Genetic diversity and resistance to anti-tuberculosis drugs of Mycobacterium tuberculosis in China

168 Pages·2017·5.64 MB·English

by  Kanglin Wan

Genetic diversity and resistance to anti-tuberculosis
drugs of Mycobacterium tuberculosis in China
Kanglin Wan
To cite this version:
Kanglin Wan. Genetic diversity and resistance to anti-tuberculosis drugs of Mycobacterium tubercu-
losis in China. Bacteriology. Université Paris Sud - Paris XI, 2007. English. ￿NNT: 2007PA112140￿.
￿tel-00734513￿
HAL Id: tel-00734513
https://theses.hal.science/tel-00734513
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UNIVERSITE PARIS-SUD XI

THESE

Pour obtenir le grade de
DOCTEUR EN SCIENCES DE L’UNIVERSITE
PARIS-SUD

Présentée par
Kanglin WAN

Study on Genetic diversity and resistance to anti-TB drugs
of Mycobacterium tuberculosis in China

Soutenue le 8 Octobre 2007 devant la commission d’examen :

Mr Dirk van Soolingen Rapporteur
Mr. Christophe Sola Rapporteur
Mr. Longfei Wu Examinateur
Mr. Gérard Leblon Président du Jury
Mr. Gilles Vergnaud Directeur de Thèse
Mr. Xu Jianguo Directeur de Thèse
At this time, feeling deeply grateful, I would like to thank every one,
teachers, friends, colleagues and families, for your kind help and
understanding in the course of my PhD thesis studies.
I would like to thank my supervisors Dr. Gilles Vergnaud and Prof.
Jianguo Xu for giving me this very precious opportunity to study and
improve my scientific knowledge.
Especially from the bottom of my heart I would like to thank Dr.
Christine Pourcel for doing a lot of work on the project design,
organization and implementation, especially for your guidance during the
thesis writing, for helping me correct the manuscript, and for many other
things. Thank you very much for your kindest help, patience and
understanding.
I would like to thank Prof. Christophe Sola for his kindest help to correct
and improve my manuscript. I would like to thank Prof. Dick van
Soolingen for his kindest guidance and help for the project and for
suggesting many valuable improvements of the thesis manuscript. I would
like to thank every Professors of the committee for accepting to be
members of my thesis jury.
I would like to thank Philippe Le Flèche, Yolande Hauck and other staff
members of GPMS, Institut de Génétique et Microbiologie, Université
Paris-Sud for their help.
I would like to thank my colleagues Liu Jinghua, Liu Zhiguang, Zhang
Yuanyuan, Lv Bing, Dong Haiyan, Jiang Yi, Zhao Xiuqin, and other staff
members and students of National Laboratory of Tuberculosis, State Key

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Laboratory for Infectious Disease Prevention and Control / National
Institute for Communicable Disease Control and Prevention, Chinese
Center for Disease Control and Prevention for their help.
I would like to thank staff members of the Tuberculosis laboratories who
provided samples and information for my thesis project in Beijing, Henan,
Jiang Su, Sichuan, Hunan, Anhui, Zhejiang, Fujian, Ji Lin, Shannxi, Gansu,
Xinjiang, Xizang, and Guangxi.
I would like to thank my wife and daughter for their help,
encouragement, patience and understanding.
This work could not have been done without the support of the
European Community through the SSA grant “Tuberculosis China” N°
012166.
Thank every one very much again.

