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UNIVERSITI PUTRA MALAYSIA
PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF GARDNERELLA VAGINALIS ISOLATED FROM WOMEN WITH BACTERIAL VAGINOSIS
FROM HOSPITAL KUALA LUMPUR, MALAYSIA
NADA KHAIRI YOUNUS AL-ASSADI
FPSK(m) 2013 13
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PHENOTYPIC AND GENOTYPIC CHARACTERIZATION
OF GARDNERELLA VAGINALIS ISOLATED FROM
WOMEN WITH BACTERIAL VAGINOSIS FROM
HOSPITAL KUALA LUMPUR, MALAYSIA
NADA KHAIRI YOUNUS AL-ASSADI
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
2013
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COPYRIGHT
All material contained within the thesis, including without limitation text,
logos, icons, photographs and all other artwork, is copyright material of
Universiti Putra Malaysia unless otherwise stated. Use may be made of
any material contained within the thesis for non-commercial purposes
from the copyright holder. Commercial use of material may only be made
with the express, prior, written permission of Universiti Putra Malaysia.
Copyright © Universiti Putra Malaysia
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PHENOTYPIC AND GENOTYPIC CHARACTERIZATION
OF GARDNERELLA VAGINALIS ISOLATED FROM
WOMEN WITH BACTERIAL VAGINOSIS FROM
HOSPITAL KUALA LUMPUR, MALAYSIA
By
NADA KHAIRI YOUNUS AL-ASSADI
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in fulfillment of the Requirements for the Degree of Master of Science
June 2013
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In the Name of God the Compassionate the Merciful
Dedicated to my loving family, my parents and sister Watffa for their unending
love, invaluable support and extraordinary courage
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Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfillment
of the requirements for the degree of Master of Science
PHENOTYPIC AND GENOTYPIC CHARACTERIZATION
OF GARDNERELLA VAGINALIS ISOLATED FROM
WOMEN WITH BACTERIAL VAGINOSIS FROM
HOSPITAL KUALA LUMPUR, MALAYSIA
By
NADA KHAIRI YOUNUS AL-ASSADI
June 2013
Chairman: Assoc. Prof. Vasanthakumari Neela, PhD
Faculty: Medicine and Health Sciences
Bacterial vaginosis (BV) is a highly prevalent, but poorly understood polymicrobial
disorder of the human vaginal microbiota. Symptoms for BV include vaginal discharge,
itching and odor. Gardnerella vaginalis which is commonly found in the vaginal mucosa
of asymptomatic women is the frequent cause of BV. BV microbiota although
dominated by G. vaginalis, it also includes a number of anaerobes. The main challenge
in the diagnosis of BV is the identification of the correct etiological agents.
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Bacterial vaginosis if untreated leads to chronic infectious/inflammatory disease with
multiple reproductive tract complications including preterm birth, neonatal infections
and suppurative lesions. Treatment options as recommended by Center for Disease
Control (CDC) include oral or intravaginal administration of metronidazole or
clindamycin. Despite treatment with these antibiotics, treatment failure and recurrent
infection rates are still rising.
Therefore, this study is aimed at identifying methods for conclusive diagnosis of BV,
and corrects laboratory techniques for isolation of G. vaginalis bacterium. Also this
study is shed the light on determining the susceptibility of G. vaginalis strains against
recommended antibiotics and determining the phenotypes and genotypes characteristics
of this bacterium with its virulence properties.
Two-hundred and seven vaginal swabs were collected from patients‟ attending the
gynecological clinic at Hospital Kuala Lumpur (HKL), Malaysia from 16th
February to
20th July 2012 and transported to the Medical Microbiology Laboratory, Universiti Putra
Malaysia. One hundred and sixty (77.2%) out of 207 samples were recorded as
symptomatic biased on clinical observation and due to the presence of vaginal discharge,
while the remaining 47 (22.7%) were recorded as asymptomatic as they did not show
any vaginal discharge.
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A patient is diagnosed for BV based on the Amsel-clinical criteria and Nugent-Gram
staining. In the present study, comparison of Amsel-clinical criteria and Nugent-Gram
stain in the diagnosis of BV showed the later to be more sensitive (91%) than the former
(53%). Among the 160 samples, 47 (73.4%) were confirmed as G. vaginalis positive by
phenotypic (culture, Gram stain and biochemical) and genotypic (16S rRNA) methods.
