MUTATIONS OF rpsL, rrs AND gidB and THEIR ASSOCIATION TO STREPTOMYCIN RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS

CLINICAL ISOLATES

BY

MISS YIN MOE HLAING

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF THE MASTER OF SCIENCE (BIOMEDICAL SCIENCES)

GRADUATE PROGRAM IN BIOMEDICAL SCIENCES

FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY

ACADEMIC YEAR 2015

COPYRIGHT OF THAMMASAT UNIVERSITY

MUTATIONS OF rpsL, rrs AND gidB and THEIR ASSOCIATION TO STREPTOMYCIN RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS

CLINICAL ISOLATES

BY

MISS YIN MOE HLAING

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE MASTER OF SCIENCE (BIOMEDICAL SCIENCES)

GRADUATE PROGRAM IN BIOMEDICAL SCIENCES

FACULTY OF ALLIED HEALTH SCIENCES THAMMASAT UNIVERSITY

ACADEMIC YEAR 2015

COPYRIGHT OF THAMMASAT UNIVERSITY

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Thesis title MUTATIONS OF rpsL, rrs AND gidB and THEIR ASSOCIATION TO STREPTOMYCIN RESISTANCE IN MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES

Author Miss Yin Moe Hlaing Degree Master of Science (Biomedical Sciences) Department/ Faculty/ University Graduate Program in Biomedical Sciences Faculty of Allied Health Sciences Thammasat University Thesis Advisor Assistant Professor Potjanee Srimanote, Ph.D. Thesis Co-Advisor Professor Pramuan Tapchaisri, Ph.D. Thesis Co-Advisor Assistant Professor Pongsri Tongtawe, Ph.D. Thesis Co-Advisor Assistant Professor Anek Pootong, Ph.D. Thesis Co-Advisor Jeeraphong Thanongsaksrikul, Ph.D. Academic Year 2015

ABSTRACT

Tuberculosis (TB) is one of the leading causes of death worldwide and the emergence and spread of drug-resistant TB is an immense threat to the global TB control programs. Being one of the 22 high TB burden countries, drug-resistant TB is an alarming issue in Thailand and molecular characterization of anti-TB drugs resistance is necessary for this region. In this study, by using PCR amplification and DNA sequencing analysis, streptomycin (SM) resistance associated mutations in rpsL, rrs and gidB genes were examined in 101 Mycobacterium tuberculosis (MTB) clinical isolates with various lineages backgrounds from Thailand (46 SM resistant and 55 SM susceptible isolates). The mutation pattern and frequency related to SM resistance and the subsequent utility for diagnostic value were determined. Their association and specificity to MTB lineages were also further analyzed. In this study, the rpsL mutations, K43R, K88R and K88T, and the gidB mutations, W45stop and G69D, were

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able to be correlated with SM resistance. The K43R was the most predominant rpsL mutations and found in 91.2% of SM resistance associated mutations and 67.4% of the SM resistant isolates. Subsequently, rpsL mutations, having the highest sensitivity (71.7%) and specificity (96.4%), were revealed to be the most reliable genetic marker for the detection of SM resistance in MTB isolates from Thailand. Surprisingly, rrs mutations associated to SM resistance were absent in this study. Combined rpsL and gidB mutations exhibited 73.9% sensitivity and 96.4% specificity for identified SM resistant isolates. In correlation with MTB lineages, rpsL mutations, especially K43R, was significantly found in Beijing family. The study further emphasized gidB E92D as a signature polymorphism for Beijing isolates. Moreover, gidB 330G>T was predicted as a promising putative maker for East-African Indian (EAI) lineages.

Keywords: Tuberculosis, Streptomycin, Drug resistance, Mutations

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ACKNOWLEDEMENTS

A debt of acknowledgment is owed to a number of people who have contributed to this thesis. I owe my first debt of sincere acknowledgement to my major advisor, Assistant Professor Dr. Potjanee Srimanote, for guiding me through the whole work by sharing her knowledge and giving me valuable comments. Without her encouragement, kindness and supervision, the completion of this study would not have been possible.

A particular debt of gratitude goes to Professor Dr. Pramuan Tapchaisri who has given me the opportunity of studying at Graduate Program in Biomedical Sciences. His wisdom and insightful advice have inspired me and have given me great strength in times of need.

I owe so much gratitude to Assistant Professor Dr. Pongsri Tongtawe for generously contributing support, guidance and expertise to my work. Her input has proved invaluable to the thesis's development.

The suggestions and technical know-how of Assistant Professor Dr. Anek Pootong and Dr. Jeeraphong Thanongsaksrikul have been of great value to me. I feel immensely indebted to their contribution as well.

I would also like to acknowledge my genuine thanks to Associate Professor Dr. Angkana Chaiprasert, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, for her kind acceptance to be the chair of the defense committee for my thesis.

My special thanks also go to all of my beloved friends in the Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, for their friendship, understanding, motivation and unconditional support throughout my research. I feel very lucky to meet such kind, outgoing and pleasant friends at this university.

A further special debt is owed to Thammasat University Scholarship for ASEAN Community and Thammasat Fiscal Year Research Grant for allowing me to pursue my Master degree and to carry out the thesis research.

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Last but not least, I must thank whole-heartedly my parents and my elder sister who have supported and encouraged me in various ways with endless love and understanding throughout my hard study.

Yin Moe Hlaing

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TABLE OF CONTENTS

Page

ABSTRACT (1) ACKNOWLEDEMENTS (3) TABLE OF CONTENTS (5) LIST OF TABLES (8) LIST OF FIGURES (9) LIST OF ABBREVIATIONS (11) CHAPTER 1 INTRODUCTION 1 CHAPTER 2 OBJECTIVES 4 CHAPTER 3 REVIEW OF LITERATURE 5

3.1 Tuberculosis (TB) 5 3.1.1 Etiological agent 5 3.1.2 Morphology and classification 5 3.1.3 Pathogenesis 6 3.1.4 Tuberculous diseases 7

3.2 Molecular characteristics of Mycobacterium tuberculosis 8 3.2.1 Genome 8

3.3 Diagnosis of active TB 10 3.3.1 AFB microscopy 10 3.3.2 TB culture 11 3.3.3 Molecular methods 11 3.3.4 Other diagnostic methods 12

3.4 Treatment of TB 13 3.4.1 WHO standard regimen for TB 13 3.4.2 Streptomycin (SM) in TB treatment 16

3.5 Drug resistance tuberculosis (DR-TB) 17 3.5.1 Emergence of DR-TB and global data 17 3.5.2 Anti-TB drug resistance mechanism 18

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TABLE OF CONTENTS (Cont.)

Page 3.6 Diagnosis of anti-TB drugs resistance 19

3.6.1 Phenotypic methods 19 3.6.2 Genotypic methods 21

3.7 Mechanism of Streptomycin action 21 3.7.1 Structure of Streptomycin 21 3.7.2 Bacterial ribosome and protein synthesis 22

3.8 SM resistance in TB 29 3.8.1 Mutations found in rpsL gene 30 3.8.2 Mutations found in rrs gene 31 3.8.3 Mutations found in gidB gene 31

3.9 TB burden in Thailand 32 CHAPTER 4 MATERIALS AND METHODS 33

4.1 Experimental outline 33 4.2 Sample size calculation 34 4.3 Mycobacterium tuberculosis clinical isolates used in this study 34 4.4 Preparation of amplified and purified Mycobacterium tuberculosis

DNA 36

4.4.1 Preparation of genomic DNA and its quantification 36 4.4.2 PCR amplification of rpsl, rrs, gidB and whiB7 DNA sequences 36 4.4.3 Agarose gel electrophoresis 37 4.4.4 Gel purification of PCR amplicons for DNA sequencing analysis 38

4.5 DNA sequencing 38 4.6 Data analysis of DNA sequencing results 4.7 Mismatched amplification mutation assay PCR (MAMA-PCR)

38 39

4.8 Verification of Beijing isolates by RD105 deletion-targeted multiplex PCR (DTM-PCR)

39

4.9 Statistical analysis 40

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TABLE OF CONTENTS (Cont.)

Page CHAPTER 5 RESULTS 42

5.1 Optimization of PCR amplification of rpsL, rrs, gidB and whiB7 genes 42 5.2 DNA sequencing analysis of the rpsL, rrs, gidB and whiB7 PCR

products derived from H37Rv MTB genomic DNA 46

5.3 Amplification of rpsL, rrs, gidB and whiB7 genes in MTB clinical isolates

53

5.4 Mutations found in rpsL, rrs and gidB 57 5.5 The presence of whiB7 mutations among SM resistant isolates

lacking of rpsL and rrs mutations 70

5.6 Association of rpsL, rrs and gidB mutations to SM resistance 72 5.7 Association of gidB sequence polymorphism and MTB lineage 74

CHAPTER 6 DISCUSSION 6.1 Mutations found in rpsL, rrs, gidB and whiB7 genes and their

association to SM resistance 6.2 Association of gidB sequence polymorphisms and MTB lineages

77 77

81

CHAPTER 7 CONCLUSION 84 REFERENCES 85 BIOGRAPHY 101

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LIST OF TABLES

Tables Page

3.1 Alternative method of grouping anti-tuberculosis agents 14 3.2 Recommended TB treatment regime 15 3.3 Frequency of drug-resistant mutants to anti-TB drugs 18 3.4 Critical proportion and drug susceptibility testing methods with

different critical concentrations 20

3.5 CLSI recommended minimal inhibitory concentrations (MIC) report and interpretations of susceptible or resistant

20

4.1 Mycobacterium tuberculosis clinical isolates used in this study 35 4.2 The lineage of Mycobacterium tuberculosis clinical isolates used in

this study 36

4.3 The primers for PCR amplification 41 5.1 Distribution of mutations in rpsL, rrs, gidB and whiB7 genes of MTB

clinical isolates 58

5.2 Sensitivity and specificity calculations of rpsL, rrs and gidB genes 73

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LIST OF FIGURES Figures Page

3.1 MTB H37Rv Genome 9 3.2 3.2 A. MTB with ZN staining seen under light microscope

3.2 B. MTB with auramine staining visualized under fluorescent microscope

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3.3 Chemical structure of SM sulfate 22 3.4 Components of a ribosome 22 3.5 The 16S rRNA binding to Shine-Dalgarno sequence helps mRNA line

up properly in the ribosome 23

3.6 Protein translation on the ribosome 24 3.7 Secondary structure diagram of 16S rRNA 26 3.8 Secondary structure of ribosomal protein S12 27 3.9 Ribosomal protein S12 (brown) interacting with the various helical

sites of 16S rRNA at the functional center of the ribosomal 30S subunit

28

3.10 Chemical structure of SM showing interactions of the various groups with specific residues of the ribosome

29

5.1 PCR amplicon of rpsL gene of MTB H37Rv 42 5.2 PCR amplicon of rrs gene of MTB H37Rv 43 5.3 PCR amplicon of gidB gene of MTB H37Rv 44 5.4 PCR amplicon of whiB7 gene of MTB H37Rv 45 5.5 BLASTN analysis of MTB H37Rv rpsL (30S ribosomal protein S12)

open reading frame (375 bp) in this study 47

5.6 BLASTN analysis of MTB H37Rv rrs (16S rRNA) fragment (1,498 bp) in this study

48

5.7 BLASTN analysis of MTB H37Rv gidB (glucose-inhibited division protein B Gid) open reading frame (675 bp) in this study

50

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Figures

LIST OF FIGURES (Cont.)

Page

5.8 BLASTN analysis of MTB H37Rv whiB7 (transcriptional regulator) open reading frame and promoter region (778 bp) in this study

51

5.9 Agarose gel electrophoresis of rpsL PCR amplicons obtained from clinical MTB isolates

53

5.10 Agarose gel electrophoresis of rrs PCR amplicons obtained from clinical MTB isolates

54

5.11 Agarose gel electrophoresis of gidB PCR amplicons obtained from clinical MTB isolates

55

5.12 Agarose gel electrophoresis of whiB7 PCR amplicons obtained from clinical MTB isolates

56

5.13 Chromatogram of rpsL sequence showing WT and mutated sequences

62

5.14 Chromatogram of rrs sequence showing WT and 1025T>C sequence 63 5.15 MAMA-PCR optimization for detection of 16T>C mutation in rrs

gene using H37Rv MTB genomic DNA as a template 64

5.16 Representative MAMA-PCR results for detection of 16T>C rrs mutation

65

5.17 Chromatogram of gidB sequence showing WT and mutated sequences

68

5.18 Chromatogram of gidB sequence showing WT and three silent mutated sequences

69

5.19 Chromatogram of whiB7 open reading frame sequence showing WT and mutated sequences

71

5.20 DTM-PCR optimization 75 5.21 Agarose gel electrophoresis of RD105 DTM-PCR amplicons of clinical

MTB isolates 76

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LIST OF ABBREVIATIONS

Symbols/Abbreviations Term % Percent

°C Degree(s) Celsius µg Microgram(s) µl Microlitre(s) µM Micromolar(s) AIDS Acquired immune deficiency syndrome bp base pair(s) CDC Centers for Disease Control and Prevention DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid EF-G Elongation factor G EF-Tu Elongation factor thermo unstable e.g. Exempli gratia (for example) et al. et alia (and others) GTP Guanosine triphosphate HIV Human immunodeficiency virus i.e. Id est (that is) kg Kilogram(s) MDR-TB Multidrug-resistant TB mg Miligram(s) min Minute(s) ml Mililitre(s) mM Milimolar(s) mRNA Messenger ribonucleic acid MTB Mycobacterium tuberculosis NCBI National center for biotechnology information ng Nanogram(s)

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LIST OF ABBREVIATIONS (Cont.) Symbols/Abbreviations Term nm Nanometre(s) no. Number(s) nt Nucelotide(s) OD Optical Density sec Second(s) SM Streptomycin TB Tuberculosis TBE Tris-Borate-EDTA tRNA Transfer ribonucleic acid U Unit UTR Untranslated region UV Ultraviolet WHO World Health Organization XDR-TB Extensively drug-resistant TB α Alpha β Beta γ Gamma

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LIST OF ABBREVIATIONS (Cont.)

List of amino acids abbreviations Symbols/Abbreviations 1 letter 3 letters Term A Ala Alanine D Asp Aspartic acid E Glu Glutamic acid G Gly Glycine H His Histidine K Lys Lysine L Leu Leucine M Met Methionine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan

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CHAPTER 1 INTRODUCTION

Tuberculosis (TB) is an infectious bacterial disease caused by

Mycobacterium tuberculosis (MTB) which is spread through the air droplet. Though TB can infect every part of the body, lungs are its predominant infected organs. Among the contagious diseases caused by a single infectious agent, it is the second greatest worldwide killer disease after HIV/AIDS1. Globally in 2014, 9.6 million people contracted TB and 1.5 million people died from this disease2. Over 95% of cases are occurring in low and middle income countries due to poor hygiene and inappropriate TB control. The WHO South-East Asian region accounts for 39% of global TB burden. Moreover, approximately 3.4 million of new TB cases occur each year particularly in five countries of this region which are Bangladesh, India, Indonesia, Myanmar and Thailand3.

Although TB can be treated with the WHO recommended standard anti-TB regimen involving isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E) or HRZE, the emergence of isolates with resistance to these drugs is a major threat to the TB control worldwide. The resistant types can be classified into various categories, i.e. mono-, poly-, multidrug- (MDR) and extensively drug- (XDR) resistances4. It was globally estimated that 3.3% of new cases and 20% of previously treated cases have MDR-TB in 2014. In the same year, there were an estimated 480,000 new cases of MDR-TB worldwide and approximately 190,000 MDR-TB patients died2.

Because of the increasing risk of drug resistance in TB treatment, the resistance molecular mechanisms in MTB were extensively studied and described5–7. Owing to the lack of horizontal gene transfer evidence, the drug resistance in MTB is mainly due to the spontaneous mutations in their genome during the inappropriate or incomplete drug therapy5,8. Intrinsic drug resistance attributed by the mycolic acid-rich lipid cell wall and efflux mechanisms of MTB has also been recognized as an

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important factor contributing the natural resistance of mycobacteria against anti-TB drugs8.

Involving in aminoglycosides group, streptomycin (SM) was the first anti-tuberculous drug that combated effectively to this infectious disease9. However, due to the usage as a sole drug for TB treatment during late 1940s, 30-40% of MTB developed the resistance to this drug10,11. Thereafter the discovery of HRZE as the combined regimen provided highly efficient TB treatment and the usage of SM in the first line anti-TB drugs group had been remarkably decreased9. Following the increasing incidence of H/R combination treatment failure (MDR-TB), SM was recommended by WHO to be reintroduced with other anti-TB drugs as the components of combined therapies for the retreatment or drug resistant TB cases12. SM binds to 16S rRNA, and ribosomal protein S12 encoded by rrs and rpsL genes, respectively. These molecules are located in the decoding center of 30S ribosomal subunit responsible for the proteins translation fidelity. SM binding to these targets causes codon misreading, thereby inhibiting protein synthesis and resulting in MTB death13. SM resistance in MTB is mediated by mutations in rpsL and rrs genes conferring SM high and intermediate resistance levels, respectively6,14,15. Moreover, the mutation in gidB, translation accessory gene which encodes a conserved 7-methylguanosine (m7G) methyltransferase specific for methylation of guanosine residue in 530-loop of 16S rRNA was found responsible for low-level SM resistance16. However, the types and the positions of mutations found in these genes were significantly associated with the geographical areas as well as the MTB lineages17,18. Therefore, a comprehensive understanding of molecular mechanism for SM resistance and their epidemiology are still required as a worldwide study. More importantly, this genotypic information can provide the specific targets for the development of rapid molecular diagnosis of SM resistance in the certain parts of the world because current diagnosis of SM resistance MTB is only available as the phenotypic drug susceptibility method which can take at least two weeks.

