Palomino – Leão – Ritacco

Tuberculosis 2007

From basic science to patient care

TuberculosisTextbook.com

First Edition

This textbook was made possible

by an unrestricted educational grant provided by

Bernd Sebastian Kamps and Patricia Bourcillier.

Tuberculosis 2007

From basic science to patient care

www.TuberculosisTextbook.com

Juan Carlos Palomino

Sylvia Cardoso Leão

Viviana Ritacco

(Editors)

Editors

Juan Carlos Palomino

Mycobacteriology Unit

Institute of Tropical Medicine

Nationalestraat, 155

20000, Antwerp

BELGIUM

Sylvia Cardoso Leão

Departamento de Microbiologia, Imunologia e Parasitologia

Universidade Federal de São Paulo

Rua Botucatu, 862 3° andar

04023-062, São Paulo, SP

BRAZIL

Viviana Ritacco

Servicio de Micobacterias, Instituto Nacional de Enfermedades Infecciosas

Carlos G. Malbrán

Av. Velez Sarsfield, 563

1281, Buenos Aires

ARGENTINA

Cover design by Pedro Cardoso Leão, BRAZIL

(pedrocardosoleao@yahoo.com.br)

Disclaimer

Tuberculosis is an ever-changing field. The editors and authors of “Tuberculosis 2007 – from

basic science to patient care” have made every effort to provide information that is accurate

and complete as of the date of publication. However, in view of the rapid changes occurring in

medical science, as well as the possibility of human error, this site may contain technical

inaccuracies, typographical or other errors. Readers are advised to check the product informa-

tion currently provided by the manufacturer of each drug to be administered to verify the

recommended dose, the method and duration of administration, and contraindications. It is

the responsibility of the treating physician who relies on experience and knowledge about the

patient to determine dosages and the best treatment for the patient. The information con-

tained herein is provided "as is" and without warranty of any kind. The contributors to this site

disclaim responsibility for any errors or omissions or for results obtained from the use of in-

formation contained herein.

Proofreading: Emma Raderschadt

Preface

This book is the result of a joint effort in response to the Amedeo Challenge to

write and publish a medical textbook on tuberculosis. This non-profit-making ini-

tiative is particularly attractive due to several reasons. First, the medium chosen for

dissemination: the book will be readily available on the internet and access will be

free to anyone. Second, its advantage over books published via traditional media is

the ease to update the information on a regular basis. Third, with the exception of

Spanish and Portuguese, no copyright is allocated and the translation of Tuberculo-

sis 2007 to all other languages is highly encouraged.

These innovations in the way of publication were translated to the organization

of the chapters in the book. This is not a classical textbook on tuberculosis diagno-

sis, management, and treatment. On the contrary, it is a multidisciplinary approach

addressing a full range of topics, from basic science to patient care. Most authors

are former members of RELACTB – a Tuberculosis Research Network for Latin

America, the Caribbean and Europe sponsored by the United Nations University −

and have worked on collaborative projects since 1995.

Classical knowledge about the disease is focused on chapters dedicated to the

history of tuberculosis, microbiology of the tubercle bacillus, description of the

disease caused by Mycobacterium tuberculosis complex members in adults, chil-

dren, and HIV/AIDS patients, conventional epidemiology, diagnostics, biosafety,

and treatment.

More recent findings, which have changed our knowledge about tuberculosis in

the last years, are detailed in chapters on the molecular evolution of the M. tuber-

culosis complex, molecular epidemiology, host genetics, immune response and

susceptibility to tuberculosis, studies on the pathogenesis of tuberculosis in animal

models, and new diagnostic and drug resistance detection approaches.

Perspectives for future research relevant to fighting the disease have also been

included in chapters focusing on the “omics” technologies, from genomics to pro-

teomics, metabolomics and lipidomics, and on research dedicated to the develop-

ment of new vaccines and new diagnostic methods, and are discussed in the last

chapter.

Nowadays, medical science should not be limited to academic circles but read-

ily translated into practical applications aimed at patient care and control of dis-

ease. Thus, we expect that our initiative will stimulate the interest of readers not

only in solving clinical topics on the management of tuberculosis but also in posing

new questions back to basic science, fostering a continuous bi-directional interac-

tion of medical care, and clinical and basic research.

Juan Carlos Palomino, Sylvia Cardoso Leão, Viviana Ritacco

Belgium, Brazil, Argentina – June 2007

Contributing Authors

Afrânio Kritski, MD, PhD

Unidade de Pesquisas em Tuberculose, Hospital Clementino Fraga Filho,

Departamento de Clinica Medica, Faculdade de Medicina, Universidade

Federal do Rio de Janeiro (UFRJ), Av Professor Rodolpho Rocco s/n – Ilha

do Fundão 4º andar, 21941-590, Rio de Janeiro, BRAZIL

Phone: ++ 55 21 2562 2426 – Fax: ++ 55 21 25506903

kritskia@gmail.com

Angel Adrián Cataldi, PhD

Centro de Investigacciones en Ciencias Veterinarias y Agrarias (CICVyA),

Instituto Nacional de Tecnologia Agropecuaria (INTA), Los Reseros y Las

Cabañas (1712) Castelar, ARGENTINA

Phone: ++ 54 11 4621 0199 – Fax: ++ 54 11 4621 0199

acataldi@cnia.inta.gov.ar

Alejandro Reyes, MSc Biology

Grupo de Biotecnología Molecular, Corporación Corpogen, Carrera 5 No

66A-34, Bogotá D.C., COLOMBIA

Phone: ++ 57 1 3484610 – Fax: ++ 57 1 3484607

alejandroreyesmunoz@gmail.com

Anandi Martin, PhD

Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat, 155,

2000, Antwerp, BELGIUM

Phone: ++ 32 3 2476334 – Fax: ++ 32 3 247 6333

amartin@itg.be

Brigitte Gicquel, PhD

Unité de Génétique Mycobactérienne, Institut Pasteur, 25 rue du Dr. Roux,

75724 Paris-Cedex 15, FRANCE

Phone: ++ 33 1 45688828 – Fax: ++ 33 1 45688843

bgicquel@pasteur.fr

8 Tuberculosis 2007

Carlos Martin, MD, PhD

Grupo de Genetica de Micobacterias, Departamento de Microbiologia,

Medicina Preventiva y Salud Publica, Facultad de Medicina, Universidad de

Zaragoza, C/ Domingo Miral sn, 50009, Zaragoza, SPAIN

Phone: ++ 34 976 761759 – Fax: ++ 34 976 761664

carlos@unizar.es

Christophe Sola, PharmD, PhD*

Unité de la Tuberculose & des Mycobactéries, Institut Pasteur de Guade-

loupe, Morne Joliviere, BP 484, 97183-Abymes, Cedex, GUADELOUPE

* Current affiliation : Unité de Génétique Mycobactérienne, Institut Pasteur,

28 rue du Dr. Roux, 75724 Paris-Cedex 15, FRANCE

Phone: ++ 33 1 40613274 – Fax: ++ 33 1 45688843

csola@pasteur.fr

Clara Inés Espitia, PhD

Instituto de Investigaciones Biomédicas, Apartado Postal 70228, Ciudad

Universitaria 04510, Mexico D.F., MEXICO

Phone: ++ 52 55 6223818 – Fax: ++ 52 55 6223369

espitia@servidor.unam.mx

Dick van Soolingen, PhD

Mycobacteria Reference Unit, Centre for Infectious Disease Control

(CIb/LIS), National Institute of Public Health and the Environment (RIVM),

P.O. box 1, 3720 BA Bilthoven, THE NETHERLANDS

Phone: ++ 31 30 2742363 – Fax: ++ 31 30 2744418

D.van.Soolingen@rivm.nl

Domingo Palmero, MD, PhD

Hospital de Enfermedades Infecciosas F. J. Muñiz, Uspallata 2272

(C1282AEN), Buenos Aires, ARGENTINA.

Phone: ++ 54 11 44326569 – Fax: ++ 54 11 44326569

djpalmero@intramed.net

Contributing Authors 9

Enrico Tortoli, ScD

Regional Reference Center for Mycobacteria. Careggi Hospital. Viale Mor-

gagni 85, 50134 Florence, ITALY

Phone: ++ 39 055 7949199 – Fax: ++ 39 055 7949010

e.tortoli@libero.it

Ernesto Montoro, MD, PhD

Instituto de Medicina Tropical ¨Pedro Kourí¨. Autopista Novia del Mediodia

Km 6 ½, La Lisa, Ciudad de La Habana, CUBA

Phone: ++ 53 7 2020651 – Fax: ++ 53 7 2046051

emontoro@ipk.sld.cu

Fabiana Bigi, PhD

Centro de Investigacciones en Ciencias Veterinarias y Agrarias (CICVyA),

Instituto Nacional de Tecnologia Agropecuaria (INTA), Los Reseros y Las

Cabañas (1712) Castelar, ARGENTINA

Phone: ++ 54 11 46211447 – Fax: ++ 54 11 46210199

fbigi@cnia.inta.gov.ar

Fernando Augusto Fiuza de Melo, MD, PhD

Instituto Clemente Ferreira, Rua da Consolação, 717, 01301-000, São

Paulo, BRAZIL

Phone: ++ 55 11 32190750 – Fax: ++ 55 11 38857827

fernandofiuza@terra.com.br

Françoise Portaels, PhD

Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat, 155,

20000, Antwerp, BELGIUM

Phone: ++ 32 3 2476317 – Fax: ++ 32 3 247 6333

portaels@itg.be

Howard E. Takiff, MD, MPH

Centro de Microbiología y Biología Celular (CMBC), Instituto Venezolano

de Investigaciones Científicas (IVIC), Km. 11 Carr. Panamericana, Cara-

cas, 1020A, VENEZUELA.

Phone: ++ 58 212 5041439 – Fax: ++ 58 212 5041382

htakiff@ivic.ve

10 Tuberculosis 2007

Iris Estrada-García, PhD

Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas,

Instituto Politécnico Nacional. Prol. de Carpio y Plan de Ayala S/N, Mexico

DF, MEXICO C.P. 11340

Phone: ++ 52 55 57296300 ext. 62507 – Fax: ++ 52 55 57296300, ext.

46211

iestrada@encb.ipn.mx and iestrada5@hotmail.com

Jacobus H. de Waard, PhD

Laboratorio de Tuberculosis, Instituto de Biomedicina, Al lado de Hospital

Vargas, San José, Caracas, VENEZUELA

Phone: ++ 58 212 8306670 – Fax: ++ 58 212 8611258

jacobusdeward@movistar.net.ve

Jaime Robledo, MD

Unidad de Bacteriología y Micobacterias, Corporación para Investigaciones

Biológicas, Carrera 72A No.78B-141, Medellín, COLOMBIA.

Phone: ++ 57 4 4410855 ext. 216 – Fax: ++ 57 4 4415514

jrobledo@cib.org.co

Jeanet Serafín-López, PhD

Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas,

Instituto Politécnico Nacional. Prol. de Carpio y Plan de Ayala S/N, Mexico

DF, MEXICO C.P. 11340

Phone: ++ 52 55 57296300, ext. 62369 – Fax: ++ 52 55 57296300 ext.

46211

jeaserafin@hotmail.com

José-Antonio Aínsa Claver, PhD

Grupo de Genética de Micobacterias, Departamento de Microbiología,

Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad de

Zaragoza. C/Domingo Miral s/n, 50009, Zaragoza, SPAIN.

Phone: ++ 34 976 761742 – Fax: ++ 34 976 761604

ainsa@unizar.es

Contributing Authors 11

Juan Carlos Palomino, PhD

Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat, 155,

20000, Antwerp, BELGIUM

Phone: ++32 3 2476334 – Fax: ++ 32 3 247 6333

palomino@itg.be

Kristin Kremer, PhD

Mycobacteria Reference Unit, Centre for Infectious Disease Control

(CIb/LIS), National Institute of Public Health and the Environment (RIVM),

P.O. box 1, 3720 BA Bilthoven, THE NETHERLANDS

Phone: ++ 31 30 2742720 – Fax: ++ 31 30 2744418

Kristin.Kremer@rivm.nl

Leiria Salazar, PhD

Departamento de Biología Estructural, Instituto Venezolano de Investiga-

ciones Científicas (IVIC), Apartado 21827, Caracas, 1020A, VENEZUELA

Phone: ++ 58 212 5041715 – Fax: ++ 58 212 5041444

lsalazar@ivic.ve

Lucía Elena Barrera, Lic Biol

Servicio de Micobacterias, Instituto Nacional de Enfermedades Infecciosas

Carlos G. Malbrán, Av. Velez Sarsfield 563 (1281) Buenos Aires, ARGEN-

TINA

Phone: ++ 54 11 43027635 – Fax: ++ 54 11 43027635

lbarrera@anlis.gov.ar

Mahavir Singh, PhD

Department of Genome Analysis, Helmholtz Center for Infection Research

(former GBF), and LIONEX GmbH, Inhoffenstr. 7, 38124 Braunschweig,

GERMANY

Phone : ++ 49 531 61815320 – Fax : ++ 49 531 2601159

msi@helmholtz-hzi.de and info@lionex.de

Maria Alice da Silva Telles, Lic Biol

Setor de Micobactérias, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355, 01246-

902, São Paulo SP, BRAZIL

Phone: ++ 55 11 30682895 – Fax: ++ 55 11 30682892

atelles@ial.sp.gov.br

12 Tuberculosis 2007

María del Carmen Menéndez, MSc, PhD

Departamento de Medicina Preventiva. Facultad de Medicina, Universidad

Autonoma de Madrid, Arzobispo Morcillo, 4, 28029-Madrid, SPAIN

Phone: ++ 34 914 975491 – Fax: ++ 34 914 975353

carmen.menendez@uam.es

María Isabel Romano, PhD

Centro de Investigacciones en Ciencias Veterinarias y Agrarias (CICVyA),

Instituto Nacional de Tecnologia Agropecuaria (INTA), Los Reseros y Las

Cabañas (1712) Castelar, ARGENTINA

Phone: ++ 54 11 46211447 – Fax: ++ 54 11 46210199

mromano@cnia.inta.gov.ar

María Jesús García, MD, MSc, PhD

Departamento de Medicina Preventiva. Facultad de Medicina, Universidad

Autonoma de Madrid, Arzobispo Morcillo, 4, 28029-Madrid, SPAIN

Phone: ++ 34 914 975491 – Fax: ++ 34 914 975353

mariaj.garcia@uam.es

Nalin Rastogi, MSc, PhD, DSc

Unité de la Tuberculose & des Mycobactéries, Institut Pasteur de Guade-

loupe, Morne Joliviere, BP 484, 97183-Abymes, Cedex, GUADELOUPE

Phone: ++ 590 590 893881 – Fax: ++ 590 590 893880

nrastogi@pasteur-guadeloupe.fr

Nora Morcillo, PhD

Reference Laboratory of Tuberculosis Control Program of Buenos Aires

Province, Hospital Dr. Cetrangolo, Vicente Lopez (1602) Buenos Aires,

ARGENTINA

Phone: ++ 54 11 4970165 – Fax: ++ 54 11 47219153

nora_morcillo@yahoo.com.ar

Patricia Del Portillo Obando, Lic Microbiol

Corporación Corpogen, Carrera 5 No 66A-34, Bogota, D.C. COLOMBIA

Phone: ++ 57 1 3484609/06 – Fax: ++ 57 1 3484607

corpogen@etb.net.co and pdelp2000@yahoo.com

Contributing Authors 13

Pedro Eduardo Almeida da Silva, PhD

Laboratório de Micobactérias, Departamento de Patologia, Fundação Uni-

versidade Federal do Rio Grande (FURG), Rua General Osório S/N, Cam-

pus Saúde, Rio Grande RS, BRAZIL

Phone: ++ 55 53 32338895 – Fax: ++ 55 53 32338860

pedre@furg.br and pedre_99@yahoo.com.br

Peter WM Hermans, PhD

Laboratory of Pediatric Infectious Diseases, Radboud University Nijmegen

Medical Centre, PO Box 9101 (internal post 224), 6500 HB Nijmegen,

THE NETHERLANDS

Phone: ++ 31 24 3666406 (office), ++ 31 24 3666407 (secr.)

Fax: ++ 31 24 3666352

P.Hermans@cukz.umcn.nl

Rodolfo Rodríguez Cruz, MD

Organização Pan-Americana da Saude, Setor de Embaixadas Norte, Lote

19, 70800-400, Brasília DF, BRAZIL

Phone: ++ 55 61 34269546 – Fax: ++ 55 61 34269591

rodolfor@bra.ops-oms.org

Rogelio Hernández-Pando, MD, PhD

Seccion de Patología Experimental, Departamento de Patologia, Instituto

Nacional de Ciencias Médicas y Nutricion Salvador Zubiran, Vasco de Qui-

roga no 15, Tlalpan, CP-14000, Mexico DF, MEXICO.