Metronidazole (MTZ), which is a broad spectrum antibiotic against candidiasis and
giardiasis is given as the first choice of drug for the treatment of BV. Among the studied
47 isolates, three showed resistance to MTZ and carried the nim gene which mediates
MTZ resistance.
A simple and reproducible scheme for identifying biotypes of G. vaginalis identified
eight biotypes based on reaction for hippurate hydrolase, ONPG test and lipase test.
Among the isolates studied, biotype 1 was the most prevalent (18/47 or 38%), followed
by biotype 7 (17/47 or 36%), biotype 5 (8/47 or 17%), and finally biotype 8 (4/47 or
8.5%). Biotypes 2, 3, 4 and 6 were not observed. The biotypes did not show any
significant association with the clinical reasons (abortion, minor surgery and vaginal
fluid) for suspecting BV.
Genotyping of the microbial pathogens is important in the management of infections as
it gives useful information about the spread of pathogens. In addition, monitoring of
endemic pathogens determines the adequacy of the general hygienic measures
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performed. All 47 G. vaginalis isolates when genotyped by amplified ribosomal DNA
restriction analysis (ARDRA) produced three different genotypes (I, II and III). Similar
to the biotypes, the genotypes also did not show any significant association with clinical
data. Twelve (25%) isolates belonged to genotype I, 22 (46%) isolates to genotype II
and 13 (27.6%) to genotype III. Isolates that were biotype 7 matched well with genotype
II. G. vaginalis isolates when further screened for virulence factors, such as vaginolysin
(vly, gene that mediate pore formation and cytotoxicity) and sialidase gene (mediate
biofilm production), it was found that 25 (53%) and 26 (55%) carried sialidase and vly
gene respectively. All G. vaginalis isolates of genotype I harbor the sialidase gene 12
(25.5%), and contain also vly gene 11 (23.4%). This result suggest of recording these
strains as a virulent strains not as a commensally strains.
In conclusion, characterization of G. vaginalis isolated from patients with BV revealed
that for the accurate identification of G. vaginalis associated BV, combination of
phenotypic and genotypic methods should be implemented in the routine diagnostic
laboratory. As the G. vaginalis strains are highly diverse, susceptibilities towards
recommended antibiotics are decreasing and frequently carry virulent and resistant genes,
routine monitoring of the G. vaginalis important for the efficient management of bacterial
vaginosis infection.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk ijazah Master Sains
FENOTIP DAN PENCIRIAN GENOTIP VAGINALIS GARDNERELLA
TERPINGGIR DARI WANITA DENGAN VAGINOSIS BAKTERIA
DARI HOSPITAL KUALA LUMPUR, MALAYSIA
Oleh
NADA KHAIRI YOUNUS AL-ASSADI
Jun 2013
Pengerusi: Profesor Madya Vasanthakumari Neela, PhD
Fakulti: Perubatan dan Sains Kesihatan
Vaginosis bakteria (BV) adalah gangguan yang sangat berleluasa, tetapi kurang difahami
polymicrobial daripada microbiota faraj manusia. Gejala untuk BV termasuk keputihan,
gatal-gatal dan bau. Gardnerella vaginalis yang biasanya ditemui di mukosa faraj wanita
asimptomatik adalah punca kerap BV. Vaginosis bakteria microbiota walaupun dikuasai
oleh G. vaginalis, ia juga termasuk beberapa anaerobes. Cabaran utama dalam diagnosis
BV adalah pengenalan ejen etiological betul.
Vaginosis bakteria jika tidak dirawat membawa kepada penyakit kronik
berjangkit/radang dengan saluran pembiakan pelbagai komplikasi termasuk kelahiran
pramatang, jangkitan neonatal dan luka-luka bernanah. Pilihan rawatan seperti yang
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disyorkan oleh Pusat Kawalan Penyakit (CDC) termasuk pentadbiran lisan atau
intravaginal metronidazole atau clindamycin. Walaupun rawatan dengan antibiotik,
kegagalan rawatan dan kadar jangkitan berulang masih meningkat.