According to WHO’s classification, Thailand is one of the 22 highest TB burden countries and approximately 120,000 of all forms of TB cases were reported in 2014. The incidence of MDR-TB was 2% in new cases and 19% in retreatment

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cases2. The molecular surveys for drug resistance to some first-line drugs and aminoglycosides except SM were carried out in Thai MTB clinical isolates19,20. Yet, the genotypic characterization mediating SM resistance has not been revealed. Therefore, this study aims to investigate the mutations in three SM resistance involving genes, rpsL, rrs and gidB, in MDR and non-MDR MTB clinical isolates and their association to the SM resistant phenotypes. Moreover, the relationship of their mutations to the MTB lineage is also determined.

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CHAPTER 2 OBJECTIVES

PRIMARY OBJECTIVE

The main objective of this thesis is to investigate the mutations in rpsL,

rrs and gidB genes and their association to streptomycin (SM) resistant phenotypes in MDR and non-MDR MTB clinical isolates from Bamrasnaradura Infectious Diseases Institute, Thailand.

SPECIFIC OBJECTIVES

1. To investigate rpsL, rrs and gidB genes sequences among MTB clinical isolates collected from Bamrasnaradura Institute during 2007-2011

2. To determine their mutation types prevalence in each different group of Thai MTB clinical isolates

3. To evaluate the relationship between SM resistance level and the mutation types 4. To correlate the SM resistance genotypes with the MTB lineages of Thai clinical

isolates

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CHAPTER 3 REVIEW OF LITERATURE

3.1 Tuberculosis (TB)

3.1.1 Etiological agent Tuberculosis (TB) is caused by a bacterium called Mycobacterium

tuberculosis (MTB). To date, more than 125 mycobacteria species have been identified including saprophytes and a few pathogens21. MTB causing TB was first demonstrated by Robert Koch in 1882. By spreading from person to person through the inhalation of MTB containing droplets, TB has become one of the oldest documented communicable respiratory diseases. Although TB disease can be found worldwide, the majority of cases are dominant in developing countries where 95% of TB deaths occur22. In 2014, there were an estimated 9.6 million cases of TB and 1.5 million people died from this disease. The 58% of world’s TB cases in 2014 occurred in the South-East Asia and the Western Pacific Regions2.

3.1.2 Morphology and classification Belonging to the member of the Actinomycete family, mycobacteria

are aerobic-to-microaerophilic bacilli and visualized under microscope as slender, slightly curved or straight, rod-shaped organisms having 0.2 to 0.6 µm × 1 to 10 µm in size. Unlike other members of the actinomycete group, they do not produce either aerial hyphae or spores. Moreover, mycobacteria grow slower than the other human bacterial pathogen requiring 2 to 6 weeks of incubation for susceptible isolates (up to 11 weeks for multidrug-resistant isolates) on complex media such as Löwenstein–Jensen (LJ) medium or Middlebrook 7H10 at 35°C to 37°C23.

As these bacteria must survive in the hostile acidic environment of the endosome and phagosome, they possess the highly unusual cell wall components. The basic structure of their cell wall resembles with that of a typical gram positive bacterium which is composed of an inner plasma membrane overlaid with a thick peptidoglycan layer without outer membrane. Being rich in proteins,

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phosphatidylinositol mannosides and lipoarabinomannan (LAM) anchored in plasma membrane, MTB’s cell wall is more complex than other bacteria notwithstanding. The peptidoglycan layer links to arabinogalactans, a branched polysaccharide consisting of D-arabinose and D-galactose to form the cell wall foundation. The terminal D-arabinose residue is esterified to hydrophobic mycolic acids with attached glycolipid surface molecules. The lipid components comprise approximately 60% of the cell wall weight. Transportation machinery proteins and porins, which are interspersed throughout the cell wall layers, account for 15% of the cell wall weight24.

This cell wall is responsible for many characteristic properties of mycobacteia (e.g., acid-fastness, slow growth, resistance to detergents and common anti-bactericidal antibodies, different antigenicity and clumping appearance). The mycobacterial cells resist staining with commonly used basic aniline dyes at room temperature. Although they take up the dye with increased staining time or application of heat, they resist decolorization with acid-ethanol. This characteristic is referred to as acid fastness and, hence, mycobacteria are called acid-fast-bacilli (AFB) distinguishing them from most other genera and species25.

MTB and closely related species are grouped as Mycobacterium tuberculosis complex (MTBC) consisting of human and animal pathogens. The typical human pathogens are MTB and Mycobacterium africanum. The animal pathogen affecting various animal speices are Mycobacterium bovis (a pathogen of cattle), Mycobacterium caprae (goats and sheep), Mycobacterium microti (voles) and Mycobacterium pinnipedii (seals and sea lions)26. Mycobacterium canettii is also a novel pathogenic taxon of MTBC although its natural reservoir and host range are still unknown27. It is medically important to differentiate MTB from all the other species of mycobacteria due to the difference in type of diseases they cause and antimicrobial agent usage for therapy.

3.1.3 Pathogenesis The pathogenesis of TB in an immune-competent person depends

on the development of cell-mediated immunity (CMI) which attacks mycobacteria and also induces the hypersensitivity28. The infection can result the pathogenic

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significances after 1-2 months of exposure by showing the two types of principle lesions, exudative type and productive type29.

3.1.3.1 Exudative type In this type, macrophages are activated to become MTB killing

cells by the T helper 1 (TH1) response to mycobacteria. The TH1 cells and activated macrophages secrete cytokines such as Interleukin-1 (IL-1), tumor necrosis factor (TNF) and gamma interferon (IFN-γ). The activated macrophages form a compact cluster or granuloma around the site of infection and they are called epitheloid cells28. This type is particularly seen in lungs tissue and may heal by resolution. During this stage, the tuberculin test becomes positive. The exudative type may lead to massive necrosis tissue or it may develop into the productive type of lesion29.

3.1.3.2 Productive type The activated macrophages fuse together to form the giant

cells that later develop into three zones: (1) a central area of large, multi-nucleated giant cells containing mycobacteria; (2) a mid-zone of pale epitheloid cells, often arranged radially; and (3) a peripherial zone of fibroblasts, lymphocytes, and monocytes. Later, peripheral fibrous tissues develop, and the central area undergoes caseous necrosis called a tubercle. MTB may persist in these tubercles for extended periods and make the lesion progressive or chronic. At the time of reactivation, a caseous tubercle may break into a bronchus, empty its contents, there by leaving a cavity in the lungs and spreading MTB to the other parts of the lungs or other organs. The cavity may subsequently heal by fibrosis or calcification29.

3.1.4 Tuberculous diseases 3.1.4.1 Site of infection

The majority of the MTB infection occurs in lungs and is referred to as pulmonary TB. However, tubercle bacilli can spread to any part of the body other than lungs (e.g., kidney, spine, brain, or lymph nodes) and it is noted as extra-pulmonary TB. This type of TB can be commonly present in individuals with human immunodeficiency virus (HIV). Miliary TB refers to the spreading of MTB to all parts of the body through the blood30.

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3.1.4.2 Active TB and Latent TB The active TB results when the immune system cannot fight

against MTB and the bacteria actively multiply at the affected organs. The infected people will show AFB positive sputum, TB symptoms and spread the bacteria to the others. On the other hand, inactive or latent TB infection (LTBI) occurs when a person carries the TB bacteria within their body without showing TB symptoms. The bacteria are present in very small numbers and hardly multiply as they are kept under control by the body’s immune system. In contrast to the active TB, LTBI shows AFB negative sputum and negative MTB cultures, and normal chest X-rays. Tuberculin skin and Interferon-gamma release assays or IGRAs (blood) tests can be positive in both types30.

3.1.4.3 Signs and symptoms of TB Common symptoms of active lungs TB are cough with

sputum and sometimes bloody sputum, chest pains, weakness, weight loss, fever and night sweats. Pulmonary TB suspect may present a productive cough for more than 2 weeks, which may be accompanied by other respiratory symptoms (shortness of breath, chest pains, haemoptysis) and/or constitutional symptoms12. 3.2 Molecular characteristics of Mycobacterium tuberculosis

3.2.1 Genome MTB H37Rv strain isolated in 1905 is widely applicable in

biomedical research as a standard reference strain because it retains full virulence in animal models of tuberculosis31 and is susceptible to all TB therapeutic drugs32. The genome of MTB H37Rv was completely sequenced in 199833. The circular genome comprises 4.4 mega base pairs (bp) and has a 65.6% of very high guanine + cytosine (G+C) content (Figure 3.1). The genome is rich in repetitive DNA, particularly remnant of insertion sequences, new multigene families and duplicated housekeeping genes. Among annotated 3,924 open reading frames, approximately 91% are predicted to encode proteins and other 50 genes are encoding for functional RNA molecules. As a consequence of high G+C content genome, although ATG (61%) is the most

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predominant translational start, GTG initiation codons (35%) are used more frequently than other bacteria. Moreover, the G+C genome also substantially influences on the bias for using the amino acids, Ala, Gly, Pro, Arg and Trp33.

Figure 3.1 MTB H37Rv Genome (The outer circle shows the scale in Mb, with 0 representing the origin of replication. The first ring from the exterior denotes the positions of stable RNA genes (tRNAs are blue, others are pink) and the direct repeat region (pink cube); the second ring inwards shows the coding sequence by strand (clockwise, dark green; anticlockwise, light green); the third ring depicts repetitive DNA (insertion sequences, orange; 13E12 REP family, dark pink; prophage, blue); the fourth ring shows the positions of the PPE family members (green); the fifth ring shows the PE family members (purple, excluding PGRS); and the sixth ring shows the positions of the PGRS sequences (dark red). The histogram (centre) represents G + C content, with <65% G + C in yellow, and >65% G + C in red) (Reproduced from33).

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As the G+C content is relatively constant throughout its genome, MTB H37Rv probably lacks of genomic or pathogenicity islands which are commonly found in other pathogenic bacteria. However, its genome is composed of ancient mobile genetic elements called insertion sequences (IS) which are present in multiple copies on its genome, with 16 copies of IS6110, 6 copies of IS1081, and 2 copies each of IS1547 and the IS-like element33. Complete genome sequence of MTB H37Rv has been widely used as powerful bioinformatics to compare with those of clinical isolates and other mycobacterial species, a process which has become known as comparative genomics34. 3.3 Diagnosis of active TB

The diagnosis of active disease (including pulmonary as well as extra-pulmonary TB) can be diagnosed bacteriologically or clinically. A patient with MTB can be bacteriologically identified from a clinical specimen by AFB smear microscopy, culture or advanced molecular methods such as molecular line probe assays (LPA) or nucleic acid amplification (NAA) tests35.

3.3.1 AFB microscopy

The sputum specimens are smeared directly onto glass slides and stained by using Ziehl-Neelsen (ZN). Under light microscope, ZN stained acid fast bacilli are visualized as pink, straight or slightly curved rods, at times having beaded appearance (Figure 3.2A). The fluorescent microscopy using auramine O or auramine-rhodamine stains can also be used and positive mycobacteria reveals as bright, yellow-orange or green bacilli against a dark background (Figure 3.2B). The sensitivity of conventional light microscopy ranged from 32 to 94% and it is greatly improved to 52 to 97% by using fluorescent microscopy36.

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A B

Figure 3.2 A; MTB with Ziehl-Neelsen (ZN) staining seen under light microscope37. B; MTB with auramine staining visualized under fluorescent microscope38.

3.3.2 TB culture The gold standard for TB diagnosis is the culture of MTB which is

more sensitive than AFB smear microscopy. There are two general types of media: solid media that take 2-6 weeks for growth of MTB and liquid media that take 9-14 days23. Löwenstein-Jensen (LJ) medium is the most commonly used egg-based solid media in resource-poor countries39. Serum albumin agar media such as Middlebrook 7H10 and 7H11 support enriched and selective condition for MTB growth23. Modified Middlebrook 7H9 broth is the most predominantly used liquid medium because it can provide better quality and faster growth of MTB39. The automated BACTECTMMGITTM 960 system containing Middlebrook 7H9 provides the rapid diagnosis of MTB in 7-8 days by using oxygen-quenched fluorochrome to detect its growth40. Other broth culture media involves the MB/BacT Alert system (bioMerieux) employing a colorimetric carbon dioxide to detect the mycobacterial growth and BACTECTM MGITTM 460 using radiometric detection39.

3.3.3 Molecular methods These techniques not only provide the rapid detection of MTB from

patient specimens and culture but also differentiate mycobacteria species. Centers for Disease Control and Prevention (CDC) recommended the performance of nucleic

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acid amplification (NAA) testing on at least one of three specimens from each patient with signs and symptoms of pulmonary TB41. The enhanced version of Amplified MTB Direct Test (AMTD, Gen-Probe, San Diego, California) is a rapid and reliable method in the diagnosis of AFB smear-negative as well as smear-positive pulmonary tuberculosis42. The Amplicor MTB test (Roche Diagnostic System, Basel, Switzerland), a PCR-based test, detects the MTBC from the AFB smear-positive respiratory specimens43.

Line probe assays (LPA) such as Inno-LiPA Mycobacteria (Innogenetics NV, Ghent, Belgium) identifies mycobacteria including MTBC by targeting the 16S - 23S rDNA spacer region amplified by Polymerase Chain Reaction (PCR) and subsequently hybridized with oligonucleotides probes arranged on a membrane strip44. The GenoType® Mycobacteria CM/AS (Hain Life Science, Nehren, Germany) differentiates MTBC and different species of NTM (Non Tuberculous Mycobacteria) in clinical isolates by targeting the 23S rRNA gene region, followed by reverse hybridization to specific oligonucleotide probes immobilized on membrane strips. Although LPAs are not suitable for direct detection on sputum smear-negative clinical specimens, they are adequately reliable for the direct testing of smear positive specimens and of mycobacterial isolates grown on cultures45.

3.3.4 Other diagnostic methods In case of lack of confirmation by the laboratory bacteriological

methods, a TB patient can be clinically diagnosed by a clinician or other medical practitioner on the basis of X-ray abnormalities or suggestive histology and extra-pulmonary cases35. Mantoux tuberculin skin test and blood test (Interferon-gamma release assays or IGRAs) can determine that a person has been infected with TB bacteria, although they cannot differentiate LTBI and active TB. The Mantoux test measures the size of hypersensitivity reactions (raised, hard area or swelling) on the skin appeared after the injection of 0.1 ml amount of fluid called tuberculin46.

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3.4 Treatment of TB

There are more than 20 drugs that are currently used for the treatment of TB and they can be categorized in five groups based on known efficiency (Table 3.1)4. According to WHO guideline, the five basic or “first-line” drugs are isoniazid (INH or H), rifampicin (RIF or R), pyrazinamide (PZA or Z), ethambutol (EMB or E) and streptomycin (SM or S)12. However, SM is classified into the second-line drugs (Group 2, 3, 4 and 5 of (Table 3.1) in the CDC Treatment of Tuberculosis, 2003 guideline47. A person who has been diagnosed of TB with either bacteriologically or clinically can receive TB treatment. New patients are defined either as those who have never had TB treatment or have taken anti-TB drugs for less than one month regardless of positive or negative sputum or culture results and they may have disease at any anatomical site12.

3.4.1 WHO standard regimen for TB treatment According to the WHO guidelines (Table 3.2), the standard

treatment regimen for drug- susceptible TB in new patients comprises a combination of isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E) during the intensive phase treatment of two months, followed by H together with R in the continuation phase of four months (2HRZE/4HR). In countries with high levels of H resistance in new TB patients, and where H drug susceptibility testing in new patients cannot be done (or results have not yet available) before the continuation phase begins, ethambutol (E) must be perpetually added until the end of six month course. TB patients returning after defaulting or relapsing from their first treatment course (retreatment TB cases) (Table 3.2), regardless of sputum smear result, may receive the treatment regimen containing 2 months of HRZES followed by 1 month of HRZE and 5 months of HRE. The regimen can be adjustable upon the availability of the DST12.

For smear positive pulmonary TB patients treated with first-line drugs, sputum smear microscopy may be necessary at the completion of the

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Table 3.1 Alternative method of grouping anti-tuberculosis agents (Reproduced from4)

Grouping Drugs Group 1 First-line oral agents Isoniazid (H); Rifampicin (R); Ethambutol (E); Pyrazinamide (Z); Rifabutin (Rfb),

Rifapentine (Rpt) Group 2 Injectable agents Kanamycin (Km); Amikacin (Am); Capreomycin (Cm); Streptomycin (S)

Group 3 Fluoroquinolones Moxifloxacin (Mfx); Levofloxacin (Lfx); Ofloxacin (Ofx), Gatifloxacin (Gfx)

Group 4 Oral bacteriostatic second-line anti-TB drugs

Ethionamide (Eto); Protionamide (Pto); Cycloserine (Cs); Terizidone (Trd); p-aminosalicylic acid (PAS), p- aminosalicylate sodium (PAS-Na)

Group 5 Anti-TB drugs with limited data on efficacy and/or long term safety in the treatment of drug-resistant TB (This group includes new anti-TB agents)

Clofazimine (Cfz); Linezolid (Lzd); Amoxicillin/clavulanate (Amx/Clv); Thioacetazone (T); Imipenem/cilastatin (Ipm/Cln); High-dose isoniazid (high-doseH); Clarithromycin (Clr), Bedaquiline (Bdq), Delamanid (Dlm), Meropenen (Mpm)

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Table 3.2 Recommended TB treatment regime (Modified from12)

Diagnostic Categories

TB patients

TB treatment regimen

Initial phase (daily)

Continuation phase (daily )

I - New patient I

- New patient II (In countries with high levels of INH resistance in new TB patients, and where INH drug susceptibility testing in new patients is not done (or results are unavailable) before the continuation phase begins)

2HRZE 2HRZE

4HR 4HRE

II Previously treated smear (+) PTB (relapse, treatment interruption, treatment failure)

2HRZES/1HRZE 5HRE

III Chronic and MDR-TB (smear positive after supervised re-treatment) Specially designed standardized or empirical regimen except H and R

(2HRZE; HRZE treatment for 2 months, 4HR; HR treatment for 4 months, 2HRZES; HRZES treatment for 2 months, 4HRE; HRE treatment for 4 months,1HRZE; HRZE treatment for one month, PTB; Pulmonary TB, EPTB; Extra-pulmonary TB, MDR-TB; Multidrug-resistant TB)

16

intensive phase of treatment, and also at the 5th and 6th month of the continuation phase. However, sputum checking at the continuation phase can be omitted if the patient is smear-negative at the start of treatment or at the second month of the treatment. If the smear is positive at second month, sputum examination must be repeated in the third month. In case of still being smear positive in month three, drug susceptibility test (DST) is recommended12.