Phone: ++ 52 55 54853491 – Fax: ++ 52 55 56551076

rhdezpando@hotmail.com and rhpando@quetzal.innsz.mx

Rommel Chacon-Salinas, PhD

Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas,

Instituto Politécnico Nacional. Prol. de Carpio y Plan de Ayala S/N, Mexico

DF, MEXICO C.P. 11340

Phone: ++ 52 55 57296300, ext. 62369 – Fax: ++ 52 55 57296300, ext.

46211

rommelchacons@yahoo.com.mx

14 Tuberculosis 2007

Sylvia Cardoso Leão, MD, MSc, PhD

Departamento de Microbioloiga, Imunologia e Parasitologia, Universidade

Federal de São Paulo (UNIFESP), Rua Botucatu 862, 3° andar, 04023-062,

São Paulo SP, BRAZIL

Phone: ++ 55 11 55764537 – Fax: ++ 55 11 55724711

sylvia@ecb.epm.br

Viviana Ritacco, MD, PhD

Servicio de Micobacterias, Instituto Nacional de Enfermedades Infecciosas Car-

los G. Malbrán, Av. Velez Sarsfield 563 (1281) Buenos Aires, ARGENTINA

Phone: ++ 54 11 43027635 – Fax: ++ 54 11 43027635

vritacco@anlis.gov.ar and vivianaritacco@gmail.com

Abbreviations

2-DE: two dimensional electrophoresis

ADA: adenosine deaminase

ADC: albumin, dextrose, catalase

AFB: acid fast bacilli

AIDS: acquired immunodeficiency syndrome

AMTD: Amplified Mycobacterium tuberculosis Direct Test

BAC: bacterial artificial chromosome

BCG: bacille Calmette-Guérin

bp: base pair

cAMP: cyclic adenosine monophosphate

CAS: Central-Asian (or Delhi)

CD4+: cluster of differentiation 4 glicoprotein

CD8+: cluster of differentiation 8 glicoprotein

CDC: Centers for Disease Control and Prevention

cfu: colony forming units

CMI: cell mediated immunity

CPC: cetylpyridinium chloride

CPF-10: culture filtrate protein 10

CR: complement receptor

CRISPR: clustered regularly interspersed palindromic repeats

CTL: cytotoxic T lymphocyte

DARQ: diarylquinoline

DAT: diacyl trehalose

DC-SIGN: dendritic cell-specific intercellular-adhesion-molecule-grabbing non-integrin

DNA: desoxyribonucleic acid

DOTS: directly observed therapy short-course

DR: direct repeat

DST: drug susceptibility test

DTH: delayed type hypersensitivity

EAI: East-African-Indian

EDTA: ethylenediaminetetraacetic acid

ELISA: enzyme-linked immunosorbent assay

ELISPOT: enzyme-linked immunospot for interferon-gamma

EMB: ethambutol

ESAT-6: 6 kDa early secretory antigenic target

ETH: ethionamide

Fc: crystallizable fraction of the Ig molecule

FDA: Food and Drug Administration

FGF-2: fibroblast growth factor 2

16 Tuberculosis 2007

G+C: guanine plus cytosine

GLC: gas-liquid chromatography

GLP: good laboratory practices

HAART: highly active anti-retroviral therapy

HEPA: high efficiency particulate air

HIV: human immunodeficiency virus

HLA: human leukocyte antigen

HPLC: high-performance liquid chromatography

Hsp: heat-shock protein

IATA: International Air Transportation Association

ICAT: isotope-coded affinity tag

IFN-γ: interferon-gamma

IFN-γR: interferon-gamma receptor

InhA: enoyl acyl carrier protein reductase

Ig: immunoglobulin

IL: interleukin

INH: isoniazid

iNOS: inducible nitric oxide synthase

IS6110 RFLP: restriction fragment length polymorphism based on insertion sequence IS6110

ITS: internal transcribed spacer

IUATLD: International Union Against Tuberculosis and Lung Disease

KasA: beta-ketoacyl ACP synthase

KatG: catalase-peroxidase enzyme

kDa: kiloDalton

LC: liquid chromatography

LSP: large sequence polymorphism

mAG: mycolyl-arabinogalactan

MALDI: matrix assisted laser desorption/ionization

MBL: mannose-binding lectin

MCP-1: monocyte chemoattractant protein-1

MDR: multidrug-resistant

MGIT: Mycobacteria Growth Indicator Tube

MHC: major histocompatibility complex

MIC: minimal inhibitory concentration

MIRU: mycobacterial interspersed repetitive units

MLST: multilocus sequence typing

MODS: microscopic observation broth-drug susceptibility assay

mRNA: messenger ribonucleic acid

MS: mass spectrometry

MSMD: mendelian susceptibility to mycobacterial diseases

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

Abbreviations 17

NEMO: NF-κB essential modulator

NF-κ-B: nuclear factor kappa B

NO: nitric oxide

NRP-1: microaerobic stage of nonreplicating persistence

NRP-2: anaerobic state

NRAMP1: natural resistance-associated macrophage protein 1

nsSNP: non-synonymous single nucleotide polymorphism

NTM: non-tuberculous mycobacteria

OADC: oleic acid, albumin, dextrose, catalase

OD: optical density

ORF: open reading frame

oriC: origin of replication

PANTA: polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocillin

PAS: para-aminosalycilic acid

PAT: penta-acyl trehalose

PCR: polymerase chain reaction

PDIM: phthiocerol dimycocerosate

PE: proteins that have the motif Pro-Glu

PGE: prostaglandin E

PGG: principal genetic groups

PGL: phenolic glycolipids

PGRS: polymorphic guanine-cytosine rich sequences

pI: isoelectric point

pks: polyketide synthase gene

PNB: para-nitrobenzoic acid

PPD: purified protein derivative

PPE: proteins that have the motif Pro-Pro-Glu

PRA: PCR-restriction enzyme analysis

PTFE: polytetrafluoroethylene

PZase: pyrazinamidase enzyme

PVNA: polymyxin B, vancomycin, nalidixic acid and amphotericin B

rBCG: recombinant BCG

RCF: relative centrifugal force

rDNA: ribosomal desoxyribonucleic acid

RD: regions of difference

REMA: resazurin microtiter assay

RFLP: restriction fragment length polymorphism

RIF: rifampicin

RNA: ribonucleic acid

RNAse: ribonuclease

rRNA: ribosomal ribonucleic acid

18 Tuberculosis 2007

rrn operon: ribosomal ribonucleic acid operon

RvD: DNA region deleted from H37Rv genome

SCG: SNP cluster group

SCID: severe combined immunodeficiency

SL: sulfolipid

SLC11A1: solute carrier family 11, member 1

SM: streptomycin

SNP: single nucleotide polymorphism

SpolDB4: fourth international spoligotyping database

sSNP: synonymous single nucleotide polymorphism

ST: shared-type in SpolDB4

STAT1: signal transducer and activator of transcription 1

TACO: tryptophan aspartate coat protein

TB: tuberculosis

TbD1: M. tuberculosis specific deletion 1

TCH: thiophene-2-carboxylic acid hydrazide

TGF-β: transforming growth factor beta

Th1: T helper 1 lymphocyte

Th2: T helper 2 lymphocyte

TL7H11: thin layer 7H11 agar

TLC: thin-layer chromatography

TLR: Toll-like receptor

TNF-α: tumor necrosis factor alpha

TOF: time of flight

TST: tuberculin skin test

US: United States

UV: ultraviolet

VDR: vitamin D receptor

VNTR: variable number tandem repeats

WHO: World Health Organization

XDR: extensively drug resistant

Content

Chapter 1: History 25

1.1. Primeval tuberculosis 25

1.2. Phthisis/consumption 29

1.3. The White Plague 30

1.4. The discovery of the tubercle bacillus 32

1.5. Sanatorium and initial therapies 34

1.6. 19

and 20

centuries 38

1.7. A global health emergency 45

References 49

Chapter 2: Molecular Evolution of the Mycobacterium

tuberculosis Complex 53

2.1. A basic evolutionary scheme of mycobacteria 53

2.2. M. tuberculosis complex population molecular genetics 57

2.3. Co-evolution of M. tuberculosis with its hosts 58

2.4. M. tuberculosis through space and time 61

2.5. Looking for robust evolutionary markers 62

2.6. Why repeated sequences were so useful at the beginning 63

2.7. Regions of differences (RDs) and SNPs in M. tuberculosis 63

2.8. Looking for congruence between polymorphic markers 69

2.9. Main lineages within the M. tuberculosis species 72

2.10. When did the bovine-human switch of M. tuberculosis take place? 78

2.11. Comparative genomics and evolution of tubercle bacilli 79

2.12. Short-term evolutionary markers and database building 80

2.13. Conclusion and Perspectives 81

References 83

Chapter 3: The Basics of Clinical Bacteriology 93

3.1. The tubercle bacillus: a continuous taxon 93

3.2. Microscopic morphology 95

3.3. Cell wall structure 97

3.4. Nutritional and environmental requirements for growth 102

3.5. Generation time 105

3.6. Metabolic and biochemical markers 106

3.7. Resistance to physical and chemical challenges 107

References 109

Chapter 4: Genomics and Proteomics 113

4.1. Impact of new technologies on Mycobacterium tuberculosis

genomics 113

4.2. M. tuberculosis genome 115

4.3. Gene expression in M. tuberculosis 127

4.4. M. tuberculosis proteome 135

4.5. An insight into M. tuberculosis metabolomics 143

4.6. Concluding remarks 146

References 147

Chapter 5: Immunology, Pathogenesis, Virulence 157

5.1. Immune response against Mycobacterium tuberculosis 157

5.2. Tuberculosis pathogenesis and pathology related to the immune

response 171

5.3. Latency and maintenance of the immune response 183

5.4. Immunotherapy for tuberculosis 184

5.5. Concluding remarks 189

References 189

Chapter 6: Host Genetics and Susceptibility 207

6.1. The difficulty in proving a genetic component for human

susceptibility 207

6.2 Search for mutations and polymorphisms that increase susceptibility 219

6.3. Candidate genes in common tuberculosis 225

6.4 Genes from mouse genetic susceptibility studies 237

6.5. The good, the bad and the maybe, in perspective 244

References 250

Chapter 7: Global Burden of Tuberculosis 263

7.1. Global epidemiology of tuberculosis 263

7.2. Tuberculosis and the interaction with the HIV epidemic 269

7.3. Progress of the DOTS strategy 272

7.4. The new Stop TB strategy 275

References 279

Chapter 8: Tuberculosis caused by Other Members of the M.

tuberculosis Complex 283

8.1. Mycobacterium bovis disease in humans 283

8.2. The BCG vaccine: adverse reactions 290

8.3. Mycobacterium africanum subtypes 293

8.4. Mycobacterium microti disease 295

8.5. Mycobacterium caprae and Mycobacterium pinnipedii 297

8.6. Identification of species within the M. tuberculosis complex 301

References 305

Chapter 9: Molecular Epidemiology: Breakthrough

Achievements and Future Prospects 315

9.1. Introduction 315

9.2. Historical context 317

9.3. Infectiousness of tuberculosis patients 319

9.4. DNA fingerprinting, contact investigation and source case finding 320

9.5. Transmission of drug resistant tuberculosis 323

9.6. Resistance and the Beijing genotype 325

9.7. Genetic heterogeneity of M. tuberculosis and multiple infections 326

9.8. The new standard genetic marker: VNTR typing 329

9.9. DNA fingerprinting to monitor eradication of tuberculosis 331

9.10. Future prospects 332

References 333

Chapter 10: New Vaccines against Tuberculosis 341

10.1. Introduction 341

10.2. Historical view 342

10.3. Genetic diversity between BCG vaccines 344

10.4. New vaccines: from the bench to clinical trials 345

10.5. Subunit vaccine candidates 348

10.6. Subunit vaccines for boosting BCG 350

10.7. Recombinant BCG vaccines 350

10.8. Live vaccines based on attenuated M. tuberculosis 352

10.9. Conclusions 354

References 356

Chapter 11: Biosafety and Hospital Control 361

11.1. Biosafety in the hospital 361

11.2. Biosafety in the laboratory 372

References 396

Chapter 12: Conventional Diagnostic Methods 401

12.1. Introduction 401

12.2. Specimen handling 402

12.3. Smear staining 406

12.4. Adenosine deaminase activity 409

12.5. Culture 410

12.6. Identification 416

References 420

Chapter 13: Immunological Diagnosis 425

13.1. Historical Overview 425

13.2. Current methods of tuberculosis diagnosis 427

13.3. Basis of immunological diagnosis 428

13.4. Serological assays 431

13.5. T cell based assays 433

13.6. Conclusions and Perspectives 437

References 438

Chapter 14: New Diagnostic Methods 441

14.1. Introduction 441

14.2. Automated culture methods 441

14.3. Nucleic acid amplification methods 450

14.4. Genetic identification methods 461

14.5. Non-conventional phenotypic diagnostic methods 472

References 479

Chapter 15: Tuberculosis in Adults 487

15.1. Introduction 487

15.2. The initial lesion 487

15.3. The inflammatory response 489

15.4. Tuberculosis infection 490

15.5. Tuberculosis disease 492

15.6. Diagnostic approaches 508

15.7. Treatment of latent tuberculosis infection 516

15.8. Contact tracing and control 518

15.9. The limits between infection and disease 519

References 519

Chapter 16: Tuberculosis in Children 525

16.1. Introduction 525

16.2. Etiology, transmission and pathogenesis 526

16.3. Primary pulmonary tuberculosis 530

16.4. Non-respiratory disease 533

16.5. Congenital tuberculosis 536

16.6. Diagnosis 537

16.7. Pediatric tuberculosis treatment 544

16.8. Vaccination 552

16.9. Prognosis of pediatric tuberculosis 553

References 554

Chapter 17: Tuberculosis and HIV/AIDS 559

17.1. Epidemiological background 559

17.2. Interactions between M. tuberculosis and HIV infection 560

17.3. Clinical characteristics 561

17.4. Multidrug-resistant tuberculosis and HIV/AIDS 568

17.5. Treatment of tuberculosis in HIV/AIDS patients 574

17.6. Immune reconstitution inflammatory syndrome 579

17.7. Treatment of latent tuberculosis infection in HIV/AIDS patients 580

17.8. Mycobacteriosis in AIDS patients 581

References 585

Chapter 18: Drugs and Drug Interactions 593

18.1. Introduction 593

18.2. Overview of existing treatment schemes 594

18.3. Drugs: structure, pharmacokinetics and toxicity 601

18.4. Drug resistance mechanisms 612

18.5. Drug interactions 617

18.6. New drugs for tuberculosis 621

18.7. Useful links 627

References 627

Chapter 19: Drug Resistance and Drug Resistance Detection 635

19.1. Introduction 635

19.2. Drug resistance surveillance 635

19.3. Methods for detection of drug resistance 640

References 655

Chapter 20: New Developments and Perspectives 661

20.1. The scenario 661

20.2. Bacillus and disease under the light of molecular epidemiology 662

20.3. New perspectives in diagnosis 665

20.4. The problem of drug resistance detection 669

20.5. On drug development 670

20.6. On vaccine development 671

20.7. Global management of research & development resources 673

20.8. Useful links 674

References 675

Chapter 1: History

Sylvia Cardoso Leão and Françoise Portaels

Nowhere in these ancient communities of the Eurasian land mass, where it

is so common and feared, is there a record of its beginning. Throughout

history, it had always been there, a familiar evil, yet forever changing,

formless, unknowable. Where other epidemics might last weeks or months,

where even the bubonic plague would be marked forever afterwards by the

year it reigned, the epidemics of tuberculosis would last whole centuries

and even multiples of centuries. Tuberculosis rose slowly, silently, seeping

into homes of millions, like an ageless miasma. And once arrived, it never

went away again. Year after year, century after century, it tightened its

relentless hold, worsening whenever war or famine reduced the peoples'

resistance, infecting virtually everybody, inexplicably sparing some while

destroying others, bringing the young down onto their sickbeds, where the

flesh slowly fell from their bones and they were consumed in the years-

long fever, their minds brilliantly alert until, in apocalyptic numbers, they

died, like the fallen leaves of a dreadful and premature autumn.

