarawak Strains closely related
In the WGS analyses for this study, a radial genetic relatedness tree was constructed
using approximately 91,000 orthologous SNPs across 23 B. pseudomallei isolates
comprising of 12 isolates from different STs from Australia and 11 isolates from 3
STs from Sarawak. Due to the high incidence of lateral gene transfer (LGT) in B.
pseudomallei (Pearson et al., 2009), core genome SNP phylogenetic analysis for B.
pseudomallei more accurately reflects genetic relatedness rather than phylogenetic
reconstruction or “reconstruction of evolutionary path”. Hence, this analysis is a
representation of the genetic relatedness between selected Sarawak strains and
Australian strains, but phylogenetic inferences made from these analyses should be
treated with caution.
As shown by the radial genetic relatedness tree in Figure 2.11, Sarawak strains are
more closely related to each other than they are to Australian strains. Within the
Sarawak strain clade, the GENs strains (ST881) cluster tightly together and appear to
be distinct from the other two GENr clusters (ST50, ST658) by approximately 16,000
SNPs. The extremely short branches of ST881 strains are remarkable as it infers that
MLST is an accurate reflection of core genome variation despite only analysing ~3kb
of the entire genome. In addition, it is speculated in this study (Section 5.2.2) that the
GENs phenotype of these strains is a result of their existence in an environment where
selective pressure imposed by the presence of aminoglycoside or macrolides seems to
be lacking. A potential change of niche for the GENs group is supported by the
inability to find environmental isolates through soil sampling efforts conducted in this
region despite considerable efforts. Future environmental sampling work should focus
on more exotic niches such as water or livestock instead of solely soil. The
possibilities of these two hypotheses are further discussed in Chapter 5. It can also be
surmised from the genetic-relatedness analysis that there appears to be a population
bottleneck resulting from a single introduction event of B. pseudomallei into Sarawak.
Further analyses are needed to investigate whether there are other mutations that
occurred in the GENs strain such as host adaptations or genome decay.
Based on the MLST data, there was a higher diversity observed in isolates from Sabah
compared with isolates collected from Sarawak. It is therefore postulated that the low
80
diversity observed in Sarawak may be due to founder effects as proposed by Pearson
et al. on their observation of low diversity in the Southeast Asian B. pseudomallei
population (Pearson et al., 2009).
Australian strains different from each other compared with Sarawak strains
Figure 2.11 also shows that Australian strains are on average very different from each
other compared with the Sarawak strains. Unsurprisingly, this observation concurred
with earlier reports that B. pseudomallei from Australia and Asia are genetically
distinct (Currie et al., 2007b, Tuanyok et al., 2007, Sitthidet et al., 2008). This
observation was further supported by Pearson et al. who conducted phylogenetic
analyses of more than 14,000 SNPs of 43 B. pseudomallei strains of different
geographical origins. Results from the study inferred that B. pseudomallei originated
from Australia and that due to the deeper gene pool in the B. pseudomallei population
from Australia, strains from Southeast Asia were a subset of Australian strains
(Pearson et al., 2009). Pearson et al. also described in the study that B. pseudomallei
strains from Australia and Southeast Asia were very distinct, consistent with the
Wallace’s Line theory (Pearson et al., 2009). Flora and fauna found on either side of
the Wallace’s Line (Figure 2.12) were noted to be very different by naturalists
including Alfred Wallace (Wallace, 1869). Malaysian Borneo B. pseudomallei strains
conformed to this theory with strains found in the region being more similar to strains
found in the rest of Southeast Asia compared to strains from Australia. For instance,
as shown in Table 2.5, ST50 was found in both Malaysian Borneo and other Asian
countries including Thailand and China. Likewise, ST46 was found in Malaysian
Borneo, Peninsular Malaysia and also other countries including Bangladesh,
Cambodia, Indonesia, Thailand and Vietnam (http://bpseudomallei.mlst.net/).
Due to copyright issues, Thai isolates were not included in this genetic relatedness
analysis. However, if Thai isolates were to be included in this analysis, it is
hypothesized that B. pseudomallei from Sarawak would be related to but still clearly
distinct from the Thai isolate. Certainly, similar genomic analysis with the inclusion
of more isolates of wider geographical origins is warranted in unravelling the
biogeography and the evolution of B. pseudomallei in the Asia-Pacific/Australasia
region.
81
Sarawak isolates
Figure 2.11 Radial genetic relatedness tree constructed using ~91,000 SNPs across 23
B. pseudomallei including selected isolates from Sarawak.
(Credit: Derek Sarovich, Erin Pice)
82
Figure 2.12 Map showing the demarcation of the Wallace’s Line.
(Source: http://theglyptodon.wordpress.com/2011/05/25/the-wallace-line/).
83
2.4 Conclusions
The various laboratory methods used in this study have confirmed the hypothesis of
this study that B. pseudomallei from Malaysian Borneo are more similar to Southeast
Asian strains and clearly distinct from Australian strains. For instance, isolates from
Malaysian Borneo were shown to all possess the YLF gene cluster and the bimABp
variant, similar to Thai B. pseudomallei (Tuanyok et al., 2007, Sitthidet et al., 2008).
MLST analyses of Malaysian Borneo isolates were consistent with the observation by
Pearson et al. that B. pseudomallei from both sides of the Wallace’s line are different
(Pearson et al., 2009). MLST also suggested that lower diversity is observed in the
Malaysian Borneo population compared with the deeper gene pool observed in the
Australian B. pseudomallei population, which shows more diversity. The lower
diversity observed in particularly Sarawak may be due to the founder effect; a result
of the radiation of few clones when B. pseudomallei was first introduced to Borneo.
Intriguingly, there appeared to be significantly higher genetic diversity of isolates
from Sabah compared with Sarawak perhaps, reflecting a more ancient introduction
event or increased human migration within Sabah or between Sabah and the islands of
southern Philippines.
Genotyping of selected isolates using the MLVA-4 showed that there were a few
clonal strains circulating within the same geographical regions where different
patients were infected with the same clonal type. Similar observations have been
previously reported in proven clonal outbreaks in communities in the Top End of
Australia (Currie et al., 2009). Findings through this method also supported previous
reports that strains with morphology variants do possess identical genomes and that
morphology variants are likely to be due to non-genomic factors (Chantratita et al.,
2007, Vipond et al., 2013).
Drug susceptibility testing has shown that while the Malaysian Borneo isolates
conformed to the MIC profile of most antibiotics tested in other countries (Dance et
al., 1989b, Jenney et al., 2001, Chan et al., 2004, Ahmad et al., 2013), up to 88% of B.
pseudomallei from Sarawak were GENs. Intriguingly, all the GENs strains appeared to
be associated with ST881 and its SLV ST997. Further work on this very rare
phenomenon is discussed in Chapter 4.
84
WGS of selected isolates from Sarawak showed that the isolates were genetically
more similar to each other than to Australian strains. The short branches within
individual STs showed that in Sarawak very little diversity exists in the core genome
of isolates with identical MLST. The WGS analysis also suggested that in this data
set, the Australian strains are very different from each other compared with the
Sarawak strains. In conclusion, more comprehensive genomic investigations are
needed to provide better insights on the biogeography and the evolution of B.
pseudomallei in the Asia-Pacific/Australasia region.
85
Chapter 3
Automated biochemical characterization of
B. pseudomallei from
Malaysian Borneo
Chapter 3: Automated biochemical characterization of
B. pseudomallei from Malaysian Borneo
Study aim
The aim of this study is to elucidate the biochemical profiles of Malaysian Borneo B.
pseudomallei isolates in comparison with Top End isolates using VITEK 2
biochemical automated system.
____________________________________________________________________
A version of this chapter has been published as:
Podin Y, Kaestli M, McMahon N, Hennessy J, Ngian HU, Wong JS, Mohana A, Wong SC,
William T, Mayo M, Baird RW, Currie BJ. Reliability of automated biochemical
identification of Burkholderia pseudomallei is regionally dependent. J Clin Microbiol. 2013
Sep;51(9):3076-8. doi: 10.1128/JCM.01290-13. Epub 2013 Jun 19.
86
3.1 Introduction
Burkholderia pseudomallei is a saprophytic soil bacterium that causes melioidosis, a
disease endemic in northern Australia and Southeast Asia affecting humans and
animals (Wiersinga et al., 2012). The clinical presentations of melioidosis range from
skin infections without sepsis to disseminated infection with sepsis and high
mortality. Pneumonia is present in around half of cases and chronic infections,
relapsed disease and activation from latency are all recognised (Cheng and Currie,
2005, Wiersinga et al., 2012).
Confirmation of diagnosis of melioidosis requires a positive culture of B.
pseudomallei from clinical samples such as blood, sputum, urine, pus, joint aspirate or
swabs from throat or rectum (Wiersinga et al., 2012). B. pseudomallei has been
identified by combining the commercial API 20NE biochemical kit (bioMérieux) with
a simple screening system involving Gram stain, oxidase reaction, typical growth
characteristics, and resistance to gentamicin (Dance et al., 1989d). Susceptibility to
amoxicillin-clavunanic acid (AMX) has also been used to differentiate B.
pseudomallei from B. cepacia which is resistant to AMX (Hodgson et al., 2009).
Unfamiliarity with B. pseudomallei and problems with inaccurate speciation using
some automated commercial biochemical identification systems have resulted in
laboratories misidentifying the bacterium as a Pseudomonas or other Burkholderia
species (Lowe et al., 2002, Koh et al., 2003, Lowe et al., 2006, Weissert et al., 2009,
Zong et al., 2012). Confirmation of B. pseudomallei identity by PCR of DNA
extracted from cultured bacterial colonies is increasingly the standard for many
laboratories (Novak et al., 2006). Various genetic targets have been published for
PCR identification of B. pseudomallei from bacterial cultures and also for direct
detection from clinical samples (Novak et al., 2006, Meumann et al., 2006, Price et
al., 2012), with a review showing the TTS1-orf2 assay to be superior in detecting B.
pseudomallei directly from clinical specimens (Kaestli et al., 2012a). Apart from
molecular methods, B. pseudomallei from cultures can also be confirmed by antigen
detection assays such as latex agglutination (Samosornsuk et al., 1999, Anuntagool et
al., 2000). More recently MALDI-TOF MS has been adapted to identify cultured
bacteria based on protein fingerprint profiles (Emonet et al., 2010, Sauer and Kliem,
2010, Dubois et al., 2012).
87
A particular problem has been the misidentification of B. pseudomallei as
Burkholderia cepacia by the VITEK 2 automated biochemical system (bioMérieux)
(Lowe et al., 2002, Koh et al., 2003, Lowe et al., 2006, Zong et al., 2012, Rossi et al.,
2013). B. cepacia belongs to a group of 17 phenotypically and genotypically similar
species which form the B. cepacia complex, with B. cepacia specifically noted as an
opportunistic pathogen infecting and causing progressive pulmonary deterioration in
patients with cystic fibrosis (Coenye et al., 2003, Vandamme and Dawyndt, 2011).
Other organisms that have been reportedly misidentified by the VITEK 2 system
include Candida albicans being misidentified as Gram-negative bacilli (Gilad et al.,
2007) and C. parapsilosis being misidentified as C. famata (Burton et al., 2011).
In this study, the VITEK 2 system biochemical profiles of confirmed B. pseudomallei
clinical strains from hospitals in Sabah and Sarawak, Malaysian Borneo were
compared with that of B. pseudomallei and B. cepacia isolates from the Royal Darwin
Hospital (RDH) in the Northern Territory, Australia.
3.2.1 Clinical isolates used for the study
A total of 68 confirmed B. pseudomallei clinical strains from hospitals in Sabah and
Sarawak, Malaysian Borneo were used for this study. In addition, 149 B.
pseudomallei and 18 B. cepacia isolates from the RDH in the Northern Territory,
Australia were included as comparison. One isolate per patient was analyzed. All
isolates were collected between September 2010 and June 2012 except for 17 isolates
collected in 1994 from Sabah.
3.2.2 VITEK 2 system
All isolates were subcultured on horse blood agar (HBA) before testing was
performed on the VITEK 2 according to manufacturer’s instructions (bioMérieux).
The VITEK 2 system utilized a panel of biochemical and enzymatic tests which
resulted in a biochemical profile that was compared against the manufacturer’s
bacterial taxa database (bioMérieux).
88
3.2.3 Confirmation of B. pseudomallei isolates by PCR
All B. pseudomallei isolates were confirmed by real-time PCR targeting B.
pseudomallei TTS1 as described in Chapter 2 (Section 2.2.10).
3.2.4 Confirmation of B. pseudomallei isolates by latex agglutination test
All B. pseudomallei isolates were confirmed by latex agglutination test as described
earlier in Chapter 2 (Section 2.2.6) and in previous studies (Samosornsuk et al.,
1999, Anuntagool et al., 2000).
3.2.5 Statistical analysis
Using Primer Version 6 software (Primer-E Ltd, Plymouth Marine Laboratory, United
Kingdom), a nonmetric multidimensional scaling (nMDS) ordination was performed
on the Euclidean distance resemblance matrix of the VITEK 2 biochemical profiles of
isolates used in this study. The same set of data was also analysed using a permutation
based nonparametric analysis of similarities (ANOSIM) and an analysis of similarity
percentages (SIMPER) by calculating the average contribution of each biochemical
test to the overall observed dissimilarity between clusters.
89
Confirmation of B. pseudomallei isolates
All B. pseudomallei isolates were confirmed by both real-time PCR targeting TTS1
(Novak et al., 2006) and by latex agglutination test (Samosornsuk et al., 1999,
Anuntagool et al., 2000). Of the isolates from Sarawak, 15/43 (35%) had been
initially identified as B. cepacia by the VITEK 2 system but were subsequently
confirmed as B. pseudomallei by both the TTS1 real-time PCR and the latex
agglutination test (Table 3.1). These 15 patients were from hospitals from different
regions in Sarawak where none had cystic fibrosis. Melioidosis was suspected
clinically in these 15 patients, with a diversity of clinical presentations including
subcutaneous infection, community acquired pneumonia and sepsis. Only 2/25 B.
pseudomallei from Sabah and 3/149 B. pseudomallei from Darwin were misidentified
as B. cepacia (Table 3.1).
Statistical analysis
Using Primer v6, a nonmetric multidimensional scaling (nMDS) ordination was
performed on the Euclidean distance resemblance matrix of the VITEK 2 biochemical
profiles of these 235 isolates. The nMDS (stress value 0.19) showed a distinct
clustering of the 15 B. pseudomallei isolates from Sarawak that were misidentified as
B. cepacia (Figure 3.1; Panel A). The nMDS ordination also revealed a tight
clustering of the correctly identified B. pseudomallei isolates regardless of country of
origin, while the B. cepacia isolates were more diverse (Figure 3.1, Panels A and C).
A permutation based, nonparametric analysis of similarities (ANOSIM) confirmed
this finding with strong evidence that the biochemical profiles of the misidentified B.
pseudomallei isolates were distinct from correctly identified B. pseudomallei (R
statistic 0.345, P<0.001).
An analysis of similarity percentages (SIMPER) calculating the average contribution
of each biochemical test to the overall observed dissimilarity between clusters
revealed that in particular two enzymatic tests, the β-N-acetyl-glucosaminidase
(BNAG)
and
β-N-acetyl-galactosaminidase
(NAGA)
which
hydrolyze
polysaccharides, were distinct between correctly and misidentified B. pseudomallei
isolates. Results showed that 88% of correctly identified B. pseudomallei isolates
contained BNAG substrates resulting in a positive test as opposed to 13% of
90
misidentified isolates. This is also evident in Figure 3.1; Panels B and D. The
exopolysaccharide (EPS) poly-β-(1-6)-N-acetyl-glucosamine (PNAG) is a substrate of
the enzyme BNAG and is produced by Burkholderia spp. PNAG has been reported to
be an important component in biofilm formation in Burkholderia species, potentially
contributing
to
multidrug
resistance
(Yakandawala
et
al.,
2011).
N-
acetylgalactosamine, a derivative of NAGA, has also been documented as one of the
basic components for EPS of B. pseudomallei (Masoud et al., 1997). The implications
for virulence and immune response of these different biochemical profiles remains
uncertain, but it has been suggested that the amount of capsular polysaccharide in B.
pseudomallei when compared to other Burkholderia species may well contribute to its
relative virulence (Cuccui et al., 2012).
As an environmental bacterium adapted to a diverse range of tropical and subtropical
habitats globally, B. pseudomallei is known to harbour a vast intra-species genomic
diversity as a result of high recombination frequency (Tuanyok et al., 2008). It is
therefore not surprising that the biochemical database of the VITEK 2 system
performs variably based on geographical location. That there was 98% accuracy for
the recent Australian strains tested in this study shows substantial improvement since
prior studies (Lowe et al., 2002, Lowe et al., 2006). The Sarawak data are supported
by the recent report from China of the same misidentification in a case of melioidosis
imported from Malaysia (Zong et al., 2012).
91
Table 3.1 Number of isolates tested with VITEK 2 system
Sample origin
Total no. B. Total no. B. No. of B. pseudomallei No. of isolates No. of B. pseudomallei No. of B. cepacia
pseudomallei
cepacia isolates isolates
correctly with
low isolates misidentified as isolates
correctly
tested
identified as
discrimination* B. cepacia**
identified
isolates§
B. pseudomalleiŦ
Sabah, Malaysian 25
Borneo
Sarawak,
43
Malaysian
Borneo
Not done
22 (88%)
1 (4%)
2 (8%)
Not done
Not done
23 (53%)
5 (12%)
15 (35%)
Not done
Darwin,
Australia
18
146 (98%)
0
3 (2%)
18 (100%)
149
§
Positive by TTS1 and agglutination tested
Ŧ
90-99% probability B. pseudomallei
* Low discrimination between B. cepacia and B. pseudomallei
* * 90-99% probability B. cepacia
(%) refers to % of total isolates of the same state/country origin
92
Figure 3.1 Nonmetric multidimensional scaling (nMDS) ordination on the Euclidean distance resemblance matrix of the VITEK 2 biochemical profile of 235
B. pseudomallei and B. cepacia isolates from Australia and Malaysian Borneo. Panel A-Samples were identified as either B. pseudomallei, B. cepacia, B.
pseudomallei misidentified as B. cepacia or isolates with low discrimination. Panel B-The bubble size reflects the presence (large) or absence (small) of
BNAG substrate in an isolate. Panel C-Analysis based on isolates from both countries, Australia and Malaysia. Panel D-The bubble size reflects the presence
(large) or absence (small) of NAGA substrate in an isolate.
*Abbreviations: Bps, B. pseudomallei; Bcep, B. cepacia; Bcep misID, B. pseudomallei misidentified as B. cepacia; BNAG, β-N-acetyl-glucosaminidase;
NAGA, N-acetylgalactosamine.
93
3.4 Conclusion
In conclusion clinicians and laboratory scientists need to be aware of continuing
potential misidentification of B. pseudomallei as B. cepacia by the VITEK 2
automated biochemical identification system, especially in patients with suspected
melioidosis acquired in exotic locations such as Malaysian Borneo. Similar
difficulties are likely to be encountered with other automated identification systems
such as MALDI-TOF MS as they are increasingly developed and utilized for patients
infected in diverse geographical locations. PCR using validated targets (Kaestli et al.,
2012a) and ultimately whole genome sequencing can confirm correct speciation.