A cured person can be denoted when a patient whose sputum smear AFB staining or culture was positive at the beginning of the treatment but whose AFB sputum smear or culture was negative in the last month of treatment and on at least one previous hospital visit. A treatment failure case is defined as a patient who completed treatment but who does not have negative sputum smear or culture result in the last month of treatment or a sputum smear or culture of a patient is positive at month five or later during the treatment. A patient whose treatment was interrupted for 2 consecutive months or more can be categorized as the default. A sum of cured and completed treatment can be excluded as a treatment success12.

3.4.2 Streptomycin (SM) in TB treatment The history of anti-tuberculous chemotherapy began in 1944 with

the discovery of streptomycin (SM) which is classified into aminoglycosides drug group48. It can also be used to treat certain bacterial infections, including brucellosis, tularemia, plague, and certain cases of drug-resistant endocarditis. Since its discovery, SM has become the drug of choice for all forms of TB. Although it was firstly used as a mono-therapy, nowadays, together with the first-line group drugs (HRZE) and other anti-TB drugs, it is an important component of combination therapy for this disease in both retreatment and in drug resistance cases (Table 3.2). SM is administered as an injectable drug, 15mg/kg daily or three times per week12. Other aminoglycosides with significant anti-mycobacterial activity include second-line kanamycin and amikacin drugs (Table 3.1).

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3.5 Drug resistance tuberculosis (DR-TB)

3.5.1 Emergence of DR-TB and global data The emergence of drug-resistant TB is not a recent phenomenon.

The mutant strains that were resistant to SM were reported in 194810 soon after its discovery. During mono-therapy of TB, drug-resistant mutants began to appear within a few months, prohibiting the success of treatment and this problem was overcome by using combinations of drugs49. However, DR-TB cases have drastically increased due to the irregular drug supply, inappropriate doctor prescription and, most importantly, poor patient adherence to treatment35. The resistant types can be classified into various categories, i.e. mono-, poly-, multidrug- (MDR) and extensively drug- (XDR) resistances4.

Over the past two decades, the emergence of multidrug-resistant TB (MDR-TB) has been registered. MDR-TB is defined as cases of TB that are infected with the strains resistant to at least both H and R. Globally, an estimated 3.3% of new cases and 20% of previously treated cases have MDR-TB in 2014. Among TB deaths in 2014, MDR-TB accounts for an estimated 190,000 cases which show a relatively high total compared with its incident cases of 480,0002. With the assistance of drug susceptibility tests, MDR-TB can be treated with empirical anti-TB treatment excluding INH and RIF (Table 3.2).

More recently in 2006, an epidemic of extensively drug-resistant TB (XDR-TB) was described in a population of HIV/AIDS patients in South Africa50. XDR-TB is defined as MDR-TB with additional bacillary resistance to fluoroquinolones (such as ofloxacin or moxifloxacin) and at least one injectable second-line drugs (capreomycin, kanamycin or amikacin). Although XDR-TB is rarely found and can be cured, the likelihood of treatment success is challenging in patients with ordinary TB or MDR-TB. XDR-TB had been reported by 105 countries by 2015 and an estimated 9.7% of people with MDR-TB have XDR-TB2.

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3.5.2 Anti-TB drug resistance mechanism 3.5.2.1 Intrinsic and acquired drug resistance

The intrinsic drug resistance in MTB may result from its unique cell wall properties, including the presence of high molecular weight mycolic acid which renders a very hydrophobic barrier responsible for the resistance to antibiotics and other toxic compounds49. The presence of β lactamase enzyme and the efflux mechanism of MTB are also responsible for its intrinsic drug resistance8. When a micro-organism obtains the resistance ability against the activity of a particular antimicrobial agent to which it was previously susceptible, this is called acquired resistance. The resistance in MTB is always linked to the spontaneous mutations in chromosomal genes during the sub-optimal drug therapy rather than horizontal gene transfer5,8.

3.5.2.2 Mutation A previous report indicated that the emergence of resistance

differs in rate for all the anti-tuberculosis agents51. The mutations that produce resistance occur at the frequency of 10-4 to10-8 for the first line group of TB drugs (Table 3.3). Table 3.3 Frequency of drug-resistant mutants to anti-TB drugs (Reproduced from51)

Drugs Frequency of drug – resistant mutants

Isoniazid 1 drug-resistant mutant for every 105-6bacilli Rifampicin 1 drug-resistant mutant for every 107-8 bacilli

Ethambutol 1 drug-resistant mutant for every 105-6 bacilli Pyrazinamide 1 drug-resistant mutant for every 102-4 bacilli

Streptomycin 1 drug-resistant mutant for every 105-6 bacilli

Since each mutation arises independently, the proportion of the double mutants equals the frequency of the proportions for each mutant

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separately. For example, the probability of a bacillus to be resistant to both INH and RIF will be 10-12 which comes from combination of the probable frequency of mutants for each drug. Consequently, the mono-therapy can easily lead to the emergence of drug resistant bacteria than the combined therapy5,51. 3.6 Diagnosis of anti-TB drugs resistance

The drug resistance of MTB resided in a patient is quite heterogeneous,

and involves the determination of low-level, moderate-level and high-level drug resistance7. ‘Sensitive’ or susceptible strains are defined as those that have never been exposed to the main anti-tuberculosis drugs (‘wild’ strains) and that respond to those drugs, generally in a remarkable uniform manner. ‘Resistance’ strains are those that differ from susceptible strains in their ability to grow in the presence of higher concentrations of a drug, i.e. higher than critical concentration52. The results of phenotypic drug susceptibility tests can be correlated to the genotypic methods.

3.6.1 Phenotypic methods

All the wild type strains of MTB carry a small proportion of the mutant that resist to the anti-TB drugs varying from 1 to 10% of the whole population in accordance with the individual TB drugs. These resistant proportions are referred to as ‘critical proportions’ and the classical proportional drug susceptibility method that can adapt to both of liquid and solid media is the current gold standard test53. It allows the quantification of the ‘critical proportion’ of resistant trait by determination of growth or no growth of MTB in the presence of a single concentration of drug called ‘critical concentration’52. Therefore, the critical concentration can be referred to as the concentration of a drug in the medium that is relevant to the success or failure outcome of the respective drug treatment. The ‘resistant’ strain is defined if the respective strain is able to grow in the medium containing the ‘critical concentration’ of TB drug more than its own ‘critical proportion’. Table 3.4 shows the critical concentrations and MTB critical proportion of first-line drugs varying to the different cultures4.

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The absolute concentration method determines the minimum inhibitory concentration (MIC) which is the lowest concentration of an anti-microbial drug that inhibits the visible growth of a micro-organism after overnight incubation. A series of drug dilution is used to determine the MIC required inhibiting the bacterial growth in vitro53. The Clinical and Laboratory Standard Institute (CLSI) recommended interpretation of MIC range of the first-line drugs except PZA is shown in Table 3.554,55. Table 3.4 Critical proportion and drug susceptibility testing methods with different critical concentrations (Modified from4,53)

Table 3.5 CLSI recommended minimal inhibitory concentrations (MIC) report and interpretations of susceptible or resistant (Modified from54,55)

Drugs MIC Range Tested (µg/ml)

MIC Tentative Interpretations (µg/ml)

Susceptible Resistant Isoniazid (H) 0.03-4 ≤ 0.25 >0.25 Rifampicin (R) 0.12-16 ≤ 1 >1 Ethambutol (E) 0.5-32 ≤ 4 >4 Streptomcyin (SM) 0.25-32 ≤ 2.0 >2.0

Drugs

Critical Proportion

(%)

DST Critical Concentrations (µg/ml) L-J Middlebrook

7H10 Middlebrook

7H11 MGIT 960

Isoniazid (H) Rifampicin (R) Ethambutol (E) Pyrazinamide (Z) Streptomycin (SM)

1 1 1 10 1

0.2 40.0 2.0 -

4.0

0.2 1.0 5.0 -

2.0

0.2 1.0 7.5 -

2.0

0.1 1.0 5.0

100.0 1.0

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3.6.2 Genotypic methods The genotypic methods determine the nucleotide mutations that

result in resistance to anti-TB drugs. Many genotypic methods have been developed in recent years to detect DR-TB rapidly. The most commonly used molecular tests are the GenoType®MTBDRplus, the INNO-LiPA Rif.TB, and the Xpert MTB/RIF assays39. The GenoType®MTBDRplus assay (Hain Life-science) identifies INH and RIF resistance by using the hybridized probes. For INH resistance, the strip is designed to detect mutations in codon 315 of katG and at position -8, -15 and -16 of the regulator of the inhA gene. The codons 506 to 533 of the rpoB gene are targeted for RIF resistance56. This method can detect the most frequent mutations designed on the probe set and the precise identification can only be obtained through sequencing of the corresponding gene. The PCR based reverse-hybridization line probe INNO-LiPA Rif. TB assay investigates RIF resistance by detecting the rpoB gene mutation57. WHO recommended gene Xpert MTB/RIF assay detects both MTBC and RIF resistance in clinical samples. This cartridge based close-system method uses molecular beacons which are nucleic acid probes that hybradize the presence or absence of the normal, RIF susceptible wild-type sequence of the rpoB gene of TB58. WHO has promoted the high accessible to this assay in the developing countries35. 3.7 Mechanism of Streptomycin Action

3.7.1 Structure of Streptomycin

Streptomycin (SM) is an antibiotic belonging to a group of water-soluble aminoglycoside originally derived from Streptomyces griseus. This aminocyclitol glycoside is an organic compound containing an aminocyclitol moiety glycosidially linked to a carbohydrate. It is marketed as the sulfate salt of SM (Figure 3.3). It has bactericidal action targeting bacterial ribosomes (Drug Bank, Accession Number DB01082 (APRD00412).

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Figure 3.3 Chemical structure of SM sulfate59.

3.7.2 Bacterial ribosome and protein synthesis

3.7.2.1 Bacterial protein synthesis The bacterial ribosome can be divided into two units, 30S

smaller subunit and 50S larger subunit (Figure 3.4). Smaller subunit of Escherichia coli ribosome is composed of 16S rRNA and 21 different small ribosomal proteins. Larger subunit is composed of 5S, 23S rRNA and 34 different ribosomal proteins60. Bacterial protein synthesis or translation can be divided into three main steps; initiation, elongation and termination. The ribosome has three sites for tRNA binding designated as the P (peptidyl), A (aminoacyl), and E (exit) sites (Figure 3.4).

Figure 3.4 Components of a ribosome61.

Initiation involves the formation of a 70S ribosome by assembling the small and large units and followed by the accurate positioning of the mRNA start codon (usually AUG), in conjunction to the initiator tRNA (typically fMet-

23

tRNA) at the ribosomal P-site. The alignment of mRNA at this site also requires an additional complementary base pairing of Shine-Dalgano sequences or ribosome binding site at approximately 7 to 10 base pairs upstream of mRNA start codon to the 3’ end of 16S rRNA for initiation of translation in both mono and polycistronic mRNA (Figure 3.5)60.

After the initiation step, the elongation starts with the binding of the elongation factor (EF-Tu-GTP) complex to assist aminoacyl tRNA (aa-tRNA) with complementary sequence (anti-codon) to the second codon in pairing at the A- site for incorporation into the growing polypeptide chain. The accuracy of protein synthesis (proofreading) is determined by a “decoding center” in the small ribosomal subunit, which recognizes correct codon-anticodon base pairs and discriminates against mismatches. In E. coli, the decoding center functions by ribosomal proteins S12, S5 and S462 together with 16S rRNA63,64. This system has been proved to be conserved among all Eubacteria including MTB65.

Figure 3.5 The 16S rRNA binding to Shine-Dalgarno sequence helps mRNA line up properly in the ribosome66.

Following the elongation step, a peptide bond can be formed between the initiator methionyl tRNA at the P-site and the second aa-tRNA at the A-site. Consequently, methionine is transferred to the second amino acid carried by the second anti-codon tRNA at the A-site, forming a peptidyl tRNA at this position and leaving the uncharged initiator tRNA at the P-site. In order to accommodate the next incoming aa-tRNA, the peptidyl tRNA is translocated from the A-site to the P-site, and

24

the uncharged tRNA from the P-site to the E-site facilitated by EF-G. The elongation continues until reaching a stop codon (UAA, UAG or UGA) (Figure 3.6). Accurate translocation is critical in maintaining reading frame and correctly translating the genetic code60. Previous data supported that ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex67. Moreover, 16S rRNA also involves in both A-site and P-site function, and significant changes in its structure occur when these sites are occupied. It interacts directly with the anti-codons of tRNAs in these sites13.

Termination release factors (RF1 and RF2) recognize the stop codons (UAA or UAG or UGA) and hydrolyze the peptidyl-tRNA bond, releasing the polypeptide chain from the ribosome. Thereafter, the post termination complex is disassembled and recycled for the next round of translation initiation60.

Figure 3.6 Protein translation on the ribosome61.

3.7.2.2 The 16S rRNA (rrs)

The 16S rRNA encoded by rrs gene is one of the components of 30S ribosomal subunit. The MTB H37Rv rrs gene is 1,537 base pair (bp) in length (GeneBank: Version; AL123456.3). The primary structure of 16S rRNA is highly conserved among bacterial kingdom. The secondary structure contains over 50 regular double helices connected by irregular single-stranded loops (Figure3.7). The helices on the 16S rRNA are numbered according to the standard helix numbering

25

H1-H45. The packing of helical elements mainly determines the overall fold of the four domains of 16S rRNA, 5’ domain, central domain, 3’ major domain and a 3’ minor domain which are shown in red, green, orange, and cyan respectively68.

Several interactions occur between specific regions of 16S rRNA and those of mRNA, aa-tRNA and ribosome during protein synthesis. The 3’ terminus of 16S rRNA interacts directly with mRNA at initiation. Some specific regions, i.e. 530 loop and 1400 to 1500, of 16S rRNA interact directly with the anti-codon regions of tRNAs in both the A-site and the P-site. The 900 loop packs closing against the Helix 44 and takes a role of conformational switch69. Consequently, any mutation in its sequence or any interfering agent that results in the change of 16S rRNA secondary structure will affect its interaction with mRNA, aa-tRNA and ribosome at A- and P-sites, thereby interfering or inhibiting the protein synthesis13,69.

3.7.2.3 gidB gene involving in the methylation of 16S rRNA In addition to having four canonical bases which are A, G, U

and C (Please see section 3.7.2.2), 16S rRNA needs to be modified to fulfill its essential role in protein translation70. This post transcriptional modification influences the ribosome functional sites. For example, modification in the H44 (around 1400 region) of 16S rRNA which forms the base of the decoding center possibly effects the aa-tRNA selection to maintain the translational fidelity71.

There are 10 known methylatable nucleosides within 16S rRNA including m7G (7-methylguanosine at position 527 of 16S rRNA)72. The gidB (Glucose-inhibited division protein B) locating on gidAB operon encodes methyltransferase specific for the guanosine base at nucleoside position 527 located in the 530 loop of 16S rRNA in E. coli. It is a single α/β domain protein containing a S-adnosyl-L-methionine (SAM) binding site folded within its quaternary structure73. SM antibiotic directly interacts to the position G527 of 16S rRNA in E. coli corresponding to position G518 in MTB69,74,75. The MTB gidB gene has 675 bp in length and codes for 224 amino acids (Gene Bank Accession; AL123456.3). Crystal structure of 30S subunit of E. coli indicated that this position locates near the proline residue at position 44 (P44) of the S12 ribosomal protein (rpsL) which interacts directly with the mRNA wobble position of the codon:anticodon at the A site during translation76,77.

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Figure 3.7 Secondary structure diagram of 16S rRNA, showing the definition of the various helical elements used through the text. The numbering and diagram correspond to the E. coli sequence. Red, 5’ domain; green, central domain; orange, 3’ major domain; cyan, 3’ minor domain (Reproduced from68).

3.7.2.4 Ribosomal protein S12 (rpsL) The ribosomal protein S12 is a part of the 30S small

ribosomal subunit and encoded by rpsL gene (ribosomal protein S L). MTB rpsL gene is 375 bp in length and codes for 124 amino acids (GeneBank Accession; AL123456.3). Locating at the decoding center of the 30S subunit, the secondary structure of the rpsL contain α- helices and β strands that are packed into β barrels. The rpsL has a globular domain at the interface side of the 30S subunit and a long terminal extension that buries and then extends though the 30S subunit to emerge on the back side to interact with other ribosomal proteins S8 and S17 (Figure 3.8)78.