The Forgotten Plague:

How the War against Tuberculosis was Won - and Lost

Frank Ryan, 1992

Tuberculosis (TB) has a long history. It was present before the beginning of re-

corded history and has left its mark on human creativity, music, art, and literature;

and has influenced the advance of biomedical sciences and healthcare. Its causative

agent, Mycobacterium tuberculosis, may have killed more persons than any other

microbial pathogen (Daniel 2006).

1.1. Primeval tuberculosis

It is presumed that the genus Mycobacterium originated more than 150 million

years ago (Daniel 2006). An early progenitor of M. tuberculosis was probably

contemporaneous and co-evolved with early hominids in East Africa, three million

years ago. The modern members of M. tuberculosis complex seem to have origi-

nated from a common progenitor about 15,000 - 35,000 years ago (Gutierrez 2005).

26 History

TB was documented in Egypt, India, and China as early as 5,000, 3,300, and 2,300

years ago, respectively (Daniel 2006). Typical skeletal abnormalities, including

Pott’s deformities, were found in Egyptian and Andean mummies (Figure 1-1) and

were also depicted in early Egyptian and pre-colombian art (Figure 1-2).

Figure 1-1: Left: Mummy 003, Museo Arqueológico de la Casa del Marqués de San Jorge,

Bogota, Colombia. Right: Computerized tomography showing lesions in the vertebral bodies of

T10/T11 (reproduced from Sotomayor 2004 with permission).

Figure 1-2: Representation of a woman with pronounced gibbus (Pott´s disease?). Momil

culture, 200 BC to 100 AD, Sinú River, Colombia (reproduced from Sotomayor 1992, with

permission).

1.1. Primeval tuberculosis 27

Identification of genetic material from M. tuberculosis in ancient tissues has pro-

vided a powerful tool for the investigation of the incidence and spread of human

TB in historic periods. It also offers potential new insights into the molecular evo-

lution and global distribution of these microbes (see Chapter 2). Research on an-

cient DNA poses extreme technical difficulties because of the minute amounts of

DNA remains, their oxidation/hydrolysis, and the extremely high risk of contami-

nation with modern DNA. For this reason, stringent criteria of authenticity for

analysis of ancient DNA were recently proposed, among them: work in physically

isolated areas, strict protocols to prevent contamination with modern DNA, the use

of negative controls, evaluation of reproducibility in different laboratories, cloning

and sequencing, and the study of associated remains (Coper 2002).

Mycobacteria are assumed to be better preserved than other bacteria due to the

resistant lipid-rich cell wall and the high proportion of guanine and cytosine in their

DNA, which increases its stability. M. tuberculosis are found only in the tissues of

an infected host, and the characteristic pathology induced by this strictly mammal-

ian pathogen tends to show residual microbial DNA contained in localized lesions.

These bacteria are, therefore, ideal microorganisms for studying ancient DNA and

were the first to be pursued. These investigations have answered important ques-

tions. They proved that TB is an ancient disease with a wide geographical distribu-

tion. The disease was widespread in Egypt and Rome (Zink 2003, Donoghue

2004); it existed in America before Columbus (Salo 1994, Konomi 2002, Soto-

mayor 2004), and in Borneo before any European contact (Donoghue 2004). The

earliest DNA-based documentation of the presence of M. tuberculosis complex

organisms was accomplished in a subchondral articular surface from an extinct

long-horned Pleistocene bison from Wyoming, US, which was radiocarbon-dated

at 17,870 +/- 230 years before the present (Rothschild 2001).

Another important achievement of the studies on ancient DNA was the confirma-

tion of the TB diagnosis in human remains that showed the typical pathology. My-

cobacterial DNA was detected in bone lesions in the spine of a male human skele-

ton from the Iron Age (400-230 BC), found in Dorset, United Kingdom (Taylor

2005); skin samples from the pelvic region of Andean mummies, carbon-dated

from 140 to 1,200 AD (Konomi 2002); and calcified pleura from 1,400 year-old

remains, found in a Byzantine basilica in the Negev desert (Donoghue 1998). DNA

techniques have also shown the presence of mycobacterial DNA, at a lower fre-

quency, in bones with no pathological changes, suggesting either dissemination of

the TB bacilli immediately prior to death or chronic milliary TB (Zink 2003).

Molecular methods other than PCR have also been used to demonstrate the pres-

ence of the tubercle bacillus in ancient remains, including mycolic acid analysis by

28 History

high performance liquid chromatography (HPLC), which is used for authentication

of positive PCR findings in calcified pleura remains (Donoghue 1998). Spoligo-

typing is a PCR-based technique used for identification and typing of M. tuberculo-

sis complex bacteria (see Chapter 9). It is a valuable tool for the study of archeo-

logical material, especially when the DNA is highly fragmented, because fragments

as small as 55-60 bp long are sufficient to provide a positive result (Donoghue

2004). Spoligotyping was the method used to study the Plesitocene remains of a

bison (Rothschild 2001) and was also applied to a subculture of the original tuber-

cle bacillus isolated by Robert Koch, confirming its species identification as M.

tuberculosis rather than Mycobacterium bovis (Taylor 2003).

Until recently, the search for mycobacterial DNA in human archeological speci-

mens failed to find evidence of the presence of M. bovis, a member of the M. tu-

berculosis complex with a remarkably wide spectrum of susceptible mammalian

hosts, and once considered a putative ancestor of M. tuberculosis (Donoghue

2004). In an up to date publication, the identification of M. bovis DNA in South

Siberian human remains was confirmed by amplification of pncA and oxyR genes

and analysis of regions of difference (RD) (for a comprehensive review on differ-

entiation of species belonging to the M. tuberculosis complex, see Chapters 2 and

8). These findings were obtained from remains that showed skeletal evidence of

TB. Carbon-dated from 1,761 to 2,199 years ago, they seem to indicate that this

population was continuously exposed to wild or domesticated animals infected with

M. bovis, which could have been reservoirs for human infection (Taylor 2007).

1.2. Phthisis/consumption 29

1.2. Phthisis/consumption

The patients suffer from a latent fever that begins towards evening and

vanishes again at the break of day. It is accompanied by violent coughing,

which expels thin purulent sputum. The patient speaks with a hoarse voice,

breathes with difficulty and has hectically flushed cheeks. The skin on the

rest of the body is ashen in color. The eyes have a weary expression, the

patient is gaunt in appearance but often displays astonishing physical or

mental activity. In many cases, wheezes are to be heard in the chest, and

when the disease spreads, sweating is seen on the upper parts of the chest.

The patients lose their appetite or suffer hunger pangs. They are often also

very thirsty. The ends of the fingers swell and the fingernails curve

greatly.

Caelius Aurelianus, 5th century AD (Herzog 1998)

The term phthisis (meaning consumption, to waste away) appeared first in Greek

literature. Around 460 BC, Hippocrates identified phthisis as the most widespread

disease of the times. It most commonly occurred between 18 and 35 years of age,

and was almost always fatal (www.tuberculosistextbook.com/link.php?id=1). He

even warned physicians against visiting consumptives in advanced stages of the

disease, to preserve their reputation! Although Aristotle (384-322 BC) considered

the disease to be contagious, most Greek authors believed it to be hereditary, and a

result, at least in part, of the individual's mental and moral weaknesses. Clarissi-

mus Galen (131-201 AD), the most eminent Greek physician after Hippocrates,

defined phthisis as an ulceration of the lungs, chest or throat, accompanied by

coughs, low fever, and wasting away of the body because of pus. He also described

it as a disease of malnutrition (Pease 1940).

The initial tentative efforts to cure the disease were based on trial and error, and

were uniformly ineffective. Heliotherapy was advocated as early as the 5

century

AD by Caelius Aurelianus. Roman physicians recommended bathing in human

urine, eating wolf livers

, and drinking elephant blood. In the Middle Ages, it was

believed that the touch of the sovereigns of England and France had the power to

cure sufferers of the King’s Evil or scrofula (scrophula or struma) - the swellings of

the lymph nodes of the neck, frequently related to TB. Depending upon the time

and country in which they lived, patients were urged to rest or to exercise, to eat or

to abstain from food, to travel to the mountains or to live underground.

30 History

1.3. The White Plague

Yet the captain of all these men of death that came against him to take him

away was consumption, for it was that that brought him down to the

grave.

The life and death of Mr. Badman, presented to the world in

a familiar dialogue between Mr. Wiseman and Mr. Attentive

John Bunyan, 1680

The TB epidemic in Europe, later known as the “Great White Plague”, probably

started at the beginning of the 17

century and continued for the next 200 years.

Death from TB was considered inevitable and, by 1650, TB was the leading cause

of mortality. The high population density and poor sanitary conditions that charac-

terized the enlarging cities of Europe and North America at the time, provided the

necessary environment, not met before in world history, for the spread of this air-

borne pathogen. The epidemic spread slowly overseas by exploration and coloni-

zation.

TB existed in America before Columbus’ arrival but was rare among the natives.

The major outbreaks of TB among the native people of North America began in

1880, after they were settled in reservations or forced to live in barracks in prison

camps. Death rates increased rapidly, and by 1886, reached 9,000 per 100,000

people (Bates 1993).

TB was also rare among Africans who lived in small remote villages. When ex-

posed to the disease by contact with Europeans, these populations experienced a

high mortality rate. Africans taken as slaves were free from TB on arrival to the

Americas. Then, cases of sub-acute fatal TB developed among them. After their

liberation from slavery and movement into the cities, TB morbidity and mortality

rose quickly, reaching 700 per 100,000 in 1912 (Bates 1993).

There is also evidence of the presence of the disease in pre-historic Asia, but it was

only toward the end of the 19

century that peaks in incidence were observed in

India and China.

In the 18

century, TB was sometimes regarded as vampirism. These folk beliefs

originated from two observations: firstly, following the death from consumption of

a family member, household contacts would lose their health slowly. This was

attributed to the deeds of the recently deceased consumptive, who returned from

the dead as a vampire to drain the life from the surviving relatives. Secondly, peo-

1.3. The White Plague 31

ple who had TB exhibited symptoms similar to what people considered to be vam-

pire traits, such as red, swollen eyes, sensitivity to bright light, pale skin, and a

blood-producing cough. They "wasted away" and "lost flesh" and at the same time

remained active, and conserved a fierce will to live. This dichotomy of lust and

"wasting away" was reflected in the vampires' desire for "food", which forced them

to feed off living relatives, who, in turn, suffered a similar wasting away (Sledzik

1994).

Precise pathological and anatomical descriptions of the disease began to appear in

the 17

century. Franciscus Sylvius de la Böe of Amsterdam (1614-1672) was the

first to identify the presence of actual tubercles as a consistent and characteristic

change in the lungs and other areas of consumptive patients. In his Opera Medica,

published in 1679, he also described the progression of the lesions from tubercles

to ulcers and cavities. The Latin word tuber means all kinds of degenerative protu-

berances or tubercles.

The English physician Richard Morton (1637-1698) confirmed that tubercles

were always present in TB of the lungs. He believed that the disease had three

stages: inflammation (tubercle formation), ulceration, and phthisis. Both Sylvius de

la Böe and Morton regarded the disease as hereditary, although Morton did not rule

out transmission by intimate contact.

Gaspard Laurent Bayle (1774-1816) definitely proved that tubercles were not

products, or results, but the very cause of the illness. The name 'tuberculosis' ap-

peared in the medical language at that time in connection with Bayle's theory. More

precisely, the name 'tuberculosis' was coined in 1839 by the German professor of

Medicine Johann Lukas Schönlein (1793-1864), to describe diseases with tuber-

cles; but he considered scrofula and phthisis to be separate entities. These ideas

were also acknowledged by Giovanne Battista Morgagni in Padua (1682-1771)

and Rudolf Virchow in Berlin (1821-1902) (Herzog 1998). In contrast, René

Théophile Hyacinthe Laënnec (1781-1826) from Paris, inventor of the stetho-

scope, and the Viennese Karl von Rokitansky (1804-1878) emphasized the uni-

tary nature of both conditions.

The earliest references to the infectious nature of TB appeared in 17

century Ital-

ian medical literature. An edict issued by the Republic of Lucca in 1699 stated that,

"… henceforth, human health should no longer be endangered by objects remain-

ing after the death of a consumptive. The names of the deceased should be reported

to the authorities and measures undertaken for disinfection" (Herzog 1998).

32 History

1.4. The discovery of the tubercle bacillus

Not bad air, not just a weakness of the infected human body’s immune

system, not any of the myriad theories that had filled the puzzled heads of

his audience all of their working lives...but a bacterium. Not just a bacte-

rium, but a bacillus the like of which had never been even suspected be-

fore, a most singular life form, with a frightening propensity to infect

every cat and chicken, pigeon and guinea pig, the white mice and rats,

oxen and even two marmosets, into which Koch had injected it.

The Forgotten Plague: How the War against Tuberculosis was Won - and Lost

Frank Ryan, 1992

The book De Morbus Contagiosus, written in 1546 by Girolamo Fracastoro

(1478-1553), explained the contagious nature of TB. He pointed out that bed sheets

and clothing could contain contagious particles that were able to survive for up to

two years. The word “particles” may have alluded to chemicals rather than to any

kind of living entity.

In his publication A New Theory of Consumptions, in 1720, the English physician

Benjamin Marten (1704-1722) was the first to conjecture that TB could be caused

by “minute living creatures", which, once they had gained entry to the body, could

generate the lesions and symptoms of phthisis. He further stated, that consumption

may be caught by a sound person by lying in the same bed, eating and drinking or

by talking together so close to each other as to “draw in part of the breath a con-

sumptive patient emits from the lungs”.

In 1865, the French military doctor Jean-Antoine Villemin (1827-1892) demon-

strated that consumption could be passed from humans to cattle, and from cattle to

rabbits. On the basis of this revolutionary evidence, he postulated that a specific

microorganism caused the disease. At this time William Budd (1811-1880) also

concluded from his epidemiological studies that TB was spread through society by

specific germs.

On the evening of March 24, 1882, in Berlin, before a skeptical audience composed

of Germany's most prominent men of science from the Physiological Society, Rob-

ert Koch (1843-1910) (www.tuberculosistextbook.com/link.php?id=2) made his

famous presentation Die Aetiologie der Tuberculose. Using solid media made of

potato and agar, Koch invented new methods of obtaining pure cultures of bacteria.

His colleague Julius Richard Petri (1852-1921) developed special flat dishes

(Petri dishes), which are still in common use, to keep the cultures. Koch also de-

1.4. The discovery of the tubercle bacillus 33

veloped new methods for staining bacteria, based on methylene blue, a dye devel-

oped by Paul Ehrlich (1854-1915)

(www.tuberculosistextbook.com/link.php?id=3), and counterstained with vesuvin.

"Under the microscope the structures of the animal tissues, such as the nucleus and

its breakdown products are brown, while the tubercle bacteria are a beautiful

blue", he wrote in the paper that followed his dramatic presentation that March

evening (Koch 1882).

He had brought his entire laboratory with him: his microscopes, test tubes, small

flasks with cultures, and slides of human and animal tissues preserved in alcohol.

Showing the presence of the bacillus was not enough. He wanted his audience to

note that bacteria were always present in TB infections and could be grown on

solidified serum slants, first appearing to the naked eye in the second week. Then,

he showed that, by inoculating guinea pigs with tuberculous material obtained from

lungs, intestines, scrofula or brains of people and cattle that have died from TB, the

disease that developed was the same, and cultures obtained from the experimental

animals were identical on the serum slopes. Koch continued his speech, proving

that whatever the dose and/or route he used, no matter what animal species he in-

oculated, the results were always the same. The animals subsequently developed

the typical features of TB. He concluded saying that “…the bacilli present in tu-

berculous lesions do not only accompany tuberculosis, but rather cause it. These

bacilli are the true agents of tuberculosis” (Kaufmann 2005).

Koch fulfilled the major prerequisites for defining a contagious disease that had, in

fact, been proposed by his former mentor Jacob Henle (1809-1885). The re-

knowned Koch's postulates (or Henle-Koch postulates) were then formulated by

Robert Koch and Friedrich Loeffler (1852-1915) in 1884, and finally polished and

published by Koch in 1890. The postulates consist of four criteria designed to es-

tablish a causal relationship between a causative microbe and a disease:

• The organism must be found in all animals suffering from the disease, but

not in healthy animals

• The organism must be isolated from a diseased animal and grown in pure

culture

• The cultured organism should cause disease when introduced into a healthy

animal

• The organism must be re-isolated from the experimentally infected animal.