Oleh itu, kajian ini bertujuan untuk mengenal pasti kaedah untuk diagnosis muktamad
G. vaginalis menyebabkan BV, faktor risiko, kecenderungan G. vaginalis terhadap
antibiotik yang disyorkan, menentukan fenotip dan genotip dan harta kebisaan.
Dua-ratus dan tujuh swab faraj dikumpulkan daripada 'pesakit menghadiri klinik sakit
puan di Hospital Kuala Lumpur (HKL), Malaysia dari 16 Februari hingga 20 Julai 2012
dan diangkut ke Makmal Mikrobiologi Perubatan, Universiti Putra Malaysia. Seratus
enam puluh (77.2%) daripada 207 sampel telah direkodkan sebagai gejala akibat
kehadiran keputihan, manakala 47 selebihnya (22.7%) telah direkodkan sebagai
asimptomatik kerana mereka tidak menunjukkan sebarang keputihan.
Seorang pesakit didiagnosis untuk BV berdasarkan kriteria Amsel klinikal dan
pewarnaan Gram Nugent. Dalam kajian ini, perbandingan kriteria klinikal Amsel dan
Nugent-Gram noda dalam diagnosis BV menunjukkan kemudian untuk menjadi lebih
sensitif (91%) daripada bekas (53%). Antara 160 sampel, 47 (73.4%) telah disahkan
sebagai G. vaginalis positif dengan kaedah fenotipik (kultur, pewarnaan Gram dan
biokimia) dan genotip (16S rRNA).
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Metronidazole, yang adalah antibiotik spektrum luas terhadap kandidiasis dan
Giardiasis, diberikan sebagai pilihan pertama dadah untuk rawatan BV. Antara 47
pencilan dikaji, tiga menunjukkan kerintangan metronidazole dan dijalankan gen nim
yang pengantara rintangan metronidazole.
Satu skim yang mudah dan diulang untuk mengenal pasti miang daripada G. vaginalis
mengenal pasti lapan miang berdasarkan reaksi untuk hippurate hydrolase, ONPG ujian
dan ujian lipase. Antara pencilan yang dikaji, biotype 1 adalah 8 yang paling lazim
(18/47 atau 38%), diikuti oleh biotype 7 (17/47 atau 36%), biotype 5 (8/47 atau 17%),
dan akhirnya biotype (4 / 47 atau 8.5%). Miang 2,3,4 dan 6 tidak dipatuhi. Miang tidak
menunjukkan apa-apa hubungan yang signifikan dengan sebab klinikal (pengguguran,
pembedahan kecil dan cecair faraj) untuk mengesyaki BV.
Mengenal pasti genotip daripada mikrob patogen adalah penting dalam pengurusan
jangkitan kerana ia memberikan maklumat yang berguna tentang penyebaran patogen.
Di samping itu, pemantauan patogen endemik menentukan kecukupan langkah-langkah
kebersihan umum dilakukan. Semua 47 pencilan untuk G. vaginalis apabila
digenotaipkan oleh dikuatkan DNA ribosomal sekatan analisis (ARDRA) menghasilkan
tiga genotip berbeza (I, II dan III). Seperti kepada miang, genotip juga tidak
menunjukkan apa-apa hubungan yang signifikan dengan data klinikal. Dua belas (25%)
mengasingkan dipunyai oleh genotip saya, 22 (46%) diasingkan untuk genotip II dan 13
(27.6%) untuk genotip III. Pencilan yang biotype 7 dipadankan dengan II genotip.
G. vaginalis diasingkan apabila terus disaring untuk faktor kebisaan, seperti vaginolysin
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(vly, gen bahawa pembentukan liang Mediate dan cytotoxicity) dan sialidase gen
(Mediate biofilm pengeluaran) , ia telah mendapati bahawa 25 (53%) dan 26 (55%)
dibawa sialidase dan gen vly. Tiada pencilan daripada II genotip terkandung gen
sialidase. Begitu juga, majoriti pencilan daripada II genotip adalah negatif gen
vaginolysin.