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Figure 3.8 Secondary structure of ribosomal protein S1278.

3.7.2.5 Involvement of 16S rRNA and ribosomal protein S12 in translational fidelity During protein synthesis, the anti-codon stem-loops (ASL) of

the A- and P- site tRNAs bind to the 30S subunit to be base paired with adjacent codons on mRNA and the decoding process occurs. The decoding of mRNA into protein requires the correct (cognate) recognition of each A-site codon by the anti-codon of the corresponding aa-tRNA with high fidelity (with an error frequency on the order of 10-3 to 10-4)13. This codon-anti-codon interaction fidelity is known as translational accuracy or proofreading which is determined by the decoding center in the 30S ribosomal subunit. The 16S rRNA in the decoding center is critical for the differentiation of cognate from near cognate tRNA. The 16S rRNA interaction partner, the rpsL (S12) also plays an important role in decoding and it has various 16S rRNA contact sites at H3, H5, H11, H12, H18, H19, H25, H27, 560 and H44 (Figure 3.9)68.

Cognate tRNA binding in the A-site induces global domain movements of the 30S subunit and alterations in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S rRNA76. Involving in the translational fidelity, G530 which is also known as ‘G530 pseudoknot’ in the 5’ domain of 16S rRNA locates at some distance from the decoding center near A1492 and A1493 in the Helix 44 of 3’ minor domain13. The rpsL is the sole protein located

Globular domain

Long terminal elongation

28

in the close vicinity of the decoding center of the 30S ribosomal subunit and its K45 loop of Thermus thermophilus (equivalent to K43 in MTB) is close to the A-site codon-anticodon helix13,69. The 16S rRNA G530 loop being stabilized by rpsL forms the shoulder domain which becomes connected directly via the codon-anticodon complex to the 16S rRNA helix 44 at the decoding center13.

Figure 3.9 Ribosomal protein S12 (brown) interacting with the various helical sites of 16S rRNA at the functional center of the ribosomal 30S subunit68.

3.7.2.6 SM inhibition of protein synthesis SM binds to 16S rRNA and ribosomal protein S12 in the

decoding center of the 30S ribosomal subunit and induces the structural rearrangement that confers the effects on miscoding. Codon misreading renders the bacteria unable to initiate proteins synthesis and leads to its death13. Specifically, SM binds to the phosphate backbone of the Thermus thermophilus 16S rRNA in the four different domains – U14 in helix 1, C526 and G527 in the loop 530, A913 and A914 in helix 27/28, and C1490 and G1491 in the helix 44 – through there forming both salt bridges and hydrogen bonds to contact with the K45 rpsL protein69 (Figure 3.10). As a result, SM disturbs the A-site function, eventually leading to the misreading of the genetic code during translation13. Many studies have shown that mutations around these sites compromise SM binding and contribute to its resistance in bacteria13,64,69.

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Figure 3.10 Chemical structure of SM showing interactions of the various groups with specific residues of the ribosome69. 3.8 SM resistance in TB

SM is the first drug successfully used for the treatment of TB. However, as

a consequence of its historical use as in a mono-therapy, the resistance to SM was found very high. The first mutants resistant to SM were reported as early as in 1946, only two years after its discovery10. The SM resistant MTB mutants can be classified into three distinct types depending on their level of resistance, high, intermediate or low-level resistance.

SM kills actively growing MTB H37Rv control strain with MIC of 4.0 ug/ml79. The Tables 3.4 and 3.5 also show the WHO and CLSI recommended critical concentration and MIC range for SM respectively. By MGIT 960 instrumentation, the MIC can be established to determine the resistance level for SM in clinical isolates by using 1.0 µg/ml followed by using 4.0 µg/ml80. According to this, 4.0 µg/ml is used as a cut-off to separate low-level from the intermediate-level drug resistance and the 20.0 µg/ml is used to categorize the high-level drug resistance. However, there are several other reports on SM resistance associated with genotypic patterns defining high, intermediate and low SM resistance with drug concentrations of ≥16 µg/ml, 8 µg/ml and ≤4 µg/ml respectively81.

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Due to its clinical importance, molecular mechanism of resistance to SM has been extensively studied in MTB. Although some mutations conferring SM resistance were elucidated, their genetic basis resistance is still not fully understood16. To date, only chromosomal mutations in rpsL, rrs and gidB genes have been significantly reported to involve SM resistance of MTB5 though some studies have tried to prove the involvement of the intrinsic resistance mechanism15,82. Mutations around the SM resistance binding sites on both rrs and rpsL result the increased translation accuracy or hyperaccuracy64,69. In contrast, gidB mutations did not prove the formation of hyperaccuracy phenotypes16.

As the site of action of SM is at the ribosomal protein S12 and the 16S rRNA of the 30S subunit of the ribosome, several investigators have found that high-level SM resistance is often linked to the mutations in the S12 protein encoding rpsL gene whereas the intermediate resistant level is responsible by the 16S rRNA encoding rrs gene6,14,15,81. The mutations in these two genes account for 52-59 % and 0-28% of SM resistance receptively5,17. On the other hands, the gidB gene conferring low-level SM resistance is the last genotype discovery in SM resistant MTB genes accounting for around 30% of the SM resistant in MTB clinical isolates16.

3.8.1 Mutations found in rpsL gene

The majority of the mutations found in rpsL gene that confer resistance to, or dependence on, SM are known as hyperaccurate phenotypes16, which compensates the effect of drug, without interfering the drug and the ribosome interaction. The most common mutation found in rpsL is lysine (K) to arginine (G) substitution at residue 43 by transition mutation14,15,17,81. However, the incidence for this mutation varies depending on geographical areas ranging from 13.2 to 89% of all rpsL mutations related to SM resistance17,18. To interest, this mutation is significantly found in MTB Beijing lineage18,83. Another common mutation is found at codon 88 and frequently reported as amino acid substitution K88R, K88T and K88Q5,14,18.

The K43R mutation invariably results the high-level SM resistance with MICs of >1000 µg/ml while K88R gave more heterogeneous MICs, ranging from 250 to >1000 µg/ml14. Although these two common mutations of rpsL gene are

31

significantly related to the high-level resistance of SM, they can rarely be found in the strains with MICs below the resistance break point17. Some other SNPs in this gene (e.g., A363G, T51C and C117T) which are not involved with resistance phenotype are also reported in the SM susceptible strain15,17.

Certain drug-resistant bacteria grow more slowly than susceptible bacteria because the mutations that confer resistance also reduce the overall fitness and growth rate of the organism and this phenomenon is known as ‘cost of resistance’. The high-level SM resistance caused by rpsL mutation did not carry a fitness cost84.

3.8.2 Mutations found in rrs gene Like rpsL mutation, a number of mutations within 16S rRNA

encoded by rrs also lead SM resistance and hyperaccuracy5. Mutations of rrs gene often causing the intermediate-level SM resistance are found in the 530 (eg., A514C or C517T) and 915 loops (e.g., A906G or A907T) of the 16S rRNA14,17. The acquired resistance of MTB after exposure to SM appeared to be caused by a ‘C’ insertion in the 530 loop85. The mutations near the upstream and downstream of the 530 loop were also detected in SM resistant clinical isolates86. Moreover, other significant mutation that is rarely reported in SM resistance, but is predominantly found in aminoglycosides, i.e. kanamycin (KM) and amikacin (AK), is A1401G SNP15. The double mutations in both rpsL and rrs genes that are responsible to SM resistant phenotypes are also found in several investigations15,86. The sequence polymorphism which is the nucleotide change at position 491 is reported and it is not associated with SM resistance in MTB87.

3.8.3 Mutations found in gidB gene Unlike rpsL and rrs, various types of mutations associated with SM

resistance MTB are found in gidB gene15–17. Even though the specific mutation spot of gidB that can confer SM resistance cannot be designated yet, many gidB mutations were found in SM resistant isolates with no rpsL and rrs mutations. On the other hand, the lineage specific polymorphism of gidB gene is significantly reported in MTB. The gidB polymorphism L16R (T47G) is exclusively present in the Latin American

32

Mediterranean (LAM) genotype, while the gidB polymorphism E92D (A276C) is associated with the Beijing genotype lineage16,88.

Furthermore, gidB gene mutation is a predisposing factor conferring the high-level SM resistance at a frequency more than 2000 times greater than that has seen in wild type strains16. However, it is not understood why SM sensitive isolates with gidB polymorphism still respond favorably to SM treatment while nearly half of those found to be SM-resistant responds poorly74. The double mutations (rpsL gidB or rrs gidB) and triple mutations (rpsL rrs gidB) were reported in rare frequency16,17.

3.9 TB burden in Thailand

Having a population of approximately 67 million, Thailand is classified as

one of the 22 high TB burden countries by WHO. In 2014, the estimated prevalence and incidence rates of all forms of TB were 236 and 171 per 100,000 populations respectively. Approximately 72,000 TB cases were notified in 2014. Among TB cases, MDR-TB was found as 2% in new TB cases and 19% in retreatment cases2. In collaboration with WHO and other non-government organizations (NGOs), Ministry of Public Health (MoPH) Thailand has been implementing the Stop TB partnership and the country has achieved full direct observed therapies (DOTs) coverage3.

Regarding with the laboratory diagnosis, in addition to the phenotypic drug susceptibility tests, newer molecular technologies such as GenoType MTBDRplus test and Xpert MTB/RIF have been introduced in Thailand3. The mutational analysis of MTB resistance genes especially to INH and RIF were also researched in Thai clinical isolates19,20. Recently, molecular survey of second-line injectable drugs such as amikacin, kanamycin and capreomycin were done in MDR/XDR strains in Thailand89. However, to date, no molecular characterization of remaining anti-TB injectable drug, SM, has been carried out. According to one phenotypic drug susceptibility test survey done in Thai clinical isolates, MTB was found more resistant to streptomycin (2.1%) than kanamycin (0.7%)90.

33

4. MATERIALS AND METHODS 4.1 Experimental Outline

DNA extraction of MTB H37Rv reference strains and Thai clinical isolates

Amplification of rpsl, rrs and gidB DNA sequences by Polymerase Chain Reaction (PCR)

Sequencing of purified DNA products

Data analysis of DNA Sequencing results for mutations

Agarose gel electrophoresis of PCR amplified DNA products

Evaluation of the prevalence of mutations to SM resistance in Thai isolates

Correlation of the SM resistant genotypes with the MTB lineages

Identification of nt 16 mutation on upstream of rrs by MAMA-PCR

RD105 DTM-PCR for 57 Beijing isolate and 18 non-Beijing lineage isolates

Mutations identified No mutation identified

whiB7 gene PCR amplification, and sequencing

34

4.2 Sample size calculation

The estimated sample size was calculated from the TB prevalence of Thailand reported in WHO global TB report 201435. The sample size necessary for the 2013 total population of Thailand is 195.92.

This data was calculated as the following formula91.

N =

N = Sample size number α = Type I error = 0.05, Zα= 1.96 P = Expected proportion = 0.1535 Q = 1 - P d = Desired precision = 0.05

According to the drug susceptibility tests done on countrywide samples,

E/SM resistance accounted for about 8% among the new case samples92. Therefore, the estimated sample size necessary for 8% of SM resistance is 15.67.

4.3 Mycobacterium tuberculosis clinical isolates used in this study

A total of 287 MTB isolates were collected during 2007 to 2011 from the

patients that had not previously been treated for TB at Bamrasnaradura Infectious Diseases Institute, Nonthaburi, Thailand. The conventional identification and drug susceptibility testing (DST) against four first-line anti-TB drugs were performed at Central Chest Disease Institute, Ministry of Public Health, Nonthaburi, Thailand. The DST was performed by using the 1% proportional absolute concentration method on Löwenstein–Jensen (LJ) medium with the following critical drug concentrations, 0.2 and 1 µg/ml for INH, and 40 µg/ml, 4 µg/ml and 2 µg/ml for RIF, SM and EMB, respectively.

Zα2PQ

d2

35

The lineage and strain type (clade) of each isolate were identified by standard spoligotyping protocol93. Our spoligotypes data were analyzed through comparison with the international SpolDB4 database (Octal codes were updated in SITVIT web application on September 4th, 2015)94 and the major TB lineages were further analyzed with TB-Lineage (TB–Insight) online software95. Among these 287 isolates, only 46 were SM resistant. Together with them, other 55 SM susceptible isolates with various susceptibility patterns were selected for comparison in this study. Forty six SM resistant isolates were composed of 20 SM mono-resistant, 16 SM and INH resistant, 1 SM and RIF resistant and 9 SM resistant MDR isolates. The drug susceptibility phenotypes of 55 SM susceptible isolates were as follows; 18 were INH mono-resistant, 7 were RIF mono-resistant, 10 were MDR and 20 were four drugs susceptible isolates. Details of anti-TB drug susceptibility phenotypes and lineage differentiation are shown in Table 4.1 and 4.2 receptively. The pan anti-TB drug-

susceptible H37Rv (ATCC27294) MTB strain was used as a reference sequence in all assays.

Table 4.1 Mycobacterium tuberculosis clinical isolates used in this study

Phenotypic susceptibility groups Number of isolates

SM resistance SM mono-resistant 20 SM and INH resistant 16 SM and RIF resistant 1 MDR with SM resistant 9 Total 46

Non-SM resistance INH mono-resistant 18 RIF mono-resistant 7 MDR with SM susceptible 10 INH, RIF, EMB, SM susceptible 20

Total 55

36

Table 4.2 The lineage of Mycobacterium tuberculosis clinical isolates used in this study

Lineage identification by LSP method* Number of isolates

East Asian (Beijing Types) 57 Indo–Oceanic 34 Euro–American 9 Unknown 1

Total 101 *According to large sequence polymorphism (LSP)-based lineage identification association obtained from TB-

Lineage95 4.4 Preparation of amplified and purified Mycobacterium tuberculosis DNA

4.4.1 Preparation of genomic DNA and its quantification One hundred and one MTB clinical isolates and MTB H37Rv strain

were grown on LJ medium and heat-inactivated in Biosafety Level-3 (BSL-3) laboratory at Bamrasnaradura Infectious Diseases Institute. MTB genomic DNA was then extracted by the cetyl-trimethyl ammonium bromide (CTAB) method as previously described96. The purity and quantity of genomic DNA was spectrophotometrically measured (NanoDrop 2000, Thermo Fisher Scientific Inc, USA) at absorbance 260 and 280 nm (A260nm and A280nm). The purity was satisfied due to the ratio of A260nm and A280nmwere between 1.5 and 2.0. For double stranded DNA, the concentration of the genomic DNA was calculated: 50 ng/ul for one OD unit at A260nm.

4.4.2 PCR amplification of rpsl, rrs, gidB and whiB7 DNA sequences Using the primers shown in Table 4.3, the entire length of two

structural genes, i.e. rpsL and gidB and a 1,589 base pairs (bp) fragment of rrs (corresponding to nucleotide position 10 of the ORF to 61 bp downstream of the ORF) were amplified with polymerase chain reaction (PCR) from extracted genomic DNA of 101 clinical MTB isolates and later sequenced. For the 12 SM resistant MTB

37

isolates that carried wild type DNA sequence of rpsL, the 810 bp containing entire length of whiB7 open reading frame and promoter region were further amplified and sequenced (Table 4.3). The oligonucleotide primers used for PCR amplification of the respective genetic loci were either based on previously described elsewhere or newly designed in the present study (Table 4.3).

The amplification mixture (25-µl) was composed of 20 ng MTB genomic DNA template, 1x Pfu PCR reaction buffer, 200 µM (each) deoxynucleotide triphosphate (Bioneer Corp, Korea), 13% (10% for rpsL amplification only) dimethyl sulfoxide, 0.4 µM of individual oligonucleotide primers (Bioneer Corp, Korea) (Table 4.3), 2.5 U of Pfu DNA polymerase (Bioneer Corp, Korea) and sterile ultra-pure water. Amplification reactions were performed under optimized thermocycling conditions on the Mastercycler Nexus PCR (Eppendorf, Germany).

The PCR condition involving the annealing temperature was optimized according to the previously described information in Table 4.3 references and by using DNAMAN software (Version 4.15, LynnonBioSoft, Canada). Amplification condition was set; an initiation step at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 50°C (rpsL) or 64°C (rrs) or 58°C (gidB) or 60°C (whiB7) for 45 second (sec) to 2 min, extension at 72°C for 1-2 min, with a final extension at 72°C for 10 min.

4.4.3 Agarose gel electrophoresis The rpsL, rrs, gidB and whiB7 DNA amplicons were investigated by

agarose gel electrophoresis. The appropriate percentage of agarose gel was prepared by mixing the agarose powder (Axygen Biosciences, Spain) with 0.5× TBE (Tris- Borate-EDTA) buffer pH 8.3 and melting in the microwave oven. The melted agasose gel mixture was slab onto the 10 cm-horizontal gel electrophoresis chamber. Each PCR amplicon was mixed with 10× loading dye, uploaded on the well of solidified agarose gel slab and run electrophoresis in 0.5× TBE buffer at 100 Volts for an approximate time. The DNA band on the gel was visualized by UV Transilluminator (Gene Flash, Syngene Bio Imaging, Japan) following ethidium bromide staining.

38

4.4.4 Gel purification of PCR amplicons for DNA sequencing analysis For DNA sequencing analysis, the rpsl, rrs, gidB and whiB7 PCR

amplicons were extracted from agarose gel and purified by using gel purification kit (Bioneer Corp, Korea). After purification, the DNA samples were verified again by running on the 1% agarose gel and visualized under UV Transiluminator.