In 1890, at the 10

International Congress of Medicine held in Berlin, Koch an-

nounced a compound that inhibited the growth of tubercle bacilli in guinea pigs

34 History

when given both pre- and post-exposure. It was called 'tuberculin' and was prepared

from glycerol extracts of liquid cultures of tubercle bacilli. Clinical trials using

tuberculin as a therapeutic vaccine were soon initiated. The results were published

in 1891 and revealed that only few persons were cured, at a rate not different from

that of untreated patients. But, although results for treatment were disappointing,

tuberculin was proven valuable for the diagnosis of TB (Kaufmann 2005).

One of Koch’s papers (Koch 1891), describing the preparation and partial purifica-

tion of tuberculin served as the first description of the production of the partially

purified derivative (PPD) of tuberculin, presently used in the Mantoux test, also

known as the Tuberculin Skin Test, Pirquet test, or PPD test (see Chapter 13).

1.5. Sanatorium and initial therapies

…not for nothing was it famous far and wide. It had great properties. It

accelerated oxidization, yet at the same time one put on flesh. It was capa-

ble of healing certain diseases which were latent in every human being,

though its first effects were strongly favorable to these, and by dint of a

general organic compulsion, upwards and outwards, made them come to

the surface, brought them, as it were, to a triumphant outburst.

- Beg pardon -- triumphant?

- Yes; had he never felt that an outbreak of disease had something jolly

about it, an outburst of physical gratification?

The Magic Mountain [Der Zauberberg]

Thomas Mann, 1924

Translated from the German by H. T. Lowe-Porter, 1953

Dialogue between Hans Castorp and consul Tienappel

The introduction of the sanatorium cure provided the first widely practiced ap-

proach to anti-tuberculosis treatment. Hermann Brehmer (1826-1889) a Silesian

botany student suffering from TB, was instructed by his doctor to seek out a

healthier climate. He traveled to the Himalayas where he studied the mountain’s

flora. He returned home cured and began to study medicine. In 1854, he presented

his medical dissertation Tuberculosis is a Curable Disease. Brehmer then opened

an in-patient hospital in Gorbersdorf, where patients received good nutrition and

1.5. Sanatorium and initial therapies 35

were continuously exposed to fresh air. This became the model for all subsequent

sanatoria, including the one depicted in Thomas Mann’s The Magic Mountain.

A young doctor named Edward Livingston Trudeau (1848-1915) established the

most famous sanatorium in the United States at Saranac Lake, in New York's Adi-

rondak Mountains (http://www.trudeauinstitute.org/info/history/history.htm). He

also suffered from TB and, in 1882, became aware of Koch's experiments with TB

bacteria and of Brehmer's sanatorium. Trudeau established the Saranac Laboratory

for the Study of Tuberculosis. It was the first institution devoted to TB research in

the United States (US).

Sanatoria, increasingly found at that time throughout Europe and the US, provided

a dual function. Firstly, they protected the general population by isolating the sick

persons, who were the source of infection. Secondly, they offered TB patients bed-

rest, exercise, fresh-air, and good nutrition, all of which assisted the healing proc-

ess. Many of them improved and returned to "life in the flatland"; many did not.

The TB specialist, the phthisiologist, was responsible for the complete physical and

mental care of the patient and the separation of TB care from the practicing clini-

cian became commonplace.

Architectural features were essential to early sanatorium design (Figure 1-3). These

included deep verandas, balconies, covered corridors, and garden shelters, fur-

nished with reclining couches for the “Cure”, the obligatory two-hour period of rest

in the open air that was frequently observed in silence (Figure 1-4). Furniture for

TB patients had to be robust, able to be thoroughly cleaned and disinfected, and

shaped with a concern for the patient’s anthropometric needs.

Alvar Aalto (1898-1976), Jan Duiker (1890-1935) and Charles-Edouard Jean-

neret (Le Corbusier) (1887-1965) were modernist architects and designers that

adapted and interpreted the ideas of functionality and rationality derived from con-

cepts used in the treatment of TB, and their designs for buildings and furniture

became icons of modernism. Aalto won the competition of Architecture, Interior

Design and Furniture Design for the constuction of the Paimio Tuberculosis Sana-

torium in 1928, and Duiker designed the Zonnestraal Sanatorium. The symbolic

association of light and air with healing made a profound influence on modernist

ideas for design. Flat roofs, balconies, terraces and reclining chairs were subse-

quently adopted for the design of fashionable buildings in rapidly expanding cities

such as Paris and Berlin (Campbell 2005).

36 History

Figure 1-3: Sanatorio Pineta del Carso, Trieste, Italy.

Figure 1-4: Sanatorio Pineta del Carso. Bed-rest, fresh air and good nutrition were the hall-

marks of sanatorium cure.

Probably, it will never be known whether sanatorium treatment was a success or a

failure, because no study was undertaken comparing the rates of mortality of sana-

torium patients with those of TB patients who were similar in age, sex, and eco-

nomic position, but who remained untreated or were treated by other methods.

1.5. Sanatorium and initial therapies 37

Nevertheless, physicians with a long and intimate experience with the disease were

unanimous in the opinion that open-air treatment was an improvement for the aver-

age consumptive (McCarthy 2001).

During the early ’60s, many sanatoria started to close. By the middle of that decade

only a few beds remained available for patients suffering from TB. Yet, the real

end of the TB sanatorium began even earlier, when the depressing era of helpless-

ness in the face of advanced TB was substituted by active therapy.

The Italian physician Carlo Forlanini (1847-1918) discovered that the collapse of

the affected lung tended to have a favorable impact on the outcome of the disease.

He proposed to reduce the lung volume by artificial pneumothorax and surgery,

methods that were applied worldwide after 1913. These and other initial therapies

are now considered dangerous and, at least, controversial:

• Artificial pneumothorax - pleural cavities were filled with gas or filtered

air, with the result of splinting and collapsing that lung (Sharpe 1931).

• Bilateral pneumothorax - only parts of the lungs were collapsed in such a

way that the patient could still live a relatively normal live. The patient suf-

fered from shortness of breath caused by the reduction in the gas exchange

surface.

• Thoracoplasty - ribs from one side of the thorax were removed in order to

collapse the infected portion of the lung permanently (Samson 1950).

• Gold Therapy - Holger Mollgaard (1885-1973) from Copenhagen intro-

duced the compound sanocrysin in 1925, which is a double thiosulphate of

gold and sodium. He tested the compound on animals and considered it

safe for human use. However, it was too toxic even in low doses. A con-

trolled trial, completed in the US in 1934, proved the toxic effects of gold

therapy. Within a year, most European countries had ceased to use it (Be-

denek 2004).

38 History

1.6. 19

and 20

centuries

There is a dread disease which so prepares its victim, as it were, for death;

which so refines it of its grosser aspect, and throws around familiar looks

unearthly indications of the coming change; a dread disease, in which the

struggle between soul and body is so gradual, quiet, and solemn, and the

result so sure, that day by day, and grain by grain, the mortal part wastes

and withers away, so that the spirit grows light and sanguine with its

lightening load, and, feeling immortality at hand, deems it but a new term

of mortal life; a disease in which death and life are so strangely blended,

that death takes the glow and hue of life, and life the gaunt and grisly form

of death; a disease which medicine never cured, wealth never warded off,

or poverty could boast exemption from; which sometimes moves in giant

strides, and sometimes at a tardy sluggish pace, but, slow or quick, is ever

sure and certain.

Nicholas Nickleby

Charles Dickens, 1870

When, in 1820, the poet John Keats (1795-1821) coughed a spot of bright red

blood, he told a friend, "It is arterial blood. I cannot be deceived. That drop of

blood is my death warrant. I must die". He died within a year, at just 25 years of

age. Keats never wrote specifically about phthisis, but his life and his works be-

came a metaphor that helped transform the physical disease "phthisis” into its

spiritual offspring, "consumption".

The central metaphor of consumption in the 19

century was the idea that the

phthisic body is consumed from within by its passions. Spes phthisica (spes - hope

+ phthisis - consumption) was a condition believed to be peculiar to consumptives

in which physical wasting led to a sense of well-being and happiness, an euphoric

blossoming of passionate and creative aspects of the soul. While the body expired

from phthisis, the prosaic human became poetic and the creative soul could be

released from the fevered combustion of the body. The paleness and wasting, the

haunted appearance, the burning sunken eyes, the perspiring skin - all hallmarks of

the disease - came to represent feminine beauty, romantic passion, and fevered

sexuality (Morens 2002).

In the 19

century, it seemed as if everyone was slowly dying of consumption. The

disease became to be viewed in popular terms, first as romantic redemption (Figure

1-5), then as a reflection of societal ills (Figure 1-6) (Morens 2002). In Alexandre

Dumas’ tale “The Lady of the Camellias”, the heroine was a courtesan regenerated

1.6. 19th and 20th centuries 39

by love and made unforgettable by progressive consumption. It was adapted to the

theatre and the movies and also inspired Giuseppe Verdi’s opera “La Traviata”.

The plot develops around the consequences of the heroine’s scandalous past, which

prevents her marriage to an honorable youngster whose father objects to the rela-

tionship. Redemption is possible only through death, and, in taking her life, con-

sumption also serves as a vehicle for punishment.

Figure 1-5: Romantic view of TB: “The Lady of the Camellias” represented by Brazilian actress

Cacilda Becker under Italian director Luciano Salce, in São Paulo, Brazil (1952).

By 1896, the cause of consumption had been discovered, and TB was definitively

linked to poverty and industrial disfigurement, child labor, and sweatshops. A con-

tagious disease and shameful indicator of class, it was no longer easily romanti-

cized in conventional artistic terms. Giacomo Puccini’s “La bohème” (1896) por-

trays TB in a new environment, affecting street artists struggling with poverty and

disease (Figure 1-6).

At the end of the 19

century, the association of TB with poor living conditions and

hygiene brought to life the differentiation and societal repulsion of diseased per-

sons, considered to be responsible for a social wickedness. Unlike the previous

image (sick people as victims), they began to be viewed as dangerous, because they

were capable of spreading the disease to those who did not share their living condi-

tions. TB was changed from a social disease to an individual one and the patient

was at the same time offender and victim of this social ailment.

40 History

A list of famous people and celebrities who had, or are believed to have had TB is

available on Wikipedia at www.tuberculosistextbook.com/link.php?id=4 and at

www.tuberculosistextbook.com/link.php?id=5

Figure 1-6: Social aspect of TB: Second act of “La bohéme”, showing Quartier Latin, with a

great crowd on the street and sellers praising their wares.

After the establishment, in the ’80s, that the disease was contagious, TB was made

a notifiable disease. A further significant advance came in 1895, when Wilhelm

Konrad von Röntgen (1845-1923) discovered X-rays

(www.tuberculosistextbook.com/link.php?id=6). After this, the progress and sever-

ity of a patient's disease could be accurately documented and reviewed.

At the beginning of the 20

century, public health authorities realized that TB was

preventable and that it was not directly inherited. Several associations were set up

to educate the community at large. Books educated people about bad food, bad air

and unhealthy drinking water. Public health reformers used illustrative posters and

stamps (see http://www.nlm.nih.gov/exhibition/visualculture/tuberculosis.html) as

a means of communication, advertisement, and persuasion. This new medium

quickly became an effective educational and fundraising tool in the widespread

campaign against TB.

Centralized official and/or non-governmental agencies for coordination and com-

munication were organized and called for conferences specifically focused on TB.

At the Central Bureau for the Prevention of Tuberculosis, which was formalized in

Berlin in 1902, Dr. Gilbert Sersiron suggested that, as the fight against TB was a

crusade, it would be appropriate to adopt the emblem of a crusader, the Duke of

1.6. 19th and 20th centuries 41

Lorraine. Godfrey of Bouillon (1060-1100), Duke of Lorraine, was the first Chris-

tian ruler of Jerusalem and his banners bearing the double-barred cross signified

courage and success to crusaders. Dr. Sersiron's recommendation was adopted and

the double-barred cross became the worldwide symbol of the fight against TB

(Figure 1-7).

Figure 1-7: double-barred cross, symbol of anti-tuberculosis crusade

Periodic international conferences systematically addressing clinical, research and

sociological aspects of TB were held until the outbreak of World War I in 1914.

After the war, in 1920, a conference on TB was held in Paris with participation of

delegates from 31 countries, among them Australia, Bolivia, Brazil, Chile, China,

Colombia, Cuba, Guatemala, Japan, Panama, Paraguay, Iran and Thailand, in addi-

tion to those of Europe and North America, thus establishing the International Un-

ion Against Tuberculosis and Lung Disease (IUATLD,

http://www.iuatld.org/index_en.phtml) in its present form.

With Edward Jenner’s (1749-1823) successful invention, showing that infection

with cowpox would give immunity against smallpox in humans, many doctors

placed their hopes on the use of M. bovis – the agent that causes bovine TB – for

the development of a vaccine against human TB. However M. bovis was equally

contagious in humans. From 1908 until 1919, Albert Calmette (1863-1933)

(http://www.pasteur.fr/infosci/archives/cal0.html) and Camille Guérin (1872-

1961) (http://www.pasteur.fr/infosci/archives/gue0.html) in France serially passed

a pathogenic strain of M. bovis 230 times, resulting in an attenuated strain called

Bacille Calmette-Guérin or BCG, which was avirulent in cattle, horses, rabbits, and

42 History

guinea pigs. BCG was first administered to humans in 1921 and it is still widely

applied today (see Chapter 10).

Then, in the middle of World War II, came the final breakthrough, the greatest

challenge to the bacterium that had threatened humanity for thousands of years -

chemotherapy. In 1943, streptomycin, a compound with antibiotic activity, was

purified from Streptomyces griseus by Selman A. Waksman (1888-1973)

(www.tuberculosistextbook.com/link.php?id=7) and his graduate student Albert

Shatz (1920-2005) (Shatz 1944a). The drug was active against the tubercle bacillus

in vitro (Schatz 1944b) and following infection of guinea pigs (Feldman 1944). It

was administered to a human patient at the end of 1944 (Hinshaw 1944). Two pio-

neering clinical studies were conducted on the treatment of TB patients with strep-

tomycin, one in Europe and the other in the US (Medical Research Council 1948,

Pfuetze 1955). A considerable improvement in the disease was observed in patients

on streptomycin therapy, but after the first months, some patients began to deterio-

rate and these pioneering studies properly interpreted such treatment failure as a

consequence of development of resistance to the drug.

In 1943, Jörgen Lehmann (1898-1989) wrote a letter to the managers of a phar-

maceutical company, Ferrosan, suggesting the manufacture of the para-amino salt

of aspirin because it would have anti-tuberculous properties (Ryan 1992). The

Swedish chemist based his theory on published information, stressing the avidity of

tubercle bacilli to metabolize salicylic acid. He realized that by changing the

structure of aspirin very slightly, the new molecule would be taken up by the bacte-

ria in just the same way, but would not work like aspirin and would rather block

bacterial respiration. Para-aminosalicylic acid (PAS) was produced and first tested

as an oral therapy at the end of 1944. The first patient treated with PAS made a

dramatic recovery (Lehmann 1964). The drug proved better than streptomycin,

which had nerve toxicity and to which M. tuberculosis could easily develop resis-

tance.

In the late ’40s, it was demonstrated that combined treatment with streptomycin

and PAS was superior to either drug alone (Daniels 1952). Yet, even with the com-

bination of the two drugs, TB was not defeated. Overall, about 80 % of sufferers

from pulmonary TB showed elimination of their germs; but 20 % were not cured,

especially those with extensive disease and cavitation (Ryan 1992).

Two further findings were very important for TB treatment. Firstly, between 1944

and 1948, the action of nicotinamide on the TB bacillus was discovered by two

different groups, but this discovery was not widely appreciated at the time. Sec-

ondly, in 1949, reports stated that the Germans had treated some 7,000 tuberculous

1.6. 19th and 20th centuries 43

patients with a new synthetic drug of the thiosemicarbazone series (Conteben),

developed by Gerhard Domagk (1895-1964)

(www.tuberculosistextbook.com/link.php?id=8), the discoverer of the first sul-

phonamide (McDermott 1969). There is a remarkable similarity between the

atomic structures

of nicotinamide, Conteben, and PAS. Conteben and PAS both

contain a chemical ring of six carbon atoms, the benzene ring, while nicotinamide

contains the pyridine ring in which an atom of nitrogen replaces one of the carbon

atoms (Fox 1953). Thus, by substituting the benzene ring in thiosemicarbazone by

this pyridine ring, a new drug, isoniazid, was developed. By mere coincidence, this

was accomplished simultaneously in three pharmaceutical companies – one in

Germany (Bayer) and two in the US (Squibb and Hoffman La Roche). Isoniazid

was soon submitted for clinical testing and because of the favorable impact of its

administration on disease evolution, the lay press headlines already told the story of

the “wonder drug” before any scientific paper was published (Ryan 1992). How-

ever, none of the three pharmaceutical companies could patent the new drug, be-

cause it had already been synthesized back in 1912 by two Prague chemists, Hans

Meyer and Joseph Mally, as a requirement for their doctorates in chemistry. Nev-

ertheless, while clinical studies were still underway, six studies showed that M.

tuberculosis readily became resistant to isoniazid (Ryan 1992).