Alternatively, laboratories with limited resources may rely on a combination of latex
agglutination and AMX susceptibility testing assists in distinguishing B. pseudomallei
from B. cepacia (Hodgson et al., 2009).
94
Chapter 4
Characterization of aminoglycoside and
macrolide susceptible B. pseudomallei from
Malaysian Borneo
Chapter 4: Characterization of aminoglycoside and
macrolide
susceptible
B.
pseudomallei
from
Malaysian Borneo
Study Aim
The aim of this study is to determine the prevalence and mechanism of gentamicin
and macrolide susceptibility in B. pseudomallei isolates from Sarawak
________________________________________________________________________
A version of this chapter has been published as:
Podin Y, Sarovich DS, Price EP, Kaestli M, Mayo M, Hii K, Ngian H, Wong S, Wong I,
Wong J, Mohan A, Ooi M, Fam T, Wong J, Tuanyok A, Keim P, Giffard PM, Currie BJ.
Burkholderia pseudomallei isolates from Sarawak, Malaysian Borneo, are predominantly
susceptible to aminoglycosides and macrolides. Antimicrob Agents Chemother. 2014
Jan;58(1):162-6. doi: 10.1128/AAC.01842-13. Epub 2013 Oct 21.
95
4.1 Introduction
Melioidosis is a potentially fatal disease endemic in Southeast Asia, northern
Australia and other tropical regions. Melioidosis is caused by the saprophytic
bacterium B. pseudomallei, commonly found in the environment of endemic regions
with infection generally occurring from contact with contaminated water or soils
(Wiersinga et al., 2012). Clinical presentations of melioidosis are highly variable and
can manifest as asymptomatic infections, localized skin abscess formation, acute or
chronic pneumonia, genitourinary, bone and joint infections or severe systemic sepsis
with or without foci of multiple abscesses in internal organs, with a mortality of >90%
in septic shock cases (White, 2003, Cheng and Currie, 2005). Due in part to the high
virulence of this organism and increased concerns for transmission by aerosolization,
B. pseudomallei was upgraded to a Tier 1 select agent by the U.S. CDC in 2012
(http://www.selectagents.gov/).
B. pseudomallei is intrinsically resistant to a wide range of antibiotics including many
β-lactams, aminoglycosides and macrolides (Eickhoff et al., 1970, Dance et al.,
1989c, Jenney et al., 2001). This array of drug resistance is conferred through a
variety of mechanisms including inactivating enzymes, cell exclusion and broad-range
efflux pumps. Although almost all B. pseudomallei strains are resistant to the
aforementioned antibiotics, there have been reports of rare (~0.1%) aminoglycoside
and macrolide susceptibility in isolates from Thailand (Simpson et al., 1999, Trunck
et al., 2009) and in a chronic-carriage patient from Australia (Price et al., 2013a).
There have been reports of BpeAB-OprB being responsible for mediating the efflux
of aminoglycosides and macrolides, and that it plays an important role in the virulence
and quorum sensing of B. pseudomallei (Chan et al., 2004, Chan and Chua, 2005,
Chan et al., 2007). However, other studies have shown that the resistance of these
drug groups in B. pseudomallei is solely conferred by the multi-drug efflux pump
AmrAB-OprA (Moore et al., 1999, Mima and Schweizer, 2010). It was also shown
that BpeAB-OprB does not play a role in quorum sensing and virulence factor, but is
in fact a broad-spectrum drug efflux system in B. pseudomallei (Mima and Schweizer,
2010).
In the current study, aminoglycoside and macrolide sensitivity in B. pseudomallei of
clinical origin isolated from a wide geographical region within Sarawak, Malaysian
96
Borneo was identified (Figure 4.1). Antibiotic susceptibility was confined to closely
related isolates based on MLST. Using WGS, a non-synonymous mutation within the
AmrAB-OprA multi-drug efflux pump was identified. Aminoglycoside and macrolide
resistance was restored through reversion of this mutation.
4.2.1 Ethics
Bacterial strain collection and research was approved by the Medical Research Ethics
Committee and registered with the National Medical Research Registrar and the
Clinical Research Centre, Ministry of Health of Malaysia (Appendix A). All clinical
isolates were from routine melioidosis laboratory diagnosis and hence no written
consent was obtained from patients.
4.2.2 Bacterial strains
The B. pseudomallei strains used in this study are listed in Table 4.2. With the
exception of MSHR7596, MSHR7597, MSHR7598 and MSHR7599, which were
derived from MSHR6808, all other strains were collected from melioidosis cases from
four hospitals located in Bintulu, Kapit, Miri and Sibu. Strains were grown at 37°C
for 24 hours on TSA (Oxoid, Australia), regular Ashdown’s agar (Oxoid, Australia)
or Ashdown’s agar without GEN containing an additional 50 μg/ml colistin.
4.2.3 MLST
MLST was performed using previously published methods (Godoy et al., 2003), and
as discussed in Section 2.2.15 of this thesis.
97
Figure 4.1 Map of study sites. Panel A shows the location of Malaysian Borneo in relation to other countries within the South East Asian
region. Panel B shows the location of the four hospitals mentioned in the study denoted by red dots. (Adapted from Google map)
98
4.2.4 Antibiotic susceptibility testing
MICs were determined using E-tests (bioMérieux, France), according to
manufacturer’s instructions and as discussed in Chapter 2 (Section 2.2.7). B.
pseudomallei strains were grown for 24 hours before MIC determination. GEN MIC
values for susceptible, intermediate, and resistant were defined as ≤4 µg/ml, 8 µg/ml
and ≥16 µg/ml respectively (Dance et al., 1989c, Jenney et al., 2001).
4.2.5 WGS and analysis
WGS was performed on six B. pseudomallei isolates (MSHR5087, MSHR5089,
MSHR5102, MSHR5104, MSHR5105, MSHR5107) using methods discussed in
Chapter 2 (Section 2.2.16).
4.2.6 Allele-specific PCR
A novel allele-specific PCR (AS-PCR) assay was designed to differentiate amrB
mutants from wild-type B. pseudomallei isolates. Allele-specific PCR was performed
as previously described (Sarovich et al., 2012b) using one allele-specific forward
primer (Invitrogen, Australia), GmS_F_mut; 5’-TCCGCGCGACGCTGATTCCGtG3’ (for mutant strains) or GmS_F_wt; 5’-TCCGCGCGACGCTGATTCCGtC-3’ (for
wildtype
strains)
and
a
common
reverse
primer
GmS_R_com;
5’-
ACGTGAGCACGACGGTGATCC-3’ in each reaction. Underlined nucleotides
represent the SNP target that matches the wild-type or mutant allele, respectively.
Lower case nucleotides represent a deliberately incorporated antepenultimate
mismatch to enhance allele specificity (Hezard et al., 1997). In a 10 μl reaction, 1 μl
of DNA template was added to 0.3 μM of each primer, 1X SYBR Green PCR Master
Mix (Life Technologies), and double-distilled water. PCR reactions were conducted
on an ABI PRISM 7900HT real-time PCR instrument (Life Technologies) at 50 °C
for 2 minutes, 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds
and 60 °C for 1 minute. A dissociation curve was performed following amplification
to confirm amplicon specificity. Figure 4.2 shows the amplification plot of this assay.
4.2.7 Restoration of aminoglycoside resistance
Aminoglycoside sensitive B. pseudomallei were grown in 50 ml TSB at 37 °C with
shaking at 180 rpm for 18 to 24 hours. The bacterial culture was centrifuged at 4,000
rpm for 10 minutes to pellet cells. The majority of the supernatant was removed and
99
the bacterial cells spread on TSA (Oxoid, Australia) containing 40 μg/ml GEN
(Pfizer, USA) and 30 μg/ml kanamycin (Sigma, Germany). Plates were incubated for
48 hours at 37 °C. Mutant colonies were purified and retained for further analysis.
4.2.8 Di-deoxy sequencing
Di-deoxy sequencing of amrB was performed on isolates by outsourcing to Macrogen
(Seoul, South Korea). The following primers were designed and used for
amplification
and
sequencing
of
this
locus:
and
GCCGGGCGTCAAGTACCAGATT-3’
AmrBseq_F;
AmrBseq_R;
5’5’-
CTGATCTGCTTCATCGCCTTCAC-3’. The nucleotide sequences have been
deposited in the GenBank database (Table 4.1).
Raw DNA sequences received from Macrogen were edited and consensus sequences
produced using ChromasPro Version 1.5 software (Technelysium Pty Ltd, Australia).
Consensus DNA sequences were then matched with DNA sequences available on the
National Centre for Biotechnology Information database using Basic Local Alignment
Search Tool (BLAST) (National Library of Medicine, USA).
4.2.9 Primer Design
All primers were designed using the Primer3 software (Koressaar and Remm, 2007,
Untergasser et al., 2012).
100
Table 4.1 Accession numbers of partial amrB gene sequences deposited in GenBank
Strain
Source
Accession number
MSHR5079 GENr isolate
KF548562
s
MSHR6808 GEN isolate
KF548563
r
MSHR7596 Spontaneous GEN mutant derived from
KF548564
MSHR6808
MSHR7597 Spontaneous GENr mutant derived from
KF548565
MSHR6808
KF548566
MSHR6808
KF548567
MSHR6808
Table 4.2 B. pseudomallei isolates used in this study
Location STa
GENr/s
Kapit
50
GENr
MSHR5084 67 yo female, osteomyelitis, blood culture.
Bintulu
658
GENr
MSHR5087* 3 yo female, abscess on scalp.
Kapit
881
GENs
MSHR5089* 45 yo female.
Kapit
881
GENs
MSHR5091 3 yo female, abscess.
Kapit
881
GENs
MSHR5093 62 yo female, community acquired pneumonia, blood
Kapit
881
GENs
MSHR5095 41 yo male, pneumonia, blood culture.
Bintulu
881
GENs
Bintulu
881
GENs
MSHR5102* 17 yo male, neutropenic sepsis.
Bintulu
658
GENr
MSHR5104* 53 yo female, blood culture.
Bintulu
881
GENs
MSHR5105* 2 yo female, pus from left cervical lymphadenitis.
Bintulu
881
GENs
MSHR5107* 18 yo male, pus from neck abscess.
Kapit
881
GENs
MSHR6381 47 yo male, shoulder abscess.
Kapit
881
GENs
MSHR6385 2 yo female, severe community acquired pneumonia, blood Kapit
881
GENs
MSHR6386 27 yo male, community acquired pneumonia, blood culture. Kapit
881
GENs
MSHR6387 89 yo male, community acquired pneumonia, blood culture. Kapit
881
GENs
MSHR6389 11 yo male, lower right eyelid and right cervical abscess,
Sibu
881
GENs
Sibu
881
GENs
MSHR6392 18 yo male, ankle abscess.
Sibu
881
GENs
Strain
Source
culture.
culture.
pus.
101
MSHR6401 61 yo male, community acquired pneumonia, blood culture. Sibu
881
GENs
MSHR6404 12 yo female, pus from lower eyelid abscess.
Sibu
881
GENs
Sibu
881
GENs
881
GENs
MSHR6410 45 yo male, knee septic abscess, blood culture.
Sibu
881
GENs
881
GENs
Sibu
881
GENs
MSHR6415 54 yo female, sepsis secondary to right leg wound.
Kapit
881
GENs
MSHR6418 15 months female, occipital abscess, culture positive from
Bintulu
881
GENs
Bintulu
50
GENr
MSHR6769 41 yo male, severe pneumonia with sepsis, blood culture.
Bintulu
881
GENs
MSHR6774 14 yo male, pus from cervical abscess.
Kapit
881
GENs
MSHR6778 11 yo female, neck abscess.
Kapit
881
GENs
MSHR6780 31 yo male, pus.
Kapit
881
GENs
MSHR6785 59 yo female, knee abscess.
Kapit
881
GENs
Kapit
881
GENs
Kapit
997
GENs
MSHR6802 3 yo female, septic shock blood culture.
Sibu
881
GENs
MSHR6803 48 yo male.
Sibu
881
GENs
Sibu
881
GENs
Sibu
46
GENr
Sibu
881
GENs
MSHR6824 50 yo female, abscess and sepsis, blood culture.
Miri
271
GENr
MSHR6828 Female, blood culture.
Bintulu
881
GENs
881
GENs
MSHR7596 Spontaneous GENr mutant derived from MSHR6808
This study ND
GENr
This study ND
GENr
This study ND
GENr
This study ND
GENr
abscess.
MSHR6808 61 yo, male, community acquired pneumonia, blood culture Sibu
a
Sequence type as determined by multilocus sequence typing
*
Whole genome sequencing was performed on this isolate
ND, not determined
GENr/s, gentamicin resistant/sensitive
102
MLST of Sarawak isolates indicates restricted geographical distribution
MLST was performed on all B. pseudomallei isolates collected in this study (Table
4.2) and ST profiles were submitted to the B. pseudomallei MLST database
(http://bpseudomallei.mlst.net/). Thirty-seven (84%) isolates were ST881 with this ST
forming the majority of samples from hospitals in the central region of Sarawak (i.e.
Bintulu, Kapit, and Sibu). ST881 has been reported once previously in a Chinese
traveller returning from working in the Malaysian jungle (Zong et al., 2012). The sole
isolate from Miri hospital in northern Sarawak was ST271. The remaining six isolates
belong to ST46, ST50, ST658 and ST997 (Table 4.2). With the exception of ST997,
which is a SLV of ST881, all other STs have been previously identified. ST658 has
been found in Laos and Thailand. ST50 has been found in China and Thailand and is a
SLV of ST46 and a double locus variant of ST271. ST46 appears to be widespread
and has been reported in historical samples collected from Peninsular Malaysia in the
1960s (McCombie et al., 2006), Bangladesh, Cambodia, India, Thailand, Vietnam and
the United States, most probably from a traveller to an endemic region. In contrast,
ST271 appears rarely and to date has only been found in Malaysian Borneo.
Sarawak is located in Malaysian Borneo, with rugged topographies resulting in areas
with limited access and the existence of subsistent indigenous communities. This
ruggedness is especially true in the central region where ST881 and ST997 were
isolated. The apparent geographic restriction associated with limited sequence
diversity of ST881 is similar to previous reports of B. pseudomallei isolates from
Papua New Guinea where low diversity of B. pseudomallei was observed in a
geographically isolated region of Western Province (Warner et al., 2008, Baker et al.,
2011a). The narrow genetic variability within the majority of the Sarawak isolates and
those from Papua New Guinea could potentially reflect recent but independent
introduction events in those two locations.
More likely, however, the observed
overlap of certain STs in Malaysian Borneo with other Asian regions supports the
hypothesis of a more ancient dispersal of B. pseudomallei to Malaysian Borneo, with
dispersal and diversity in Sarawak restricted by its remoteness and potentially also by
limited anthropogenic activities (Baker et al., 2011a).
103
ST881 and ST997 isolates are susceptible to the aminoglycosides and macrolides
All ST881 and ST997 isolates (n=38) were shown to be susceptible to the
aminoglycoside, GEN, with MICs ranging from 0.5 to 1.5 μg/ml (Table 4.3). In
contrast, GEN MICs for wild-type B. pseudomallei were approximately 128 μg/ml
(Simpson et al., 1999). GEN is a selective component of the Ashdown’s agar media
used for B. pseudomallei detection and isolation in endemic regions (Ashdown,
1979a). In addition, the ST881 and ST997 isolates were sensitive to KAN (also an
aminoglycoside) and AZM (a macrolide) with MICs of 1.0 and 2.0 μg/ml,
respectively (Table 4.3). All ST881/997 isolates were susceptible to the antibiotics
used in treating melioidosis, notably CAZ, MEM, DOX, AMX and SXT,
demonstrating MICs similar to other wild-type B. pseudomallei strains. NonST881/997 isolates collected in this study had MIC values similar to other wild-type
B. pseudomallei strains with sensitivity to the clinically relevant antibiotics and
resistance to GEN, kanamycin and azithromycin (Table 4.3).
WGS of four GENs isolates revealed a novel cytosine (C) to guanine (G) transition
located within amrB (amrB-C1102G), which encodes the membrane spanning
component of the resistance-nodulation-division family (RND) efflux pump, AmrABOprA. This non-synonymous single nucleotide polymorphism (SNP) conferred a
threonine to arginine substitution (T367R) in a highly conserved region of the protein.
This substitution was predicted to be deleterious using PROTEAN which is a software
for protein sequence analysis and prediction (Plasterer, 2000)(17). This mutation has
not been previously reported in any other sequenced B. pseudomallei isolate to date
(NCBI database as of 15 May 2013; Figure 4.3). To rapidly interrogate the amrB1102G SNP in the larger collection of isolates obtained in this study, a novel AS-PCR
assay was designed. Using the AS-PCR, the SNP state at amrB-1102G was rapidly
and accurately determined where it was confirmed that this mutation was restricted to
B. pseudomallei isolates with the GENs phenotype. All strains in this study that were
GENr were shown to possess the wild-type nucleotide at this position.
104
Reversion of the C1102G mutation restored aminoglycoside and macrolide resistance
To verify the role of amrB-1102G in aminoglycoside and macrolide sensitivity, GENr
mutant derivatives of a GENs strain (MSHR6808) were selected and the amrB-1102G
SNP in the mutant strains was interrogated. Of 95 GENr mutants, four mutants that
potentially mutated at amrB-1102 were identified using AS-PCR. DNA sequence
analysis confirmed that only MSHR7599 reverted to the wild-type sequence at amrB
(amrB-G1102C) (R367T). Surprisingly, MSHR7596, MSHR7597 and MSHR7598
were shown to possess a novel thymine transversion at this position (amrB-G1102T),
which altered the codon to a third configuration, resulting in a methionine substitution
(R367M). The GEN MICs for all four mutants were ≥256 μg/ml whereas the original
strain (MSHR6808) had an MIC of 0.75 μg/ml (Table 4.3). Similarly, the MIC for
kanamycin increased from 1.0 μg/ml to 128 μg/ml and the MIC of azithromycin
increased from 1.5 μg/ml to between 32 and 128 μg/ml (Table 4.3). These MIC data
indicated that methionine is an effective substitute for threonine at this position, and
demonstrated that B. pseudomallei has at least two different ways to develop GENr at
amrB-1102. Intriguingly, 91 of the 95 mutants were unchanged at amrB-1102; thus,
multiple mechanisms exist for acquiring GENr in B. pseudomallei. The genetic bases
for resistance in these mutants were not characterized in the current study.
Importantly, the restoration of the amrB-1102C allele in one mutant, concomitant with
acquisition of aminoglycoside and macrolide resistance, is evidence that that the
C→G substitution in ST881 and ST997 confers aminoglycoside and macrolide
susceptibility.
Clinical and environmental significance of aminoglycoside and macrolide sensitive B.
pseudomallei in Sarawak
The emergence and persistence of ST881 and ST997 in Sarawak is not fully
understood. This study has demonstrated that ST881 and ST997 can cause diseases in
humans despite being susceptible to aminoglycosides and macrolides, suggesting that
the loss of aminoglycoside and macrolide resistance has little consequence for
virulence of these strains.