4.5 DNA sequencing

Approximately 500-750 ng of purified rpsl, rrs, gidB and whiB7 amplicons were sent for DNA sequencing with the BigDye Terminator Cycle Sequencing Kit version 3.1 kit (Applied Biosystems, Foster City, CA, USA) in an ABI 3730XL DNA Analyzers (Life Technologies, Applied Biosystems, USA).

4.6 Data analysis of DNA sequencing results

To confirm the presence of mutations, sequencing was done in both directions with the same primer pairs as those used in the amplification reactions and occasionally, additional primers were used as specified in Table 4.3. Sequence data were assembled and analyzed with Sequence Scanner v2.0 (Applied Biosystems) and DNAMAN (Version 4.15, LynnonBioSoft, Canada) software. The quality of chromatogram was determined by having average raw signal intensity (>50), average noise (<20) and average raw signal to noise ratio (>20). The qualified forward and reverse sequences were trimmed and assembled in DNAMAN software. The resulting assembled sequences were aligned against the wild type sequences of the respective genetic loci of MTB reference strain H37Rv (NCBI Entrez nucleotide sequence database accession no. AL123456.3 [updated on February 15, 2015]) using the nucleotide blast algorithm and align two sequence tools97. The mutations with the corresponding amino acid changes were identified by DNAMAN (LynnonBioSoft) software.

39

4.7 Mismatched amplification mutation assay PCR (MAMA-PCR) MAMA-PCR primers were designed in this study for the rapid detection of

an allele-specific alteration from T to C at nucleotide position 16 (16T>C) (previously annotated at nt position 15) of rrs gene98. As shown in Table 4.3, the mutant allele-specific forward primer for rrs 16 position carried specific mismatch nucleotide ‘C’ at the 3’ end (rrs-16 mt). To intensify the 3’ mismatch effect, another nucleotide alteration, ‘C’ was introduced at the second position from the 3’ end of rrs-16 mt primer. The conserved wild type forward primer without any base alteration (rrs-16 wt) was also designed for amplification of the wild type sequence. The amplification of mutant allele occurred only in reactions with mutant forward primer (rrs-16 mt). The 264 primer was used as a reverse primer.

The amplification mixture (25-µl) was composed of 20 ng MTB genomic DNA template, 1x Taq PCR reaction buffer, 200 µM (each) deoxynucleotide triphosphate (Bioneer Corp, Korea), 0.2 µM of individual oligonucleotide primers (Bioneer Corp, Korea) (Table 4.3), 5 U of Taq DNA polymerase (Thermo Fisher Scientific, Inc., USA), 1.5 mM MgCl2 (Thermo Fisher Scientific, Inc., USA) and sterile ultra-pure water. Amplification reactions were performed under optimized thermocycling conditions on the Mastercycler Nexus PCR (Eppendorf, Germany). The PCR optimization was achieved by setting the initial step at 94°C for 10 min and it was followed by 25 thermocycling with denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 45 sec.

4.8 Verification of Beijing isolates by RD105 deletion-targeted multiplex PCR

(DTM-PCR)

In order to verify the spoligotyping results, RD105 DTM-PCR was performed for all Beijing (East Asian) isolates (n=57) and representative of non-Beijing isolates (n=18; 15 Indo-Oceanic and 3 Euro-American) using three primers as previously described99 (Table 4.3). The non-Beijing lineage isolates carrying intact RD105 yielded 1,466 bp (1,495 bp by MTB H37Rv accession no. AL123456.3 database)

40

amplicons while the Beijing isolates of MTB (including Ancestral, Modern and W family of Beijing lineage) contained a 3,467 bp (3,468 bp by the MTB H37Rv accession no. AL123456.3 database and DNAMAN software) deletion corresponding to RD105 region yielded only 761 bp (785-bp by MTB H37Rv accession no. AL123456.3 database) DTM-PCR product.

The amplification mixture (20-µl) was composed of 20 ng MTB genomic DNA template, 1x ProFi Taq PCR reaction buffer, 250 µM (each) deoxynucleotide triphosphate (Bioneer Corp, Korea), 0.5 µM of individual oligonucleotide primers (Bioneer Corp, Korea) (Table 4.3), 5 U of ProFi Taq DNA polymerase, 2 mM MgCl2

(Bioneer Corp, Korea), 10% dimethyl sulfoxide and sterile ultra-pure water. Amplification reactions were performed under optimized thermocycling conditions on the Mastercycler Nexus PCR (Eppendorf, Germany). The PCR optimization was achieved by setting the initial step at 95°C for 5 min and it was followed by 40 thermocycling with denaturation at 95°C for 20 sec, annealing at 68°C for 30 sec and extension at 68°C for 4 min. The final extension was at 68°C for 3 min. 4.9 Statistical analysis

The association between mutations and SM resistance as well as MTB lineage was assessed with GraphPad Prism 6 software. The p-value designated at <0.05 was calculated by Fisher exact test to associate the significance of identified mutations with phenotypic SM drug resistance as well as with MTB lineages.

Odds ratio (OR), 95 % confidence intervals (CI), sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were also calculated to indicate the association between the genotypic and phenotypic characteristics and to exclude the most related genotypic markers that confer SM resistance.

41

Table 4.3 The primers for PCR amplification

Target Primer name Primer sequence (5’ 3’) PCR product (bp) Nucleotide positionb Tac(°C) Reference(R)

rpsL S13 GGCCGACAAACAGAACGT 504 781514 - 782017 50

R100 S16 GTTCACCAACTGGGTGAC R100

rrs 285 GAGAGTTTGATCCTGGCTCAG

1589 1471855 - 1473443 64

R101

rrs-1R ACAGACAAGAACCCCTCACG R15

264a TGCACACAGGCCACAAGGGA R101

gidB gidB3 GAACGGAAGATCGTCCAC 977 4407459 - 4408435 58

R83

gidB4 CGATAGTTGAAGCCTGGC R83

whiB7 F UTR-whiB7 GCTGGTTCGCGGTCGGACCT 810 3568321 - 3569130 60

R89 R-whiB7 AGGAGCTGATCCCGGGTTTC This work

MAMA-PCR rrs rrs-16 wt TGGGTTTTGTTTGGAGAGTT

1041 1471842 - 1472882 56 This work

rrs-16 mt TGGGTTTTGTTTGGAGAGCC This work 264 TGCACACAGGCCACAAGGGA R101

DTM-PCR RD105 P1 GGAGTCGTTGAGGGTGTTCATCAGCTCAGTC

4253 78876 – 83128 (P1-P2) 68

R99 P2 CGCCAAGGCCGCATAGTCACGGTCG R99 P3 GGTTGCCCACTGGTCGATATGGTGGACTT 1495 78876 – 80370 (P1-P3) R99

a Primer used for sequencing only bDNA sequencing co-ordinated according to the nucleotide sequence accession number AL123456.3 cTa, annealing temperature

42

CHAPTER 5 RESULTS

5.1 Optimization of PCR amplification of rpsL, rrs, gidB and whiB7 genes

The optimum annealing temperature for amplification of H37Rv MTB

rpsL, rrs, gidB and whiB7 were 50, 64, 58 and 60° C, respectively. The PCR amplicon of the respective genes are shown in Figures 5.1-5.4.

Figure 5.1 PCR amplicon of rpsL gene of MTB H37Rv. Amplicon of MTB H37Rv rpsL shows as a specific band at 504 bp on the 1% agarose gel (as indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, rpsL amplicon derived from MTB H37Rv genomic DNA Lane 2, No template control

43

Figure 5.2 PCR amplicon of rrs gene of MTB H37Rv. Amplicon of MTB H37Rv rrs shows as a specific band at 1,589 bp on the 1% agarose gel (as indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, rrs amplicon derived from MTB H37Rv genomic DNA Lane 2, No template control

44

Figure 5.3 PCR amplicon of gidB gene of MTB H37Rv. Amplicon of MTB H37Rv gidB shows as a specific band at 977 bp on the 1% agarose gel (as indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, gidB amplicon derived from MTB H37Rv genomic DNA Lane 2, No template control

45

Figure 5.4 PCR amplicon of whiB7 gene of MTB H37Rv. Amplicon of MTB H37Rv whiB7 shows as a specific band at 810 bp on the 1% agarose gel (as indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, whiB7 amplicon derived from MTB H37Rv genomic DNA Lane 2, No template control

46

5.2 DNA sequencing analysis of the rpsL, rrs, gidB and whiB7 PCR products derived from H37Rv MTB genomic DNA

The PCR products of rpsL, rrs, gidB and whiB7 of MTB H37Rv were sent for

DNA sequencing and the so-obtained sequencing data were initially examined and cleaned up using Sequence Scanner 2 software (ABI). The forward and the reverse DNA sequence of the individual genes were then assembled and trimmed using DNAMAN software (LinnonBiosoft). The nucleotide length following the assembly and trimming are as follows: rpsL; 447 bp, rrs; 1,498 bp, gidB; 938 bp and whiB7; 778 bp. Thereafter, assembled sequences of the respective genes were analyzed using BLASTN search. It was found that all four fragments were identical to their reference sequences in the genome of MTB H37Rv (accession numbers AL123456.3 or CP009480.1). The homology results are shown in Figures 5.5-5.8.

47

Mycobacterium tuberculosis H37Rv, complete genome

Sequence ID: gb|CP009480.1|Length: 4396119

Score Expect Identities Gaps Strand

693 bits(375) 0.0 375/375(100%) 0/375(0%) Plus/Plus

Features: 30S ribosomal protein S12

30S ribosomal protein S7

Query 1 ATGCCAACCATCCAGCAGCTGGTCCGCAAGGGTCGTCGGGACAAGATCAGTAAGGTCAAG 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 780966 ATGCCAACCATCCAGCAGCTGGTCCGCAAGGGTCGTCGGGACAAGATCAGTAAGGTCAAG 781025

Query 61 ACCGCGGCTCTGAAGGGCAGCCCGCAGCGTCGTGGTGTATGCACCCGCGTGTACACCACC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 781026 ACCGCGGCTCTGAAGGGCAGCCCGCAGCGTCGTGGTGTATGCACCCGCGTGTACACCACC 781085

Query 121 ACTCCGAAGAAGCCGAACTCGGCGCTTCGGAAGGTTGCCCGCGTGAAGTTGACGAGTCAG 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 781086 ACTCCGAAGAAGCCGAACTCGGCGCTTCGGAAGGTTGCCCGCGTGAAGTTGACGAGTCAG 781145

Query 181 GTCGAGGTCACGGCGTACATTCCCGGCGAGGGCCACAACCTGCAGGAGCACTCGATGGTG 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 781146 GTCGAGGTCACGGCGTACATTCCCGGCGAGGGCCACAACCTGCAGGAGCACTCGATGGTG 781205

Query 241 CTGGTGCGCGGCGGCCGGGTGAAGGACCTGCCTGGTGTGCGCTACAAGATCATCCGCGGT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 781206 CTGGTGCGCGGCGGCCGGGTGAAGGACCTGCCTGGTGTGCGCTACAAGATCATCCGCGGT 781265

Query 301 TCGCTGGATACGCAGGGTGTCAAGAACCGCAAACAGGCACGCAGCCGTTACGGCGCTAAG 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 781266 TCGCTGGATACGCAGGGTGTCAAGAACCGCAAACAGGCACGCAGCCGTTACGGCGCTAAG 781325

Query 361 AAGGAGAAGGGCTGA 375

|||||||||||||||

Sbjct 781326 AAGGAGAAGGGCTGA 781340

Figure 5.5 BLASTN analysis of MTB H37Rv rpsL (30S ribosomal protein S12) open reading frame (375 bp) in this study.

48

Mycobacterium tuberculosis H37Rv, complete genome

Sequence ID: gb|CP009480.1|Length: 4396119

Score Expect Identities Gaps Strand

2767 bits(1498) 0.0 1498/1498(100%) 0/1498(0%) Plus/Plus

Features:rRNA-16S ribosomal RNA

Query 1 TGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGTCTCTTCGGAGATACTCGAGTG 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468483 TGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAAGGTCTCTTCGGAGATACTCGAGTG 1468542

Query 61 GCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAAC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468543 GCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAAC 1468602

Query 121 TGGGTCTAATACCGGATAGGACCACGGGATGCATGTCTTGTGGTGGAAAGCGCTTTAGCG 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468603 TGGGTCTAATACCGGATAGGACCACGGGATGCATGTCTTGTGGTGGAAAGCGCTTTAGCG 1468662

Query 181 GTGTGGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTGACGGCCTACCAAGGCGAC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468663 GTGTGGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTGACGGCCTACCAAGGCGAC 1468722

Query 241 GACGGGTAGCCGGCCTGAGAGGGTGTCCGGCCACACTGGGACTGAGATACGGCCCAGACT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468723 GACGGGTAGCCGGCCTGAGAGGGTGTCCGGCCACACTGGGACTGAGATACGGCCCAGACT 1468782

Query 301 CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGC 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468783 CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGC 1468842

Query 361 CGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCATCGACGAAGGTCCGGG 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468843 CGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCACCATCGACGAAGGTCCGGG 1468902

Query 421 TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGC 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468903 TTCTCTCGGATTGACGGTAGGTGGAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGC 1468962

Query 481 GGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGTGG 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1468963 GGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGTGG 1469022

Query 541 TTTGTCGCGTTGTTCGTGAAATCTCACGGCTTAACTGTGAGCGTGCGGGCGATACGGGCA 600

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469023 TTTGTCGCGTTGTTCGTGAAATCTCACGGCTTAACTGTGAGCGTGCGGGCGATACGGGCA 1469082

Query 601 GACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATC 660

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469083 GACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATC 1469142

(Please see over leaf)

49

Query 661 AGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAA 720

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469143 AGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAA 1469202

Query 721 GCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTA 780

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469203 GCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTA 1469262

Query 781 GGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGG 840

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469263 GGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGG 1469322

Query 841 GGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGA 900

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469323 GGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGA 1469382

Query 901 GCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGAC 960

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469383 GCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGAC 1469442

Query 961 GCGTCTAGAGATAGGCGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAG 1020

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469443 GCGTCTAGAGATAGGCGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAG 1469502

Query 1021 CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCATGTTGC 1080

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469503 CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCATGTTGC 1469562

Query 1081 CAGCACGTAATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGG 1140

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469563 CAGCACGTAATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGG 1469622

Query 1141 ATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGT 1200

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469623 ATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGT 1469682

Query 1201 ACAAAGGGCTGCGATGCCGCGAGGTTAAGCGAATCCTTAAAAGCCGGTCTCAGTTCGGAT 1260

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469683 ACAAAGGGCTGCGATGCCGCGAGGTTAAGCGAATCCTTAAAAGCCGGTCTCAGTTCGGAT 1469742

Query 1261 CGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGC 1320

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469743 CGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGC 1469802

Query 1321 TGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAA 1380

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469803 TGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAA 1469862

Query 1381 CACCCGAAGCCAGTGGCCTAACCCTCGGGAGGGAGCTGTCGAAGGTGGGATCGGCGATTG 1440

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469863 CACCCGAAGCCAGTGGCCTAACCCTCGGGAGGGAGCTGTCGAAGGTGGGATCGGCGATTG 1469922

Query 1441 GGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTTCT 1498

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 1469923 GGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTTCT 1469980

Figure 5.6 BLASTN analysis of MTB H37Rv rrs (16S rRNA) fragment (1,498 bp) in this study.

50

Mycobacterium tuberculosis str. Haarlem, complete genome

Sequence ID: gb|CP001664.1|Length: 4408224

Score Expect Identities Gaps Strand

1247 bits(675) 0.0 675/675(100%) 0/675(0%) Plus/Plus

Features: chromosome partitioning protein ParA

glucose-inhibited division protein B Gid

Query 1 TCACGCCGTCCCTCCACTCGCCATCCGTGCCGACCCTCGGGCGATCTGCTTTCCACGTCG 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404321 TCACGCCGTCCCTCCACTCGCCATCCGTGCCGACCCTCGGGCGATCTGCTTTCCACGTCG 4404380

Query 61 TGCGAACACCACGGTCGCGGGCGGACGCAAATAGTTCGCGCCACATGTCACCACCCTGAC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404381 TGCGAACACCACGGTCGCGGGCGGACGCAAATAGTTCGCGCCACATGTCACCACCCTGAC 4404440

Query 121 ATCAACCGCGCCCGATGCGATCATCACACGCCGGTGCTCCCGTACTTCGTCGTGAGCCCG 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404441 ATCAACCGCGCCCGATGCGATCATCACACGCCGGTGCTCCCGTACTTCGTCGTGAGCCCG 4404500

Query 181 CTCGCCTTTGATGGCGAGCATTCGCCCGTTCGGCCGTATCAACGGCATGCTCCATTTCGT 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404501 CTCGCCTTTGATGGCGAGCATTCGCCCGTTCGGCCGTATCAACGGCATGCTCCATTTCGT 4404560

Query 241 CAACTTGTCCAACGCGGCCACCGCCCGTGACACCGCAGCGTCGCTGCCGCCCAATTGGTC 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404561 CAACTTGTCCAACGCGGCCACCGCCCGTGACACCGCAGCGTCGCTGCCGCCCAATTGGTC 4404620

Query 301 CTGCACCCAGGACTCCTCGGCGCGCCCCCGCACGATCTCAACGGCCACGCCCAGATCTGT 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404621 CTGCACCCAGGACTCCTCGGCGCGCCCCCGCACGATCTCAACGGCCACGCCCAGATCTGT 4404680

Query 361 CACCATCTCTCGAAGAAACTCGGTGCGGCGCAGTAGCGGTTCTAGGAGAACTACCTGGAG 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404681 CACCATCTCTCGAAGAAACTCGGTGCGGCGCAGTAGCGGTTCTAGGAGAACTACCTGGAG 4404740

Query 421 GTCCGGCCGCGCTATCGCCAATGGCACGCCCGGCAACCCGGCTCCGCTACCGATATCCAC 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404741 GTCCGGCCGCGCTATCGCCAATGGCACGCCCGGCAACCCGGCTCCGCTACCGATATCCAC 4404800

Query 481 GACCCGGTCACCGCGTTCGAGGAGCTCACCGATCACGGCGCAGTTCAGTAGATGCCGGTC 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404801 GACCCGGTCACCGCGTTCGAGGAGCTCACCGATCACGGCGCAGTTCAGTAGATGCCGGTC 4404860

Query 541 CCATAGCCTACCGACTTCGCGGGGTCCCACCAGCCCCCGCTCCACACCGGGTCCCGCCAA 600

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404861 CCATAGCCTACCGACTTCGCGGGGTCCCACCAGCCCCCGCTCCACACCGGGTCCCGCCAA 4404920

Query 601 CGCTTCGGCGTACCGCCGAGCAAGGCCAAGCCGCGGTCCGAAGATCGCAGACGCCGCGGG 660

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 4404921 CGCTTCGGCGTACCGCCGAGCAAGGCCAAGCCGCGGTCCGAAGATCGCAGACGCCGCGGG 4404980

Query 661 CTCGATCGGAGACAT 675

|||||||||||||||

Sbjct 4404981 CTCGATCGGAGACAT 4404995

Figure 5.7 BLASTN analysis of MTB H37Rv gidB (glucose-inhibited division protein B Gid) open reading frame (675 bp) in this study.