In the view of many doctors in those early stages of chemotherapy, the role for

drug therapy was to bring the disease under sufficient control to allow surgeons to

operate the diseased organs. John Crofton (1912-)

(www.tuberculosistextbook.com/link.php?id=9), working at the University of Ed-

inburgh, developed a protocol that resulted in a breakthrough in TB treatment and

control. With his “Edinburgh method” based on meticulous bacteriology and appli-

cation of the available chemotherapy, a 100 percent cure rate for TB was a reason-

able objective. With the success rate obtained by using three drugs together,

(streptomycin, PAS, and isoniazid) TB was completely curable, making surgical

treatment redundant.

Dr. Crofton believed that the conquest of the disease would also imply other meas-

ures, such as pasteurization of milk, tuberculin testing in cattle, BCG vaccination,

mass radiography screening for early diagnosis of disease, isolation of infectious

cases, and general population measures, including reduction of overcrowding and

general improvement of the standard of living.

The “Madras Experiment” was carried out in India in 1956 to test a totally different

concept of therapy, by comparing the results of treatment in a sanatorium with

treatment at home with daily PAS and isoniazid for a year. After a 5-year period of

44 History

follow up, the proportion of persons clear of disease in the two groups was similar

and approached 90 %.

The spirit of optimism that followed was encouraged by the discovery of a series of

new anti-tuberculosis drugs. The drug company Lepetit discovered that the mold

Streptomyces mediterranei produced a new antibiotic, Rifamycin B. Chemical

manipulation of this compound by CIBA resulted in the production of rifampicin,

which has a remarkable potency against M. tuberculosis. Other compounds with

anti-tuberculosis activity were discovered: pyrazinamide, ethambutol, cycloserine,

and ethionamide.

At the end of the ’70s, the primary care of TB patients moved from specialized

institutions to general hospitals and ambulatory care services. At that time, many

hospitals were reluctant to assume such responsibility for fear of spreading the

disease to other patients and to hospital personnel. To overcome their apprehen-

sion, rational safety measures were introduced for the provision of primary care to

TB patients in those settings. Earlier studies on TB transmission performed by

Wells and Riley provided an insight into the characteristics of TB transmission and

set the basis for its containment (Gunnels 1977, see Chapter 11). By applying the

experimental design of his mentor William Firth Wells, Richard Riley pioneered

the study that first documented the role of the droplet nuclei in the transmission of

TB (Riley 1962). The experiments were carried out using guinea pigs lodged in

chambers above wards where TB patients were hospitalized. Only particles small

enough to be carried by the air reached the animals, which, as a result of the inha-

lation of these particles, became infected with the same strains as those infecting

the patients. This could be confirmed by comparison of drug susceptibility patterns.

Indeed, the conclusions of those investigations still stand strong. During coughing,

sneezing, talking or singing, sputum smear-positive TB patients can eliminate large

or small droplets of moisture containing viable bacilli. Large droplets tend to settle

quickly onto the floor and, if inhaled, are trapped in the upper airways and de-

stroyed by local mucocilliary defenses. Smaller droplets (1-10 µm) remain sus-

pended in the air for prolonged periods of time. Evaporation of moisture leaves a

residue – the droplet nucleus. This frequently contains only one or a few bacteria,

which are the infectious units of TB. It was thus established that the risk of TB

transmission is proportional to the concentration of droplet nuclei in the environ-

ment.

Infectivity was also found to be associated with environmental conditions and the

characteristics of the disease in each individual case, such as the bacillary content

of sputum, the presence of cavitation, the frequency of cough, and the presence of

1.7. A global health emergency 45

laringeal TB (see Chapter 11). Therapy with anti-tuberculosis drugs was identified

as the most effective measure for controlling patient´s production of infectious

particles and thus readily reversing infectivity (Gunnels 1977). Therefore, patients

should only require isolation while they were sputum positive and before initiation

of specific therapy. Hospitalization was either abolished or reduced to a few weeks

for most patients (Kaplan 1977). Once a patient´s diagnosis and treatment program

had been defined, physicians who had no particular expertise in chest medicine

could maintain a quality treatment program in most instances. That was the end of

the phthisiologist´s era.

1.7. A global health emergency

 Someone in the world is newly infected with TB bacilli every second.

 Overall, one-third of the world's population is currently infected with

the TB bacillus.

 5-10 % of people who are infected with TB bacilli (but who are not

infected with HIV) become sick or infectious at some time during their

life. People with HIV and TB infection are much more likely to de-

velop TB.

World Health Organization

Fact sheet N°104

Revised March 2006

In Europe and in the US, the general improvement in public health helped to reduce

the burden of TB well before the arrival of specific drugs. TB program activities,

reinforced by successful chemotherapy, resulted in a pronounced reduction of in-

fection and death rates. The disease became greatly controlled but it never quite

disappeared. Then, in around 1985, cases of TB began to rise again in industrial-

ized countries. Several inter-related forces drove this resurgence, including increase

in prison populations, homelessness, injection drug use, crowded housing and in-

creased immigration from countries where TB continued to be endemic. Above all,

the decline in TB control activities and the human immunodeficiency vi-

rus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic were two major

factors fueling each other in the reemergence of TB.

TB programmes had become loose in industrialized countries because the disease

was considered close to elimination. A study performed in 1991 showed that 89 %

46 History

of 224 patients discharged on TB treatment were lost to follow-up and failed to

complete therapy. More than a quarter were back in hospital within a year, still

suffering from TB (Brudney 1991). This study reflected the occurrence of incon-

sistent or partial treatment, which was going on everywhere (Clancy 1990). Pa-

tients cease to take all their medicines regularly for the required period for different

reasons: they start to feel better, doctors and health workers prescribe the wrong

treatment regimens, or the drug supply is unreliable. Uncompliance frequently

results in the emergence of bacteria resistant to drugs and ultimately in the emer-

gence of a “superbug”, resistant to all effective drugs (Iseman 1985). Multidrug-

resistant TB, or MDR-TB, refers to M. tuberculosis isolates that are resistant to at

least both isoniazid and rifampicin, the two most powerful anti-tuberculosis drugs.

MDR-TB takes longer to treat with second-line drugs, which are more expensive

and have more side-effects (see Chapters 18 and 19).

In the early ’90s, an extensive outbreak of highly resistant TB affected more than

350 patients in New York City. The strain was resistant to all first-line anti-

tuberculosis drugs and almost all patients had HIV/AIDS. The hospital environ-

ment was the setting where more than two thirds of the patients acquired and

transmitted the infection. As a consequence, this outbreak affected mainly HIV-

infected patients and health care workers (Frieden 1996). At that time, New York

City became the epicenter of drug-resistant TB, where one in three new cases were

found resistant to one drug and one in five to more than one drug. Important

HIV/AIDS related hospital outbreaks of MDR-TB similar to the one occurred in

New York were described also in non-industrialized countries like Argentina (Ri-

tacco 1997).

Indeed, the HIV/AIDS epidemic has produced a devastating effect on TB control

worldwide. While one out of ten immunocompetent people infected with M. tuber-

culosis will fall sick in their lifetimes, among those with HIV infection, one in ten

per year will develop active TB. In developing countries, the impact of HIV infec-

tion on the TB situation, especially in the 20-35 age group, is overwhelming.

While wealthy industrialized countries with good public health care systems can be

expected to keep TB under control, in much of the developing world a catastrophe

awaits. In poorly developed countries, TB remains a significant threat to public

health, as incidences remain high, even after the introduction of vaccination and

drug treatment (Murray 1990). The registered number of new cases of TB world-

wide roughly correlates with economic conditions: highest incidences are seen in

the countries of Africa, Asia, and Latin America with the lowest gross national

products (see Chapter 7).

1.7. A global health emergency 47

Supervised treatment, including sometimes direct observation of therapy (DOT),

was proposed as a means of helping patients to take their drugs regularly and com-

plete treatment, thus achieving cure and preventing the development of drug resis-

tance. The Directly-Observed Treatment, Short-course (DOTS,

http://www.who.int/tb/dots/whatisdots/en/index.html) strategy was promoted as the

official policy of the WHO in 1991 (see Chapter 7).

The World Health Organization estimates that eight million people get TB every

year, of which 95% live in developing countries. An estimated two million people

die yearly from TB. World Health Organization (http://www.who.int/tb/en) de-

clared TB a global health emergency in 1993 (World Health Organization 2006).

In 1998, the IUATLD joined with the WHO and other international partners to

form the Stop TB Initiative

, a defining moment in the re-structuring of global ef-

forts to control TB. The original Stop TB Initiative has evolved into a broad Global

Partnership, Stop TB Partnership (http://www.stoptb.org), with partners gathered in

Working Groups to accelerate progress in seven specific areas: DOTS Expansion,

TB/HIV, MDR-TB, New TB Drugs, New TB Vaccines, New TB Diagnostics, and

Advocacy, Communications and Social Mobilization.

The World Health Assembly of 2000 endorsed the establishment of a Global Part-

nership to Stop TB and the following targets:

• By 2005: 70% of people with infectious TB will be diagnosed and 85% of

them cured.

• By 2015: the global burden of TB disease (deaths and prevalence) will be

reduced by 50% relative to 1990 levels.

• By 2050: The global incidence of TB disease will be less than one per mil-

lion population (elimination of TB as a global public health problem)

In spite of these global efforts, TB continues to pose a dreadful threat. A notorious

example is the sudden emergence in 2005, in a rural hospital located in Kwa-Zulu-

Natal, a South African province, of a deadly form of TB associated with

HIV/AIDS. This outbreak illustrates the devastating potential of what came to be

called extensively drug resistant TB (XDR-TB) (Gandhi 2006). XDR-TB was de-

fined as MDR-TB with further resistance to second-line drugs (see Chapter 19).

XDR-TB can develop when these second-line drugs are also misused or misman-

aged and, therefore, also become ineffective (Raviglione 2007). The menace of

XDR-TB is not restricted to that remote African setting. A recent survey, per-

formed by 14 supra-national laboratories, on drug susceptibility testing results from

48 countries confirmed this. From 19.9 % of identified MDR-TB isolates, 9.9 %

48 History

met the criteria for XDR-TB. These isolates originated from six continents, con-

firming the emergence of XDR-TB as a serious worldwide public health threat

(Shah 2007).

Nowadays, treating TB is feasible and effective, even in low income countries, if

based on reliable public health practice, including good laboratory infrastructure,

appropriate treatment regimens, proper management of drug side-effects and re-

sources to maintain adherence and prevent spread. The emergence of XDR-TB

should stimulate the improvement of these basic control measures.

It is also crucially important to intensify research efforts devoted to developing

effective TB vaccines, as well as shortening the time required to ascertain drug

sensitivity, improving the diagnosis of TB, and creating new, highly effective anti-

tuberculosis medications. Without supporting such efforts, we still run the risk of

losing the battle against TB.

Disease names related to different clinical forms of TB

Name Clinical form

Phthisis Original Greek name for TB

Lung Sickness TB

Consumption TB

Lupus vulgaris TB of the skin

Mesenteric disease TB of the abdominal lymph nodes

Pott’s disease TB of the spine

Scrofula TB of the neck lymph nodes

King´s evil TB of the neck lymph nodes

White Plague TB especially of the lungs

White swelling TB of the bones

Milliary TB Disseminated TB

Acknowledgement: This chapter is dedicated to Professor Pino Pincherle (1893-1996),

radiologist, founder and director of the Sanatorio Pineta del Carso in Trieste, Italy. Since the

establishment of the Sanatorium, in 1933, Professor Pincherle was responsible for all

physiotherapy treatment and radiologic exams, but after only five years he was compelled to

sell his part in the Sanatorium due to racial laws. In 1939 the family emigrated to Brazil. He

is the grandfather of Sylvia Cardoso Leão, author of this chapter.

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case of tuberculosis in Britain. J Clin Microbiol 2005; 43: 2236-40.

50. World Health Organization (WHO). Frequently asked questions about TB and HIV.

Retrieved 6 October 2006.

51. Zink AR, Grabner W, Reischl U, Wolf H, Nerlich AG. Molecular study on human tuber-

culosis in three geographically distinct and time delineated populations from ancient

Egypt. Epidemiol Infect 2003; 130: 239-49.

52 History

Chapter 2: Molecular Evolution of the Mycobacte-

rium tuberculosis Complex

Nalin Rastogi and Christophe Sola

2.1. A basic evolutionary scheme of mycobacteria

Mycobacteria are likely to represent a very ancient genus of bacteria. Probably, the

Mycobacterium genus originates from a common ancestor whose offspring spe-

cialized in the process of colonizing very different ecological niches. The evolu-

tionary relationships between organisms of the genus Mycobacterium have been

investigated on the basis of the analysis of derived similarities (“shared derived

traits”, synapomorphies).

Since no contemporary living species may directly stem from another contempo-

rary species, it is advisable to speak of «common ancestors», by building clado-

grams rather than genealogical trees when comparing a monophyletic group. Such

cladistic analysis (the word clade is derived from the ancient Greek κλάδος, klados,

meaning branch) forms an ideal basis for modern systems of biological classifica-

tion. Cladograms so generated are invariably dependent on the amount of informa-

tion selected by the researcher.

An ideal approach takes into account a wide variety of information in order to form

a natural group of organisms (clade) which share a unique ancestor that is not

shared with other organisms on the tree, i.e., each clade comprises a series of char-

acteristics specific to its members (synapomorphies), and absent from the group of

organisms from which it diverged. Such distinction involves the notion of out-

groups (organisms that are closely related to the group but not part of it). The

choice of an outgroup constitutes an essential step, since it can profoundly change

the topology of a tree. Similarly, much attention is needed to distinguish between

characters and character states prior to such analysis (e.g., “blue eyes” and

“black eyes” are two character states of the character “eye-color”). A character

state of a determined clade which is also present in its outgroups and its ancestor is

designated as plesiomorphy (meaning “close form”, also called ancestral state).

The character state which occurs only in later descendants is called an apomorphy

(meaning “separate form”, also called the “derived” state). As only synapomor-

phies are used to characterize clades, the distinction between plesiomorphic and

synapomorphic character states is made by considering one or more outgroups.

A collective set of plesiomorphies is commonly referred to as a ground plan for

the clade or clades they refer to; and one clade is considered basal to another if it

54 Molecular Evolution of the Mycobacterium tuberculosis Complex

holds more plesiomorphic characters than the other clade. Usually, a basal group is

very species-poor in comparison to a more derived group. Thus, conservative

(apomorphic) branches, defined as anagenetic branches represent species whose

characteristics are closer to those of the ancestor than others.

Possibly, the founder of the genus Mycobacterium was a free-living organism and

today’s free-living mycobacterial species (and also some saprophytic species?)

represent the conservative branches of founding mycobacteria. The more distant

organisms are probably the ones that live in association with various multicellular

organisms. It has been suggested that the mycobacteria that created a long-lasting

association with marine animals (probably placoderms) are at the root of this phy-

logenetic branch. Thus, Mycobacterium marinum would stem from the conserva-

tive branch, whereas other vertebrate-associated mycobacteria would build the

anagenetic branch. Grmek speculates that the association of a mycobacterial spe-

cies with a marine vertebrate may have occurred during the superior Devonian (300

million years ago) (Grmek 1994). Figure 2-1 shows the phylogenetic position of

the Mycobacterium tuberculosis complex species within the genus Mycobacterium

based on a tree of the gene coding for the 16S ribosomal ribonucleic acid (rRNA).

Figure 2-1: Phylogenetic position of the tubercle bacilli within the genus Mycobacterium (re-

produced with permission from Gutierrez et al. 2005)

2.1. A basic evolutionary scheme of mycobacteria 55

In the past, mycobacterial systematics used to rely on phenotypic characters; more

recently, however, genetic techniques have boosted taxonomic studies (Tortoli

2003). The first natural characters used to distinguish between mycobacterial spe-

cies were growth rate and pigmentation. Rapid growers (< 7 days) are free, envi-

ronmental, saprophytic species, whereas slow growers are usually obligate intra-

cellular, pathogenic species. The slow-fast grower division, which virtually always

relies on the possession of one or two rRNA operons (rrn operon) (Jy 1994), was

shown to be phylogenetically coherent (Stahl 1990, Devulder 2005).