However, it cannot be ruled out that this mutation
enhanced environmental survival of ST881 and ST997. Despite the aminoglycoside
and macrolide sensitivity of the B. pseudomallei strains in this study, patients infected
with these strains had similar clinical presentations and outcomes to those infected
with GENr B. pseudomallei strains in this study (Table 4.2) and in other studies
105
(Wiersinga et al., 2012, Hassan et al., 2010b, How et al., 2009, Limmathurotsakul et
al., 2010a). This observation is consistent with Trunck and co-workers, who reported
unaltered virulence in rare GENs isolates from clinical cases (Trunck et al., 2009).
Furthermore, this study has shown that the distribution of the GENs strains was not
confined to a small geographic region; presumptive locations of B. pseudomallei
acquisition for the patients infected with ST881/997 strains span a large region within
Malaysian Borneo, with an area of approximately 60,000 km2 encompassing the
Bintulu, Kapit and Sibu regions (Figure 4.1). Although the GENs phenotype has been
previously observed in B. pseudomallei, it has been putatively a result of niche
adaptation and genome decay and is very rare (Trunck et al., 2009, Price et al.,
2013a). Currently, no other instances of clear phenotypic change have been associated
with a specific B. pseudomallei lineage, possibly with the exception of B. mallei, the
causative agent of glanders. B. mallei is a clone of B. pseudomallei, which has
undergone genome reduction and has become aminoglycoside sensitive (Moore et al.,
2004, Nierman et al., 2004). Unlike B. mallei, which has lost the entire AmrAB-OprA
efflux pump operon, a unique mechanism leading to aminoglycoside and macrolide
susceptibility in ST881 and ST997 was identified in this study. Further genomic
analysis of these strains would be useful to determine whether they have undergone
early stages of genome decay similar to that seen in isolates from chronic-carriage
patients (Price et al., 2013a). In addition, the existence or persistence of these strains
in the environment, and whether susceptibility to aminoglycosides and macrolides
affect their survival in the environment, warrant further investigation.
4.4 Conclusions
In this study, I analysed B. pseudomallei clinical isolates from Sarawak, Malaysian
Borneo, to determine both the prevalence and the molecular basis of GENs. The
majority of these isolates were a single ST and were sensitive to aminoglycosides and
macrolides, a phenotype that is very rare in B. pseudomallei from other endemic
regions. Aminoglycoside sensitivity was the result of a non-synonymous SNP within
amrB. Most selective media for B. pseudomallei growth contains gentamicin as a
selective agent (Ashdown, 1979a, Howard and Inglis, 2003, Francis et al., 2006). Use
of these media in Sarawak as a sole diagnostic tool for melioidosis is problematic as,
based on findings from this study, approximately 86% of cases would not be
106
diagnosed. Alternative selective media without gentamicin (for example Ashdown’s
media supplemented with colistin instead of gentamicin) may be a more appropriate
option for laboratory diagnosis of suspected melioidosis cases in Sarawak. Laboratory
scientists and clinicians in central Sarawak and surrounding regions should exercise
caution when confirming B. pseudomallei infection using selective media containing
gentamicin. Findings from this study have significant implications for laboratory
diagnosis and environmental sampling of B. pseudomallei, particularly in Malaysian
Borneo and other potentially endemic regions where B. pseudomallei has yet to be
uncovered.
107
GENS B. pseudomallei is amplified
Figure 4.2 Amplification plot of GENs allelic-specific real-time PCR. The pink line
as indicated by the arrow shows amplification of an isolate possessing the GENs allele
whereas the blue line shows no amplification.
Figure 4.3 Alignment of the amrB hyperconserved membrane spanning domain in
selected B. pseudomallei isolates. An amrB-C1102G single nucleotide polymorphism
was observed within the Malaysian clinical strains MSHR5087 and MSHR6808,
conferring gentamicin sensitivity. In contrast, a novel amrB-C1102T single nucleotide
polymorphism in the laboratory-generated mutants MSHR7596, 7597 and 7598
resulted in aminoglycoside resistance. The Malaysian clinical strain MSHR5079
shares the wild-type amrB sequence (amrB-1102C) with other gentamicin-resistant B.
pseudomallei (K96243 and MSHR0668), and was generated in vitro in the
MSHR7599 mutant, confirming the role of amrB-1102G in gentamicin sensitivity.
108
Table 4.3 Minimum inhibitory concentrations for B. pseudomallei isolates obtained in
this study
Minimum inhibitory concentration (μg/ml)*
Strain
AMX
AZM
CAZ
DOX
GEN
KAN
MEM
SXT
MSHR5079
3
16
1.5
1.5
96
24
0.75
0.75
MSHR5084
3
ND
1.5
3
32
16
0.75
0.5
MSHR5087
3
ND
1.5
1.0
1.0
2
0.5
3
MSHR5089
3
ND
1.5
1.5
1.0
2
0.5
4
MSHR5091
6
ND
3
1.5
1.0
1.5
0.75
4
MSHR5093
6
ND
4
1.5
1.0
1.5
1.0
3
MSHR5095
3
ND
1.0
1.5
1.5
2
0.5
0.75
MSHR5097
3
ND
1.0
1.5
1.0
2
0.5
0.5
MSHR5102
3
24
2
3
≥256
128
2
0.125
MSHR5104
2
ND
0.75
1.0
1.5
2
0.19
0.75
MSHR5105
3
ND
1.5
1.5
1.5
1.5
0.5
4
MSHR5107
3
ND
1.0
1.5
1.0
1.5
0.5
1.0
MSHR6381
3
ND
2
0.5
1.0
2
1.0
3
MSHR6385
3
ND
2
0.75
1.0
1.5
0.75
2
MSHR6386
3
ND
2
0.38
1.0
1.5
0.75
2
MSHR6387
3
ND
3
0.5
1.0
2
0.75
2
MSHR6389
3
ND
2
0.38
1.5
2
0.75
1.0
MSHR6390
3
ND
1.5
0.5
1.0
2
0.75
2
MSHR6392
2
ND
1.5
0.25
0.75
2
0.75
1.5
MSHR6401
3
ND
4
0.75
1.5
2
1.0
1.5
MSHR6404
3
ND
2
0.5
1.5
2
1.0
2
MSHR6408
3
ND
2
0.5
1.0
1.5
0.75
2
MSHR6409
2
ND
1.5
0.5
0.5
2
0.5
2
MSHR6410
3
1.0
2
0.5
0.75
2
0.75
2
MSHR6411
3
ND
2
0.5
1.0
1.5
0.75
2
MSHR6414
1.5
ND
0.75
0.5
0.75
ND
0.5
0.5
MSHR6415
3
ND
1.5
0.75
0.5
2
0.75
3
MSHR6418
3
ND
2
0.75
1.0
1.5
0.75
2
MSHR6419
2
24
2
2
48
24
1.0
2
MSHR6769
2
0.75
1.5
0.75
1.0
2
0.5
0.5
MSHR6774
8
ND
4
0.75
0.5
1.5
1.0
2
MSHR6778
1.5
ND
1.0
0.5
0.5
ND
0.5
2
109
MSHR6780
3
ND
2
1.5
0.75
2
0.75
1.5
MSHR6785
3
ND
1.5
0.5
0.5
1.5
0.75
3
MSHR6789
3
ND
1.5
0.5
0.5
1.5
0.75
1.5
MSHR6793
3
1.0
1.5
0.75
0.75
2
0.75
2
MSHR6802
3
ND
2
0.5
1.5
2
0.5
1.5
MSHR6803
3
ND
1.5
0.5
0.75
2
0.75
4
MSHR6806
2
ND
3
0.75
1.0
2
1.0
3
MSHR6816
3
24
3
1.0
96
32
0.75
1.5
MSHR6822
3
1.0
1.5
0.5
0.75
2
0.75
1.5
MSHR6824
2
ND
1.0
1.0
16
12
0.75
3
MSHR6828
3
ND
1.0
1.0
0.75
2
1.0
2
MSHR6808
3
1.0
3
0.5
0.75
1.5
1
1-2
MSHR7596
ND
48
ND
ND
≥256
≥256
ND
ND
MSHR7597
ND
48
ND
ND
≥256
≥256
ND
ND
MSHR7598
ND
32
ND
ND
≥256
96
ND
ND
MSHR7599
ND
128
3
0.5
≥256
≥256
1
1-2
Abbreviations: AMX, amoxicillin-clavulanic acid; AZM, azithromycin; CAZ, ceftazidime;
DOX, doxycycline; GEN, gentamicin; KAN, kanamycin; MEM, meropenem; SXT,
trimethoprim-sulfamethoxazole; ND, Not determined.
* The definition of MIC breakpoint values for susceptible (s), intermediate (i), and
resistant (r) for AMX (s ≤ 8/4, i 16/8, r ≥ 32/16), CAZ (s ≤ 8, i 16, r ≥ 32), DOX (s ≤ 4,
i 8, r ≥ 16), MEM (s ≤ 4, i 8, r ≥ 16) and SXT (s ≤ 2/38, r ≥ 4/76) were based on
recommendations by CLSI and the pathology laboratory of the Royal Darwin Hospital
approved guidelines (CLSI, USA). The MIC breakpoint values of AZM (s ≤ 2, i 4, r ≥
8), GEN (s ≤ 4, i 8, r ≥ 16) and KAN (s ≤ 16, i 32, r ≥64) were based on
manufacturer’s
interpretive
guidelines
110
for
aerobes
(bioMérieux,
France).
Chapter 5
Environmental investigation of
B. pseudomallei and other Burkholderia spp.
in Sarawak
Chapter
5:
B. pseudomallei
Environmental
and
other
investigation
Burkholderia
spp.
of
in
Sarawak
Study aims
The major aims of this study were to determine if B. pseudomallei could be isolated
from central Sarawak soil samples, and to assess the relatedness between any such
isolates and clinical isolates from the same region. This will provide information that
will contribute to the understanding of B. pseudomallei environmental hazards in
central Sarawak.
5.1 Introduction
5.1.1 Burkholderia cepacia complex
To date, the Burkholderia genus has over 60 species, which reside in diverse
ecological niches including free living in soil or in association with a host, be it plants,
amoeba, fungi or animals (Coenye and Vandamme, 2003, Vandamme and Dawyndt,
2011)(http://www.bacterio.net/burkholderia.html). Burkholderia species (spp.) are
traditionally known to be plant pathogens or endosymbionts and soil bacteria
(Coenye and LiPuma, 2003, Coenye and Vandamme, 2003, Mahenthiralingam et al.,
2005, Coenye and Vandamme, 2007), with the exception of B. pseudomallei and B.
mallei which are also mammalian pathogens (Coenye and Vandamme, 2003, Coenye
and Vandamme, 2007). B. pseudomallei causes disease in humans and animals,
(Dance, 1991, Wiersinga et al., 2012), whereas B. mallei is a host-adapted pathogen
which causes glanders mainly in horses, mules and donkeys (Coenye and Vandamme,
2003, Coenye and Vandamme, 2007). There are 17 closely related Burkholderia spp.
that are categorised as Burkholderia cepacia complex (Bcc). Studies have shown that
some members of the Bcc; namely Burkholderia ambifaria, Burkholderia anthina,
111
Burkholderia arboris, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia
contaminans, Burkholderia diffusa, Burkholderia dolosa, Burkholderia lata,
Burkholderia
multivorans,
Burkholderia
pyrrocinia,
Burkholderia
seminalis,
Burkholderia stabilis, Burkholderia ubonensis and Burkholderia vietnamiensis, were
isolated from both humans and the environment, whereas Burkholderia latens and
Burkholderia metallica were only to date isolated from humans (Vandamme and
Dawyndt, 2011). While some Bcc members are considered plant pathogens, others are
plant commensals by producing protective antimicrobial compounds and perform
nitrogen fixation while colonizing roots. In addition, Bcc bacteria have also been
shown to be potential bioremediation agents by degrading environmental pollutants
(Mahenthiralingam et al., 2005). In addition to their roles in plants and soil, some Bcc
have been reported to cause opportunistic infection in immunocompromised patients,
cystic fibrosis (CF) patients and others suffering from chronic lung disease (Coenye
and Vandamme, 2003, Mahenthiralingam et al., 2005, Coenye and Vandamme, 2007,
Vandamme and Dawyndt, 2011).
5.1.2 Ecology of B. pseudomallei
B. pseudomallei is primarily found in the soil and freshwater sources of endemic areas
(Chambon, 1955). Due to its presence in the environment and its association with
melioidosis cases in humans and animals (Currie, 2000, Cheng and Currie, 2005),
knowledge of the ecology and epidemiology of B. pseudomallei in the environment is
important to inform public health.
The prevalence and distribution of B. pseudomallei in the environment are dynamic,
influenced by a wide range of factors. Soil water content has been shown to influence
the isolation rate of B. pseudomallei with the recovery rate being higher in moist as
compared to dry soil (Tong et al., 1996). While rainfall has been shown to have a
positive impact on near surface cell numbers of B. pseudomallei in soil by increasing
the moisture content (Wuthiekanun et al., 1995, Brook et al., 1997, Currie and Jacups,
2003, Kaestli et al., 2009), higher isolation of B. pseudomallei from soil is also
112
dependent on other factors such as land use (Kaestli et al., 2009). In the Northern
Territory of Australia for instance, soil collected from residential areas in the dry
season was shown to have higher percentage of B. pseudomallei positive samples as
compared to other areas which was reversed in the wet season. It was hypothesized
that this is due to the use of water reticulation and irrigation systems in gardens which
were only operational in the dry season and which might contribute to improved
survival conditions and the spread of B. pseudomallei (Kaestli et al., 2009).
Earlier works showed that soil texture also influences the prevalence of the organism
(Strauss et al., 1969c, Thomas et al., 1979). For instance, studies have shown that clay
loam which is often nutrients-rich, harboured more B. pseudomallei as compared to
heavy clay. It was hypothesized that this was due to clay loam being less dense,
creating a habitat for B. pseudomallei which is more aerated and contains more
nutrients and also allows for more root penetration (Chen et al., 2003, Kaestli et al.,
2009).
Other parameters correlated with B. pseudomallei numbers in the soil include higher
acidity (Tong et al., 1996, Chen et al., 2003, Draper et al., 2010), less salinity (Chen et
al., 2003), and higher iron content (Kaestli et al., 2009, Draper et al., 2010, Baker et
al., 2011b). Also, higher nitrate soil content has been shown to enhance soil
acidification (Haynes and Williams, 1992, Clegg, 2006, Rooney et al., 2006, Kaestli
et al., 2009) and to allow the bacteria to survive anaerobically by using nitrate as
electron terminal acceptor (Hamad et al., 2011).
Vegetation has also been reported to impact on the distribution of B. pseudomallei in
the environment (Inglis et al., 2001, Levy et al., 2003, Kaestli et al., 2012b), where B.
pseudomallei has been shown to colonize roots and aerial parts of specific grasses
(Kaestli et al., 2012b). As well, human activities such as construction or excavation
(Inglis et al., 2001, Cheng and Currie, 2005) and agricultural activities, use of
fertilizers (M. Kaestli, manuscript in preparation), and animal rearing also had direct
113
and indirect impacts on the ecology and distribution of the genus Burkholderia
particularly B. pseudomallei (Haynes and Williams, 1992, Salles et al., 2006, Clegg,
2006, Rooney et al., 2006, Kaestli et al., 2009). Although it has been hypothesized
that transferring top soil from one area to another may have contributed to the
distribution of B. pseudomallei, there is currently no published evidence supporting
this hypothesis.
Biological antagonists have also been reported to influence the distribution of
B. pseudomallei, particularly certain Burkholderia spp. such as Burkholderia
thailandensis, Burkholderia ubonensis and other members of Bcc have been
demonstrated
to
possess
antagonistic
activities
towards
B. pseudomallei
(Trakulsomboon et al., 1999, Salles et al., 2004, Warner et al., 2008, Kaestli et al.,
2009, Marshall et al., 2010, Lin et al., 2011).
5.1.3 Environmental surveys of B. pseudomallei
Environmental surveys of B. pseudomallei have been conducted in various countries
including Singapore (Thin et al., 1971, Ong et al., 2013), Taiwan (Chen et al., 2010),
Malaysia (Strauss et al., 1967, Ellison et al., 1969, Strauss et al., 1969b, Strauss et al.,
1969c, Inglis, 2002, Hassan et al., 2004), Lao PDR (Wuthiekanun et al., 2005,
Vongphayloth et al., 2012), Papua New Guinea (Warner et al., 2008, Baker et al.,
2011a), Brazil (Rolim et al., 2009), Madagascar
(B. de Smet, personal
communication), with most results thus far from Thailand (Wuthiekanun et al., 1995,
Trakulsomboon et al., 1999, Vuddhakul et al., 1999, Limmathurotsakul et al., 2010b,
Limmathurotsakul et al., 2013a) and Australia (Thomas et al., 1979, Inglis et al.,
2004, Kaestli et al., 2009, Corkeron et al., 2010, Baker et al., 2011b, Hill et al., 2013).
Most of these studies showed that B. pseudomallei was present in the environment
and that the distribution varied between wet and dry seasons.
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5.1.4 Unchlorinated water supply harbouring B. pseudomallei
Previous studies have also shown that unchlorinated water supplies contained B.
pseudomallei (Inglis et al., 1999, Currie et al., 2001, Draper et al., 2010, Hampton et
al., 2011, Baker et al., 2011b, Mayo et al., 2011, McRobb et al., 2013). In the
Northern Territory, Currie et al., Mayo et al. and McRobb et al. reported clusters of
melioidosis cases in several remote communities caused by a common water supply
containing B. pseudomallei where genotyping methods revealed that the clinical
isolates and the isolates from the water source were clonal (Currie et al., 2001, Mayo
et al., 2011, Hampton et al., 2011, McRobb et al., 2013). Similarly in Townsville, B.
pseudomallei was shown to be abundant in groundwater seeps in Castle Hill where
MLST analysis revealed that exposure to the groundwater may have contributed to the
clusters of clinical cases (Baker et al., 2011b). Consistent with favourable parameters
of B. pseudomallei in soil discussed previously, the presence of B. pseudomallei in
water was significantly associated with soft water (water with low content of calcium,
magnesium and other metal cations), low salinity, higher iron content and acidity
(Draper et al., 2010). In light of public health risks of presence of B. pseudomallei in
water supplies, preventive measures such as proper maintenance of chlorination
systems and more recently installation of UV filters have been proposed to reduce
such risks (Inglis et al., 2000, McRobb et al., 2013).
In Malaysia, apart from earlier environmental investigations in the 1960s (Strauss et
al., 1967, Ellison et al., 1969, Strauss et al., 1969b, Strauss et al., 1969c), currently,
there is no other published systematic environmental study on B. pseudomallei
conducted in Malaysia including Malaysian Borneo. The data available are sporadic,
with B. pseudomallei reportedly isolated from Johor, Kedah, Perak and Perlis in
Peninsular Malaysia (Hassan et al., 2013). In their study, Hassan et al. inoculated soil
samples into tryptic soy broth (TSB) containing 10 mg/L gentamicin for 48 hours at
37 °C. The broth was then subcultured onto B. pseudomallei selective agar (BPSA)
(Howard and Inglis, 2003), and incubated at 37 °C for 48 hours (Hassan et al., 2013).
Other environmental investigations of B. pseudomallei were conducted in response to
115
outbreaks in Sarawak in 2005 (Sarawak Health Department, unpublished data) and
Pahang in 2010 (Hin et al., 2012, Sapian et al., 2012).
