UNIVERSITI PUTRA MALAYSIA PHENOTYPIC AND GENOTYPIC ... · A simple and reproducible scheme for identifying biotypes of G. vaginalis identified eight biotypes based on reaction for

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


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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By


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





By


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


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



(Internal Examiner)

Examiner2, PhD

Professor



(Internal Examiner)

External Examiner, PhD

Professor



(External Examiner)

PROF.DR. SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


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


(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


(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


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.


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




3.4 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




4.4 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


4.7 Study 4: To determine the genotypic characteristics of Gardnerella


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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UNIVERSITI PUTRA MALAYSIA PHENOTYPIC AND GENOTYPIC ... · A simple and reproducible scheme for identifying biotypes of G. vaginalis identified eight biotypes based on reaction for