51

Mycobacterium tuberculosis H37Rv, complete genome

Sequence ID: gb|CP009480.1|Length: 4396119

Score Expect Identities Gaps Strand

1437 bits(778) 0.0 778/778(100%) 0/778(0%) Plus/Plus

Features: ABC transporter ATP-binding protein

transcriptional regulator

Query 1 GCCGAGCCCGACGCGATCGTCGTCTGAGCCGGCTCGCGCCGGCGGGCGCACCATCGCGGG 60

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561486 GCCGAGCCCGACGCGATCGTCGTCTGAGCCGGCTCGCGCCGGCGGGCGCACCATCGCGGG 3561545

Query 61 CTATGCAACAGCATCCTTGCGCGGACGTCCGCGCGGACGCTTGTGACTCACGATCGAGCC 120

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561546 CTATGCAACAGCATCCTTGCGCGGACGTCCGCGCGGACGCTTGTGACTCACGATCGAGCC 3561605

Query 121 TTGGTCGAATATCTCACCACCCCAAACGCCCCAGGGTTCAGCCCGCTGAAGCGCCGCGGC 180

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561606 TTGGTCGAATATCTCACCACCCCAAACGCCCCAGGGTTCAGCCCGCTGAAGCGCCGCGGC 3561665

Query 181 CAAGCACTGCCGCCTGATCGGGCAGCTCACACACAGTGTCTTGGCTACCTCGAGACCGGC 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561666 CAAGCACTGCCGCCTGATCGGGCAGCTCACACACAGTGTCTTGGCTACCTCGAGACCGGC 3561725

Query 241 CGGGGTATCGGCGAACCACAGATCGGGATCACCGACGTGGCACGGCAAAACCGGCAATCT 300

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561726 CGGGGTATCGGCGAACCACAGATCGGGATCACCGACGTGGCACGGCAAAACCGGCAATCT 3561785

Query 301 TTGTCTGGGGGTCTGTCTGGGGACTGTCAGTACCGACACGTCCTGTTTCACCTGCTTCCT 360

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561786 TTGTCTGGGGGTCTGTCTGGGGACTGTCAGTACCGACACGTCCTGTTTCACCTGCTTCCT 3561845

Query 361 GGTCTGGTGGCGGTTCTTCGAAAGTGATCCGGACCAGGGATGCTGCGGTGGGCAGATGTC 420

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561846 GGTCTGGTGGCGGTTCTTCGAAAGTGATCCGGACCAGGGATGCTGCGGTGGGCAGATGTC 3561905

Query 421 CCGAAAGTTTGGCCACGGATCCTGTGACTTCGGGTCCGTGGCCATCTGGCGAAACGGGGC 480

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561906 CCGAAAGTTTGGCCACGGATCCTGTGACTTCGGGTCCGTGGCCATCTGGCGAAACGGGGC 3561965

Query 481 TGATTACGTAGCGCTTACGTAGAGCCCCGCTCCACGGACTCGTCAGTCGCGGCGGCGACA 540

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3561966 TGATTACGTAGCGCTTACGTAGAGCCCCGCTCCACGGACTCGTCAGTCGCGGCGGCGACA 3562025

Query 541 CGGTTCTTGCTATGGGGGGTTCCCGCGGTTGGCACCGCGGCAGCCGCGCCGACACCAAAT 600

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3562026 CGGTTCTTGCTATGGGGGGTTCCCGCGGTTGGCACCGCGGCAGCCGCGCCGACACCAAAT 3562085

(Please see over leaf)

52

Query 601 GCGTTGTTGTCAATCACCGCGGCCGCCCTCCTCTCGTGTCGCGCGCGGTTGCCAGCCCCC 660

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3562086 GCGTTGTTGTCAATCACCGCGGCCGCCCTCCTCTCGTGTCGCGCGCGGTTGCCAGCCCCC 3562145

Query 661 CAATGCCATCTCCAGGCTGGCAGCAGAATGCGACCTGGAGGTTAACCGGTGGCAGCAGCT 720

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3562146 CAATGCCATCTCCAGGCTGGCAGCAGAATGCGACCTGGAGGTTAACCGGTGGCAGCAGCT 3562205

Query 721 GACCACAACCGATTTTCTGACCTGCGCGTTTGCCGGTACAGGCCCGGTTCAGGTCCGA 778

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

Sbjct 3562206 GACCACAACCGATTTTCTGACCTGCGCGTTTGCCGGTACAGGCCCGGTTCAGGTCCGA 3562263

Figure 5.8 BLASTN analysis of MTB H37Rv whiB7 (transcriptional regulator) open reading frame and promoter region (778 bp) in this study.

53

5.3 Amplification of rpsL, rrs, gidB and whiB7 genes in MTB clinical isolates After obtaining the optimal annealing temperature for amplification of

rpsL, rrs, gidB and whiB7 in Section 5.1, the conditions for the individual gene were employed to amplify the respective gene fragments from 101 isolates (46 SM resistant and 55 SM susceptible). The representative PCR amplicons of each gene are shown in Figures 5.9-5.12.

Figure 5.9 Agarose gel electrophoresis of rpsL PCR amplicons obtained from clinical MTB isolates (504 bp was indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, No. 218 (MDR) Lane 2, No. 235 (MDR) Lane 3, No. 281 (MDR) Lane 4, No. 294 (MDR + SM resistance) Lane 5, No. 194 (Susceptible) Lane 6, No. 197 (Susceptible) Lane 7, No. 220 (Susceptible) Lane 8, No. 256 (Susceptible) Lane 9, No. 263 (Susceptible) Lane 10, No. 275 (Susceptible) Lane 11, No. 296 (Susceptible) Lane 12, No. 11 (RIF mono-resistance) Lane 13, No. 18 (RIF mono-resistance) Lane 14, No. 87 (RIF mono-resistance) Lane 15, No. 72 (SM mono-resistance) Lane 16, No. 84 (SM mono-resistance) Lane 17, No. 90 (SM mono-resistance) Lane 18, No. 95 (SM mono-resistance) Lane 19, No template control

54

Figure 5.10 Agarose gel electrophoresis of rrs PCR amplicons obtained from clinical MTB isolates (1,589 bp, was indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Numbers at the left are DNA sizes in base pairs (bp). Lane 1, No. 11 (RIF mono-resistance) Lane 2, No. 18 (RIF mono-resistance) Lane 3, No. 87 (RIF mono-resistance) Lane 4, No. 72 (SM mono-resistance) Lane 5, No. 84 (SM mono-resistance) Lane 6, No. 90 (SM mono-resistance) Lane 7, No. 95 (SM mono-resistance) Lane 8, No. 194 (Susceptible) Lane 9, No. 197 (Susceptible) Lane 10, No. 220 (Susceptible) Lane 11, No. 256 (Susceptible) Lane 12, No. 263 (Susceptible) Lane 13, No. 275 (Susceptible) Lane 14, No. 296 (Susceptible) Lane 15, No. 218 (MDR) Lane 16, No. 235 (MDR) Lane 17, No. 281 (MDR) Lane 18, No. 294 (MDR + SM resistance) Lane 19, No template control

55

Figure 5.11 Agarose gel electrophoresis of gidB PCR amplicons obtained from clinical MTB isolates (977 bp was indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, No. 51 (SM mono-resistance) Lane 2, No. 59 (INH mono-resistance) Lane 3, No. 61 (MDR) Lane 4, No. 68 (SM mono-resistance) Lane 5, No. 77 (INH mono-resistance) Lane 6, No. 78 (INH mono-resistance) Lane 7, No. 80 (MDR) Lane 8, No. 131 (Susceptible) Lane 9, No. 149 (Susceptible) Lane 10, No. 153 (Susceptible) Lane 11, No. 267 (INH + SM resistance) Lane 12, No. 6 (Susceptible) Lane 13, No. 19 (Susceptible) Lane 14, No. 31 (Susceptible) Lane 15, No. 36 (Susceptible) Lane 16, No. 48 (Susceptible) Lane 17, No. 56 (Susceptible) Lane 18, No template control

56

Figure 5.12 Agarose gel electrophoresis of whiB7 PCR amplicons obtained from clinical MTB isolates (810 bp, as indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, No. 25 (SM mono-resistance) Lane 2, No. 68 (SM mono-resistance) Lane 3, No. 72 (SM mono-resistance) Lane 4, No. 217 (SM mono-resistance) Lane 5, No. 297 (SM mono-resistance) Lane 6, No. 4 (MDR + SM resistance) Lane 7, No. 15 (MDR + SM resistance) Lane 8, No. 176 (MDR + SM resistance) Lane 9, No. 1 (SM + INH resistance) Lane 10, No. 39 (SM + INH resistance) Lane 11, No. 250 (SM + INH resistance) Lane 12, No template control

57

5.4 Mutations found in rpsL, rrs and gidB

Coding sequences of rpsL, rrs and gidB amplified and sequenced from 101 isolates (46 SM resistant and 55 SM susceptible) were analyzed. Detail mutation results are shown in Table 5.1. The rpsL DNA sequence analyses indicated that the majority of SM resistant isolates, 31 out of 46, carried an A to G transition mutation at nucleotide (nt) position 128 (128A>G), resulting in substitution of lysine by arginine at codon 43 (K43R) as shown in Figure 5.13A (middle panel). The additional two different missense mutations were found in the codon 88 of other two SM resistant MDR isolates. One carried 263A>G substitution conferring K88R amino acid alteration and another carried 263A>C transversion conferring K88T, respectively (Figure 5.13B).

Interestingly, the chromatogram of one SM mono-resistant isolate (isolate no. 68) revealed mixed rpsL wild type (WT) and mutated 128A>G in the DNA sequencing data of both forward and reverse sequencing data (Figure 5.13A bottom panel).

In the non-SM resistant group, none of the rpsL mutation was found in either INH or RIF mono-resistant and non-SM resistant MDR isolates. However, in the remaining of the four first-line drug susceptible isolates (susceptible to INH, RIF, EMB and SM, respectively), rpsL DNA sequencing analyses indicated that two strains carried K43R mutation. Subsequently, to verify their SM susceptible character, the drug susceptibility test was repeated and the results confirmed their susceptibilities at SM critical concentration of 4 µg/ml by using 1% proportional absolute concentration method.

Surprisingly, none of the mutation in 16S rRNA coding sequence (rrs) was detected among the 46 SM resistant isolates in this study. Only one isolate belonging to the four first-line drug susceptible group showed 1025T>C polymorphism (Figure 5.14).

58

Table 5.1 Distribution of mutations in rpsL, rrs, gidB and whiB7 genes of MTB clinical isolates Resistance pattern (Total no. of isolates)

LSP-based Lineages* (no. of isolates)

Spoligotyping Clades (no. of isolates)

Mutations in nucleotide position [amino acid position] (no. of isolates)

SM Other drug resistance

rpsL rrs gidB whiB7β

Resistance (46)

Mono- resistance (20)

East Asian (15)

Beijing (15) 128A>G [K43R] (14) WT (1)

WT (14) WT (1)

276A>C [E92D], 615A>Gα (14)

276A>C [E92D], 615A>Gα (1)

ND WT (1)

Indo-Oceanic (4)

EAI5 (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1) WT (1)

EAI2–Nonthaburi (1) 128A>G [K43R]ǂ (1) WT (1) 276A>C [E92D]ǂ, 330G>Tǂ,

615A>Gα (1)

ND

Manu1 (1) WT (1) WT (1) 615A>Gα (1) WT (1)

Unknown (1) WT (1) WT (1) WT (1) WT (1)

Euro-American (1)

T2 (1) WT (1) WT (1) 134G>A [W45stop] (1) WT (1)

INH (16) East Asian (13)

Beijing (13) 128A>G [K43R] (12) WT (1)

WT (12) WT (1)

276A>C [E92D], 615A>Gα (12)

276A>C [E92D], 615A>Gα (1)

ND 242G>A [R81H], 246_247insTT [P82fs] (1)

Indo-Oceanic (3)

EAI2–Nonthaburi (3) WT (3) WT (3) 330G>Tα, 615A>Gα (3) 188delG [W63fs] (3)

59

Table 5.1 Distribution of mutations in rpsL, rrs, gidB and whiB7 genes of MTB clinical isolates (Continued)

Resistance pattern (Total no. of isolates)

LSP-based Lineages* (no. of isolates)

Spoligotyping Clades (no. of isolates)

Mutations in nucleotide position [amino acid position] (no. of isolates)

SM Other drug resistance

rpsL rrs gidB whiB7β

Resistance (46)

RIF (1) East Asian (1) Beijing (1) 128A>G [K43R] (1) WT (1) 276A>C [E92D], 615A>Gα (1) ND

MDR (9) East Asian (3) Beijing (3) 128A>G [K43R] (2) 263A>G [K88R] (1)

WT (2) WT (1)

276A>C [E92D], 615A>Gα (2)

276A>C [E92D], 615A>Gα (1)

ND

Indo-Oceanic (4)

EAI5 (2) 128A>G [K43R] (1) WT (1)

WT (1) WT (1)

276A>C [E92D], 615A>Gα (1)

276A>C [E92D], 615A>Gα (1) ND WT (1)

EAI1–SOM (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1) WT (1)

Manu1 (1) WT (1) WT (1) 330G>Tǂ , 615A>Gα (1) WT (1)

Euro-American (2)

T1 (1) 128A>G [K43R] (1) WT (1) WT (1) ND T3-ETH (1) 263A>C [K88T] (1) WT (1) 206G>A [G69D] (1)

Susceptible (55)

Susceptible to INH, RIF, SM & EMB (20)

East Asian (9) Beijing (9) 128A>G [K43R] (2) WT (6) WT (1)

WT (2) WT (6) WT (1)

276A>C [E92D], 615A>Gα (2)

276A>C [E92D], 615A>Gα (6)

330G>Tα, 615A>Gα (1)

ND

60

Table 5.1 Distribution of mutations in rpsL, rrs, gidB and whiB7 genes of MTB clinical isolates (Continued) Resistance pattern (Total no. of isolates)

LSP-based Lineages* (no. of isolates)

Spoligotyping Clades (no. of isolates)

Mutations in nucleotide position [amino acid position] (no. of isolates)

SM Other drug resistance

rpsL rrs gidB whiB7β

Susceptible (55)

Susceptible to INH, RIF, SM & EMB (20)

Indo-Oceanic (8)

EAI5 (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1) ND

EAI1-SOM (2) WT (1) WT (1)

1025T>C (1) WT (1)

330G>Tα, 615A>Gα (1)

330G>Tα, 615A>Gα (1) EAI2-Manila (2) WT (2) WT (2) 330G>Tα, 615A>Gα (2) EAI2-Nonthaburi (2) WT (2) WT (2) 330G>Tα, 615A>Gα (2) Manu3 (1) WT (1) WT (1) WT (1)

Euro- American (3)

T1 (1) WT (1) WT (1) WT (1)

H3 (2) WT (2) WT (2) WT (2) INH mono-resistance (18)

East Asian (7) Beijing (7) WT (7) WT (7) 276A>C [E92D], 615A>Gα (7) ND

Indo-Oceanic (9)

EAI5 (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1)

EAI1-SOM (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1)

EAI2-Manila (3) WT (3) WT (3) 330G>Tα, 615A>Gα (3)

EAI6-BGD1 (4) WT (4) WT (4) 330G>Tα, 423G>Aα, 615A>Gα (4) Euro-American (2)

T3-OSA (1) WT (1) WT (1) WT (1)

H3 (1) WT (1) WT (1) WT (1)

61

Table 5.1 Distribution of mutations in rpsL, rrs, gidB and whiB7 genes of MTB clinical isolates (Continued) Resistance pattern (Total no. of isolates)

LSP-based Lineages* (no. of isolates)

Spoligotyping Clades (no. of isolates)

Mutations in nucleotide position [amino acid position] (no. of isolates)

SM Other drug resistance

rpsL rrs gidB whiB7β

RIF mono-resistance (7)

East Asian (3) Beijing (3) WT (3) WT (3) 276A>C [E92D], 615A>Gα (3) ND

Indo-Oceanic (3)

EAI1-SOM (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1)

EAI2-Nonthaburi (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1)

Manu_ancestor (1) WT (1) WT (1) 615A>Gα (1) Unknown (1) Unknown (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1)

MDR (10) East Asian (6) Beijing (6) WT (5) WT (1)

WT (5) WT (1)

276A>C [E92D], 615A>Gα (5)

330G>Tα, 615A>Gα (1)

ND

Indo-Oceanic (3)

EAI5 (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1) EAI2-Nonthaburi (1) WT (1) WT (1) 330G>Tα, 615A>Gα (1) EAI6-BGD1 (1) WT (1) WT (1) 330G>Tα, 423G>Aα, 615A>Gα (1)

Euro-American (1)

H3 (1) WT (1) WT (1) WT (1)

*According to large sequence polymorphism (LSP) lineage identification association obtained from TB-Lineage95

α Synonymous amino acid change ǂ Isolate with mixed population WT – Wild Type ND – Not Determined

βOnly 12 samples with no rpsL mutants were sequenced for whiB7 gene.