5.1.5 Landscape use in central Sarawak and statement of the working hypothesis
Central Sarawak, which includes Kapit and Belaga where most melioidosis cases
were reported, has an approximate size of 45,000 km2. The climate in the region is
equatorial with an average temperature of 27 °C. Although the rainy season is
typically between November and February, it rains all year round in the region
(Figure 5.1) (Malaysian Meteorological Department). The harsh topography of the
region consisting of mountain ranges intertwined with rivers causes it to be almost
inaccessible by land travel. Hence, many communities are isolated, with travelling on
the river and the occasional helicopter flights for emergency medical situations being
the two most reliable travelling options. Due to its relatively large area and remote
location, many communities live a subsistent lifestyle, relying on small-scale farming
of rice and other edible crops, rearing of livestock, and hunting-gathering of jungle
produce. As for water supplies, households in this region rely on gravity-fed springs
which are often unchlorinated and the river water for consumption as well as bathing.
In this region, the last 20 years have seen extensive logging, dam constructions and
proliferation of oil palm plantations. It is common for rural communities to be living
in close proximity to these activities. As shown in Figure 5.6, a high percentage of
melioidosis cases are from areas where these land use activities are widespread.
5.1.6 Statement of working hypothesis
Primary hypothesis

There is more diversity in environmental B. pseudomallei as compared to
clinical B. pseudomallei in Sarawak.
Secondary hypotheses
Hypothesis 1 and the initial objectives (Objectives 1, 2 – see below) of the study
were not achieved due to no environmental B. pseudomallei being isolated from
116
Sarawak despite several field trips for soil and water collection. Hence, the following
hypotheses were postulated to explain why no B. pseudomallei were detected in the
soil in Sarawak:

Antagonistic activities by other Burkholderia spp. towards B. pseudomallei

GENs B. pseudomallei isolates from Sarawak are less robust than GENr B.
pseudomallei in competing with the organisms present in the environment

Localized or point source dissemination of B. pseudomallei in the environment
of central Sarawak such as water and animals, rather than widespread presence
of B. pseudomallei throughout the environment of the region.
Primary objectives:
1. To isolate environmental B. pseudomallei isolates from sites in Sarawak with high
prevalence of melioidosis reported.
2. To conduct molecular comparison of environmental and clinical B. pseudomallei
isolates found in Sarawak.
* These two objectives were not achieved because no B. pseudomallei were isolated
from the environmental samples collected.
Secondary objectives
3. To characterize other non-B. pseudomallei Burkholderia spp. isolated from the
environmental samples.
4. To investigate potential causes for the lack of environmental B. pseudomallei
isolated from Sarawak:

Reduced fitness of GENs B. pseudomallei in the environment

Antagonistic activities among Burkholderia spp.

Point source dissemination of B. pseudomallei from a non-soil habitat or
mammalian reservoir
The major aims of this study were to detect environmental B. pseudomallei in central
Sarawak soil samples, and to assess the relatedness between any such isolates and
117
clinical isolates from the same region. This would contribute to the understanding of
the risk of exposure to B. pseudomallei in the environment of central Sarawak. Since
the primary objectives were not achieved as no B. pseudomallei was isolated from the
environmental samples collected, the study proceeded with descriptions of other
Burkholderia spp. isolated from the environmental samples collected. The remainder
of the study encompassed the investigations of the secondary hypotheses and
objectives outlined above.
118
5.2 Isolation and epidemiology of environmental B. pseudomallei and
Burkholderia spp. in Sarawak
Study objectives
The first two objectives of the study discussed in this chapter are to isolate
environmental B. pseudomallei from Sarawak and to compare the molecular
characteristics of environmental and clinical B. pseudomallei from the state. In order
to maximise the clinical relevance, environmental samples were collected from sites
close to where melioidosis cases had emerged.
5.2.1 Materials and methods
5.2.1.1 Environmental sampling strategies
The procedure for environmental sampling site selection was a modification of the
methods described by Kaestli et al. (Kaestli et al., 2009). The sampling sites were:
1. Case associated sites – Sites where reported melioidosis patients have been in
contact with the environment including rice farms, yards around the house,
school fields.
2. Targeted sites – Disturbed sites with higher probability of isolating B.
pseudomallei based on previously published studies such as rice farms (not
necessarily associated with melioidosis cases), construction sites, plantations,
freshly dug area.
3. Opportunistic sites – Randomly selected sites whenever opportunities to
collect samples occurred.
5.2.1.2 Sample size power calculation
Based on feasibility (manpower, budget, shipment, laboratory space), the aim was to
collect a maximum of 200 soil samples in the first round of sampling. The sampling
strategy was to first cover a larger area aiming to sample more sites with less number
of samples per site followed by a more concentrated sampling in an area once a
positive area was identified. As long as the environmental distribution and habitat of
119
B. pseudomallei in Sarawak was still unknown, covering a larger area and different
habitats increased the probability to detect the bacteria. With the assumption of a 10%
detection rate of environmental B. pseudomallei in melioidosis hotspot areas
considering the observed local melioidosis case rate and with a collection of 10
samples per site, the probability to have at least one sample positive for B.
pseudomallei (if it was present) was 65% with a 95% CI not crossing zero with 0.2 to
44%. Based on this calculation, the aim was to collect 10 soil samples per site and to
cover at least 20 sites across different areas and habitats in the first sampling round.
However, for some sites, only five samples were collected due to feasibility
limitations.
5.2.1.3 First environmental sampling
Although it rains all-year round in Sarawak (Figure 5.1), the first environmental
sampling trip was conducted between April and June 2011, which typically has lower
rainfall than other times of the year. Districts that were selected for the first sampling
were Kapit, Bintulu, Belaga, Serian and Kuching as shown in Figure 5.2. Table 5.1
(page 130) shows the sampling sites and the categories of the different sampling sites
with a total of 141 soil samples collected.
5.2.1.4 Second environmental sampling
Due to the negative results of the first environmental sampling round, a second
environmental sampling trip was conducted in December 2011, which is a high
rainfall time of year. The second round was based on intense sampling conducted at
the farm of a deceased melioidosis patient in Kapit where five samples were initially
collected in the first sampling trip. In order to increase the probability to detect B.
pseudomallei, a total of 100 soil samples were collected from the site within an area
of 85 X 60 m2 where each sample was 3 m apart in a grid-like manner as previously
described (Limmathurotsakul et al., 2013a). During this sampling trip, two
melioidosis cases were reported in Kuching. Hence, 16 additional soil samples from
two case associated sites were collected in Kuching.
120
Mean rainfall for selected districts in Sarawak
800
700
Rainfall in mm
600
500
400
300
200
100
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Month
Kapit
Bintulu
Sibu
Kuching
Figure 5.1 Mean annual rainfall for the years 1995-2009 in Sarawak districts selected
for the environmental survey for B. pseudomallei. (Source: Malaysian Meteorological
Department)
Figure 5.2 Map showing the environmental sampling sites denoted by red dots.
(Adapted from Google Earth, Google Inc.)
121
5.2.1.5 Permission and consent
Prior to sampling, permission and consent were obtained to collect soil samples from
either private owners or the relevant authorities responsible for the sampling sites.
5.2.1.6 Soil sample collection
Soil samples were collected from sites described in the preceding section using
previously described methods (Kaestli et al., 2007). Upon arrival, sampling sites were
surveyed to identify locations that were reported to be associated with potentially
higher presence of B. pseudomallei such as animal pens, water irrigation, closer
vicinity to creek and agricultural activities (Kaestli et al., 2009). Daily human
activities carried out at the sites such as farming, bathing and recreational sports were
also taken into account during the survey. Holes were dug within the vicinity of such
locations to increase chances of B. pseudomallei recovery. Depending on the size and
the topography of the sampling site, five to six holes were selected per site. Holes
were dug using a local equivalent of auger called ‘sandak’ (Figure 5.3) to 30 cm
depth. A hand trowel was then used to transfer soil into labelled zip-lock bags. The
bags of soil were protected from direct sunlight as UV light had been shown to kill B.
pseudomallei (Dance, 2000a). The landscape (eg. water run-off, water-logging,
distance to stream, elevation) and vegetation descriptions were recorded and the point
location of each hole was recorded using a handheld global positioning system (GPS)
unit (GPS Extrex, Garmin, USA). In between holes, the ‘sandak’ and the hand trowel
were cleaned with water and sterilized with 70% ethanol to prevent crosscontamination of B. pseudomallei between holes. All sealed bags of soil samples were
transported back to the laboratory analysis.
5.2.1.7 Water sample collection
Whenever the opportunity arose, water samples were collected in clean 600 ml water
bottles and kept away from exposure to sunlight. Water bottles were sealed and
packed into padded bags before being transported back to the laboratory analysis.
122
5.2.1.8 Recovery of B. pseudomallei from soil samples
The soil samples were tested for viable B. pseudomallei as described previously
(Kaestli et al., 2007). Soil of 20 g was added to 20 ml of deionized water in a 50 ml
tube (Falcon, BD Biosciences, USA) and incubated at 37 °C shaking at 240 rpm for
up to 39 hours. After incubating at room temperature without shaking for at least 30
minutes, 100 μl of the supernatant of the mixture were spread onto modified
Ashdown’s agar plate (denoted by Plate A) containing tryptone, glycerol, 0.1% crystal
violet, 1% neutral red, 40 μg/ml gentamicin and incubated at 37 °C.
Another 10 ml of the supernatant was inoculated into 30 ml of Ashdown’s broth
containing 15 g/L tryptone, 5 ml/L 0.1% crystal violet, 50 μg/ml colistin (Ashdown,
1979b) and incubated at 37 °C in air. On day 2 post-inoculation, one 10 μl loopful of
surface liquid culture was further streaked onto Ashdown’s agar, referred as Plate B.
On day 7 post-inoculation, another 10 μl loopful of the surface liquid culture was
streaked onto Ashdown’s agar, referred as Plate C. Figure 5.4 provides an overview
of the procedures involved in culturing B. pseudomallei from soil.
5.2.1.9 Recovery of B. pseudomallei from water samples
Similar to B. pseudomallei recovery from soil, 100 μl of water sample was inoculated
directly onto Ashdown’s agar (Plate A). Another 10 ml of the water sample was added
into 30 ml of Ashdown’s broth and incubated at 37 °C without shaking. On day 2
post-inoculation, 10 μl loopful of the surface liquid culture was further streaked onto
Ashdown’s agar, referred as Plate B. On day 7 post-inoculation, another 10 μl loopful
of the surface liquid culture was streaked onto Ashdown’s agar, referred as Plate C. In
addition, 500 ml of the water sample was filtered through a 0.2 μm nitrocellulose
membrane and the membrane submerged in 30 ml Ashdown’s broth and incubated at
37 °C. On day 2 post-inoculation, 10 μl loopful of the surface liquid culture was
further streaked onto Ashdown’s agar, referred as Plate D. On day 7 post-inoculation,
another 10 μl loopful of the surface liquid culture was streaked onto Ashdown’s agar,
referred as Plate E (Figure 5.5). Plates were observed for up to 14 days for growth.
123
5.2.1.10 Use of Ashdown’s agar with gentamicin and with colistin
Upon confirming that all GENs B. pseudomallei were not susceptible to colistin (as
discussed in Section 2.2.8), 40 μg/ml of gentamicin in the Ashdown’s agar recipe was
substituted with 50 μg/ml colistin for the second round of sampling. Therefore, the
bacterial cultures of the environmental samples from the first field trip were based on
Ashdown’s agar with gentamicin while the samples of the second round were cultured
using media without gentamicin but with colistin.
Figure 5.3 ‘Sandak’, a locally available tool that was used to dig holes
124
+ dH2O(20 ml)
100µl
37°C/240 rpm/39hrs
10ml
37°C
Plate A
30ml Ashdown’s broth
Day 7
Day 2
37°C
Subculturing to get
pure culture
Plate B
Plate C
Figure 5.4 Procedures for culturing B. pseudomallei from soil samples
Water sample
37°C
100µl
10ml
0.2 μm filter
Plate A
Day 2
Plate B
Day 7
Plate C
Day 2
Plate D
Subculturing to get pure culture
Figure 5.5 Procedures for culturing B. pseudomallei from water samples
125
Day 7
Plate E
5.2.1.11 Morphological identification of B. pseudomallei from culture
Ashdown’s agar plates (plates A, B and C) that were inoculated were observed every
day for up to 14 days for growth of the classical purple dry, wrinkled colonies of B.
pseudomallei (Ashdown, 1979a). Colonies with morphologies resembling B.
pseudomallei were subcultured onto fresh Ashdown agar plates. Upon sufficient
growth, the pure culture was marked for storage and for transportation to Menzies
School of Health Research for final identification.
All the work described in Sections 5.2.1.1 to 5.2.1.11 was done in a PC2 laboratory at
the Institute of Health and Community Medicine, UNIMAS before a loopful of the
plates A, B, C, and pure cultures of suspected B. pseudomallei were transported to the
laboratory at Menzies School of Health Research.
5.2.1.12 Transport of isolates from UNIMAS laboratory to Menzies School of
Health Research
Sweeps of plates (Plate A, B and C) and pure culture of suspected B. pseudomallei by
morphological identification were inoculated into agar slants containing TSB and agar
(Oxoid) in screw cap tubes. Tubes containing inoculated agar slants were sealed
tightly with parafilm (Parafilm, Sigma-Aldrich, USA) and following standard
operation procedures transported from UNIMAS to Menzies School of Health
Research using authorized courier service at room temperature. The total
transportation time was up to 10 days. Export permits were obtained from the
Ministry of Health of Malaysia and the import permit from Australian Quarantine and
Inspection Service (AQIS) as shown in Appendix A.
5.2.1.13 Revival of sweeps and isolates transported from UNIMAS to Menzies
School of Health Research
Upon arrival at Menzies School of Health Research laboratory, bacterial culture in
agar slants were revived by scraping the agar slant with a 10 μl loop and streaking
onto Chocolate agar and Ashdown’s agar before incubating the plates at 37 °C. Plates
126
were observed for growth and morphological identification of culture was done
similar to methods previously discussed in Section 5.2.1.11. All cultures were
subjected to DNA extraction using 10% w/v Chelex® 100 resin (Bio-Rad
Laboratories Inc., USA) as described in Section 5.2.1.15 and the DNA extract was
subjected to real-time polymerase chain reaction (PCR) discussed in Section 5.2.1.16.
5.2.1.14 Storage of bacterial isolates
Pure cultures were streaked onto Chocolate agar (Oxoid, Australia) and incubated at
37 °C for up to two days before stored in trypticase soy broth with 10% glycerol stock
at -80 °C. Plates were kept for up to two weeks before being discarded.
5.2.1.15 DNA extraction of sweeps of bacterial culture using Chelex ® 100
In order to minimize the labour and waiting time for positive culture by focusing
resources on PCR positive samples, a sweep of plate A was collected on day 2 postinoculation and subjected to DNA extraction using 10% w/v Chelex® 100 resin in
sterile deionized water (Bio-Rad Laboratories Inc., USA). Briefly, a loopful of culture
was collected evenly across the plate using a disposable 10 μl loop and resuspended
into 500 μl of 10% w/v Chelex® 100 resin (Bio-Rad Laboratories Inc., USA). Upon
vortexing for up to 15 seconds, the cell suspension was incubated in a heat block
(Ratek Strument Pty Ltd, Australia) set at 95 °C for 20 minutes. The suspension was
then centrifuged at 8,000 rpm for 10 minutes (Eppendorf, Germany), after which, 60
μl DNA extract was transferred into fresh sterile tubes and stored at 4 °C until further
use.
5.2.1.16 Type III secretion system real-time PCR
The DNA extracts of B. pseudomallei-suspected cultures were subjected to a PCR
targeting 115-base pair (bp) in the open reading frame 2 (orf2) of the TTS1 of B.
pseudomallei (Novak et al., 2006, Meumann et al., 2006, Kaestli et al., 2007). As
previously described, in a 10 μl PCR reaction, 0.5 ng template DNA was added to
900nM primers, 200nM probe and 1X Applied Biosystems genotyping master mix
127
(Life Technologies, USA) with thermal cycling condition at 50 °C for 2 minutes, 95
°C for 10 minutes, and 30 cycles of 95 °C for 15 seconds and 58 °C for 1 minute on
an ABI PRISM 7900HT real-time PCR instrument (Life Technologies, USA) (Kaestli
et al., 2012a).
5.2.1.17 recA PCR
Isolates that tested negative to the TTS1 real-time PCR were subjected to the recA
PCR that amplifies 385-bp fragment of the recA gene of Burkholderia spp using
primers
BUR3;
5’-GA(AG)AAGCAGTTCGGCAA-3’,
and
BUR4;
5’-
GAGTCGATGACGATCAT-3’ (Payne et al., 2005). Each 20 μl PCR reaction
contained 0.5 ng template DNA, 1 U Hot Star Taq polymerase (Life Technologies,
USA), 250 μM of deoxynucleoside triphosphates A, T, C and G (dNTPs), 1x PCR
buffer, 1.5 mM MgCl2 and 10 ρmol of each primer. Thermal cycling was performed
using Palm-Cycle version 2.4 PCR instrument (Corbett Research Pty Ltd, Australia)
for 40 cycles of 94 °C for 1 minute, 48 °C for 1 minute, 72 °C for 1 minute, followed
by another 10 minutes of 72 °C. PCR product was visualized by gel electrophoresis in
a 1% agarose gel.
5.2.1.18 recA Sequencing
Non-B. pseudomallei isolates that tested positive to the primer pairs BUR3/BUR4 of
the recA gene were subjected to recA sequencing. DNA sequencing of the recA gene
reveals the Burkholderia spp. (Payne et al., 2005). In order to prepare the samples,
sequencing
PCR
was
performed
using
GATCGA(AG)AAGCAGTCGGCAA-3’,
and
primers
BUR1;
BUR2;
5’5’-
TTGTCCTTGCCCTG(AG)CCGAT-3’ which amplified an 869-bp fragment flanking
the region amplified by primer pairs BUR3/BUR4 (Payne et al., 2005) using thermal
cycling condition similar as above. Upon confirmation of the correct PCR
amplification size using gel electrophoresis, recA sequencing of PCR products was
outsourced to Macrogen Inc. (Seoul, South Korea).
128
5.2.1.19 16S ribosomal DNA sequencing
Non-B. pseudomallei isolates that were not successfully identified by recA sequencing
were subjected to 16S ribosomal DNA (rDNA) sequencing which had been shown to
be useful in speciation of
non-fermenting gram-negative bacilli including the
Burkholderia spp. (Bosshard et al., 2006). Previously described primers were used,
BAK11w;
5’-AGTTTGATC(A/C)TGGCTCAG-3’
and
BAK2;
5’-
GGACTAC(C/T/A)AGGGTATCTAAT-3’ amplifying a region of about 700-bp in
fragment size (Bosshard et al., 2006). The thermal cycling condition was performed
using Palm-Cycle version 2.4 PCR instrument (Corbett Research Pty Ltd, Australia)
for 45 cycles of 94 °C for 1 minute, 48 °C for 1 minute, 72 °C for 1 minute, followed
by another 10 minutes of 72 °C. PCR product was visualized by gel electrophoresis in
a 1% agarose gel. Upon confirmation of the correct PCR amplification size using gel
electrophoresis, 16S rDNA sequencing of the PCR products was outsourced to
Macrogen Inc. (Seoul, South Korea).