Kesimpulannya, pencirian G. vaginalis diasingkan daripada pesakit dengan BV
mendedahkan bahawa untuk mengenal pasti yang tepat G. vaginalis bersekutu BV,
gabungan kaedah fenotip dan genotip perlu dilaksanakan di dalam makmal diagnostik
rutin.
Sebagai strain G. vaginalis adalah sangat pelbagai, mudah dipengaruhi ke arah antibiotik
yang disyorkan semakin berkurangan dan sering membawa getir dan tahan gen,
pemantauan rutin G. vaginalis penting bagi pengurusan yang cekap jangkitan bakteria
vaginosis.
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ACKNOWLEDGEMENTS
First and foremost, I would like to express my gratitude to my beloved father Sir
Khairi Younus Al-Assadi and to my mother Hind Ahmed AL-Hafid and to my sisters
and brother for extending their great support, encouragement and blessing throughout
my life.
I also take this opportunity to express a deep sense of gratitude to my husband
Dr. Ihsan Muneer Ahmed for his cordial support, valuable information and guidance,
which helped me in completing this task through various stages. And to my two
daughters Sara, Aya and to my lovely son Abdul-Rahman.
I take this opportunity to express my profound gratitude and deep regards to my guide
(Associate professor, Vasanthakumari Neela) for her exemplary guidance, monitoring
and constant encouragement throughout the course of this thesis. My sincere gratitude to
my co-supervisor, Dr. Renu Gopinath, Dr. Syafinaz Amin Nordin and Dato Dr.
Ravindran Jegasothy. I also like to thank Dr. Zulida Rejali for translating this thesis
abstract to Malay language.
My special thanks to my special friends Shakira, liew, Hamed, Shahram, Seenu and
Mohamed Fahmi for their valuable support. Lastly, I thank almighty, my friends
Dr. Yassamin Sahib and Zena for their constant encouragement without which this
assignment would not be possible.
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I certify that a Thesis Examination Committee has met on 11.6.2013 to conduct the final
examination of Nada Khairi Younus on her thesis entitled “Phenotypic and Genotypic
Characterization of Gardnerella vaginalis Isolated from Women with Bacterial
Vaginosis from Hospital Kuala Lumpur” in accordance with the Universities and
University College Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A)
106] 15 March 1998. The committee recommends that the student be awarded the Master of
Science.
Members of the Thesis Examination Committee were as follows:
Chairman, PhD
Professor
Name of faculty/ institute
Universiti Putra Malaysia
(Chairman)
Examiner1, PhD
Professor
Name of faculty/ institute
Universiti Putra Malaysia
(Internal Examiner)
Examiner2, PhD
Professor
Name of faculty/ institute
Universiti Putra Malaysia
(Internal Examiner)
External Examiner, PhD
Professor
Name of faculty/ institute
Universiti Putra Malaysia
(External Examiner)
PROF.DR. SEOW HENG FONG, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the senate of Universiti Putra Malaysia and has been
accepted as fulfillment of requirement for degree of Master of Science. The members of
the Supervisory Committee were as follows:
VasanthaKumari Neela, PhD
Associate professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Renu Gopinath, PhD
Senior medical lecturer
Department of Obstetrics and Gynecology
Hospital Kuala Lumpur (HKL)
(Member)
Syafinaz Amin Nordin, PhD
Senior medical lecturer
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Dato Ravendran Jegasothy, PhD
Head and senior consultant,
Department of Obstetrics and Gynecology
Hospital Kuala Lumpur (HKL)
(Member)
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for quotations and citation which
have been duly acknowledged. I also declare that it has not been previously and is not
concurrently, submitted for other degree at Universiti Putra Malaysia or other
institutions.