In the ’50s, the hypothesis of co-evolution, or parallel evolution, between hosts and

mycobacteria looked no more likely than the alternative hypothesis of «multiple,

casual (furtive) introductions» of various saprophytes into different hosts. The

traditional epidemiological belief for tuberculosis (TB) is that the anthropozoonosis

due to M. tuberculosis may find its origin in a zoonotic agent, i.e., Mycobacterium

bovis (Cockburn 1963). This view is still sustained by some authors (Smith 2006a).

However, genetics brought some new clues into the debate (Brosch 2002). For

example, the sequencing of the Mycobacterium leprae genome, by its defective

nature, confirmed the previous history-driven hypothesis that M. leprae was a

younger pathogen than M. tuberculosis (Cole 1998, Cole 2001). In the case of the

M. tuberculosis complex, comparative genomics has also shown that the M. bovis

genome is smaller than the M. tuberculosis genome, opening the way to a new

scenario for the evolution of the tubercle bacillus (Brosch 2002). M. bovis genomic

reduction (loss of genes) indeed suggests that it could be a younger pathogen than

M. tuberculosis or, in other words, that human TB disease preceded bovine disease

(Brosch 2002, Cockburn 1963). Figure 2-2 shows that the common ancestor of

members of the M. tuberculosis complex is close to three of its branches: “Myco-

bacterium canettii”, Mycobacterium africanum and the ancestral East-African-

Indian (EAI) clade. However, according to Smith et al., “until it is demonstrated

that strains of M. africanum subtype I can be maintained in immunocompetent

cells, the host-association of the most recent common ancestor of the M. tuberculo-

sis complex remains unsolved” (Smith 2006b).

56 Molecular Evolution of the Mycobacterium tuberculosis Complex

Figure 2-2: Scheme of the proposed evolutionary pathway of the M. tuberculosis bacilli illus-

trating successive loss of DNA in certain lineages (reproduced with permission from Brosch et

al. 2002)

Ancient humans, bovids and mastodons experienced erosive diseases caused by M.

tuberculosis. As an alternative to the classical hypothesis of TB spread being

driven by human migration, bovids, mastodons, or simply diet might well be con-

sidered to be the natural epidemiological vehicle of TB. In this way, a poorly

pathogenic environmental or animal Mycobacterium spp. would have progressively

acquired some human-specific virulence traits (Rotschild 2001, Rotschild 2006a).

The association of hyperdisease and endemic stability may have promoted a

smooth and long-term transition from zoonosis to anthropozoonosis (Coleman

2001, Rotschild 2006b). Other complex anthropological parameters, such as the

history of agriculture and livestock domestication, may also have been mediators of

TB spread (Smith 1995, Bruford 2003). In this sense, it is also logical to compare

the pathogenicity of the various M. tuberculosis complex members in various host

species. Interestingly, it has been observed that M. africanum apparently elicits a

more attenuated T cell response to the 6 kiloDalton (kDa) early secreted antigen

2.2. M. tuberculosis complex population molecular genetics 57

(ESAT-6) than M. tuberculosis in patients with TB. M. africanum could thus be

considered to be an opportunistic human pathogen. If confirmed, these findings are

new evidence that strain differences affect human interferon-based T cell responses

(de Jong 2006). Strain-related differences in lymphokine (including interferon-

gamma) response in mice with experimental infection were also reported in 2003

(Lopez 2003).

2.2. M. tuberculosis complex population molecular genetics

Until recently, the question of individual genetic variation within the M. tuberculo-

sis complex gained little attention and most research on M. tuberculosis was or-

ganism- rather than population-centered. The advent of molecular methods, and

their widespread use in population studies, introduced both new conceptual and

new technological developments. The inference of phylogenies from molecular

data goes back to the early ’90s with the development of software such as PHYLIP

and PAUP (Felsenstein 1993, Swofford 1990, Swofford 1998). In particular, the

study of the M. tuberculosis complex phylogeny closely followed the development

of increasing numbers of sophisticated genotyping methods. The way was opened

by M. tuberculosis fingerprinting by restriction fragment length polymorphism

based on insertion sequence IS6110 (IS6110 RFLP) (van Embden 1993). However,

the use of IS6110 RFLP in evolutionary genetics discovery was of limited value for

many reasons:

• fast variation rate of this evolutionary marker (de Boer 1999)

• complexity of forces driving its transposition and risk of genetic conver-

gence (Fang 2001)

• nature of experimental data produced which requires sophisticated soft-

ware for analysis

• difficulty to build large sets of data (Heersma 1998, Salamon 1998)

The discovery in 1993 of the polymorphic nature of the Direct Repeat (DR) locus,

and the subsequent development of the spoligotyping method based on DR locus

variability, introduced more modern concepts and tools for M. tuberculosis com-

plex genotyping (Groenen 1993, Kamerbeek 1997). Our research group bet that the

highly diverse signature patterns observed by spoligotyping could indeed contain

phylogenetical signals, and the construction of a diversity database was started de

novo (Sola 1999). Today, a total of 62 M. tuberculosis complex clades/lineages are

detailed in the Fourth International Spoligotyping Database (SpolDB4) which de-

58 Molecular Evolution of the Mycobacterium tuberculosis Complex

scribes 1,939 shared-types representing a total of 39,295 M. tuberculosis strains

from 122 countries (Brudey 2006). This database is available on the internet at

SITVIT (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo). Some of the ma-

jor M. tuberculosis complex clades and their spoligotype signatures are described

below under section 2.9. The assumption that the DR locus was neutral still re-

mains speculative; however, the finding of other clustered regularly interspersed

palindromic repeats (CRISPR) loci in both Archae and Bacteriae has become a hot

issue (Jansen 2002, Pourcel 2005, Makarova 2006). Spoligotyping was immedi-

ately followed by the discovery of tandem repeat loci in the M. tuberculosis com-

plex and the Variable Number of Tandem Repeats (VNTR) genotyping technique

(Frothingham 1998). Later, the Mycobacterial Interspersed Repetitive Units

(MIRU) technique (Supply 2001) was developed, which is also designated as Mul-

tiple Locus VNTR analysis (MLVA). Multi-Locus Sequence Typing (MLST) was

introduced as an alternative method (Baker 2004). More recently, systematic Single

Nucleotide Polymorphism (SNP) genotyping (Filliol 2006, Gutacker 2006) was

described followed by Large Sequence Polymorphism (LSP), the latter performed

either by microarray or real-time Polymerase Chain Reaction (PCR) (Mostowy

2002, Tsolaki 2005).

2.3. Co-evolution of M. tuberculosis with its hosts

Simulation models reported in 1988 suggested that a social network with a size of

180 to 440 persons is required for TB to occur with endemicity. In such conditions,

host-pathogen coexistence would be maintained in populations (McGrath 1988).

The concept of endemic stability, already mentioned above, suggests that an infec-

tious disease may reach an epidemiological state in which the clinical disease is

scarce, despite high levels of infection in the population (Coleman 2001). Clearly,

this concept may apply to TB since it is most likely to have been a vertically trans-

mitted disease before being responsible for large outbreaks.

The question of how many isolated communities of between 180 to 440 persons

may have experienced, sequentially or concomitantly, the introduction of one or

more founding genotypes of M. tuberculosis complex (each one with its own spe-

cific virulence), in other words, how TB was “seeded” is of paramount importance.

To provide the initial conditions of a dynamic epidemic system we must understand

how these early founding genotypes spread in low demographic conditions. Today,

we can observe a phylogeographically structured global epidemic, built as a result

of millennia of evolution. Some clones are extinct, others have an increased risk of

emergence (Tanaka 2006). The evolution rate of TB is likely to have been succes-

2.3. Co-evolution of M. tuberculosis with its hosts 59

sively slow (human and cattle migration and low endemicity or hyperendemicity

but little or no disease), then moderate (five centuries of post-Columbus sail-based

migration) with important anthropological changes that may have created bursting

conditions linked to demographic growth and migration, and lastly, fast (since the

introduction of air transportation), i.e. within the five decades of increasing move-

ments of strains and people, concomitantly to new outbreaks in demographically

active and resource-poor countries where the great majority of cases is now pres-

ent.

Consequently, the worldwide bacterial genetic snapshot of the TB epidemic is the

result of a combination of slow, medium, and fast evolving superimposition pic-

tures of various outbreak histories. Such a jigsaw puzzle will be difficult, if not

impossible, to reconstruct. However, looking for rare and isolated genotypes, which

may have undergone a slower evolution, as well as searching for ancient desoxyri-

bonucleic acid (DNA) may constitute two complementary scientific strategies in

attempting to reach this goal.

One recent success of the first strategy is exemplified by the finding of a peculiar

highly genetically diverse “M. canettii” in the Horn of Africa. “M. canettii” was

likely to be the most probable source species of the M. tuberculosis complex, rather

than just another branch of it (Fabre 2004). Further results confirm that, despite its

apparent homogeneity, the “M. canettii” or “M. prototuberculosis” genome is a

composite assembly resulting from horizontal gene transfer events predating clonal

expansion. The large amount of synonymous single nucleotide polymorphism

(sSNP) variation in housekeeping genes found in these smooth strains of “M. pro-

totuberculosis” suggests that the tubercle bacilli were contemporaneous with early

hominids in East Africa, and may have thus been evolving with their human host

much longer than previously thought. These results open new perspectives for

unraveling the molecular bases of M. tuberculosis evolutionary success (Gutierrez

2005).

The second strategy has also provided interesting results that support the notion of

TB’s ancient origin. The isolation and characterization of ancient M. tuberculosis

DNA from an extinct bison, dated 17,000 years B.C., suggest the presence of TB in

America in the late Pleiostocene (Rotschild 2001). The extensive infection of many

individuals of the Mammut americanum species with the M. tuberculosis agent also

suggests that, apart from Homo sapiens, mastodons and bovids may have spread

the disease during the Pleistocene (Rotschild 2006a, Rotschild 2006b). When

looking at human remains, several DNA studies served to trace back the presence

of TB to Egyptian mummies, where M. tuberculosis and also M. africanum geno-

types were identified (Zink 2003). Figure 2-3 shows an ancient Egyptian clay arte-

60 Molecular Evolution of the Mycobacterium tuberculosis Complex

fact with a traditional kyphosis suggestive of Pott’s disease. The presence of TB in

America before the arrival of the Spanish settlers is also well demonstrated both by

paleopathological evidence and studies on ancient DNA (Salo 1994, Arriaza 1995).

Recent paleopathological evidence also suggests the presence of leprosy and TB in

South East Asian human remains from the Iron Age (Tayles 2004). Taken together,

these results may argue that the limited number of different genogroups that we

observe today are likely to stem from those that were seeded in the past, have re-

mained isolated by distance during millennia, and have had time to co-evolve inde-

pendently before gaining reasonable statistical chances to meet.

Figure 2-3: Egyptian clay artefact of an emaciated man with a characteristic angular kyphosis

suggestive of Pott’s disease (reproduced from TB, Past, Present, 1999, TB Foundation)

2.4. M. tuberculosis through space and time 61

2.4. M. tuberculosis through space and time

The concept of phylogeography was originally introduced by Avise (Avise 1987),

as “the history of processes that control the geographic distribution of genes and

lineages by constructing the genealogies of populations and genes”. The term was

introduced as a way to bridge population genetics and molecular ecology and to

describe geographically structured signals within species. This concept might well

be applied to studies on the global spread of M. tuberculosis through time. If the

ancestor of M. tuberculosis adapted specifically and slowly to human beings, it

may have had the time to develop, via an extreme clonality, a deeply rooted and

peculiar phylogeographical structure reflecting both the demographic history and

the history of TB spread.

The geographic distribution of bacteriophage types was the only method to detect

the geographic subdivision of the M. tuberculosis complex species during the ’70s

and the ’80s (Bates 1969, Sula 1973); however, no phylogenetic relationships could

be inferred at that time using mycobacteriophages. A numerical analysis of M.

africanum taxonomy also suggested differences between isolates from West and

East Africa (David 1978). The naming of two M. africanum variants (subtype I and

II) created confusion and the status of M. africanum as a homogeneous sub-species

of M. tuberculosis complex is still uncertain. The existence of some major geo-

graphical and epidemiological significant genetic variants of the M. tuberculosis

complex was also recognized as early as 1982 (Collins 1982). Among these were

the Asian, the bovine and the classical variants, in addition to africanum I and

africanum II variants.

Lateral genetic transfer was presumed to be minor in M. tuberculosis, and the clo-

nal structure of the M. tuberculosis complex was formally demonstrated by the

finding of strong linkage disequilibrium within MIRU loci (Supply 2003). Only

recently has the issue of M. tuberculosis complex lateral genetic transfer gained

interest, particularly in regard to its links to genetic diversity and to potential acqui-

sition of virulence (Kinsella 2003, Rosas-Magallanes 2006, Alix 2006). The im-

portance of lateral genetic transfer in one species’ history is of primary importance

to better understand its specificity. As for the members of the M. tuberculosis com-

plex, with the exception of M. canettii, there is no evidence for this kind of transfer

or for housekeeping gene recombination (Smith 2006a). Indeed, recent evidence

argues in favor of the existence of lateral genetic transfer in the precursor of the M.

tuberculosis complex, and in favor of environmental mycobacteria being the source

of certain genetic components in the M. tuberculosis complex. These findings rein-

force the idea that the ancestor of the M. tuberculosis complex was an environ-

62 Molecular Evolution of the Mycobacterium tuberculosis Complex

mental Mycobacterium (Rosas-Magallenes 2006). Another source of exogenous

DNA may be plasmids that have been shown to be present in modern species of

mycobacteria, and sometimes to carry virulence genes (Le Dantec 2001, Stinear

2000, Stinear 2004). The mosaic nature of the genome of ancestral “M. prototuber-

culosis” species also argues in favor of numerous gene transfer events and/or ho-

mologous recombination within ancient species of the M. tuberculosis complex

(Gutierrez 2005).

2.5. Looking for robust evolutionary markers

When looking for robust evolutionary markers, the evolutionist will first choose

markers that are assumedly neutral in order to avoid debates on function or poten-

tial selection, whether positive or stabilizing. For the M. tuberculosis complex, the

very existence of an obligate intracellular life, which provides a stable chemical

and metabolic environment, suggests that a classical metabolic selection scheme

must have played a minimal role in the evolution of the M. tuberculosis complex

genome (Musser 2000). Host specialization and niche adaptation may have been

more important. Changes towards acquisition of an intracellular life style may also

be responsible for loss of function and hence, loss of genes.

Silent mutations in housekeeping genes were the first candidates to be selected as

evolutionary markers. However, the amount of genetic diversity found in the genes

selected in that original study was unexpectedly low, which led to the hypothesis

that TB had spread only recently from a unique precursor. Indeed, the rate of ge-

netically neutral synonymous mutations (sSNP) was shown to be as low as

1/10,000 whereas the rate of non-synonymous mutations (nsSNP) outnumbered

sSNPs by almost 2 to 1 (Sreevatsan 1997).

As for spoligo- and MIRU typing, at first glance it seems reasonable to consider

these markers as neutral. No evident role for the DR locus, a member of CRISPR

sequences, has been proven yet; however, there is an increased interest in CRISPR

and the CRISPR-associated genes cas, which may mean to the bacterial world what

silencing RNAs means for the eukaryotic world (Makarova 2006). Apart from the

senX3-regX3 double component system, which was presumably involved in viru-

lence, the function of MIRU loci remains poorly investigated (Parish 2003). In all

cases, the phylogenetical information content obtained by studying the DR and the

VNTR loci was previously shown to be rich (D. Falush 2003 - Prague, European

Concerted Action Meeting, unpublished data).

2.6. Why repeated sequences were so useful at the beginning 63

2.6. Why repeated sequences were so useful at the beginning

The description of repeated sequences goes back to the early age of molecular

biology (Britten 1968). Their role in the selection of new vital functions in life is

indeed of paramount importance for genetic evolution (Britten 2005). In the M.

tuberculosis complex, repetitive DNA sequences were used as probes and showed

to be useful for fingerprinting strains in epidemiological studies (Eisenach 1988).