5.2.1.20 Analysis of DNA sequences
DNA sequences received from Macrogen were edited and consensus sequences
produced using ChromasPro Version 1.5 software (Technelysium Pty Ltd, Australia).
Consensus DNA sequences were matched with DNA sequences available on the
National Centre for Biotechnology Information database using the Basic Local
Alignment Search Tool (BLAST) (National Library of Medicine, USA).
129
5.2.2 Results and discussions
No recovery of B. pseudomallei from environmental samples
Out of 241 soil samples and 20 water samples collected from both the first and second
sampling trips, none was found to be positive for B. pseudomallei. Table 5.1 shows
the number of soil samples collected from the different districts excluding the 100 soil
samples collected in the grid-sampling in Kapit. Considering the various measures
taken to increase the probability of B. pseudomallei detection from these
environmental samples including the selection of strategic sites, the unsuccessful
recovery of B. pseudomallei from samples collected from these sites was rather
unexpected.
Table 5.1 Number of soil samples collected from the different districts in Sarawak
Number of soil samples
District
Case associated sites
Targeted sites
Opportunistic sites
Total
Belaga
9
3
6
18
Bintulu
15
10
16
41
Kapit
10
20
15
45
Kuching
26
3
8
37
Total
60
36
45
141
* The 100 soil samples from the grid-sampling in Kapit were not included in this table
As indicated in Figure 5.6, the melioidosis incidence rate was highest in Kapit,
Belaga and Tatau districts (Panel D) where the melioidosis cases from these districts
were presented to the hospitals in Kapit, Sibu and Bintulu. Kapit and Bintulu were
selected for environmental samplings based on the high number of cases that were
presented to the hospitals in these two districts. Although some of the environmental
sampling sites selected were case associated, no B. pseudomallei was isolated. There
is a possibility that the majority of melioidosis cases in the region were from very
interior sites, as suggested by the positive association between incidence rate of
melioidosis and the locations of the dams, oil palm plantations and secondary
130
vegetations in central Sarawak (Figure 5.6; Panels A, B, C). As proposed earlier and
shown in previous studies, there is a strong correlation between human activities such
as construction or excavation, agricultural activities and use of fertilizers on the
ecology of Burkholderia spp. including B. pseudomallei (Haynes and Williams, 1992,
Inglis et al., 2001, Salles et al., 2004, Clegg, 2006, Rooney et al., 2006, Kaestli et al.,
2009)(M. Kaestli, manuscript in preparation). The sites that were sampled in this
study were at least 70 km radius away from the areas which had positive correlation
with the land use activities. Hence, in future studies on the environmental hazard of B.
pseudomallei in Sarawak, it may be worth conducting more rigorous environmental
sampling in central Sarawak at epidemiologically relevant sites closer to the dams,
plantations and secondary vegetation.
131
A-Primary forest, secondary vegetation and
non-forest areas;
B-Oil palm plantations;
C-Types of vegetations;
D-Incidence rate of melioidosis in Sarawak.
A
B
C
D
Figure 5.6 Map of Sarawak overlaid with human activities (Sources: http://www.bmfmaps.ch/EN/composer/#maps/1001 and Sarawak Health Department)
132
GENs B. pseudomallei lacks competitiveness compared with GENr
Due to the unsuccessful recovery of environmental B. pseudomallei and the discovery
of GENs in Sarawak (as discussed in Chapter 2 and 4), I postulated in this study that
GENs B. pseudomallei may not compete as well as GENr strains in the environment
and may not survive the competition with other bacteria during recovery process from
soil samples. In order test the hypothesis that GENs B. pseudomallei did not compete
as well as their GENr counterparts, an experiment was designed to mimic the
survivability of GENs B. pseudomallei in the environment in the presence of other
organisms in the soil. Section 5.3 describes the experiment and the recovery rate of B.
pseudomallei-inoculated soils at two time-points.
Timeliness of environmental sample processing
While timeliness in environmental sample processing may very well be one of the
confounding factors in the unsuccessful recovery of the organism in this study, B.
pseudomallei had been shown to be culturable from soils that were stored for more
than a year after collection (M. Kaestli, personal communication). In the present
study, the samples collected were not processed immediately. The longest duration
between collection and processing was up to two weeks after collection due to the
travelling time from some of the sampling sites back to the laboratory in UNIMAS.
Although previous studies have shown that the distribution of bacteria in the soil is
dynamic and highly influenced by physico-chemical soil parameters such as soil pH,
water content, temperature or salt and nutrient concentration (Tong et al., 1996, Chen
et al., 2003, Salles et al., 2004, Clegg, 2006, Rooney et al., 2006, Kaestli et al., 2009,
Hamad et al., 2011), these factors were not investigated in this present study.
Isolation of non-B. pseudomallei Burkholderia spp.
Other Burkholderia spp. were successfully isolated using both types of Ashdown’s
agar, either with gentamicin or colistin. As shown in Table 5.2, there was not much
difference in the type of Burkholderia spp. isolated using either gentamicin or colistin
133
as the selective agent in Ashdown’s agar. The only apparent difference in the
organisms isolated from both types of media was evidence for a higher detection rate
of B. ubonensis in one sample set which was cultured with Ashdown’s agar with
colistin (9/116 samples) as compared to another sample set cultured with Ashdown’s
agar with gentamicin (2/125 samples) (Fisher’s Exact test, P=0.029). As these sample
sets were not collected at the same sites, this difference in detection rate could be due
to an actual difference in abundance of B. ubonensis rather than growth preference in
the different selective media. Alternatively, this difference could also be attributed to
the possibility that some B. ubonensis in Sarawak are gentamicin sensitive. Although
the recovered B. ubonensis from Sarawak were not subjected to Etest for gentamicin
susceptibility, these isolates grew well in regular Ashdown’s agar containing 40 µg/ml
of gentamicin. This is an indication that the B. ubonensis that were isolated were
gentamicin resistant. Other than B. ubonensis, B. thailandensis and B. multivorans
were isolated from only two samples each when cultured using gentamicin and B.
diffusa was isolated from one sample using colistin as the selective agent. In general,
the results indicated that both selective agents were effective and selective in isolating
environmental Burkholderia spp.
Table 5.3 shows the soil samples collected from different districts in Sarawak that
were positive for Burkholderia spp. The isolation rate of Burkholderia spp. appeared
to be highest in case associated and targeted sites if the 100 additional soil samples
from the second sampling round were not taken into account in the analysis.
Burkholderia spp. that were successfully isolated included B. ubonensis, B.
thailandensis, B. multivorans, B. diffusa and B. cepacia (Table 5.4). There were also
several environmental isolates that were unable to be identified by either recA or 16S
rDNA sequencing despite being recognised as Burkholderia spp. These isolates were
categorised as Burkholderia spp.
134
Table 5.2 Comparison of culture yields using Ashdown’s agar with gentamicin and
colistin
Selective agent used in Soil samples
Soil
Ashdown’s agar
Burkholderia spp. (%)
125
Gentamicin
samples
with Burkholderia spp. (n)
20 (16)
B. pyrrocinia (10)
B. ubonensis (2)
B. cepacia (1)
B. multivorans (2)
B. thailandensis (2)
Burkholderia spp. (5)
116
Colistin
19 (16.4)
B. ubonensis (9)
B. pyrrocinia (8)
B. cepacia (1)
B. diffusa (1)
Table 5.3 Soil samples positive for Burkholderia spp. by culture
Case associated
District
Targeted
Opportunistic
Soil
Soil samples
Soil
Soil samples with
Soil samples
Soil samples
samples
with
samples
Burkholderia
collected
with
Burkholderia
spp. (%)
Burkholderia
spp. (%)
spp. (%)
Belaga
9
0
3
1 (30)
6
0
Bintulu
15
3 (20)
10
3 (30)
16
2 (13)
Kapit
10
2 (20)
20
6 (30)
15
4 (27)
Kuching
26
13 (50)
3
0
8
0
Total
60
17 (30)
36
10 (27)
45
6 (13)
* This table does not include 100 samples from Kapit collected in the second
sampling
135
Table 5.4 Type of Burkholderia spp. isolated from the different types of sampling sites
Type of sampling site
Soil samples
Soil samples with
Burkholderia spp. (n)
Burkholderia spp. (%)
Case associated
60
15 (25)
B. pyrrocinia (9)
B. ubonensis (6)
B. multivorans (1)
B. diffusa (1)
Case associated (Kapit
100
6 (6)
B. ubonensis (4)
100 grid sampling from
B. pyrrocinia (1)
second trip)
B. cepacia (1)
Ralstonia spp. (1)
Targeted
36
7 (19)
B. pyrrocinia (2)
B. ubonensis (2)
B. multivorans (1)
B. thailandensis (2)
B. cepacia (1)
Opportunistic
45
7 (16)
B. pyrrocinia (6)
B. ubonensis was mostly isolated from case associated sites (6/8 samples with B.
ubonensis), followed by targeted sites and none from opportunistic sites. The same
observation applied to other Burkholderia spp. where isolation rates were higher for
case associated sites. This was not due to sampling bias as the number of
environmental samples collected for all three categories was similar (Table 5.1). In
light of more B. ubonensis being isolated from case associated sites with no B.
pseudomallei isolated, the lack of detected B. pseudomallei might be explained by
antibiosis activities by B. ubonensis towards B. pseudomallei as previously shown by
Marshall and colleagues (Marshall et al., 2010). This hypothesis was further explored
in Section 5.4 of this thesis.
136
In this present study, B. thailandensis was isolated only from two sampling sites and
all within the Kapit region. The prevalence of B. thailandensis in the environment has
been shown to be inversely proportionate to that of B. pseudomallei (Trakulsomboon
et al., 1999, Warner et al., 2008). Surprisingly, results from this study suggested that
B. thailandensis in Sarawak was not as widespread as in Thailand. That both B.
thailandensis and B. pseudomallei were not present or were uncommon (B.
thailandensis) at the sites sampled may be an indication that the environmental niches
of both organisms in these sites are different from what has been shown in other
studies (Trakulsomboon et al., 1999, Warner et al., 2008).
B. pyrrocinia was commonly found across all types of sampling sites. B. pyrrocinia is
a member of the B. cepacia complex that is mostly described as an environmental
bacterium (Vandamme et al., 2002) but had also been isolated from cystic fibrosis
patients (Storms et al., 2004). Therefore, it is not surprising that B. pyrrocinia was
isolated from environmental samples in Sarawak.
Some Burkholderia spp. colonies isolated from this study showed great resemblance
to B. pseudomallei. As depicted in Figure 5.7, B. cepacia (Panel A) and B.
thailandensis (Panel B) possessed dry, wrinkled colonies, similar to the typical
morphotypes B. pseudomallei (Panel D). In addition, B. ubonensis (Panel F) and
Ralstonia spp. (Panel B) were found to resemble the mucoid morphology of B.
pseudomallei (Panel E). Previous studies have also shown that some Burkholderia
spp. resembled B. pseudomallei on Ashdown’s agar (Price et al., 2013b).
Morphotype-switching in B. pseudomallei is common where B. pseudomallei has
been shown to possess the ability to switch from the classical dry, wrinkled
morphotype to the smooth mucoid morphotype depending on various factors such as
growth temperature at either 37 °C or 42 °C, starvation, pH and iron content of media
(Chantratita et al., 2007). Hence, it is not surprising that some of the Burkholderia
spp. colonies were mistaken for B. pseudomallei based on solely morphological
identification.
137
B. cepacia
A
B. pseudomallei
D
Ralstonia spp
B
B. pseudomallei
E
B. thailandensis
C
B. ubonensis
F
Figure 5.7 Comparison of different morphologies of selected non-B. pseudomallei
environmental and B. pseudomallei isolates.
138
In addition to Burkholderia spp., other species of bacteria were also isolated including
Ralstonia spp. (one isolate from case associated and two isolates from targeted sites)
and Achromobacter xylosoxidans (one isolate from opportunistic site). Ralstonia spp.
was formerly included in the Pseudomonas genus (Yabuuchi et al., 1995, Vandamme
and Dawyndt, 2011) while A. xylosoxidans is an environmental bacterium that is
increasingly recognized as an opportunistic pathogen for immunosuppressed patients
(Spear et al., 1988, Ciofu et al., 2013).
Out of the 20 water samples collected from this study, one sole water sample yielded
Burkholderia spp. which could not be further identified by either recA or 16S rDNA
sequencing.
In short, B. pseudomallei was not recovered from environmental samples collected
from Sarawak. Nonetheless, other Burkholderia spp. were isolated from the
environmental samples. It was hypothesized that the lack of recovery of
environmental B. pseudomallei in Sarawak was potentially due to one or more of the
following: lack of fitness of GENs B. pseudomallei; the presence of antibiosis
activities against B. pseudomallei; and point source dissemination of B. pseudomallei
in Sarawak. These hypotheses were further explored in the following sections of this
chapter.
139
5.3 Soil inoculation experiment
Objectives
The objective of this experiment was, in view of the lack of success in isolating B.
pseudomallei from environmental samples, to determine whether the GENs B.
pseudomallei isolates from Sarawak were able to compete with other organisms in the
soil as well as GENr B. pseudomallei. Two sets of soil were used in this experiment,
one using sterilized soil and one unsterilized soil.
5.3.1.1 Modified Ashdown’s agar with 50 μg/ml colistin
As discussed in Section 2.2.8, all Ashdown’s agar plates used in this experiment were
prepared using 50 μg/ml of colistin as the selective agent.
5.3.1.2 Sterilization of soil and quality control
Sandy clay loam from the Top End that had been confirmed negative for B.
pseudomallei by culture on Ashdown’s agar and direct molecular detection (Kaestli et
al., 2007) was used in this experiment. Half of the soil was subjected to heatsterilization at 120 °C for two hours. A quality control was carried out to make sure
that the soil was properly sterilized by incubating the soil with water at 37°C in 240
rpm for up to 39 hours. 100 μl of the supernatant of the soil/water mixture was further
inoculated onto Ashdown agar at 37 °C and observed for growth for up to a week and
recorded as no growth if so. Soil samples were then weighed into 100 g aliquots and
transferred into clean containers for inoculation.
5.3.1.3 Preparation of B. pseudomallei isolates
Bacterial isolates were streaked onto Chocolate agar plates (Oxoid, Australia) and
incubated at 37 °C for up to 18 hours. Cell suspensions of all B. pseudomallei isolates
were prepared by resuspending scraped bacterial cells from the plates with 0.85%
sodium chloride saline (Pfizer, USA). Optical densities were measured at 600 nm
using WPA CO-7000 Medical Colorimeter (Biochrom Ltd, UK). Serial dilutions were
140
prepared according to previously described methods to achieve cell densities of
approximately 360 CFU/g of soil (Kaestli et al., 2007).
5.3.1.4 Soil inoculation with selective B. pseudomallei isolates
Each container of 100 g of either sterilized or unsterilized soil was inoculated with
20ml suspension of selected B. pseudomallei isolates at approximately 360 CFU/g of
soil based on previously described method (Kaestli et al., 2007). A total of six B.
pseudomallei isolates from different locations were used in this experiment, consisting
of three GENs isolates from Sarawak (MSHR6793, MSHR6808, MSHR6822), a
GENr isolate each from Sarawak (MSHR5099), Sabah (MSHR6369) and the Top End
(MSHR668). Soil samples were inoculated in duplicates and incubated at 37 °C until
testing at week 5 and week 14 post-inoculation.
5.3.1.5 Soil DNA extraction
As previously described, soil DNA extraction was done by incubating 20 g of soil
with 20 ml of Ashdown’s Broth for 39 hour shaking at 37 °C (Kaestli et al., 2009).
The soil supernatant was then centrifuged twice and the pellet processed using the
PowerSoil Kit (MoBio Laboratories, USA). Modifications of the protocol included
the addition of 0.8 mg of aurintricarboxylic acid (ATA) to inhibit nucleases and 20 μl
of proteinase K (w/v 20 mg/ml).
5.3.1.6 PCR of B. pseudomallei inoculums into soil
Upon incubation, a semi-quantification of B. pseudomallei load in the inoculated soil
was done. A real-time PCR targeting TTS1 was conducted using primers described in
the previous section. Each sample was done in duplicates. A final volume of 20 μl
PCR reaction contained 4 μl DNA template, 4.5 mM MgCl2, 0.40 μg/μl of bovine
serum albumin (BSA), 1 U Hot Star Taq polymerase (Life Technologies, USA), 250
μM of deoxynucleoside triphosphates A, T, C and G (dNTPs), 0.25 U uracil DNA
glycosylase (Invitrogen, USA),1x PCR buffer and 10 ρmol of each primer. In order to
141
bind PCR inhibitors such as humic acids present in the soil DNA extracts,
nonacetylated bovine serum albumin (BSA) at a final concentration 0.40 μg/μl was
added to the PCR reaction. Real-time PCR was performed using a Rotor-Gene 2000
(Corbett Research Pty Ltd, Australia) with thermo cycling conditions starting with a
uracil DNA glycosylase incubation step at 37 °C for 10 min followed by 94 °C for 10
min and then 40 cycles of 94 °C for 15 s and 60 °C for 1 min. The threshold cycle
(Ct) values of the real-time PCR reaction were compared by setting the real-time PCR
threshold at the same level (Threshold=0.03) for runs of soil samples harvested 5
weeks and 14 weeks post-inoculation. B. pseudomallei gDNA extracts of known
concentration in three dilutions were used as standards for normalization of Ct values
of the samples. Statistical analyses of the Ct values were 2-tailed and considered
significant if P values were less than 0.05 (P<0.05).
5.3.1.7 Recovery of viable B. pseudomallei from inoculated soil samples
The recovery of viable B. pseudomallei from the inoculated soil samples was achieved
as described in Section 5.2.1.8 and in Figure 5.4 in the preceding section.
5.3.1.8 DNA extraction and TTS1 real-time of sweeps from plates A, B and C
DNA extractions of sweeps of Ashdown’s agar plates A, B and C were done as
described earlier in Section 5.2.1.15. Similarly, the real-time PCR targeting the TTS1
gene was also performed using previously described methods in Section 5.2.1.16.
5.3.1.9 Experimental limitations
Ideally, soil samples from central Sarawak should have been used in this experiment.
However, due to cross-border biological control where unsterilized soil cannot be
brought into Australia, a soil type in the Northern Territory that had properties as
close to Sarawak soil as possible was selected for this experiment.
142
Table 5.5 shows the mean Ct values of duplicate TTS1 real-time PCR reactions of
DNA extracts of soil and culture plates A, B and C that were each done in duplicates.
In order to compare the growth of GENs and GENr isolates in sterilized and
unsterilized soil, sterilized and unsterilized soil was each inoculated with one of three
GENs or GENr isolates in duplicates. Soil DNA was extracted 5 and 14 weeks post
inoculation and TTS1 real-time PCR was performed on the DNA. The difference in
Ct values (∆Ct) between sterilized and unsterilized soil was calculated for each isolate
for 5 and 14 weeks. A T-test was conducted on ∆Ct between GENs and GENr
inoculated soil samples at 5 and 14 weeks as well as a multivariate permutational
Permanova analysis performed with replicates as random factors nested in the fixed
effect, the resistance to gentamicin.