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Table of Contents
1. INTRODUCTION ............................................................................................................... 10
1.1. Introduction on tuberculosis ............................................................................................. 11
1.2. The epidemiology of tuberculosis .................................................................................... 13
1.2.1. Nomenclature and definitions of TB epidemiology ......................................... 13
1.2.2. The General prevalence situation of TB in the world ...................................... 16
1.2.3. The general prevalence situation of TB in China ............................................. 16
1.3. Drug resistance and drug resistance detection .................................................................. 23
1.3.1. Conventional phenotypic methods ................................................................... 24
1.3.2. Genetic methods ............................................................................................... 24
1.4. Main methods of M. Tuberculosis Genotyping ................................................................ 26
1.4.1. IS6110-RLFP ................................................................................................... 27
1.4.2. Spacer oligonucleotide typing (Spoligotyping) ............................................... 29
1.4.3. Multiple Loci VNTR Analysis (MLVA) .......................................................... 29
1.4.4. single-nucleotide polymorphisms .................................................................... 33
1.4.5. Genomic deletion analysis ............................................................................... 34
1.5. Application of Genetic Markers for the phylogeny of M. tuberculosis ............................ 34
1.5.1. The major genetic groups based on mutations at katG and gyrA .................... 35
1.5.2. Deletion analyses ............................................................................................. 36
1.5.3. Definition of spoligotype signatures for the identification of clades ............... 38
1.5.4. The W/Beijing family ...................................................................................... 41
1.6. The transmission of drug-resistant strains ........................................................................ 43
1.7. Molecular Epidemiology study of TB in China ............................................................... 43

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1.8. Aim of the thesis project: organisation of a nationwide study of the M. tuberculosis drug
resistance and genetic diversity in the People’s Republic of China ............................................... 46
1.8.1 Analysis of antibiotic resistance ........................................................................ 47
1.8.2 Genetic diversity study...................................................................................... 47
2. METHODS .......................................................................................................................... 48
2.1. M. tuberculosis strains ...................................................................................................... 49
2.1.1. Standard strain H37Rv ..................................................................................... 49
2.1.2. Collection and Identification of Clinical strains in China................................ 49
2.2. Preliminary drug-resistance research ............................................................................... 50
2.2.1. The Drug sensitivity test .................................................................................. 50
2.2.2. Primary study on the mutation characteristics of Rifampicin (RFP)- and Isoniazid
(INH)-resistance relationship genes of M. tuberculosis .................................................... 50
2.3. A survey of the genetic diversity in 12 Provinces ............................................................ 52
2.3.1. DNA Sample Preparation, Extraction from Bacterial Cells ............................. 52
2.3.2. Spacer oligonucleotide typing .......................................................................... 53
2.3.3. Genomic deletion analysis in strains of the Beijing family ............................. 55
2.3.4. Multiple Loci VNTR Analysis (MLVA) .......................................................... 56
2.4. Main bioinformatic analysis softwares used in this study. ............................................... 58
3. RESULTS ............................................................................................................................ 59
3.1. Randomized drug-resistance research: identification of mutations in drug resistant genes
........................................................................................................................................................ 60
3.1.1. The Drug sensitivity test .................................................................................. 60
3.1.2. Preliminary study on the mutation characteristics of Rifampicin-resistance
relationship gene rpoB ...................................................................................................... 62

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3.1.3. Preliminary study on the mutation characteristics of INH-resistance relationship
genes.................................................................................................................................. 68
3.2. The genetic diversity of M. tuberculosis strains in China ................................................ 73
3.2.1. Preliminary study (Publication N° 3) ............................................................... 73
3.2.2. Extended analysis of the DR/CRISPR locus in Beijing-family strains ........... 74
3.2.3. VNTR locus selection for MLVA ..................................................................... 76
3.2.4. A survey of diversity in 12 Provinces .............................................................. 79
3.2.5. The Beijing family strains ................................................................................ 80
3.2.6. The non-Beijing strains .................................................................................... 88
3.2.7. Repartition of the different families in each province ..................................... 92
4. DISCUSSION AND CONCLUSION ............................................................................... 100
4.1. drug-resistance research ................................................................................................. 101
4.1.1. Conventional phenotypic resistance test ........................................................ 102
4.1.2. Genotypic resistance test ................................................................................ 103
4.2. Genetic diversity study of M. tuberculosis in China ...................................................... 105
4.2.1. Geographical distribution of M. tuberculosis Beijing family in 12 provinces 106
4.2.2. The origin of the Beijing family .................................................................... 108
4.2.3. The age distribution of ancestral Beijing-family strains ................................ 109
4.2.4. Genetic diversity of Beijing-family isolates as assessed by MLVA................ 110
4.2.5. Molecular epidemiological Characteristics of non-Beijing isolates ............... 110
4.3. Association of M. tuberculosis Beijing genotype with BCG vaccination and
drug-resistance .............................................................................................................................. 112
4.3.1. Role of BCG vaccination in the dissemination of the Beijing family ............ 112
4.3.2. The role of Drug-resistance in the dissemination of the Beijing family of M.
tuberculosis. ..................................................................................................................... 113