Shortly after the characterization of the insertion sequence IS6110 (Thierry 1990),

an international consensus method IS6110 RFLP was adopted almost concomi-

tantly to the World Health Organization declaration of TB as a public health emer-

gency (van Embden 1993). IS6110 RFLP changed the traditional belief that no

more than 10 % of TB cases were due to recent transmission, and sparked a new

hope for disease eradication by contributing to the adequate surveillance and pre-

vention of TB transmission (Alland 1994, Small 1994). For diverse reasons, how-

ever, the use of IS6110 was of little help in solving the phylogenetic structure of

the M. tuberculosis complex because it turned out to be a poor phylogenetic marker

(Fleischmann 2002). A rapidly emerging issue was that IS6110 was ineffective in a

large part of the world, including South-East Asia (Fomukong 1994). Another

insertion sequence, IS1081, was also suggested as an interesting potential phyloge-

netic marker; however, its generalized use in M. tuberculosis complex population

genetics was also hampered, among other reasons, by the RFLP format (van Sool-

ingen 1997, Park 2000).

2.7. Regions of differences (RDs) and SNPs in M. tuberculosis

One approach to understanding the molecular evolution of the M. tuberculosis

complex and looking for virulence genes is to identify regions of difference (RD)

between M. tuberculosis complex genomes (Inwald 2003) or to look for Single

Nucleotide Polymorphisms (SNPs). Substractive genomic hybridization was ini-

tially used to identify three distinct genomic regions between virulent M. bovis, M.

tuberculosis, and the avirulent M. bovis bacille Calmette-Guérin (BCG) strain,

designated respectively as RD1, RD2, and RD3 (Mahairas 1996). One of these

regions, RD1, was shown to contain important virulence genes including the two

immunodominant T-cell antigens ESAT6 and culture filtrate protein 10 (CFP10)

(Pym 2002). In another study (Gordon 1999), restriction-digested bacterial artificial

chromosome (BAC) arrays of H37Rv strain were used to reveal the presence of 10

regions of difference between M. tuberculosis and M. bovis (RD1 to 10); 7 of

which (RD4-RD10) were deleted in M. bovis. The deletion pattern of M. africanum

is closer to that of M. tuberculosis than to the pattern of M. bovis (Gordon 1999).

64 Molecular Evolution of the Mycobacterium tuberculosis Complex

Brosch et al. analyzed the distribution of 20 variable regions resulting from inser-

tion-deletion events in the genome of the tubercle bacilli in one hundred strains

belonging to all sub-species of the M. tuberculosis complex (Brosch 2002). The

authors showed that the majority of these polymorphisms resulted from ancient

irreversible genetic events in common progenitor cells, the so-called Unique Event

Polymorphisms (UEP). Based on the presence or absence of an M. tuberculosis

specific deletion 1 (TbD1, a 2 kb sequence), M. tuberculosis can be divided into

“ancient” TbD1 positive and “modern” TbD1 negative strains. This classification

superimposes well with the previous principal genetic group (PGG) classification

(Sreevatsan 1997); however, only two groups of strains, the EAI and the M. africa-

num strains are TbD1 positive. The RD9 deletion identifies an evolutionary lineage

represented by M. africanum, M. microti and M. bovis that diverged from the pro-

genitor of the present M. tuberculosis strains before TbD1 occurred (Brosch 2002).

These findings contradict the long-held belief that M. tuberculosis evolved from a

precursor of M. bovis, suggesting a new evolutionary scenario of the M. tuberculo-

sis complex. Since M. canettii and other ancestral M. tuberculosis complex strains

lack none of these regions, they are supposed to be direct descendants of the tuber-

cle bacilli that existed before the M. africanum-M. bovis lineage separated from the

M. tuberculosis lineage (Brosch 2002). This scenario was confirmed in a follow-up

study in which in silico and macroarray based hybridization experiments confirmed

the existence of a core set of 219 conserved genes shared by M. leprae and M.

tuberculosis. Among these new phylogenetical markers is the pks 15/1 gene, which

encodes one of the polyketide synthase enzymes required for the lipid metabolism

of cell wall building. All modern strains show a 7-base pair (bp) frameshift deletion

in this gene that induces a knock-out of the enzyme. M. canettii, most PGG1 an-

cestral EAI, and Beijing strains add two amino acids that do not interfere with pks

function, whereas strains in the M. bovis lineage bear a 6-bp DNA deletion that

involves deletion of these two extra amino acids (Constant 2002).

Three recent studies provide landmarks in TB molecular and phylogenetic popula-

tion studies. The first one suggests the existence of six phylogeographical lineages,

each associated with specific sympatric human populations (Gagneux 2006). These

observations show that mycobacterial lineages are adapted to particular human

populations. Whether these results are considered from either a “splitter” or from a

“gatherer” perspective, they endorse the idea that there are probably just a small

number of founding genogroups of the M. tuberculosis complex. Also, these results

support previous results on M. tuberculosis complex genetic diversity and our hy-

pothesis that M. tuberculosis complex is an ancient pathogen that co-evolved with

its hosts (Sola 2001a, 2001b, Sebban 2002).

2.7. Regions of differences (RDs) and SNPs in M. tuberculosis 65

Two SNP-population-based phylogenies also provided similar results, i.e. a limited

number of M. tuberculosis complex phylogeographical genogroups (Figure 2-4).

According to a study led by Musser’s group, eight deeply branching genetic groups

(I to VIII) were found; however, this was still not representative of the worldwide

genetic diversity of M. tuberculosis because of a biased sampling, e.g., lack of

Central Asian (CAS) strains (Gutacker 2002). A second study corrected this bias by

creating one new subgroup for the CAS lineage (Gutacker 2006). This lineage is

close to the root, which suggests that the Indian subcontinent played a major role in

TB evolution and expansion.

Figure 2-4 Phylogenetic tree obtained on SNPs, adapted from Gutacker et al. 2006 and sup-

plemental data. In blue: spoligotyping-based nomenclature or characteristics. In red: IS6110-

based clade nomenclature with some characteristics IS6110 copy number or molecular weight

data. In green: Musser’s principal genetic group (Sreevatsan 1997). In black: SNP-based

designation of clades with some characteristics strains (CDC1551, H37Rv, strain 210).

Similar results were obtained independently by Alland et al., reinforcing the idea

that unrelated lineages may acquire the same number of IS6110 by homoplasia

(Alland 2003). The same group recently analyzed 212 SNPs in correlation with

MIRU and spoligotyping on a worldwide representative collection of clinical iso-

lates. Their results are illustrated in Figures 2-5 (A to C). The M. tuberculosis com-

plex tree presented four main branches containing six SNP cluster groups (SCG1 to

SCG6) and five subgroups as depicted in Figure 2-5 B (Filliol 2006). These results

provide good congruence with spoligotyping and, to a lesser extent, with MIRU12,

66 Molecular Evolution of the Mycobacterium tuberculosis Complex

endorsing the latest genetic diversity studies on spoligotyping (Brudey 2006). Still,

it can be argued that in both SNP-based studies, identical bias could have been

introduced since the SNPs analyzed in both cases were selected based on the four

M. tuberculosis complex genome sequences available to date: M. tuberculosis

strains 210, CDC1551, H37Rv and M. bovis strain AF2122.

Figure 2-5, A to C: (From Filliol et al. 2006 J. Bacteriol., reproduced with permission). A: a

distance-based neighbor-joining tree on 159 sSNPs resolves the 219 M. tuberculosis complex

isolates in 56 sequence types (ST). STs are indicated by a dot with numerical value and color

code for SNP Cluster Group (SCG) belonging. B: Model-based neighbor-joining tree based on

a data set with 212 SNPs, which resolves 327 M. tuberculosis complex isolates into 182 ST

with identical cluster (compare with A). SNP Cluster Groups are indicated by colors. Principal

Genetic Groups (1 to 3) are also highlighted. C: distribution of the spoligotype clades on the

SNP-based phylogeny.

2.7. Regions of differences (RDs) and SNPs in M. tuberculosis 67

68 Molecular Evolution of the Mycobacterium tuberculosis Complex

Table 2-1 provides a nomenclature correlation between M. tuberculosis complex

groups defined by spoligotyping and those defined by sSNPs. As shown in this

table, the most ancient clade, EAI defines SCG 1 or sSNP-I according to Alland’s

or to Musser’s designation, respectively. SCG 2 and sSNP-II define the Beijing

lineage. SCG 3a or sSNP-IIa defines the CAS or Delhi genogroup. SCG 3b or

sSNP-III defines the Haarlem family of strains. SCG 3c and SCG 4, or sSNP-IV

and sSNP-V, define the “IS6110 European low-banders” or X genogroup (Sebban

2002, Dale 2003, Warren 2004). SCG 5 or sSNP-VI is mainly constituted by the

Latin American and Mediterranean (LAM) genogroup (Sola 2001a). SCG 6a and

SCG 6b (sSNP-VII and sSNP-VIII) define the poorly characterized Principal Ge-

netic group 3 lineage that also includes some ill-defined T genotypes (Filliol 2002).

Last but not least, SCG 7 defines the bovine and seal M. tuberculosis complex

subspecies whereas no counterpart is provided in Musser’s classification (Filliol

2006).

Table 2-1: Comparison of spoligotype and SNP terminology

PGG

(Sreevatsan 1997)

Spoligotyping-based

(Filliol 2003)

SCG-based

(Filliol 2006)

SNP-based

(Gutacker 2006)

PGG EAI SCG 1 sSNP-I

PGG1 Beijing SCG 2 sSNP-II

PGG1 CAS SCG 3a sSNP-IIA

PGG 1 Bovis SCG 7 M. tuberculosis complex

PGG2 Haarlem SCG 3b sSNP-III

PGG2 X1 SCG 3c sSNP-IV

PGG2 X1,X2,X3 SCG 4 sSNP-V

PGG2 LAM SCG 5 sSNP-VI

PGG3 T (Miscellaneous) SCG 6 sSNP-VII

sSNP-VIII

PGG = Principal Genetic Group EAI = East African Indian

SCG = SNP cluster group CAS = Central Asian (or Delhi)l

SNP = Single nucleotide polymorphism

2.8. Looking for congruence between polymorphic markers 69

2.8. Looking for congruence between polymorphic markers

The concept of molecular clock, attributed to Zuckerkandl and Pauling in 1962,

was originally based on hemoglobin evolution and later generalized to DNA evo-

lution (Zuckerkandl 1987). As for M. tuberculosis, we are dealing with polymor-

phic markers, i.e. repeated sequences, which are physically linked to the chromo-

some and therefore transmitted together with it. Concomitantly, these sequences are

evolving at their own pace and hence possess more than one molecular clock. Al-

though the combination of various molecular clocks of different paces in a single

analysis may be criticized (Wilson 2003), this approach was used successfully in

the past to detect the EAI and the LAM clades by observing congruence between

spoligotyping and VNTR data (Figure 2-6, extracted from Sola 2001b).

Figure 2-6 : Close-up on a spoligotyping-based neighbor-joining (NJ) phylogenetical tree, built

on SpolDB2 database showing the EAI and LAM branches. The superimposition of spoligo-

typing, VNTR and Principal Genetic grouping shows congruence between various markers

(extracted from Sola 2001b) in blue boxes: Spoligotyping shared-type n°/VNTR allele: ETR-A

to E from left to right). In red boxes: SpolDB-shared-type n°/Soini’s spoligotyping number (see

Soini et al. 2000)/Principal Genetic Group (see Sreevatsan 1997). In the blue boxes of the

upper figure, the third number is the strain identification number. In circles: spoligotyping

shared-type number.

70 Molecular Evolution of the Mycobacterium tuberculosis Complex

IS6110 RFLP was recognized very early to evolve faster than spoligotype since

more RFLP than spoligo genotypes are present when a single set is analyzed

(Kremer 1999). The mutation rate of IS6110 was estimated recently to be 0.287 per

genome per year for a strain with a typical number of 10 copies (Rosenberg 2003).

Using the infinite allele model and the same set of data (Kremer 1999), the relative

mutation rate of spoligotype is calculated to be 13.5 % of the rate of IS6110 (Ta-

naka 2005). This corresponds to a spoligotype mutation rate of around 0.039 events

per year. A more complex model was recently developed, which assumes that the

mutation rate of a given spoligotype is proportional to the number of spacer units

present in the DR region. This new model allows the detection of emerging strains

of M. tuberculosis (Tanaka 2006).

Population bottlenecks are important in biology since they create genetic conditions

that favor founder effect and speciation. Among many bottleneck hypotheses, the

one ascribed to the late Pleistocene is very attractive. It involves volcanic winter

and differentiation of modern humans at a time comprised between 50,000 and

15,000-25,000 years ago (Ambrose 1998). These events may have created envi-

ronmental conditions favoring the spread of M. tuberculosis. We may hypothesize

on the global spreading of a single clone (Kapur 1994), or of a limited number of

clones, based on the expansion of the surviving re-founders, preserved in various

small refuges located in tropical areas (Ambrose 1998). The ample human genetic

diversity observed today in Africa (as well as the apparently ample M. tuberculosis

genetic diversity) may be due either to a longer evolutionary period, or to the pres-

2.8. Looking for congruence between polymorphic markers 71

ervation of such ample diversity in this continent during the bottleneck event. Con-

sequently, for the M. tuberculosis complex, we can hypothesize that the high ge-

netic diversity observed in “M. prototuberculosis” could be a remnant of this bot-

tleneck event, with a strong resilience and hence a high preservation of the previous

genetic diversity inside these tropical refuges. This ecological perspective is also

supported by data suggesting that human beings migrated back to Africa after the

demographic expansion into the South-East Asian peninsula (Cruciani 2002). Thus,

if demographic and epidemic factors are considered in addition to evolutionary and

genetic factors, the modern tubercle bacilli are more likely to find their origin in

India or South-East Asia rather than in Africa. The fact that the TbD1 positive

East-African-Indian strains, which are likely to have disseminated when adequate

demographical conditions were fulfilled, are genetically the closest to the M. ca-

netti- “M. prototuberculosis” strains argues in favor of this hypothesis.

Given the astonishingly reduced SNP diversity observed initially in the M. tuber-

culosis complex (Sreevatsan 1997, Musser 2000), the bottleneck hypothesis is

seducing. However, the 15,000- to 25,000-year time frame was calculated by com-

putation of synonymous mutation rates based on Escherichia coli and Salmonella

divergence, i.e. based on a uniform calibration rate for nucleotide substitution (the

basic molecular clock). This choice of independency from growth rate (doubling

time) and other parameters, such as mutation rate and population size, may be criti-

cized. The doubling time of E. coli is 20 min and that of M. tuberculosis is 20

hours. If we logically assume that sSNPs acquisition is related to DNA metabolism,

then, a ratio of 60x should be applied to the computation presented in Kapur’s

paper, thus providing a much larger time-frame (900,000-1,500,000 years) for the

presence of M. tuberculosis complex bacilli on earth, an hypothesis that is consis-

tent with the latest results obtained on “M. prototuberculosis”, which shows an

unusually high SNP diversity (Gutierrez 2005).

According to a recent multigenic phylogenetic approach, the speciation process in

mycobacteria might have been progressive and relatively homogenous across the

whole genome (Devulder 2005). When comparing substitution rates of fast and

slow growing mycobacteria by means of a relative rate test, non-significant differ-

ences were observed. These findings suggest that the two groups evolved at the

same rate. In other words, the evolutionary rate does not necessarily correlate to the

number of generations. This framework fits with the strictly clonal evolution of M.

tuberculosis and the co-evolution hypothesis that suggests adaptation between

particular mycobacterial lineages and particular human populations (Supply 2003,

Gagneux 2006). However more recent genetic studies using SNPs analysis suggests

that some genes such as the ones coding for the PE-PGRS and PPE proteins that

72 Molecular Evolution of the Mycobacterium tuberculosis Complex

have the motifs Pro-Glu (PE) and Pro-Pro-Glu (PPE), thought to be critical in host-

pathogen interactions, are prone to recombination and gene conversion events

(Karboul 2006, Liu 2006).

2.9. Main lineages within the M. tuberculosis species

Within the scope of this chapter is the description of the results of the molecular

population approach that allowed the definition of genetically homogenous clusters

of M. tuberculosis complex, which are now shown to be preferentially linked to

some human hosts (Brudey 2006, Gagneux 2006). Table 2-2 provides the latest

description of statistically, epidemiologically or phylogeographically relevant clo-

nal complexes of the M. tuberculosis complex based on spoligotyping signatures

described in the SpolDB4 database (a high resolution image can be downloaded

at: http://www.biomedcentral.com/1471-2180/6/23/figure/F1?highres=y; from

Brudey 2006).