For soils harvested 5 weeks post-inoculation, there was evidence for both GENs and
GENr isolates to show considerably higher growth in sterilized as compared to
unsterilized soil. GENs showed an average reduction of 12.2 Ct values (95% CI -14.0
to -10.4) in unsterilized soil and GENr isolates an average reduction of 10.3 Ct values
(95% CI -12.4 to -8.2). At 5 weeks, there was no evidence that the GENr isolates
coped better in unsterilized soil as compared to the GENs isolates with an average
difference of 1.9 ∆Ct (95% CI -4.2 to 0.5, P=0.115) . This was supported by the lack
of significant difference performing the Permanova analysis (Pseudo-F 2.8, df(10),
P=0.21).
At 14 weeks post-inoculation, GENs isolates showing a significantly larger decline in
unsterilized as compared to sterilized soil with an average reduction of -26.7 (95% CI
-39.4 to -14.1), which compared to an average reduction of -15.5 (95% CI -25.6 to 5.3). However, while there was suggestion of decreased growth in unsterilized soil of
GENs in comparison with GENr isolates, this was still not statistically significantly
different (average difference 11.3 ∆Ct (95% CI -25.3 to 2.8, P=0.105)(Permanova
Pseudo-F 2.9, df(10), P=0.19).
143
There was evidence for the GENs isolates showing a significant decline in unsterilized
soil from 5 to 14 weeks inoculation (average -14.6 ∆Ct, 95% CI -25.6 to -3.4,
P=0.015). This was not the case for GENr isolates (average -5.1 ∆Ct, 95% CI -14.1 to
3.9, P=0.24).
This analysis supports but does not provide conclusive evidence for the hypothesis
that GENs isolates did not survive as well as GENr isolates in unsterilized soil,
potentially due to lack of fitness in competing with other organisms present in the
unsterilized soil.
Comparisons between isolate growth in sterilized and unsterilized soil were also done
using the ∆Ct of TTS1 real-time PCR reactions of DNA extracts of sweeps of culture
plates A, B and C (see Table 5.5) inoculated with the soil samples. Consistent with
the above results of real-time PCR on DNA extracted directly from the inoculated
soil, DNA extracts of culture plates also showed higher Ct values if the soil samples
were inoculated with GENs as compared to GENr B. pseudomallei. Although not
statistically significant (P=0.4), the ∆Ct values between plates inoculated with GENs
and GENr loaded soil ranged from 0.1 to 10.0 for samples harvested 5 weeks postinoculation. In plates inoculated with soil harvested 14 weeks post-inoculation, the
∆Ct values between plates from GENs and GENr loaded soil was higher and plates
with GENs B. pseudomallei inoculated soil were mostly real-time PCR negative for B.
pseudomallei (∆Ct values 19.1-22.4). These results suggest that in comparison with
GENr strains, GENs B. pseudomallei showed less ability to compete with the microbes
naturally present in the unsterilized soil during the culture recovery step and this
inability became more apparent with longer incubation.
Intriguingly, a Sarawak GENr strain (MSHR5099), seemed to also lack the ability to
compete in the unsterilized soil. This might be attributed to the fact that this particular
strain was isolated from a chronic patient. Similar to what was observed in a patient
with chronic infection with B. pseudomallei in the Darwin Prospective Melioidosis
144
Study, MSHR5099 may have adapted to survival in its human host and diminished its
ability to compete in the environment (Price et al., 2013a).
Ideally, this experiment could provide more information if the incubation period of
the soil was further prolonged. An extended incubation was however not possible due
to limited time and resources for additional DNA extractions. A replicated experiment
using soil types from Sarawak would also be useful in elucidating the survival rate of
the
different
B.
pseudomallei
strains
145
in
different
soil
types.
Table 5.5 Ct values of TTS1 real-time PCR of soil extracts and plates A, B and C.
Soil inoculum
Blank-S
Blank-U
668-S (NT GENr)
668-U (NT GENr)
5099-S (Sarawak GENr)
5099-U (Sarawak GENr)
6369-S (Sabah GENr)
6369-U (Sabah GENr)
6793-S (Sarawak GENs)
6793-U (Sarawak GENs)
Soil extract
PCR NEG
PCR NEG
16.2
24.6
16.8
29.1
15.0
25.2
17.9
30.0
16.8
29.8
16.8
28.2
5 weeks post-inoculation
Plate A
Plate B
No growth
No growth
PCR NEG
PCR NEG
16.1
11.7
17.1
18.7
15.8
11.0
23.7
17.5
16.4
9.3
20.0
12.4
16.2
10.9
22.7
16.5
16.2
10.3
24.6
24.1
16.4
11.2
20.4
28.8
14 weeks post-inoculation
Plate C
Soil extract
Plate A
Plate B
No growth
PCR NEG No growth No growth
PCR NEG
PCR NEG PCR NEG PCR NEG
11.4
14.2
12.3
13.8
20.8
22.9
13.6
17.9
11.5
15.6
14.1
11.9
14.9
11.9
14.1
12.2
12.5
13.9
25.8
14.4
23.1
11.7
14.8
12.5
13.0
21.9
11.5
16.3
12.6
16.4
26.3
11.2
16.7
12.7
15.8
31.0
U=Unsterilized soil; S=Sterilized soil
Note: Ct values presented here are means of duplicates of real-time PCR reactions and also of duplicate soil samples
146
Plate C
No growth
PCR NEG
13.8
19.8
12.5
PCR NEG
12.2
15.5
12.7
PCR NEG
12.6
PCR NEG
12.6
PCR NEG
5.4 Antibiosis activities of Burkholderia spp. towards aminoglycoside susceptible
B. pseudomallei from Sarawak
Objectives
This study addressed research question 3 as mentioned at the beginning of this chapter
with the objective of establishing whether the lack of success in isolating
environmental B. pseudomallei from Sarawak was due to antibiosis activities by other
Burkholderia spp. towards B. pseudomallei, as previously described (Trakulsomboon
et al., 1999, Warner et al., 2008, Kaestli et al., 2009, Marshall et al., 2010, Lin et al.,
2011). In order to address this question, an experiment was designed with a panel of
B. pseudomallei being challenged by a panel of Burkholderia spp. from various
geographical origins.
5.4.1.1 Antagonism screening
This is a modification of previous work by Marshall et al. (Marshall et al., 2010)
where “target” strains were strains spread on agar plates as bacterial indicator lawns.
“Producer” strains on the other hand were defined as bacterial suspensions that were
spotted onto the indicator lawn.
5.4.1.2 Bacterial isolates used
Target isolates used for this experiment were comprised of a panel of B. pseudomallei
from various geographic origins namely Sarawak, Sabah and the Northern Territory,
Australia with different gentamicin susceptibility profiles.
Producers consisted of non-B. pseudomallei Burkholderia spp. environmental isolates
including B. pyrrocinia (n=2), B. ubonensis (n=13), B. thailandensis (n=2), B. cepacia
(n=1) all of which were from Sarawak and one single B. ubonensis (MSMB112) from
the Northern Territory. Sweeps of the original agar plates where some of the panel of
Sarawak environmental strains were isolated from were also used as producers in this
experiment. Table 5.6 provides complete information of all the isolates used. All
isolates were either speciated by whole genome sequencing, MLST, 16S rDNA or
recA sequencing.
147
Table 5.6 Isolates used for antagonistic screening
Isolate number
Speciation
GENr/GENs
Geographical Origin
Indicator/Producer
MSHR668
B. pseudomallei
GENr
NT, Australia
Indicator
B. pseudomallei
GEN
r
Sarawak, Malaysia
Indicator
s
Sarawak, Malaysia
Indicator
MSHR5079
MSHR5087
B. pseudomallei
GEN
MSHR5099
B. pseudomallei
GENr
Sarawak, Malaysia
Indicator
MSHR6369
B. pseudomallei
GEN
r
Sarawak, Malaysia
Indicator
MSHR6418
B. pseudomallei
GENs
Sarawak, Malaysia
Indicator
MSHR6793
B. pseudomallei
GEN
s
Sarawak, Malaysia
Indicator
MSMB1649
B. cepacia
GENr
Sarawak, Malaysia
Producer
r
Sarawak, Malaysia
Producer
MSMB1291
B. pyrrocinia
GEN
MSMB1645
B. pyrrocinia
GENr
Sarawak, Malaysia
Producer
MSMB1381
B. thailandensis
GENr
Sarawak, Malaysia
Producer
MSMB1365
B. thailandensis
GEN
r
Sarawak, Malaysia
Producer
MSMB112
B. ubonensis
GENr
NT, Australia
Producer
MSMB1397
B. ubonensis
GEN
r
Sarawak, Malaysia
Producer
MSMB1417
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1423
B. ubonensis
GEN
r
Sarawak, Malaysia
Producer
MSMB1643
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1647
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1648
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1650
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1651
B. ubonensis
GEN
r
Sarawak, Malaysia
Producer
MSMB1653
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1655
B. ubonensis
GEN
r
Sarawak, Malaysia
Producer
MSMB1656
B. ubonensis
GENr
Sarawak, Malaysia
Producer
MSMB1675
B. ubonensis
GEN
r
Sarawak, Malaysia
Producer
MSMB1677
B. ubonensis
GENr
Sarawak, Malaysia
Producer
Sweep 20 of MSMB1417
Not applicable
Not done
Sarawak, Malaysia
Producer
Not applicable
Not done
Sarawak, Malaysia
Producer
Not applicable
Not done
Sarawak, Malaysia
Producer
Sweep 23 of MSMB1648 &
Not applicable
Not done
Sarawak, Malaysia
Producer
Not applicable
Not done
Sarawak, Malaysia
Producer
Not applicable
Not done
Sarawak, Malaysia
Producer
Sweep 26 of MSMB1655 &
Not applicable
Not done
Sarawak, Malaysia
Producer
Not applicable
Not done
Sarawak, Malaysia
Producer
Not applicable
Not done
Sarawak, Malaysia
Producer
MSMB 1649
MSMB1656
148
5.4.1.3 Preparation of bacterial culture
Bacterial lawns of indicator isolates were prepared as described earlier in Section
2.2.8. Then, 10 μl of a producer strain was inoculated onto the lawn. Each plate was
divided into four quadrants and each quadrant was spotted with a different producer
strain as shown in the diagram in Figure 5.8. Plates were incubated at 37 °C and
observed daily for up to five days.
Lawn of
indicator*
bacteria
Producers
Figure 5.8 Set-up of plates for antagonism experiment
*Indicator is also referred to as “target”
149
Two different types of antagonistic effects observed
Table 5.7 summarizes the overall results of this experiment. Three types of
antagonistic effects were observed in this experiment; 1) Zone of inhibition or halos
around producers (Figure 5.9), 2) clearings in the midst of the producers’ growth
patch (Figure 5.10), and 3) producers being overgrown by indicators (Panels A and
D of Figure 5.11).
Zones of inhibition were only observed around two producers, an NT B. ubonensis
(MSMB112) and a Sarawak B. cepacia (MSMB1649) on indicator lawns of
MSHR5087 (GENs), MSHR6418 (GENs), MSHR6793 (GENs), MSHR6369 (GENr)
and MSHR 668(NT GENr) (Figure 5.9).
As for the clearings within the producers’ vicinity (Figure 5.10), it was most
prominent in plates that were inoculated with B. pseudomallei indicator strains
MSHR5079 and MSHR5099 across the whole panel of producers. These clearings
visually resemble cell lysis or phage plaques caused by bacteriophages (Cox, 2012).
MSHR5079 was a GENr isolate from Kapit, Sarawak whereas MSHR5099 was a
GENr isolate from a chronic melioidosis patient in Bintulu. Other than these
observations, there were no other clear antagonistic activities by the challengers
against Sarawak GENs B. pseudomallei.
As shown in Panels A and D of Figure 5.11, the producers were overgrown by the
indicator. This suggests that the producers did not possess any antibiosis properties
against the B. pseudomallei indicators, neither did the producers possess any form of
defence to stop being overgrown by the indicators. In contrast, Panels B and C
showed that both the producers and indicators were confined within what appeared to
be a ‘mutually accepted boundary’.
150
MSHR5087 [GENs]
- - - - - - - s
MSHR6418 [GEN ]
- - - - - - - MSHR6793 [GENs]
- - - - - - - r
MSHR5079 [GEN ]
- • • • • • • •
MSHR5099 [GENr]
- - - - - - - MSHR6369 [GENr]
- - - - - - - r
MSHR688 [GEN ]
- - - - • • - Note:
(-) = No antagonistic activities observed
(+) = Zones of inhibition around producers observed
(•) = Cell lysis of producers observed
(▼) = Producers taken over by B. pseudomallei lawn
•
•
-
•
•
-
•
•
-
-
•
-
+
+
+
+
+
151
+
+
+
•
+
+
•
•
•
•
•
•
•
-
•
•
-
-
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
▼
+
+
+
•
+
+
-
+
+
+
+
Sweep of MSMB1677
S9
Sweep of MSMB1650
Sweep of MSMB1651
Sweep of MSMB1655
Sweep of MSMB1675
MEM182
K1
K5
S4
Sweep of MSMM1417
Sweep of MSMB1643
Sweep of MSMB1647
Sweep of MSMB1648
& MSMB1649
MEM5
MEM139
MEM164
MEM180
B. pyrrocinia
B. thailandensis
MSMB1656
MSMB1675
MSMB1677
MSMB112
MSMB1649
MSMB1381
MSMB1365
MSMB1291
MSMB1645
B. pseudomallei
MSMB1423
MSMB1643
MSMB1647
MSMB1648
MSMB1650
MSMB1651
MSMB1653
MSMB1655
Lawn of indicator
MSMB1397
MSBB1417
B. ubonensis
(Sarawak)
B. ubonensis (NT)
B. cepacia
Table 5.7 Overall results of antagonistic experiment
+
•
+
Sarawak B. ubonensis did not have antibiosis effect towards B. pseudomallei
No antibiosis effect by Sarawak B. ubonensis isolates towards any B. pseudomallei
was observed. These included B. pseudomallei either from Sarawak, Sabah or the
Northern Territory. Likewise, B. thailandensis and B. pyrrocinia did not seem to
possess antibiosis properties towards any B. pseudomallei. The sole B. ubonensis from
the Northern Territory used in the experiment showed strong antibiosis activities
towards B. pseudomallei (Figure 5.9). The observation that Sarawak B. ubonensis did
not show any antibiosis effects against B. pseudomallei was unexpected because
strong antibiosis of B. ubonensis towards B. pseudomallei was previously reported in
more than 50% of Australian strains (Marshall et al., 2010). Since Burkholderia spp.
antibiosis towards B. pseudomallei was not the focus of this study, no further work
was done beyond this experiment. The results from this experiment suggest that the
antibiosis attributes of the B. ubonensis strain from the Northern Territory were
different from those of Sarawak strains. This observation and the question of whether
this difference occurs for all B. ubonensis from the Northern Territory and Sarawak
warrant further investigations. It will also be useful to isolate and characterize the
antibiosis compounds secreted by the different B. ubonensis from the Northern
Territory and Sarawak.
As shown in the results of this experiment, the only Burkholderia spp. from Sarawak
that demonstrated antagonistic effects towards B. pseudomallei was B. cepacia
MSHR1649. Although no other studies have to date demonstrated antibiosis of B.
cepacia towards B. pseudomallei, one study demonstrated B. cepacia having
antibiosis effects against plant pathogenic fungi (Li et al., 2007)
Curiously, two of the sweeps (K1 and S4) showed consistent antagonism activities
across almost all the panel of B. pseudomallei except for MSHR5079 and MSHR5099
(Table 5.7). While B. ubonensis was the only Burkholderia spp. isolated from these
sweeps, this result suggests that there were other uncharacterized organisms in the
sweep responsible for the antagonistic effect against B. pseudomallei. Further work is
needed to isolate and characterize the other organisms in the sweep that contributed to
the antagonistic effect.
152
As for the clearings depicted in Figure 5.10, the Burkholderia spp. challengers
seemed lysed by what appeared to be bacteriophages induced from B. pseudomallei
MSHR5079 and MSHR5099. B. pseudomallei had been reported to produce
bacteriophages spontaneously (DeShazer, 2004, Sariya et al., 2006, Gatedee et al.,
2011). Consistent with a previous report, the two isolates of B. pseudomallei in this
study seemed to be resistant towards infections by their own phages (Sariya et al.,
2006) and the phages seemed more efficient in infecting B. ubonensis and B.
pyrrocinia from Sarawak. Bacteriophage activities towards B. cepacia complex had
also been reported elsewhere (Lynch et al., 2010, Golshahi et al., 2011, Lynch et al.,
2012). Further work is required to isolate and characterize these phages.
Unlike the B. ubonensis isolates from Balimo, Papua New Guinea, as previously
reported (Marshall et al., 2010), the overall results from this experiment showed that
the Sarawak B. ubonensis strains tested thus far did not possess antibiosis properties
against B. pseudomallei. However, a single B. cepacia isolate and several mixed
environmental cultures from Sarawak were shown to possess antagonistic effects
against B. pseudomallei. Although the actual strains responsible for the antibiosis
effect in the mixed culture were not identified, this experiment provided evidence of
antibiosis effects against B. pseudomallei in the samples from Sarawak. Nevertheless,
limited Sarawak Burkholderia spp. isolates were tested in this study and hence, it
cannot be concluded that all Sarawak B. ubonensis do not possess antibiosis properties
against B. pseudomallei. It can neither be concluded on whether other Burkholderia
spp. isolates from Sarawak possess any antibiosis activities against B. pseudomallei.
153
Figure 5.9 Zones of inhibition around the producers (arrows). Caused by MSHR112
and MSMB1649 against most B. pseudomallei lawns except MSHR5099 and
MSHR5079.
Figure 5.10 Lysis of inoculated producers. As indicated by the black arrows, this
form of clearing is more noticeable in plates with lawns of MSHR5079 and
MSHR5099, both are GENr B. pseudomallei isolates from Sarawak.
154
Lawn of Indicator
Producers
A
C
B
D
Figure 5.11 Other forms of antibiosis. Panels B and C show no antibiosis activities
by either the target cells or the challengers. Panels A and D show that the producers
were taken over by the indicator.
155
5.5 Summarized discussions and conclusions for Chapter 5
Initially, the negative isolation of B. pseudomallei from environmental samples was
attributed to the presumption of GENs strains being abundant in the environment as
evident in the clinical isolates from central Sarawak. As discussed in Chapter 2, up to
88% of clinical isolates from central Sarawak were found to be gentamicin sensitive, a
rare occurrence with only 1:1000 reported in Thailand (Trunck et al., 2009). Having a
majority of clinical isolates being GENs in Sarawak is an unusual and unique
occurrence. Hence, it was hypothesized that the use of gentamicin in Ashdown’s agar
may have contributed to the lack of recovery of B. pseudomallei from the
environmental samples collected in the first sampling round. The Ashdown’s agar was
modified by replacing gentamicin with colistin, but there was still no recovery of B.
pseudomallei in the second sampling trip. In spite of no environmental recovery of B.
pseudomallei from Sarawak, other Burkholderia spp. were isolated. A consensus
guideline for environmental investigation of B. pseudomallei was published in 2013
which recommended that the selective media TBSS containing 50 μg/ml colistin
(TBSS-C50) be used as the primary enrichment medium with Ashdown broth be used
as an alternative (Limmathurotsakul et al., 2013a). The use of TBSS-C50 should
certainly be adopted and used together with Ashdown’s agar in further environmental
investigations of B. pseudomallei in Malaysian Borneo where the medium has been
shown to be more sensitive than Ashdown’s broth in study sites in Thailand.