62

Panel A Panel B Figure 5.13 Chromatogram of rpsL sequence showing WT and mutated sequences. Panel A, rpsL WT (top), 128A>G (middle) and mixed WT/128A>G (bottom) Panel B, rpsL WT (top), 263A>G (middle) and 263A>C (bottom)

Wild Type

128A>G (K43R)

Wild Type

263A>G (K88R)

A and G overlapping at nt 128 263A>C (K88T)

63

Figure 5.14 Chromatogram of rrs sequence showing WT and 1025T>C sequences.

As rrs sequencing primers in this study could not reveal the first 39 nucleotides located on the 5’-end of the coding sequence, MAMA-PCR was developed to detect the newly identified 16T>C mutation of rrs sequence98. MAMA- PCR was optimized using H37Rv MTB genomic DNA as a template (Figure 5.15). Following the optimization, MAMA-PCR was performed with the genomic DNA of the 12 SM resistant isolates lacking rpsL mutations and the results elucidated that none of them carried 16T>C mutation (Figure 5.16).

Wild Type

1025T>C (A>G for reverse)

64

Figure 5.15 MAMA-PCR optimization for detection of 16T>C mutation in rrs gene using H37Rv MTB genomic DNA as a template (as indicated by arrow). Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, PCR amplicon of MTB H37Rv genomic DNA revealed a rrs specific band with

1,041 bp amplified by rrs-16 wt forward primer (primer for detection of wild type sequence) as indicated by arrow

Lane 2, No template control amplified with rrs-16 wt forward primer Lane 3, PCR product of MTB H37Rv genomic DNA amplified with rrs-16 mt forward

primer (primer for detection of 16T>C mutated sequence) showed no amplification

Lane 4, No template control amplified with rrs-16 mt forward primer

65

Figure 5.16 Representative MAMA-PCR results for detection of 16T>C rrs mutation. The WT rrs MAMA-PCR amplicons (1,041 bp) obtained from the representative of SM resistant clinical MTB isolates lacking of rpsL mutation are indicated by arrow. Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, SM resistant clinical MTB isolates lacking of rpsL mutation isolate no. 5 (SM

mono-resistance) amplified by rrs – 16 wt forward primer Lane 2, SM resistant clinical MTB isolates lacking of rpsL mutation isolate no. 5 (SM

mono-resistance) amplified by rrs – 16 mt forward primer Lane 3, SM resistant clinical MTB isolates lacking of rpsL mutation isolate no. 252

(SM + INH resistances) amplified by rrs – 16 wt forward primer Lane 4,. SM resistant clinical MTB isolates lacking of rpsL mutation isolate no. 252

(SM + INH resistance) amplified by rrs – 16 mt forward primer Lane 5, H37Rv control amplified by rrs – 16 wt forward primer Lane 6, H37Rv control amplified by rrs – 16 mt forward primer Lane 7, No template control amplified by rrs – 16 wt forward primer Lane 8, No template control amplified by rrs – 16 mt forward primer

66

Six different types of gidB single nucleotide polymorphisms (SNPs) were found in this study. Among them, two mutations were exclusively found in SM resistant isolates. The former was 134G>A transition rendering a premature stop codon (W45stop) (Figure 5.17A bottom panel) and it was carried by a SM mono- resistant isolate which harbored no mutation in both rpsL and rrs sequences. The latter was 206G>A (G69D) mutation (Figure 5.17B bottom panel) found in a SM resistant MDR isolate which was previously known to carry 263A>C rpsL (K88T) in this study.

Other four additional gidB SNPs were presumably not involved in SM resistance as a consequence of they were evident in both SM resistant and susceptible isolates. Firstly, the 276A>C missense mutation, corresponding to E92D, was found in 57 of 101 isolates (Figure 5.17C middle panel). Among them, 34 were from SM resistant isolates (34/46) and 23 were non SM resistant isolates (23/55). The three remaining variants were all silent mutations conferring synonymous amino acids, i. e. 330G>T (V110V), 423G>A (A141A) and 615A>G (A205A). They were identified in 29, 5 and 90 clinical MTB isolates, respectively (Figure 5.18A, B, C). Majority of the above mentioned isolates carried either double or triple silent mutations. Only four isolates were identified to have a single gidB, 615A>G silent mutation. Fifty-seven isolates carrying 276A>C (E92D) and 29 isolates carrying 330G>T isolates also harbored 615A>G mutation resulting in gidB double mutations. Five isolates simultaneously carrying gidB triple silent mutations were also found.

In addition, mixed population pattern between wild type and mutated gidB alleles was seen in the chromatograms obtained from two out of four isolates with a single gidB silent mutation (615A>G). One of these isolates, no. 68, which had been previously known to possess mixed wild type and mutated rpsL sequences, carried two double peaks chromatogram at positions 276 (A/C overlapping) as shown in Figure 5.17C and 330 (G/T overlapping) as shown in Figure 5.18A, while another isolate, no. 176, carried G/T overlapping peaks at position 330.

Only nine (two SM resistant and seven SM susceptible isolates) MTB isolates did not carry any mutation in gidB sequence. Therefore, among three tested

67

SM resistance associated genes, the mutations in gidB sequence were the highest frequencies and varieties in MTB isolates of this study.

Additionally, double mutations of rpsL and gidB, were found in 32 of 46 of SM resistant isolates and two out of 55 SM susceptible isolates (Table 5.1). The mutations in two loci, i.e. in rrs and gidB, were also identified in a single SM susceptible isolate.

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Panel A Panel B

Panel C Figure 5.17 Chromatogram of gidB sequence showing WT and mutated sequences.

Panel A, gidB WT (top) and gidB 134G>A (bottom) Panel B, gidB WT (top) and gidB 206G>A (bottom) Panel C, gidB WT (top), gidB 276A>C (middle) and mixed WT/276A>C

(bottom)

Wild Type

134G>A (W45stop) 206G>A (G69D)

Wild Type Wild Type

276A>C (E92D)

A and C overlapping at nt 276

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Panel B Panel C

Panel A Figure 5.18 Chromatogram of gidB sequence showing WT and three silent mutated sequences.

Panel A, gidB WT (top), gidB 330G>T (middle) and mixed WT/330G>T (bottom)

Panel B, gidB WT (top) and gidB 423G>A (bottom) Panel C, gidB WT (top) and gidB 615A>G (bottom)

Wild Type

330G>T (V110V)

G and T overlapping at nt 330

Wild Type Wild Type

423G>A (A141A) 615A>G (A205A)

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5.5 The presence of whiB7 mutations among SM resistant isolates lacking of rpsL and rrs mutations

To determine SM resistance phenotype of MTB isolates induced by

intrinsic mechanism, promoter and structural (open reading frame) regions of transcriptional regulator gene, whiB7, were amplified and sequenced (Table 4.3) from the 12 SM resistant isolates lacking of rpsL and rrs mutations. No mutation was elucidated in the untranslated promoter region of this whiB7 gene. Analysis of the structural sequence of whiB7 gene indicated that three isolates exhibited a frame shift mutation (W63fs) caused by G deletion at the nucleotide position 188 (188delG) shown in Figure 5.19 A. Interestingly, these isolates were all EAI2-Nonthaburi lineages based on spoligotyping. These ‘G’ deleted nucleotide sequences were identical to the whiB7 authologous protein, LJ80_17410 of MTB strain 96121 (GenBank Accession: CP009427.1) belonging to Manila family (EAI2-Manila) of Indo Oceanic lineage102. In the EAI2-Manila genome sequence, this whiB7 authologue was also annotated as ‘disrupted transcriptional regulator’; therefore, this nucleotide deletion was unlikely to be responsible for SM resistant phenotype of the isolates.

Furthermore, other two point mutations were found in whiB7 coding region in a single SM and INH combined resistant isolate (isolate no.1), i.e. 242G>A (R81H) and 246_247insTT (two ‘T’ insertions between nucleotide 246 and 247) causing a frameshift translation (P82fs). Moreover, all whiB7 mutations demonstrated co-appearance with those of gidB (Table 5.1).

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Panel A Panel B Figure 5.19 Chromatogram of whiB7 open reading frame sequence showing WT and mutated sequences.

Panel A, whiB7 wild type sequence (top) and the nucleotide G deletion at position 188 (188delG) (bottom)

Panel B, whiB7 wild type sequence (top) and 242G>A and TT insertion between nucleotide 246 and 247 (reverse sequences are shown) (bottom)

Wild Type Wild Type

G deletion at nt 188

Wild Type

246_247 2insTT (AA for reverse)

242G>A (R81H) (C>T for reverse)

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5.6 Association of rpsL, rrs and gidB mutations to SM resistance

Of three tested genes, rpsL mutants had a significant association with SM resistant phenotype (OR = 67.3, 95% CI = 14.3 – 317.3, p= <0.0001). With a p-value less than 0.0001 and extremely high odd ratio, rpsL 128A>G (K43R) was the most frequent mutation associated with the SM resistance and accounted for 94% of all rpsL mutations. The remaining mutations, K88R and K88T, had lower odd ratio but marginally substantial correlation to the SM resistance (OR = 3.7, 95% CI = 0.1 – 92.1, p = 0.4554) with SM resistance. In addition, due to the rrs 1025T>C mutant was found in the SM susceptible isolate, it had no relationship to SM resistance as seen by the statistically unlikely associated (OR = 0.39, 95% CI = 0.02 – 9.8, p = 1.0).

No correlation was demonstrated in case of three gidB silent mutations (330G>T, 423G>A and 615A>G) and one missense mutation (E92D) to SM resistance as they had been found in both SM resistant and susceptible isolates. Other gidB mutations, 206G>A (G69D) and 134G>A (W45stop) were found exclusively in SM resistant isolates and their association to SM resistant phenotype were marginally but significant correlated (OR = 3.7, 95% CI = 0.1 – 92.1, p = 0.4554).

By excluding the gidB and rrs polymorphisms which were not associated to the SM resistance, the sensitivity and specificity of remaining rpsL and gidB mutations were calculated in order to demonstrate their positive and negative proportion in SM resistant and sensitive isolates (Table 5.2). The detection of three rpsL mutations had a high sensitivity (71.7 %) and specificity (96.4%) for their presence in SM resistant isolates and this gene could be regarded as a reliable genetic marker for SM resistance. Individually, the rpsL K43R exhibited the satisfactory correlation with 67.4% sensitivity and 96.4% specificity while rpsL codon 88 and gidB mutations had very low sensitivity because of their presence in a very low frequency. However, they were confined to SM resistance with 100% specificity. Combined all three rpsL and two gidB mutations had the highest detection capability at specificity 73.9% and specificity 96.4% for identification of SM resistant genotype from Thailand. Among these, the rpsL K43R was responsible for 91.2% of SM resistance associated mutations (Table 5.2).

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Table 5.2 Sensitivity and specificity calculations of rpsL, rrs and gidB genes

Genes and mutation types

SM resistance (no. of mutations/no. of isolates)

SM sensitive (no. of mutations/no. of isolates)

p-value Odds Ratio (OR)

95% Confidence Interval (CI) for OR

Sensitivity (%)

Specificity (%)

Accuracy

(%) PPV (%)

NPV (%)

rpsL (K43R) 31/46 2/55 <0.0001 54.8 11.7 – 255.7 67.4 96.4 83.2 93.9 77.9

rpsL (K88R) 1/46 0/55 0.4554 3.7 0.1 – 92.1 2.2 100 55.4 100 55

gidB (W45stop) 1/46 0/55 0.4554 3.7 0.1 – 92.1 2.2 100 55.4 100 55

rpsL and gidB (K88T and G69D)

1/46 0/55 0.4554 3.7 0.1 – 92.1 2.2 100 55.4 100 55

rpsL (K43R, K88R and K88T)

33/46 2/55 <0.0001 67.3 14.3 – 317.3 71.7 96.4 85.1 94.3 80.3

rpsL& gidB 34/46 2/55 <0.0001 75.1 15.8 – 356.4 73.9 96.4 86.1 94.4 81.5

PPV – Positive Predictive Value NPV – Negative Predictive Value

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5.7 Association of gidB sequence polymorphisms and MTB lineages

According to LSP-based identification that was mapped to our spoligotyping data, 57, 34, 9 and 1 were East Asian (Beijing), Indo-Oceanic, Euro-American and unknown lineages respectively among 101 MTB isolates in this study (Table 5.1). Thirty two out of 34 Indo-Oceanic lineage and eight out of nine Euro- American lineage isolates in this study could be further sub-classified by using spoligotyping database (Table 5.1).

Interestingly, 276A>C (E92D) together with 615G>A silent mutations in gidB gene were presented in 55 Beijing and 2 EAI5 lineage isolates (Table 5.1). Two Beijing isolates did not possess 276A>C (E92D) but instead harbored 330G>T and 615G>A. The previous studies indicated that this sequence variation (276A>C [E92D]) in gidB was a signature polymorphism for MTB Beijing isoates16,18,88. In order to assess the discrepancies between gidB SNPs and spoligotyping in the above mentioned four isolates, RD105 DTM-PCR was carried out for the 57 and 18 spoligotyping-based Beijing and non-Beijing isolates respectively.

Figure 5.20 shows the DTM-PCR optimization on H37Rv MTB (as non- Beijing lineage control) and MTB no. 19 (as Beijing lineage control). The results of RD105 DTM-PCR in the clinical strains showed RD105 deletion type amplification in those 55 Beijing isolates and confirmed their spoligotying based-lineage identification (Figure 5.21).

The RD105 deletion type amplification specific for Beijing was also seen in two isolates of EAI5 lineage which containing Beijing-type gidB E92D alterations. However, intact RD105 was amplified in one of two Beijing isolates lacking E92D-Beijing signature. Interestingly, the remaining one isolate simultaneously showed the mixed presence of both intact and deletion of RD105 amplification. Furthermore, this Beijing and non-Beijing mixed pattern was also found in one EAI2-Nonthaburi (no.68) isolate and one Manu1 (no. 176) isolate which had mixed chromatogram for rpsL and/or gidB.

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Figure 5.20 DTM-PCR optimization Intact RD105 region (amplified from MTB H37Rv) was seen as 1,495 bp while the deleted RD105 region (amplified from no.19 MTB Beijing lineage) was seen as 785 bp as indicated by arrows. Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1, RD105 DTM-PCR amplicon of H37Rv MTB (non-Beijing lineage) Lane 2, RD105 DTM-PCR amplicon of no. 19 MTB (Beijing lineage) Lane 3, No template control

Except two isolates with E92D and two mixed WT/330G>T isolates, the

remaining (East-African Indian) EAI clades of Indo-Oceanic lineage MTB isolates in this study appeared to carry 330G>T and 615G>A synonymous variants of gidB. Strikingly, among these EAI group, all five isolates of EAI6-BGD1 clade had an additional silent mutation, 423G>A. Therefore, 330G>T alteration could be regarded as the potential signature polymorphism for all EAI group of Indo-Oceanic lineage. In addition, 330G>T and 423G>A could potentially be a putative signature marker for EAI6-BGD1 sublineage. Similarly to the reference H37Rv MTB, all nine Euro-American lineage isolates in this study lacked those three gidB synonymous mutations.

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Figure 5.21 Agarose gel electrophoresis of RD105 DTM-PCR amplicons of clinical MTB isolates. Intact RD105 region was seen as 1,495 bp while the deleted RD105 region was seen as 785 bp as indicated by arrows. Numbers at the left are DNA sizes in base pairs (bp). Lane M, GeneRuler 100 bp DNA ladder Lane 1-2, DTM-PCR amplicon of Beijing lineage isolates by spoligotyping Lane 3-4, DTM-PCR amplicon of non-Beijing lineage isolates by spoligotyping Lane 5-6, DTM-PCR amplicon of two non-Beijing lineage isolates (EAI5) by

spoligotyping showing RD105 deletion Lane 7, DTM-PCR amplicon of a Beijing lineage isolate by spoligotyping showing

both intact and deleted RD105 Lane 8, DTM-PCR amplicon of a Beijing lineage isolate by spoligotyping showing

intact RD105 Lane 9-10, DTM-PCR amplicon of non-Beijing lineage isolate (EAI2-Nonthaburi and

Manu1) by spoligotyping showing both intact and deleted RD105 Lane 11, DTM-PCR amplicon of H37Rv (non-Beijing [H3] lineage) by spoligotyping

showing intact RD105 Lane 12, RD105 DTM-PCR amplicon of no. 19 MTB isolate (Beijing lineage) showing

RD105 deletion Lane 13, No template control

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CHAPTER 6

DISCUSSION

6.1 Mutations found in rpsL, rrs, gidB and whiB7 genes and their association to SM resistance

As the sites of action of SM are the ribosomal protein S12 and the 16S

rRNA of the 30S subunit of the ribosome, several investigators have found that high-level SM resistance is often linked to the mutations in the S12 protein encoded by rpsL gene whereas the intermediate resistant level is responsible by the 16S rRNA encoded by rrs gene5,14,81. Additionally, the gidB gene conferring low-level SM resistance is the last prominent genotype discovery for SM resistant MTB genes16.