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4.4. Future work .................................................................................................................... 113
4.5. Conclusion ...................................................................................................................... 115
ANNEXES ............................................................................................................................ 116
5. PUBLICATIONS ............................................................................................................... 138
5.1. Publication N°1 (Translation from Chinese) Study on the mutation characteristics of M.
tuberculosis Rifampicin-resistance gene (rpoB) .......................................................................... 139
5.2. Publication N°2 (Translation from Chinese) Study on genetic diversity of 65
Mycobacterium tuberculosis strains from three provinces of China based on 7 variable number of
tandem repeats .............................................................................................................................. 144
Publication N°3 Mycobacterium tuberculosis diversity in 5 Chinese provinces as assessed by
MLVA and spoligotyping. ............................................................................................................. 150
6. REFERENCES .................................................................................................................. 156

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List of Abbreviations
ADR Secondary Resistance or acquired drug resistance
AFB Acid-Fast Bacilli
AFLP amplified fragment length polymorphism
BAC bacterial artificial chromosome
BCG Bacillus Calmette-Guerin
bp base pairs
CRISPR clustered regularly interspaced short palindromic repeats
DNA deoxyribonucleic acid
dNTP Deoxynucleoside tRFPhosphate
DR direct repeat
EAI the East African Indian
EDTA Ethylene diamine tetraacetic acid
EMB Ethambutol
ETR exact tandem repeat
g Gram
HGDI Hunter-Gaston diversity index
IDR Initial drug resistance
INH Isoniazid
IS6110 insertion sequence 6110
IS insertion sequence
kb kilobase
kg Kilogram
LAM the Latin-American and Mediterranean
LSP Large-sequence polymorphisms
M Mole
M. africanum Mycobacterium africanum
M. bovis Mycobacterium bovis
M. canettii Mycobacterium canettii
M. caprae Mycobacterium caprae
MDR multi-drug resistance
MDR-TB multi-drug resistance TB
mg Milligram
MIC minimal inhibitory concentrations
Min Minute
MIRU mycobacterial interspersed repetitive units
MLVA multiple loci VNTR analysis
ml Millilitre
M. microti Mycobacterium microti
mM Millimole
M. pinipedii Mycobacterium pinipedii
MPTR major polymorphic tandem repeats
M. tuberculosis Mycobacterium tuberculosis
NAP P-nitro-alpha-acetylamino-beta-hydroxypropiopheneone
nsSNP nonsynonymous single-nucleotide polymorphisms
NTM non-tuberculous mycobacteria
PCR polymerase chain reaction
PDR primary drug resistance
PFGE pulsed field gel electrophoresis
PPD purified protein derivative
SNP single nucleotide polymorphism
RAPD the DNA polymorphism of randomly-amplified-PCR
RD Region of deletions

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RDH Reverse-line Blot Hybridation
RFP Rifampicin
RFLP restriction fragment length polymorphism
rpm revolutions per minute
rpoB the RNA polymerase B subunit
PNB 4-Nitrobenzoic acid
SDS Sodium dodecyl sulphate
SM Streptomycin
SNP single nucleotide polymorphism
SPIDR the spacer interspersed DR
Spoligotyping spacer oligonucleotide typing
sSNP synonymous single-nucleotide polymorphisms
TB Tuberculosis
TbD M. tuberculosis-specific deletion
TCH 2-Thiophencarboxtylic acid hydrazide
Tris Tri hydroxymethyl aminomethance
µg Microgram
μl Microlitre
µM Micromole
VNTR variable number of tandem repeats
WHO the World Health Organization
XDR Extensively drug-resistant

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