Three possible reasons for the negative recovery of environmental B. pseudomallei
were postulated and explored in this study; 1) antibiosis activities against B.
pseudomallei, 2) GENs B. pseudomallei strains from Sarawak lack competitiveness in
the environment, and 3) point source dissemination of B. pseudomallei from animals
or a specialised environmental niche.
In the antibiosis experiment described in Section 5.4, B. ubonensis and B.
thailandensis isolates from Sarawak did not possess antagonistic properties against B.
pseudomallei unlike previous reports from Australia (Marshall et al., 2010) and
Thailand (Trakulsomboon et al., 1999). However, a B. cepacia isolate from Sarawak
showed antagonistic activities against B. pseudomallei. In addition, some of the
sweeps or mixed cultures demonstrated antagonistic activities towards B.
pseudomallei; indicating the presence of unidentified bacterial strains possessing
156
antibiosis properties against B. pseudomallei. Although the actual strains responsible
for the antibiosis effect in the mixed culture were not identified (as it was not the main
focus of this study), this experiment provided evidence of antibiosis effects against B.
pseudomallei in the samples from Sarawak. Surprisingly, results from this experiment
also suggested that some of the B. pseudomallei from Sarawak possess bacteriophages
that had lytic effect on some of the Burkholderia spp. tested. Further isolation and
characterization of the phages will be useful.
Albeit soil not of Sarawak origin was used, the experiment discussed in Section 5.3
provided an insight into the competitiveness of GENs and GENr B. pseudomallei in
soil. Compared to GENr B. pseudomallei, Sarawak GENs B. pseudomallei strains
tested were shown to have a lower survivability and recovery rate in the presence of
other organisms in the soil samples, especially in the soils that were harvested 14
weeks post-inoculation. This lack of competitiveness might contribute to the lower
recovery rate of GENs B. pseudomallei from environmental samples whereby they
were outgrown by presumably other Burkholderia spp. and other bacteria present in
the unsterilized soils. However, the results of this experiment did not explain the
unsuccessful isolation of GENr B. pseudomallei from the environmental samples in
Sarawak. Hypothetically, GENr strains should be able to be isolated from Sarawak
soil samples since GENr clinical isolates were also isolated from patients in the region.
However, this was not the case; suggesting that the negative environmental B.
pseudomallei recovery from Sarawak may very well be due to other factors. There
could be a combination of very low prevalence of GENr in the environment (as
reflected in the clinical isolates), with the existence of a localized source of
dissemination for both GENs and GENr B. pseudomallei.
Possible culturable and non-culturable environmental B. pseudomallei
Inglis et al. in an environmental surveillance study in the Northern Territory
suggested that their failed attempt in isolating B. pseudomallei from soil samples may
be attributed to 2 reasons; 1) presence of viable but non-culturable B. pseudomallei in
the environment, 2) culturable B. pseudomallei that is not as widespread in the
environment (Inglis et al., 2004). In this present study, soil was enriched in
Ashdown’s broth to amplify B. pseudomallei enough to be detected by direct soil
DNA extraction and TTS1 real-time PCR. However, the negative results indicated
157
that no B. pseudomallei were amplified during the enrichment process. Nevertheless,
it cannot be surmised whether the negative results from the soil enrichment was due to
either absence of culturable B. pseudomallei or absence of viable but non-culturable
B. pseudomallei.
Possible localized environmental niche of B. pseudomallei in central Sarawak
While results from the experiments discussed earlier did not fully support or disprove
their respective hypotheses, these results suggest that B. pseudomallei in Sarawak
might be either host-adapted to an animal host leading to a zoonotic transmission, or
present in a specialised environmental niche.
Animals may be a possible reservoir for B. pseudomallei in Sarawak. WGS analysis
on GENs B. pseudomallei as discussed in Chapter 2 did not suggest massive gene
loss as shown in B. mallei whose adaptation to an equine host had occurred over a
long period of time (Nierman et al., 2004, Moore et al., 2004, Losada et al., 2010).
Although animal melioidosis has been reported previously involving orang-utans in
Malaysian Borneo (de Silva, 1971, Peters et al., 1976, Ouadah et al., 2007) (Sarawak
Veterinary Department, unpublished data), there was no widespread disease reported
among orang-utans. In addition, the population of orang-utans in Malaysian Borneo is
very small and only confined to sanctuaries that are not within the regions where
human melioidosis are reportedly prevalent. Apart from orang-utans, animal
melioidosis has been seen in gibbons and goats where occurrence is very rare (Ouadah
et al., 2007)(Sarawak Veterinary Department, unpublished data). If proven, the hostadaption theory proposed in the current study seems to be a more recent introduction
due to the absence of large regions of gene loss and the low genetic diversity between
GENs isolates as discussed earlier. As such, the host-adaption theory does not explain
the lack of success in the recovery of environmental GENr B. pseudomallei. The
possibilities of both, GENs and GENr B. pseudomallei being host-adapted or sharing
the same environmental niche in Sarawak are yet to be proven or disproved.
Hence, a particular environmental reservoir appeared to be the more plausible
explanation. Localised or point distribution of B. pseudomallei has been previously
reported in Western Province, Papua New Guinea (PNG) and Torres Straits Islands
(Warner et al., 2008, Baker et al., 2011a, Baker et al., 2013). Despite being endemic
158
for melioidosis, low environmental prevalence of B. pseudomallei has been reported
in the Western Province, PNG (Warner et al., 2008, Baker et al., 2011a) and in Torres
Straits Islands (Baker et al., 2013) where low diversity of STs were found in localized
communities (Baker et al., 2011a, Baker et al., 2013). These studies suggested that the
organism has a very specific niche on these islands. Similar to the scenarios in Balimo
and Badu Island, communities in central Sarawak are also subsistent farmers due to
geographic isolation and the genetic diversity of B. pseudomallei in the region was
limited where 86% of the clinical isolates belonged to ST881 (as discussed in
Chapter 2). The discovery of a limited genetic diversity combined with the unique
GENs trait in B. pseudomallei in Sarawak, suggested low selective pressure against
GENs B. pseudomallei which could be achieved through dwelling in a habitat with no
competing other microbes. Due to absence or low selective pressure in a
specialised/localised niche, B. pseudomallei in central Sarawak may have lost its GEN
resistance trait over time. In addition to no recovery of environmental B. pseudomallei
in this present study, previous attempts to isolate B. pseudomallei from environmental
samples from central Sarawak had very low success (Inglis, 2002, Hassan et al.,
2004). As such, these scenarios provide compelling evidence of the existence of a
possibly localized source of environmental B. pseudomallei in central Sarawak yet to
be sampled.
Water as possible source of B. pseudomallei distribution
Since no B. pseudomallei was detected in soil samples that were collected from
various districts in Sarawak, one possible B. pseudomallei reservoir in the region is a
water source. Households in the region rely on natural water sources such as rivers,
streams and water springs, all of which are not chlorinated and filtered. Previous
studies have shown that B. pseudomallei was found in abundance in groundwater
seeps after an intense rainfall in Castle Hill of Townsville (Baker et al., 2011b) and
unchlorinated water supplies as sources of localized clonal outbreaks in several
communities in Australia (Inglis et al., 1999, Currie et al., 2001, Draper et al., 2010,
Hampton et al., 2011, Mayo et al., 2011, McRobb et al., 2013). In addition, high
recovery rates of B. pseudomallei from domestic water supplied have been reported in
studies in Australia and Thailand (Limmathurotsakul et al., 2013b, McRobb et al.,
2013). The water samples tested in this study were mostly collected from either rivers
and stagnant water used by the households or from water run-offs within the sampling
159
sites, where the collection of water samples was not done systematically. Although
the water samples collected from this study were negative for B. pseudomallei
isolates, one water sample grew other Burkholderia spp. isolate. Hence, a more
vigorous environmental sampling strategy including more extensive testing of water
samples is necessary to validate this hypothesis.
In conclusion, although the two hypotheses (antibiosis activities against B.
pseudomallei and lack of fitness of Sarawak GENs B. pseudomallei strains in the
environment) discussed were not fully supported or disproved by the experiments, my
results suggested a third hypothesis; the possibility of water as an environmental
reservoir. While it appears a plausible explanation for the lack of recovery of B.
pseudomallei from soil in Sarawak, further studies are required to support this
hypothesis.
160
Chapter 6
Concluding chapter: General discussions
and
Future directions
Chapter 6: Concluding chapter
6.1 General discussions
Melioidosis is endemic in Malaysia and was first documented in 1918 at the Institute
of Medical Research in Kuala Lumpur (Stanton and Fletcher, 1921, Stanton and
Fletcher, 1925). A previous study has suggested that Malaysian Borneo is the ‘hyperendemic’ foci for melioidosis in the country (Puthucheary 2001). Despite this
recognition, the magnitude of the epidemiology of melioidosis and B. pseudomallei in
Malaysian Borneo remains unclear. B. pseudomallei is a soil bacterium which
incidentally also infects humans and animals (Nandi et al., 2010). Understanding the
genotypic and phenotypic characteristics of B. pseudomallei strains that have caused
human disease is equally as important as investigating the ecology of B. pseudomallei
in communities that have been affected by melioidosis. Hence, this study had five
aims:

To characterize the phenotypic and molecular attributes of clinical B.
pseudomallei from Malaysian Borneo.

To elucidate the biochemical profiles of Malaysian Borneo B. pseudomallei
isolates in comparison with Top End isolates using VITEK 2 biochemical
automated system.

To determine the prevalence and mechanism of gentamicin and macrolide
susceptibility in B. pseudomallei isolates from Sarawak.

To determine if B. pseudomallei could be isolated from central Sarawak soil
samples.

To assess the relatedness between environmental B. pseudomallei isolates and
clinical isolates from the same region.
Genotyping of B. pseudomallei clinical isolates from Malaysian Borneo using various
methods discussed in Chapter 2 showed that Malaysian Borneo B. pseudomallei were
more related to other Southeast Asian strains than to Australian strains; consistent
with previous work (Cheng et al., 2004, Currie et al., 2007b, Tuanyok et al., 2007,
Sitthidet et al., 2008, Pearson et al., 2009). Isolates from Malaysian Borneo were
shown to all possess the YLF gene cluster and the bimABp variant, similar to Thai B.
161
pseudomallei (Tuanyok et al., 2007, Sitthidet et al., 2008). Consistent with the
observation by Pearson et al. that B. pseudomallei from the two sides of Wallace’s
line are different, MLST analyses showed that Malaysian Borneo isolates clustered
with other Southeast Asian strains and not with Australian strains (Pearson et al.,
2009). The analyses also suggested that lower diversity is observed in the Malaysian
Borneo population compared with the deeper gene pool observed in the presumably
more ancient Australian B. pseudomallei population, which has considerably more
diversity. The genetic diversity of B. pseudomallei from Sarawak was also shown to
be lower than that of Sabah, possibly due to a founder effect with radiation of only a
few clones when B. pseudomallei was first introduced to Borneo. I postulated that the
higher B. pseudomallei diversity observed in Sabah is potentially due to Sabah’s close
proximity to the islands of southern Philippines compared with Sarawak where inland
geographic isolation limits human movements. It is possible that individuals with
melioidosis were infected in southern Philippines and received treatment in Sabah.
MLST analysis on the only four B. pseudomallei isolates from the Philippines
revealed that two (ST57, ST99) out of the four were similar to isolates from Sabah
(ST58, ST232), sharing at least five of the seven loci interrogated in the analysis.
While MLST data from more isolates of the Philippines are needed to confirm the
human movement hypothesis, the preliminary data from this study suggest that this
hypothesis is plausible. MLVA-4 analyses showed some of the melioidosis cases in
Sabah and Sarawak were caused by clonal strains where cases were not necessarily
epidemiologically linked, suggesting a lower gene pool of B. pseudomallei in the
region compared to the Top End of the Northern Territory. Consistent with MLST
analysis, WGS results confirmed that Malaysian Borneo isolates were more closely
related to each other than to Australian strains. Genotyping of B. pseudomallei
isolates in this study has provided an insight into the molecular epidemiology and the
population structure of B. pseudomallei from Malaysian Borneo in relation with
strains from the rest of the world.
Drug susceptibility testing showed that 88% of collected B. pseudomallei from
Sarawak were susceptible to GEN and were associated with ST881 and its SLV,
ST997. As shown in Chapter 4, this study confirmed the mechanism to be a novel
non-synonymous mutation within amrB, an essential component of the AmrAB-OprA
multi-drug efflux pump. Subsequent reversion experiments demonstrated two ways by
162
which GENs B. pseudomallei ST881 was able to revert back to wild-type GENr; by
guanine to cytosine transition at amrB-1102 which result in R367T and guanine to
thymine transition at the same nucleotide position resulting in R367M. In addition to
restoring GEN resistance, these two reversions were also shown to restore resistance
to macrolides in ST881. B. pseudomallei is usually intrinsically GEN resistant which
results in GEN being used in different B. pseudomallei selective media worldwide
(Ashdown, 1979a, Ashdown, 1979b, Galimand and Dodin, 1982, Wuthiekanun et al.,
1990, Howard and Inglis, 2003, Francis et al., 2006). Hence, this discovery has
significant implications for clinical and laboratory diagnosis of melioidosis and
environmental sampling for B. pseudomallei, particularly in Malaysian Borneo but
also in other potentially endemic regions where B. pseudomallei has yet to be
uncovered.
In Chapter 3, biochemical testing using the VITEK 2 system showed that 25% of the
68 B. pseudomallei isolates tested were misidentified as B. cepacia. Two particular
enzymatic tests, NAGA and BNAG, were shown to be the main differential enzymes
responsible for the misidentification. These enzymes are reportedly associated with
the basic components of exopolysaccharide which have been suggested to be
associated with relative virulence of B. pseudomallei. Findings from this study have
raised awareness of continuing potential misidentification of B. pseudomallei as B.
cepacia by the VITEK 2 automated biochemical identification system, especially in
patients with suspected melioidosis acquired in exotic locations such as Malaysian
Borneo.
The relatedness between environmental B. pseudomallei isolates and clinical isolates
from Malaysian Borneo was addressed in Chapter 5. However, the primary aims
were not achieved because no B. pseudomallei was isolated from two environmental
sampling trips conducted in Sarawak. The first sampling trip coincided with the
discovery that the majority of clinical isolates from Sarawak were GENs. Based on
this, it was postulated that the use of gentamicin in selective media may have resulted
in the lack of B. pseudomallei isolation from the environment in Sarawak. Therefore,
gentamicin was replaced with colistin in selective media used in further culture efforts
for all environmental and clinical isolates from Sarawak in the second trip. However,
despite this modification still no B. pseudomallei was isolated.
163
Although no B. pseudomallei was isolated from Sarawak in this study, other
Burkholderia spp. such as B. thailandensis, B. ubonensis, B. pyrrocinia, B. cepacia. B.
multivorans and other untypable Burkholderia spp. were isolated. B. ubonensis has
been shown to have antibiosis activities against B. pseudomallei and B. thailandensis
has been reported to be inverse proportionate to B. pseudomallei in some environment
niches (Trakulsomboon et al., 1999, Warner et al., 2008, Kaestli et al., 2009, Marshall
et al., 2010). Building on this premise, I hypothesized that no B. pseudomallei was
isolated from the environmental sampling in Sarawak in part because of antagonistic
activities by other Burkholderia spp. against B. pseudomallei. In view of the
prevalence of GENs B. pseudomallei in clinical isolates collected from the
environmental sampling sites, I also postulated that GENs B. pseudomallei may be
less competitive compared with GENr B. pseudomallei in the environment and this
may also have contributed to no B. pseudomallei being isolated from the
environmental samples. I also proposed that there is possibly a localized niche (either
environmental or possibly even an animal reservoir) for B. pseudomallei in central
Sarawak separate from the sites sampled in this study, further contributing to why no
B. pseudomallei were isolated from the environmental samples I collected.
In summary therefore, three hypotheses were formulated to explain why no B.
pseudomallei was isolated from environmental samples collected from Sarawak
during this study; 1) Antagonistic activities by other Burkholderia spp. towards B.
pseudomallei, 2) GENs B. pseudomallei isolates from Sarawak are less robust than
GENr B. pseudomallei in competing with the organisms present in the environment, 3)
A localized niche of B. pseudomallei exists in central Sarawak such as water or
animals.
In testing the hypothesis of antibiosis activities of other Burkholderia spp. against B.
pseudomallei, experiments were conducted using a panel of B. pseudomallei and other
Burkholderia spp. isolates from Sarawak and the Top End. Unlike previous reports, B.
ubonensis and B. thailandensis isolates from Sarawak in this study did not show any
antibiosis activities against B. pseudomallei (Trakulsomboon et al., 1999, Marshall et
al., 2010). However, results also showed that there were antagonistic effects by other
untypable Burkholderia spp. towards B. pseudomallei. This study also suggested that
164
some of the Sarawak B. pseudomallei strains possessed bacteriophages that showed
lytic effects against some of the Burkholderia spp. tested. Hence, results from this
study were not conclusive enough to support the hypothesis of antibiosis activities of
other Burkholderia spp. against B. pseudomallei.
In order to address the second hypothesis stating a lack of competitiveness for GENs
B. pseudomallei, an experiment was performed to compare the competitiveness of
GENs and GENr B. pseudomallei in soil. A panel of GENs and GENr B. pseudomallei
isolates from Sarawak, Sabah and the Top End were inoculated into sterilized and
unsterilized soil samples. Inoculated soils were harvested and cultured 5 weeks and 14
weeks post-inoculation to understand the fitness of GENs B. pseudomallei strains in
the presence of other soil microorganisms. While Sarawak GENs B. pseudomallei
strains tested were shown to have a lower survival in the presence of other
microorganisms in the soil, the experiments did not address or explain the
unsuccessful isolation of GENr B. pseudomallei from the environmental samples in
Sarawak. Theoretically, GENr strains should be able to be isolated from Sarawak soil
samples since GENr clinical isolates were also isolated from patients in the region.
Hence, results suggested that the negative environmental B. pseudomallei recovery
from Sarawak may be due to several factors; a combination of very low prevalence of
GENr in the environment (as reflected in the clinical isolates), with the existence of a
localized source of dissemination for both GENs and GENr B. pseudomallei.
As proposed in Chapter 2, the GENs phenotype of B. pseudomallei from Sarawak
may be a result of their existence in an environment where selective pressure imposed
by the presence of aminoglycoside or macrolides is lacking. The inability to isolate B.
pseudomallei from the environment as discussed in Chapter 5 did not support this
theory since no B. pseudomallei was isolated despite no gentamicin was used in
culturing samples from the second trip. Hence, the most likely niche for B.
pseudomallei in Sarawak is either a host-adapted situation such as animal hosts, or a
specialised environmental niche such as water. Although the possibility of animal
reservoirs for B. pseudomallei in Sarawak was discussed, WGS analysis on GENs B.
pseudomallei (Chapter 2) did not suggest massive gene loss as shown in B. mallei
whose adaptation to an equine host had occurred over a long period of time (Moore et
al., 2004, Nierman et al., 2004, Losada et al., 2010). Animal melioidosis has been
165
reported previously in Sarawak (Sarawak Veterinary Department, unpublished data).