In this study, SM resistance related rpsL, rrs and gidB mutations in 46 SM resistant and 55 SM susceptible isolates (total 101 isolates) were investigated. The majority of the SM resistant MTB isolates was contributed by the rpsL mutants (71.7 %). These data coincided with the SM resistance of MTB worldwide of which the mutations in rpsL were responsible for approximately 60%5. Notwithstanding, its magnitude of significance was variable depending on the geographical areas. Recently, the mutation rates of this gene were found at 76.5% in China103. Moreover, the South-East Asian countries (SEA) surveillance indicated that 81% and 90.2% of rpsL mutation were responsible for SM resistant MTB in Vietnam81 and Singapore18, respectively. Whereas in other East Asian countries such as Japan and Korea and European countries, i.e. Latvia and Portugal, the rpsL mutation in SM resistant MTB had been reported between averagely 60-67%16,104–106. Furthermore, the presence of rpsL mutants in SM resistant MTB isolates in other countries such as Mexico, Spain, Brazil, Poland, India, Pakistan was significantly lower than the mentioned above countries (24-42%.)17,88,107–110.

The K43R (128A>G) mutation is reported worldwide as the most common rpsL mutation found among SM resistant MTB5. Recently, the screening of MTB SM resistance in Vietnam and Singapore demonstrated that K43R was responsible for

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76.6% and 89% of the rpsL mutants respectively18,81. In this study, the frequency of rpsL K43R alteration in SM resistant rpsL mutants was found extremely high up to 94%. It was also responsible for 91.2% of all SM resistance associated mutations and 67.4% of all SM resistant isolates in this study. Moreover, K43R mutation was reported to be remarkably associated with the Beijing lineage MTB especially in East Asia and SEA18,81. Thailand also burdened the high prevalence of Beijing lineage genotype111.

The second frequently found rpsL mutation is at codon 88. The amino acid lysine at codons K43 and codon K88 are the key SM binding sites on the ribosomal protein S12 (coded by rpsL). Therefore, any substitution to lysine such as K88R (or K43R or K88T) would ultimately result in the SM resistance of the bacteria112. In this study, K88T was found in a non-Beijing genotype similar to that of the previous reports in SEA and other countries18,81. Following the 3D structures comparisons of K43R, K88R and K88T with those of the wild type using Phyre2 protein homology online software113, no major structural difference of the SM binding sites was found. Therefore, the reduction in SM binding activities of the mutants might be compromised by the impact of cognate amino acid alterations.

In the previous studies regarding to phenotypic SM resistant level with rpsL mutations, codon 43 alteration was strongly linked to the high-level of SM resistance over than 1000 µg/ml whereas codon 88 alteration yielded more heterogeneous MIC level14. A study from Vietnam showed that all rpsL K43 mutated MTB isolates were examined to have high SM resistance level (≥ 16 µg/ml) while rpsL K88 mutations were found in isolates exhibited both intermediate (≥8 µg/ml) and high-levels81. The 71.7% rpsL mutations and 94% prevalence of codon 43 alteration portrayed that MTB isolates in the present study were significantly related to the high-level SM resistance, albeit rpsL K43R presented in two susceptible isolates. Some previous studies also reported this rarely found incidence in the SM susceptible MTB isolates17,114. In one previous study, the MTB rpsL mutation discrepancy between the genotypic and phenotypic results was verified by repeating the DST on the MTB strains isolated from the follow-up samples and it indicated that the newly isolated MTB were indeed SM resistant114. In this study, the DST of those

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original isolates was re-examined and it was found that they remained SM susceptible at the absolute concentration, 4 µg/ml. However, the isolates form the follow-up samples were inaccessible and minimal inhibitory concentrations (MICs) could not be performed. In order to validate these discrepancy results, performing MIC which was lower than the designated absolute concentration was still necessary.

Locating in the decoding center of the 30S ribosome together with ribosomal protein S12, the 16S rRNA encoded by rrs gene essentially involves in the protein synthesis. Its pseudonot 530 loop (Helix 18) and 1400 region (Helix 44), take part in the selection of the cognate tRNA attachment to mRNA13,69. The 900 region (Helix 27) packs closing against the Helix 44 and takes a role of conformational switch. SM binding to all of these functional sites consequently leads to the misreading of genetic code during translation13,69. Many reports have demonstrated that the mutations clustered around 530 and 900 regions of rrs are related to SM resistance of MTB up to 28%5,17. The mutation around 1400 region, especially rrs 1401A>G, was mainly associated to other aminoglycosides, i.e. kanamycin and amikacin, rather than SM15,89. In this surveillance, nevertheless, almost entire rrs covering all those three important regions was sequenced. Surprisingly, no rrs mutation was found in all SM resistant MTB isolates except one polymorphism which was not associated to SM susceptibility and identified in a single susceptible isolate. More recently, a report from Singapore revealed a new nt 16T>C mutation (at nt position 15 with the previous annotation of H37Rv genome) in one SM resistant strain98. Therefore, it was further attempted to examine this specific mutation in 12 isolates lacking rpsL alteration by developing MAMA-PCR. No mutation in this particular position was identified in those isolates. Similarly, a previous report from India indicated that there was no mutation found in the loop at 530 or 900 in SM resistance isolates115. In addition, the prevalence of rrs gene mutations responsible to SM resistance in MTB may be related to geographical areas. Averagely less than 10% of rrs polymorphism was associated with SM resistance in East Asian and SEA countries16,18,81,103,104 whereas European and Latin American countries reported the higher prevalence of rrs mutations (13-28%)17,88,107,110. As the predominance of MTB lineages differs according to the geographical areas, the significance of rrs mutations

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in MTB probably relates to the different MTB lineages as well. A recent study from Thailand reported only rrs 1401A>G mutation correlating to a specific hotspot for kanamycin and amikacin resistance in nearly 73% of MDR/XDR-TB isolates89. Although the phenotypic drug susceptibility test did not perform for KM in our study, the absence of nt position 1401 mutation indicated that all 101 tested MTB isolates in this study might be kanamycin and amikacin susceptible.

It was previously known that mutations in gidB rendering the low-level SM resistance could be discovered in MTB strains lacking the rpsL and rrs mutations16,88. In this study, each of a missense and non-sense (resulting in premature stop codon) gidB mutations were demonstrated in the SM resistant isolates. Though G69D gidB mutation was present with rpsL K88T mutation, W45stop gidB mutation stood out as a sole SM resistant conferring mutation without any changes in other screened SM resistant responsible genes. To the best of my knowledge, G69D mutation found in this study is novel although the previous studies reported mutations at W45 position88.

As mutations in rpsL, rrs and gidB were absent in approximately 26% of SM resistant isolates in this study, the resistance in these isolates may be contributed from the intrinsic mechanism. A transcriptional regulator encoded by whiB7 was previously reported to confer intrinsic drug resistance in MTB by activating the expression of its regulon composed of tap (Rv1258c) efflux pump which specifically transports SM and other antibiotics116. The mutations in the 5’ untranslated region (UTR) of whiB7 caused overexpression of this transcriptional regulator and subsequently increased the expression of eis (aminoglycoside acetyltransferase-encoding gene) and tap, conferring the low-level resistance to KM and cross-resistance to SM, respectively82. Consequently, the entire promoter and structural regions of whiB7 were sequenced in 12 SM resistant isolates devoid of rpsL mutations. No nucleotide variation was detected in the untranslated promoter region. However, interestingly, in the whiB7 open reading frame, double mutations of a missense and frameshift which might be the consequence for SM resistance was found in one isolate. In addition, an additional whiB7 frameshift mutation due to ‘G’ deletion at position 188 (188delG) was exclusively shown in all three EAI2-Nonthaburi

81

lineages. BLAST search for this 188delG whiB7 sequence was identical to whiB7 authologue transcriptional regulator LJ80_17410 of MTB EAI2-Manila lineage 96121. However, this gene was annotated as ‘disrupted’. Thus, 188delG probably confined to these particular EAI2-Nonthaburi and EAI2-Manila lineages rather than SM resistance of MTB. However, additional investigations are still required to confirm the role of whiB7 as a SM resistant associated gene.

6.2 Association of gidB sequence polymorphisms and MTB lineages

Previous study reported that lineage specific polymorphisms found in gidB were useful as phylogenetic surrogate makers of MTB. E92D could be presumptive as a phylogenetic marker for Beijing family whereas L16R for Latin American Mediterranean (LAM) clades16,88,117. Moreover, gidB A80P could also be useful for the detection of MIRU-VNTR genetic cluster Q1, a dominant cluster of M/XDR-TB isolates in Lisbon, Portugal118. Similar to the previous investigations, the gidB E92D variant was found in almost all Beijing isolates in this study. However, this alteration was absent in two Beijing isolates. Furthermore, the gidB E92D mutation was identified in two isolates belonging to EAI5 genotype. Moreover, another isolate which had EAI2-Nonthaburi lineage (no.68) showed the mixed gidB wild type and E92D patterns. Consequently, the Beijing family spoligotyping results were validated with a rapid and reliable Beijing genotype identification test, DTM-PCR99. This test targets the unique large sequence polymorphism (LSP), RD105, which is deleted in all Beijing/W strains and can be used as a reliable marker to define the isolates of this family119. DTM-PCR confirmed the 55 spoligotyped Beijing isolates with gidB E92D to be Beijing lineage isolates. It also showed the absence of RD105 in two EAI5 isolates carrying E92D characteristic of Beijing genotypes, indicating the convergent evolution of these isolates belonging to EAI5 to delete RD105. The RD105-deleted non-Beijing and Beijing carrying the RD105 region were previously reported but these finding were not discussed120,121. For the two Beijing isolates lacking lineage specific gidB E92D, RD105 region was amplified in one of them indicating that Beijing isolate could rarely carry RD105.

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The discrepancy between spoligotyping and lineage specific PCR was also demonstrated in a study in which L16R polymorphism, another gidB phylogenetic indicator for LAM genotype, was congruent with LAM-specific PCR but not with spoligotyping in a few isolates88. It was also shown that phylogenetic accuracy of Direct Repeats (DRs) locus based genotyping methods such as spoligotyping and MIRU-VNTR exhibited lesser discriminatory power when compared to a gold standard DNA sequencing122. These contradict issues come from the highly changeable or convergent evolution which can result identical genetic characters between phylogenetically unlinked strains. Accordingly, SNPs which bear almost no homoplasy have become dependable genetic markers for phyogenetic differentiation in MTB122,123. The identification of E92D in Beijing isolates of this study also consolidated its usefulness as a reproducible marker for Beijing genotype.

Recently, a study revealed that phylogenetic polymorphisms in antibiotic resistant genes in MTB and gidB 330G>T was shown to be EAI specific signature among many different types of MTB lineages from different countries124. In our study, the specific SNP for EAI subdivision, an additional silent mutation, 423G>A were exclusively found in all EAI6-BGD1 isolates. The last silent mutation, 615A>G, was widely dispersed in the majority of lineages other than the Euro-American group. Hence, all of these finding may not yet be conclusive and required further studies or these polymorphisms may be specific to the particular geographic regions.

Apart from the significant proportion of rpsL mutation types, the frequency of mutations found in gidB was very low and most importantly, no mutation was observed in rrs gene among SM resistant strains in this study. This might probably link to phylogenetic evolution of Beijing family sublineages in this area. The modern Beijing strain ST-10 is the major sublineage evolved in Thailand125 and it was determined to be highly clonal and stable over a certain period126. The SM resistant samples with Beijing genotype dominance did not carry diversified mutations in three characterized genes. But the samples were not yet determined for ST type.

Strikingly, the mixed population pattern in two isolates was found in this study. Mixed infection or infection with different genotypes can arise when a patient

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is exposed with more than one strain of MTB before, during or after the TB treatment127,128. According to the DNA sequencing, significant genetic heterogeneity for SM resistant rpsL mutation and/or MTB lineage was investigated in two isolates, no. 68 and no. 176, respectively. The mixed WT/mutant pattern of rpsL and gidB was identified in no. 68 whereas that of only gidB was found in no. 176. The mixed patterns were also identified in RD105 PCR of these two isolates. This could indicate that the prevalence of mixed infection in this region needs to be further studied.

Thailand is classified by WHO as one of the 22 high TB burden countries and the MDR-TB was notified up to 29% of retreatment cases in 20142. According to the WHO TB treatment guideline12, SM is prescribed for the retreatment or relapse TB cases in Thailand. As drug resistance due to INH and RIF is the most common, SM has become considered as a drug of choice of MDR-TB. Therefore, knowing the genotypic SM resistant pattern is necessary. In a retrospective cohort study conducted using nationwide TB surveillance data from 2004 to 2008, the phenotypic EMB and/or SM resistance was identified in 8.0% of new cases.92 In this study, with the data from 2007 to 2011 and represented the samples from central of Thailand, the phenotypic SM resistance was found in 16.0% of the total. These might be due to the more extensively screening for anti-TB drug resistance as reported by WHO report 20152 in Thailand. Therefore, diagnosis or determination of SM drug susceptibility at both genotypic and phenotypic levels is essential before its prescription.

In the examination of the three significant SM resistant genes, two of them, rpsL and gidB, were accountable for 73.9% of the SM resistance and the majority of the SM resistant MTB isolates in this study could be predicted to have the high-level resistance. Moreover, further investigation on the whiB7 sequence of the 12 SM resistant isolates lacking of rpsL and rrs alterations (26%) revealed that one of them had mutation in this gene that could be associated to SM resistance. Therefore, other eleven isolates (24%) resisted to the SM with unknown mechanism. The mutation data in this study would further lead to the development of rapid molecular diagnosis for SM resistant genotypes in clinical Thai MTB isolates for effectively facilitating the TB treatment in Thailand.

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CHAPTER 7

CONCLUSION

In summary, rpsL, rrs and gidB genes in 101 MTB clinical isolates with various phenotypic backgrounds were successfully sequenced and their predominant mutations conferring SM resistance were determined. Thereafter, the contribution of combined mutations that could be responsible for SM resistance was calculated. Subsequently, the SM resistant genotypes and MTB lineages were also correlated. The E92D mutation of gidB was ascertained to be a phylogenetic signature for Beijing genotype. In addition, 330G>T, the synonymous gidB mutation which could be promisingly useful as a surrogate maker for MTB East-African Indian (EAI) clades differentiation, was identified.

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BIOGRAPHY

Name Miss Yin Moe Hlaing Date of birth August 3rd, 1983 Education Attainment 2005: B. M. Tech. (Medical Laboratory Technology)

University of Medical Technology Yangon, Myanmar

Scholarship 2013: Thammasat University Scholarship for ASEAN Community and Thammasat Fiscal Year Research Grant

Conference attended 1. The 1st International Allied Health Sciences Conference 2014; International Conference on Medical Innovation for Health, November 4th – 6th 2014, Rama Gardens Hotel, Bangkok, Thailand 2. 2014 1st Semester Progress Seminar of Graduate Program in Biomedical Sciences, December 12th 2014, Faculty of Allied Health Sciences, Thammasat University (Rangsit Center), Pathumtani, Thailand 3. The 2nd Joint Symposium of Thammasat University and BK21 PLUS of CUK, January 21st – 23rd, 2015, Faculty of Allied Health Sciences, Thammasat University (Rangsit Center), Pathumtani, Thailand 4. 2014 2nd Semester Progress Seminar of Graduate Program in Biomedical Sciences, June 20th 2015, Kantary Hotel, Ayuttaya, Thailand Oral Presentations 1. Hlaing YM. Association of rpsL, rrs and gidB genes mutations with Streptomycin resistance in Mycobacterium tuberculosis clinical isolates from Thailand. 2014 1st Semester Progress Seminar of Graduate Program in Biomedical

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Sciences, December 12th 2014, Faculty of Allied Health Sciences, Thammasat University (Rangsit Center), Pathumtani, Thailand 2. Hlaing YM, Mutations of rpsL, rrs and gidB and their association to Streptomycin resistance in Mycobacterium tuberculosis clinical isolates. 2014 2nd Semester Progress Seminar of Graduate Program in Biomedical Sciences, June 20th 2015, Kantary Hotel, Ayuttaya, Thailand Poster presentations 1. Hlaing YM, Srimanote P, Tongtawe P, Thanongsaksrikul J. Mycobacterium tuberculosis rpsL Mutation among Thai Clinical Isolates. The 1st International Allied Health Sciences Conference 2014; International Conference on Medical Innovation for Health, November 4th – 6th 2014, Rama Gardens Hotel, Bangkok, Thailand 2. Hlaing YM, Srimanote P, Tongtawe P, Thanongsaksrikul J. Mycobacterium tuberculosis rpsL Mutation among Thai Clinical Isolates. The 2nd Joint Symposium of Thammasat University and BK21 PLUS of CUK, January 21st – 23rd, 2015, Faculty of Allied Health Sciences, Thammasat University (Rangsit Center), Pathumtani, Thailand