NADA KHAIRI YOUNUS AL-ASSADI
Date: 11th
June 2013
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TABLE OF CONTENTS
Page
ABSTRACT iii
ABSTRAK vii
ACKNOWLEDGEMENT xi
APPROVAL xii
DECLARATION xiv
LIST OF TABLES xix
LIST OF FIGURES xx
LIST OF ABBREVIATIONS xxii
CHAPTER
1 INTRODUCTION 1
1.1 Problem statement 4
1.2 The objectives of this study 5
2 LITERATURE REVIEW 6
2.1 History on the nomenclature 6
2.2 Taxonomy of Gardnerella vaginalis 8
2.3 Prevalence of Gardnerella vaginalis 10
2.4 Pathogenesis and clinical presentation 11
2.5 Risk factors for bacterial vaginosis 12
2.6 Diagnosis of bacterial vaginosis 13
2.6.1 Amsel-clinical criteria 13
2.6.2 Nugent-Gram stain 13
2.6.3 Statistical analysis 15
2.7 Bacteriological characteristics of Gardnerella vaginalis 16
2.7.1 Morphologic characteristics 16
2.7.2 Culture media and colony characteristics 16
2.7.3 Biochemical characteristics 18
2.7.4 Molecular identification 19
2.7.5 Antimicrobial susceptibility 20
2.7.6 Biotyping scheme 22
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2.7.7.Genotyping by amplified ribosomal DNA restriction analysis
(ARDRA) 24
2.7.8 Virulence factors 25
3 MATERIALS AND METHODS 26
3.1 Clinical samples 26
3.2 Study 1: To determine the prevalence of Gardnerella vaginalis isolated
from women with bacterial vaginosis from Hospital Kuala Lumpur
(HKL) 27
3.2.1 Diagnosis of bacterial vaginosis 27
3.2.1.1 Amsel-clinical criteria (wet mount) 27
3.2.1.2 Nugent-Gram stain 28
3.2.1.3 Statistical analysis 31
3.3 Bacteriological characteristics of Gardnerella vaginalis 32
3.3.1 Culture media and colony characteristics 32
3.3.2 Biochemical characteristics 32
3.4 Molecular identification 33
3.4.1 DNA preparation (extraction) 33
3.4 2 PCR for Gardnerella vaginalis 16S rRNA gene 35
3.4.3 Agarose gel electrophoresis of DNA 36
3.4.4 DNA purification 37
3.4.5 DNA sequencing 38
3.5 Study 2: To determine the prevalence of metronidazole resistance
Gardnerella vaginalis strains 39
3.5.1 Antibiotic susceptibility test (AST) 39
3.5.2 Polymerase chain reaction to detect nim gene resistant to
metronidazole 40
3.6 Study 3: To determine the biotypic characteristics of
Gardnerella vaginalis among the clinical isolates 41
3.6.1 Hippurate hydrolysis test 42
3.6.2 O-nitrophenol-β-d-galactopyranoside (ONPG) 42
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3.6.3 Lipase test 43
3.7 Study 4:.To determine the genotypic characteristics of Gardnerella
vaginalis among the clinical isolates 44
3.8 Study 5: To assess the distribution and presence of virulence factors of
Gardnerella vaginalis 46
4 RESULTS 48
4.1 Clinical samples 48
4.2 Study 1: To determine the prevalence of Gardnerella vaginalis isolated
from women with bacterial vaginosis from Hospital Kuala Lumpur 48
4.2.1 Diagnosis of bacterial vaginosis 49
4.2.1.1 Amsel-clinical criteria (wet mount) 49
4.2.1.2 Nugent-Gram stain 50
4.2.1.3 Statistical analysis 51
4.3 Bacteriological characteristics of Gardnerella vaginalis 53
4.3.1 Culture media and colony characteristics 53
4.3.2 Biochemical characteristics 55
4.4 Molecular identification 55
4.5 Study 2: To determine the prevalence of metronidazole resistance
Gardnerella vaginalis strains 58
4.5.1 Antibiotic susceptibility test (AST) 58
4.5.2 Determination of metronidazole resistant Gardnerella vaginalis
isolates by PCR 63
4.6 Study 3: To determine the biotypic characteristics of Gardnerella
vaginalis among the clinical isolates 64
4.7 Study 4: To determine the genotypic characteristics of Gardnerella
vaginalis among the clinical isolates 65
4.8 Study 5: To assess the distribution and presence of virulence factors of
Gardnerella vaginalis 67
4.8.1 Virulence genes detection 67
4.8.2 Sequence of sialidase and vly genes 68
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5 DISCUSSION 73
6 CONCLUSION AND RECOMMENDATIONS 83
REFERENCES 85
APPENDICES 92
BIODATA OF STUDENT 104
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