If proven, the host-adaption theory proposed in the current study seems to be a more
recent introduction due to the absence of large regions of gene loss and the low
genetic diversity between GENs isolates as discussed earlier.
The other possibility is water sources as environmental niches for B. pseudomallei.
Households in the central Sarawak rely on natural water sources such as rivers,
streams and water springs which are not chlorinated and filtered. Previous studies
have shown that B. pseudomallei was highly prevalent in groundwater seeps after an
intense rainfall (Baker et al., 2011b) and unchlorinated water supplies as sources of
localized clonal outbreaks in several communities in Australia (Inglis et al., 2011,
Currie et al., 2001, Draper et al., 2010, Hampton et al., 2011, Mayo et al., 2011,
McRobb et al., 2013). In this study however, the collection of water samples was not
done systematically and the few water samples tested in this study did not yield B.
pseudomallei. A more systematic water sampling strategy is certainly needed to
investigate this hypothesis. As such, the possibilities of both, GENs and GENr B.
pseudomallei being host-adapted or sharing the same environmental niche in Sarawak
are yet to be proven or disproved in this study.
In conclusion, knowledge gained from answering the research questions outlined in
this thesis has provided insight into the population structure of B. pseudomallei in
Malaysian Borneo and also contributed to current knowledge of the worldwide
population structure. This study has also proven the mechanism of gentamicin and
macrolide susceptibility in B. pseudomallei isolates in Sarawak which has significant
implications for laboratory diagnosis and environmental sampling of B. pseudomallei
in Malaysian Borneo and other potentially endemic regions where B. pseudomallei
has yet to be found. While the potential environmental hazards of B. pseudomallei in
Malaysian Borneo remains to be unravelled, this study has created more research
questions to be explored in the future.
166
6.2 Future directions
Findings from this study have set a path for several future research pursuits in
genomics, molecular epidemiology, biogeography and microcosm of B. pseudomallei
in Malaysian Borneo and the whole Southeast Asian region.
Further genomics analyses of GENs and mutants
WGS analyses in this study have shown that GENs B. pseudomallei isolates from
Sarawak are very closely related. While the mechanism of the GENs phenotype has
been attributed to a novel non-synonymous mutation within the amrB gene of the
AmrAB-OprA multi-drug efflux pump, further analyses are needed to investigate
whether there are other mutations that occurred in the GENs strains such as host
adaptations or genome decay as reported in B. mallei (Moore et al., 2004, Nierman et
al., 2004, Losada et al., 2010) and in isolates from chronic-carriage patients (Price et
al., 2013a). Similar analyses would also be useful for ST997 which is an SLV of
ST881 which also possesses the GENs phenotype. Although this study has proven that
GENs strain ST881 were able to restore gentamicin resistance via two different
nucleotide substitutions, the study has also revealed that the majority of the reverted
mutants (from GENs to GENr) were unchanged at amrB-1102. This suggests that
multiple mechanisms exist for acquiring GENr in B. pseudomallei. It would be
worthwhile to study the mechanisms which conferred aminoglycoside and macrolide
resistance of the remaining mutants that were not further characterized in this study.
Bacteriocin compounds against B. pseudomallei and bacteriophages of B.
pseudomallei
In the experiment to determine antagonistic effect of Burkholderia spp. against B.
pseudomallei, several unidentified Burkholderia spp. isolates from Sarawak were
shown to possess antibiosis elements against B. pseudomallei. The speciation of the
Burkholderia spp. that possessed antibiosis activities against B. pseudomallei and
identification of the antagonistic substance released by these bacteria are worth
pursuing. It has been suggested that bacteriocin compounds released by Burkholderia
spp. could be used as biocontrol against B. pseudomallei in melioidosis endemic
regions (Marshall et al., 2010). In addition to focusing on B. pseudomallei, there
should also be further studies on the ecological niche of Burkholderia spp. in
167
melioidosis endemic areas in Borneo to understand the interactions between B.
pseudomallei and other Burkholderia spp.
Findings from this study also suggested that some of the Sarawak B. pseudomallei
possessed bacteriophages which should be identified. Studies have shown that
bacteriophages can potentially be used in the treatment of Bcc respiratory infections in
cystic fibrosis patients particularly in antibiotic resistance cases (Carmody et al.,
2010, Golshahi et al., 2011, Semler et al., 2011). Hence, it may be useful in
understanding the roles of bacteriophages in B. pseudomallei such as organism
virulence, survivability in the environment, host-adaptation and intracellular survival
(Ronning et al., 2010).
Specific niche for B. pseudomallei in central Sarawak
As postulated in this study, water is a possible environmental niche for B.
pseudomallei in central Sarawak since no B. pseudomallei was isolated from soil
samples collected in this study. Future work should focus on more vigorous water
sampling strategies in the region instead of solely on soil. In order to address the
concern of the timeliness of water samples getting transported back into the laboratory
for further analysis, new sampling method should be employed. In a pilot study in
Laos, B. pseudomallei sampling from surface water was done using Moore swabs
which were found to be suitable for sampling large volumes of water and were
effective for detection of B. pseudomallei if the contamination is transient or in
flowing water with expected low bacterial density (Vongphayloth et al., 2012). A
similar strategy can be adopted in water sampling in Sarawak since this method has
been shown to be simple and cost-effective.
Animal host adaptation was postulated as a possible niche for B. pseudomallei in
Sarawak and this hypothesis was not supported or refuted by this study. Further work
is certainly needed including the analysis of the GENs B. pseudomallei genomes to
investigate whether there are other mutations that occurred in these strains that
contribute to host adaptations or genome decay as seen in B. mallei upon adaptation to
its mammalian hosts (Moore et al., 2004, Nierman et al., 2004, Losada et al., 2010). B.
mallei has been shown to have evolved from a single strain of B. pseudomallei
ancestor through erosion of nonessential genomes responsible for environmental
168
survival and has therefore become completely host-adapted while causing glanders in
equids (Moore et al., 2004, Nierman et al., 2004, Losada et al., 2010). It was also
shown that the genome of B. mallei is approximately 1.5 Mbp smaller than B.
pseudomallei (Godoy et al., 2003, Nierman et al., 2004, Losada et al., 2010). Hence,
the possibility of animals being contributors to the dispersal of B. pseudomallei in the
environment of Malaysian Borneo is not discounted.
Previous reports on animal melioidosis have been sporadic and most cases involved
goats and primates in Sarawak (Sarawak Veterinary Department, unpublished data).
Rural communities in central Sarawak have been known to keep livestock such as
chickens and pigs, and melioidosis has been reported previously in birds (Thomas et
al., 1980, Low Choy et al., 2000, Hampton et al., 2011) and pigs (Ketterer et al., 1986,
Dance, 1991, Mustaffa Babjee and Nor Aidah, 1994). In addition, rodents which live
in close proximity to humans have a long history of association with melioidosis
(Whitmore, 1913, Stanton and Fletcher, 1925). It would be worthwhile to attempt to
isolate B. pseudomallei from rectal swabs and faecal samples from animals found
within these communities in central Sarawak. Animal droppings have been suggested
to play a role in the dispersal of B. pseudomallei in the environment (Kaestli et al.,
2012b), and the bacteria have been detected in scats from marsupials (Kaestli et al.,
2012b) and B. pseudomallei has been isolated from the large intestine of a goat
(Shanta, 1960). A study has also shown that B. pseudomallei preferentially colonizes
the gastrointestinal tract of orally inoculated mice which likely serves as a reservoir
for dissemination of infection to extra-intestinal sites (Goodyear et al., 2012). Thus,
apart from being the least invasive sample collection method, rectal swabs and faecal
samples would be the most suitable samples to collect in elucidating the carriage of B.
pseudomallei in animals.
In addition to domesticated animals, the possibility of wildlife as hosts for B.
pseudomallei should also be explored. Increased human encroachment, dam
constructions, logging and oil-palm plantations over the past two decades have
resulted in massive reduction and destruction of wildlife habitats in Malaysian
Borneo. Most of the surviving wildlife is confined to fragmented forests that are less
than sufficient in sustaining their food needs (Caldecott, 1988, Goossens et al., 2005).
Due to limited food sources, it is common for some wildlife to forage on crops in
169
farms that are within proximity of these fragmented forests (Hambali et al., 2012).
This results in increased interaction between humans and wildlife (such as wild boars,
primates) which may have also affected the dispersal of B. pseudomallei in the area.
Wild boars which have been considered as food source as well as crop pests, have
been hunted widely by the communities of central Sarawak and the seasonal
migration of wild boars has been documented in the region (Caldecott 1988).
Although melioidosis cases have been documented in domesticated pigs (Ketterer et
al., 1986, Dance, 1991, Mustaffa Babjee and Nor Aidah, 1994), no melioidosis in wild
boars has been reported thus far. Other than wild boars, the distribution of primates
has also been affected by the change in the ecology. There are up to 10 species of
primates such as orang-utans, gibbons, macaques, monkeys, langurs, tarsiers and loris
in Malaysian Borneo where their distribution is dependent on their diets at the
different natural habitat types such as mangrove forest, freshwater swamps, peat
swamp forest, tropical evergreen forest, and riverine forest, and also on the seasons of
the different fruits in these habitats (Meijaard and Nijman, 2003). Due to these
ecological changes and conservation efforts by the government, the orang-utans have
been living in fragmented forests that have since been gazetted as their sanctuaries
(Goossens et al., 2005). Melioidosis has been documented in orang-utans and gibbons
in semi-wildlife sanctuaries in Malaysian Borneo (de Silva, 1971, Peters et al., 1976,
Ouadah et al., 2007)(Sarawak Veterinary Department, unpublished data), and wildcaught macaques possibly from Indonesia (Dance et al., 1992, Ritter et al., 2013).
Sampling of wildlife may be another possible study to investigate the role of animals
in the dissemination of B. pseudomallei. However, it can be foreseen that sample
collection from wildlife would be logistically challenging due to accessibility of their
habitats and also because some of the species are highly endangered. Hence,
coordination with the conservation agencies within the region will be helpful in
identifying the habitats of the different wildlife, in collecting the relevant samples
from the animals and also in handling the animals if necessary.
In addition to collecting samples from non-symptomatic or healthy animals to
understand B. pseudomallei carriage in animals, it would also be worthwhile to collect
and characterize B. pseudomallei isolates from clinically ill animals. Although reports
of animal melioidosis have been sporadic, it does not mean it is not common in
170
Malaysian Borneo. Rather, it may reflect a combination of a low level of suspicion of
melioidosis among veterinarians and animal owners in the region, and that
melioidosis cases that occurred in wildlife would be undetected. Thus, for better
coverage of animal cases, coordination with the veterinary department and private
veterinary practices is crucial in ensuring quality samples are collected from
veterinary cases for bacterial culture and good clinical data are available for
epidemiological analysis.
Molecular epidemiology of B. pseudomallei in the Borneo and Southeast Asia
While this study has unravelled the molecular epidemiology of B. pseudomallei from
Malaysian Borneo in relation to isolates collected worldwide by using MLST, the
question remains whether the inclusion of more isolates from Malaysian Borneo and
Peninsular Malaysia would provide a better insight into the population structure of B.
pseudomallei in the region. The majority of isolates from Peninsular Malaysia
currently on the MLST database were from historical isolates collected in the 1960s
(McCombie et al., 2006). Hence, analysis of more B. pseudomallei isolates from
Peninsular Malaysia will add more value to the current data available.
Furthermore, determining the geographic attribution of B. pseudomallei isolates from
Brunei Darussalam and Kalimantan located in Indonesian Borneo using genotyping
methods (i.e. BTFC/YLF cluster assay, MLST) would add further value in depicting
the population structure of B. pseudomallei in the region. Melioidosis cases have been
documented in Brunei Darussalam (Luqman et al., 1999, Chong et al., 2010, Lim and
Chong, 2010, Pande and Kadir, 2011), a country with an estimated population of
390,000 in an area of 5,756 km2 divided into two pieces of land surrounded by
northern Sarawak (http://www.brunei.org.au/bruneidarussalam.php). Studies at a
tertiary hospital in the country have shown that most melioidosis cases had multiorgan involvement and relapse cases were common (Chong et al., 2010, Lim and
Chong, 2010, Pande and Kadir, 2011). Human movements between Brunei
Darussalam, Sabah and Sarawak are high because land travellers to-and-fro Sarawak
and Sabah need to cross the two pieces of lands belonging to the Brunei Darussalam
kingdom and Bruneians often cross the border to Malaysian Borneo for recreation.
Hence, the question remains whether there are similarities between the B.
pseudomallei strains found in Brunei Darussalam and Malaysian Borneo. No MLST
171
data on B. pseudomallei isolates from Brunei Darussalam has been available thus far
(http://bpseudomallei.mlst.net/ - Accessed on 12 February 2014).
Malaysian Borneo especially Sarawak shares a long and porous border with West
Kalimantan of Indonesia. Other than the initial historical Dutch literature of
melioidosis in Indonesia (Snijders, 1933), reports of melioidosis cases in the country
have been sporadic thus far involving several cases of exposure to B. pseudomallei
during the tsunami in Aceh (Athan et al., 2005, Othman et al., 2007, Arzola et al.,
2007), individuals who may have been infected while travelling in Indonesia (Beeker
et al., 1999, Schindler et al., 2002, Arzola et al., 2007) and in animals imported from
the country (Dance et al., 1992, Ritter et al., 2013). The only documentation of
melioidosis in Indonesian Borneo thus far involved an American traveller who was
supposedly exposed to B. pseudomallei while he was in Kalimantan (Schindler et al.,
2002). Other than these reports, there has not been any other documentation of
melioidosis being diagnosed in Indonesia including Kalimantan. This may be
attributed to possibly low and geographically restricted presence of B. pseudomallei
combined with a low identification due to limited diagnostic resources (Currie et al.,
2008). This raises the question of the magnitude of melioidosis in Kalimantan. The
discovery that clinical B. pseudomallei isolates in central Sarawak are predominantly
GENs in this study and that central Sarawak is close to the border to Kalimantan
(albeit separated by a mountain range) beg the questions of whether GENs B.
pseudomallei is as common across the border and whether environmental B.
pseudomallei in West Kalimantan may be as elusive as their counterparts in Sarawak.
As discussed earlier, B. pseudomallei isolates from the Philippines seem to have
genetic and epidemiological links to isolates from Sabah. Currently, there are not
many reports of melioidosis in the Philippines where most reports involved imported
animals and also cases of travellers returning from the Philippines (John, 1976, Fuller
et al., 1978, Dance et al., 1992, Turner et al., 1994, Duplessis and Maguire, 2009).
Similar to the melioidosis situation in Indonesia; it is possible that the low reported
numbers are due to limited diagnostic resources which result in low identification of
cases of melioidosis. Hence, more vigorous collection of epidemiological data of
melioidosis patients in Sabah is needed to establish whether some may have been
travelling to southern Philippines prior to presenting with symptoms. Genotyping of
172
isolates from these patients will also provide insight into the hypothesis that B.
pseudomallei from Sabah may be similar to those from the Philippines.
In conclusion, while these studies have answered many of the research questions
regarding the molecular epidemiology and biogeography of B. pseudomallei in
Malaysian Borneo that were proposed initially, directions were modified in light of
some of the unexpected early findings and more studies are now required to pursue
new hypotheses resulting from this thesis.
173
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APPENDIX A:
Summary of Research permits and ethics approval for melioidosis study in
Malaysian Borneo
Agency
Administrative level
The Economic Planning Unit, the Prime Minister’s
Department of Malaysia
Malaysian
Government
Ministry of Health of Malaysia
o National Medical Research Register (NMRR)
o Medical Research Ethics Committee (MREC)
o National Institute of Health (NIH)
Malaysian
Government
o
Sarawak Health Department
Kapit Hospital, Sarawak
State Government of
Sarawak
State Government of
Sarawak
State Government of
Sarawak
District of Kapit
Sibu Hospital, Sarawak
District of Sibu
Sarawak Veterinary Department
Sarawak Biodiversity Centre, Sarawak State Government
Bintulu Hospital, Sarawak
District of Bintulu
Miri Hospital, Sarawak
District of Miri
Queen Elizabeth Hospital, Sabah
Centre of Disease Control, Ministry of Health of Malaysia –
For exportation of infectious materials
Australian Quarantine and Inspection Service (AQIS),
Department of Agriculture, Fishes and Forestry – For
importation of infectious materials
205
State Government of
Sabah
Malaysian
Government
Australian
Government
APPENDIX B:
Soil sampling form for B. pseudomallei
Study form – Burkholderia pseudomallei soil sampling
Date (Day, Month, Year)
Study No's
m
m
y
_____ to
_____
y
Area
General location parameters
GIS (codes)
Address
Diagram of place + collection sites
General comments to place
Pics taken?
Any waterlogging < ~25m?
If yes, where?
Pic of hole+soil/veg
+ study number
Pic of area:
1(yes), 0 (no), 2 (in wetseason), 3 (don’t know)
where?
Distance to stream / run off
1(<10m), 2 (10-50m), 3 (50-100m), 4
(>100m), 0 (don't know)
comments
Sun accessibility
0(shade most of day),
1(shade half a day), 2(in full sun)
comments
Vegetation around site
1 (grass), 2 (low bush), 3 (crops),
4 (single trees), 5 (open forest),
6 (forest), 7 (other)
comments
206
APPENDIX B (continued)
In Slope? / steepness?
1(flat)
2
3
0 (flat), 1(slightly rising), 2 (steep)
4
Signs of environmental change?
any obvious irrigation?
1(yes), 0 (no), 3 (don’t know)
where?
specify
Used regularly by humans?
Used regularly by animals?
what animals?
Soil sample parameters
Depths of samples (cm)
A: shallow
B: deeper
Soil Water Status
0 (dry), 1 (moderately moist),
2 (moist),
3 (wet), 4 (soaked)
Soil Texture
1(sand), 2(sandy loam),
3(loam), 4(sandy clay loam),
5(clay), 6(organic material),
7(gravels), 8(roots rich),
9(other), 10 (sandy clay),
11(clay loam)
(in lab, use soil texture chart)
Color of moist soil
1 (white), 2 (grey), 3 (yellow),
4 (pale red), 5 (dark red),
6 (pale brown), 7(dark brown),
8 (black),
(in lab, use Munsell
Soil Color Handbook)
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
207
APPENDIX C: PUBLISHED PAPERS FROM THIS THESIS
1.
PODIN, Y., KAESTLI, M., MCMAHON, N., HENNESSY, J., NGIAN, H. U.,
WONG, J. S., MOHANA, A., WONG, S. C., WILLIAM, T., MAYO, M.,
BAIRD, R. W. & CURRIE, B. J. 2013a. Reliability of automated biochemical
identification of Burkholderia pseudomallei is regionally dependent. J Clin
Microbiol, 51, 3076-3078.
2.
PODIN, Y., SAROVICH, D. S., PRICE, E. P., KAESTLI, M., MAYO, M., HII,
K., NGIAN, H., WONG, S., WONG, I., WONG, J., MOHAN, A., OOI, M.,
FAM, T., TUANYOK, A., KEIM, P., GIFFARD, P. M. & CURRIE, B. J. 2014.
Burkholderia pseudomallei isolates from Sarawak, Malaysian Borneo are
predominantly susceptible to aminoglycosides and macrolides. Antimicrob Agents
Chemother, 58, 162-166.
208
209
210
211
212
213
214
215
216

Podin-PhD thesis - CDU eSpace

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