2.9.1. Principal lineages of the Genetic group 1

2.9.1.1. The East African-Indian (EAI) lineage

This lineage was first described in Guinea-Bissau (Källenius 1999) and was shown

to be frequent in South-East Asia, India, and East Africa (Kremer 1999). This

group of strains is characterized by a low number of IS6110 copies. A subgroup of

these strains harboring a single copy of IS6110 was shown to be widespread in

Malaysia, Tanzania, and Oman (Fomukong 1994). In combined datasets (i.e.

pooled datasets characterized by one or more methods), this lineage demonstrated

congruence between spoligotypes (absence of spacers 29-32, presence of spacer 33,

absence of spacer 34), VNTR [exact tandem repeat A (ETR-A) allele ≥4], katG-

gyrA grouping (Group 1), and later the presence of the TbD1 sequence (Soini

2000, Sola 2001b). More recently, the presence of an oxyR C37T transition was

shown to be specific to the lineage (Baker 2004). This lineage was shown to belong

to cluster group 1 or Cluster I (Filliol 2006, Gutacker 2006). It harbors a specific

region of difference, RD239 and was renamed as Indo-Oceanic in the work of

Gagneux et al. (Gagneux 2006). It is speculated that this lineage, which is endemic

in South-East Asia, South-India, and East-Africa, may have originated in Asia,

where TB could have historically found favorable spreading conditions. The Ma-

nila family was first identified by Douglas in 1997, and was later thoroughly char-

acterized by the same group (Douglas 2003). This genotype was identified based

on the prevalence of clustered strains isolated from Philippino immigrants in the

2.9. Main lineages within the M. tuberculosis species 73

United States (US) and was only later shown to be prevalent in the Philippines. The

Manila family bears ST19 as prototypic spoligo-signature and is actually identical

to EAI-2 (Filliol 2002). ST89, which defines the Nonthaburi (Thailand) group of

strains, is a derived clone (Namwat 1998). In this family, specific variants have

been also described for Vietnam (ST139 or EAI-4), Bangladesh (ST591, ST1898 or

EAI-6 and 7) and Madagascar (ST109, EAI-6).

We have no precise idea about the prevalence of the EAI lineage in India and

China, although it is evident that this genotype is more specifically linked with

South-East Asia and South India than with Northern China. This may be due to

differences in civilization and agriculture histories between North and South China

(Sola 2001b). It is also very difficult to analyze what links these clones may have

with strains in the major genetic group 2, given the presence of the spacer 33 in this

group of strains (a spacer that is absent in groups 2 and 3). A striking discovery

related to these strains was made recently when analyzing medieval human remains

discovered in an English parish. TB was confirmed by amplifying multiple M.

tuberculosis loci and EAI genotypes were apparently identified by spoligotyping

(Taylor 1999). Whether these spoligotyping results obtained on medieval remains

are reliable or not should be confirmed independently; however, the possibility of

the presence of EAI genotypes in 13

century England should not be excluded.

2.9.1.2. The Beijing lineage

The Beijing genotype belongs to the principal genetic group 1 of Sreevatsan, and

its specific spoligotype signature (absence of spacer 1-33, presence of spacer 34-

43) was discovered in 1995 (van Soolingen 1995). However, a notorious outbreak

due to a multidrug resistant clone of one of its offspring (New York W strain) had

been characterized earlier, at the beginning of the ’90s (Plikaytis 1994, Bifani

2002). The emergence of this family of related genotypes continues to pose a seri-

ous threat to TB control due to its high virulence and frequent association with

multidrug resistance. It was hypothesized that this genotype emerged successfully

in East Asia due to mass BCG vaccination during the 20

century (van Soolingen

1995, Abebe 2006). However, Beijing should also be considered as a group of

variant clones that evolved from a common precursor at an undefined time, maybe

during the Genghis Khan reign or before (Mokrousov 2005).

These strains are characterized by the presence of an inverted IS6110 copy within

the DR region, an IS6110 element at a particular insertion site (within the origin of

replication) and one or two IS6110 copies in a DNA region called NTF (Plikaytis

1994, Kurepina 1998). A characteristic Beijing lineage-defining SNP (G81A in

Rv3815c) has been reported by Filliol et al. According to SNP analysis, the Beijing

74 Molecular Evolution of the Mycobacterium tuberculosis Complex

cluster was designated as SCG 2 or sSNP-II (Filliol 2006, Gutacker 2006). Other

characteristic sSNPs of the Beijing lineage were described in putative DNA repair

genes (Rad 2003).

More recently, new phylogenetically-informative specific LSP markers were found,

such as RD105, which is present in all Beijing/W or RD142, RD150 and RD181. It

allows a further division of the Beijing lineage into four monophyletic subgroups

(Tsolaki 2005). The Beijing lineage was recently renamed as the East Asian Line-

age by other authors (Gagneux 2006). Its most frequent VNTR signature is 42435

(Kremer 1999).

Recent evidence points to an early dispersal of the Beijing genotype in correlation

to genetic haplotype diversity of the male Y chromosome (i.e. in correlation with

human phylogeography). These results suggest that the spreading history of Beijing

has a molecular evolutionary history that is much more intricate and more deeply

rooted to human history than initially thought. Using the Beijing genotype as a

model, and comparing its phylogeography to Y-chromosome-based phylogeogra-

phy, Mokrousov et al. hypothesized that two events shaped the early history of this

genotype: (1) its upper Paleolithic origin in the Homo sapiens sapiens K-M9 cluster

in central Asia, and (2) a primary dispersal of the secondary Beijing NTF:: IS6110

lineage by Proto-Sino-Tibetan farmers within East-Asia (human O-M214/M122

haplogroup) (Mokrousov 2005).

2.9.1.3. The Central-Asian (CAS) or Delhi lineage

The presence in India of a specific lineage of the M. tuberculosis complex was

concomitantly and independently reported by two different groups using IS6110

RFLP and spoligotyping, respectively (Bhanu 2002, Filliol 2003). This lineage was

also shown to be endemic in Sudan, other sub-Saharan countries and Pakistan

(Brudey 2006). Using IS6110 RFLP, the Delhi lineage shows a characteristic band

pair in the high molecular weight region (12.1 and 10.1 kilobase pairs) and its spe-

cific spoligotype signature is formed in the absence of spacers 4-27 and 23-34. This

spoligo-signature shows numerous variants and several subgroups such as CAS1-

Kili (for Kilimanjaro) and CAS1-Dar (for Dar-es-Salaam), which have already

been defined on the basis of new spoligotype-signatures that are specific for each

new clonal complex (Mc Hugh 2005, Eldholm 2006). Still, more results using other

polymorphic markers should complement these data. VNTR signatures of M. tu-

berculosis complex clinical isolates from South-Asian immigrants in London and

native patients in Rawalpindi, Pakistan, were identical (allele combination 42235)

and correlated with the CAS spoligotype (Gascoyne-Binzi 2002, Brudey unpub-

lished results).

2.9. Main lineages within the M. tuberculosis species 75

This genotype family could be the ancestor of the Beijing family since it clusters

close to Beijing when analyzed by a combination of MIRU, spoligotyping and

VNTR (Sola 2003). In India, its frequency varies from one region to another: it is

more prevalent in the North than in the South, where the EAI family predominates

(Suresh 2006). An outbreak strain named CH was recently reported in Leicester,

United Kingdom. It belongs to the CAS family and harbors a specific deletion

(Rv1519). In broth media, this strain was found to grow more slowly and to be less

tolerant to acid and H

than two laboratory reference strains, CDC1551 and

H37Rv. Nevertheless, its ability to grow in human monocyte-derived macrophages

was not impaired. This strain induced more anti-inflammatory IL-10, more IL-6

gene transcription/secretion from monocyte-derived macrophages, and less protec-

tive IL-12p40 than CDC1551 and H37Rv strains. Thus, this strain seems to com-

pensate the microbiological attenuation by skewing the innate response toward a

phagocyte deactivation. The complementation of Rv1519 reversed its ability to

elicit anti-inflammatory IL-10 production by macrophages. These results suggest

that the Rv1519 polymorphism confers an immune subverting M. tuberculosis

phenotype that might contribute to the persistence and outbreak potential of this

lineage (Newton 2006).

2.9.2. Lineages belonging to the Principal Genetic groups 2 and 3

2.9.2.1. The Haarlem family

The Haarlem family was described in the Netherlands in 1999 (Kremer 1999). On

IS6110 RFLP, these strains harbor a double band at 1.4 kb. Their spoligotype is

characterized by the absence of the spacer 31, which is due to the presence of a

second copy of IS6110 in the DR region (Groenen 1993). Due to an asymmetric

insertion within the DR locus, this second IS6110 copy hinders the detection of

spacer 31 (Filliol 2000, Legrand 2001). Three main spoligotype-signatures define

the variants H1 to H3 (Filliol 2002). However, many Haarlem clonal complexes

may harbor other Haarlem-based spoligo-signatures that are, as yet, poorly charac-

terized. Another characteristic of the Haarlem lineage is the frequent VNTR pattern

33233 (Kremer 1999). The Haarlem family is highly prevalent in Northern Europe.

It is present in the Caribbean to a lesser extent and is also prevalent in Central Af-

rica, where it is believed to have been introduced during the European colonization

(Filliol 2003). This family, which is highly diverse, merits further studies to better

understand its evolutionary history. A SNP in the mgt gene of the M tuberculosis

Haarlem genotype was discovered recently (Alix 2006). More SNPs are expected

to be specific of the Haarlem lineage.

76 Molecular Evolution of the Mycobacterium tuberculosis Complex

2.9.2.2. The Latin American and Mediterranean (LAM) family

The LAM family was defined by the finding of linkage disequilibrium between the

absence of spacers 21-24 in the spoligotyping and the presence of an ETR-A allele

equal to 2 (Sola 2001b). However, this genotype family is more diverse and its

study is more complicated than initially thought. Strains belonging to the

LAM3/F11 family and the S/F28 family harbor identical spoligotypes of the shared

type ST4, revealing the existence of genetic convergence between spoligotypes

(Warren 2002). This phenomenon seems, however, to be rare and highly dependent

on the structure of the observed spoligotype. The absence of spacers 21-24 may

also have occurred more than once in tubercle bacilli evolution although no genetic

evidence has suggested such a convergence event until now. Many sub-motifs -

LAM1 to LAM12 - have been suggested according to the latest international spoli-

gotype database project SpolDB4 (Brudey 2006). However, the phylogenetic sig-

nificance of the common absence of spacers 23-24 has not been demonstrated in

this lineage. In this sense, some genotypes that show strong geographical specific-

ity (for example the LAM10-Cameroon or the LAM7-Turkey) were initially la-

beled as LAM, although there is no evidence of their phylogenetical relation to

other LAM spoligo-signatures (Niobe-Eyangoh 2003, Zozio 2005). Recently, a

specific deletion designated as RD

RIO

was shown to be linked to certain LAM

spoligo-signatures present in Rio de Janeiro, Brazil (L Lazzarini, R Huard, JL Ho

personal communication).

The LAM clade is frequent in Mediterranean countries and its presence in Latin

America is supposed to be linked to the Lusitanian-Hispanian colonization of the

New World. Conversely, it may have been endemic in Africa and/or in South

America, spreading to Europe later. At this stage, we must highlight that paleopa-

thological and ancient DNA data support the existence of TB before the arrival of

Spanish settlers to Latin America in the 15

century (Arriaza 1995, Salo 2001).

2.9.2.3. The X family: the European IS6110 low banders

The X family of strains is defined by two concomitant features, a low number of

IS6110 copies and the absence of spacer 18 in the spoligotyping (Sebban 2002).

This latter is indeed an important characteristic common to at least three spoligo-

type shared types: ST119, ST137, and ST92. Both characteristics are present in the

CDC1551 strain, which was once suggested to be highly virulent. The X family

was also the first group identified in Guadeloupe (Sola 1997) and the French Poly-

nesia (Torrea 1995). Specific epidemic variants of this genotype family were de-

scribed in South Africa (Streicher 2004). The absence of spacer 18 bears phyloge-

netical significance because it is improbable that this spacer was deleted more than

2.9. Main lineages within the M. tuberculosis species 77

once in the evolution of M. tuberculosis. The distribution of the X family appears

to be linked to Anglo-Saxon countries (Dale 2003). It is also highly prevalent in

South Africa and to a lesser extent in the Caribbean. Currently, it is only poorly

documented in India. The strong presence of this genotype family in Mexico could

be explained by its close proximity to the USA.

2.9.2.4. The T families and others

The «ill-defined» T group is characterized «by default». It includes strains that

miss spacers 33-36 and can hardly be classified in other groups. This is a general

characteristic of strains belonging to the principal genetic groups 2 and 3, together

with the absence of an intact pks 15/1 gene (Marmiesse 2004). The presence of

intact polyketide synthase genes, active in the synthesis of the specific lipid com-

plex of the M. tuberculosis complex is now known to be linked to virulence (Con-

stant 2002). Conversely, the 7 bp frameshift deletion in pks15/1 may be considered

as a phylogenetical marker specific for the modern M. tuberculosis strains (Gag-

neux 2006) and may define the recently designated Euro-American lineage. It is

expected that the combination of spoligotype and improved MIRU signatures will

be the best way to precisely define epidemiological clonal complexes (Supply

2006). Alternatively, RDs and/or SNPs may also improve the taxonomic definition

of these clones.

Table 2-2 shows the nomenclature correspondence between the main spoligotyp-

ing-based M. tuberculosis complex lineages and those recently described by

MLST-SNPs (Baker 2004) and LSP (Gagneux 2006). As shown, spoligotyping

appears to be more discriminative than the other two typing systems since it is able

to resolve clinical isolates within the branch of the modern strains that are not

solved by LSP. Specific RDs are described for many individual spoligotype-

signatures; however, no Table is yet available for LSP and/or SNP synthetic corre-

spondence with spoligotype.

Even if there is consensus in the fact that the main branches of the genetic tree of

the M. tuberculosis complex have now been found, many uncertainties still remain

with regard to the chronology of the evolution of the M. tuberculosis complex. For

example, Gagneux et al. suggest that West African 2 diverged from an ancestral

branch of M. bovis, whereas West African 1, characterized by a deletion of

RD711, did not (Gagneux 2006).

78 Molecular Evolution of the Mycobacterium tuberculosis Complex

Table 2-2 Comparison of spoligotype, Multi Locus Sequence Typing (MLST) and Large Se-

quence Polymorphism (LSP) nomenclature

Spoligotyping-based

(Filliol 2003)

MLST

(Baker 2004)

LSP

(Gagneux 2006)

Comment

East-African-Indian

(EAI)

IV Indo-Oceanic Prevalent in South East Asia,

East Africa and South India

Beijing I East-Asian Prevalent in China, Japan,

South East Asia, Russia

Central-Asian (CAS) III

East-African-

Indian

Prevalent in North India,

Pakistan, Libya, Sudan

X, Haarlem, LAM II Euro-American Ubiquitous

M. africanum NA West African 1 Nigeria, Ghana

M. africanum NA West African 2 Senegal, Gambia

Recent results in our laboratory have shown that, in certain cases, it should be pos-

sible to reconstruct the past evolutionary history of some modern clones of the M.

tuberculosis complex belonging to the principal groups 2 and 3. As an example, a

striking identity was found recently between the MIRU typing results of the main

LAM7-Turkey clonal complex (Zozio 2005) and the Japanese group T3-OSA (Ano

2006) (Millet et al. unpublished results). The meaning of this identity is under

investigation and there is no reason to believe that it is due to convergence. Simi-

larly, an endemic clone found in Nunavik (Nguyen 2003) was shown to be related

to a clone found to be prevalent in central Europe (Poland and Germany) (Sola et

al. unpublished results). Once again, we are trying to analyze how and when such

movement of strains took place and whether they are representative of a deeply

rooted anthropological structure or from modern outbreaks.

2.10. When did the bovine-human switch of M. tuberculosis

take place?

The question of the molecular evolution of M. bovis provides an interesting frame-

work for comparison with that of M. tuberculosis (Smith 2006a). In particular,

Smith et al. discuss in detail how population bottlenecks and selective sweeps

deeply affect the population structure of strictly clonal pathogens, such as members

of the M. tuberculosis complex. Using the genetic diversity of M. bovis in the

United Kingdom as a model, these authors demonstrate that all M. bovis genotypes

derive from a single clonal complex that is likely to have emerged as a result of the

actions of bovine TB control programs, which have been in force for the last 100

years. These authors also suggest that comparative genomics between two selected

genomes that have gone through very different selection pressures (H37Rv and M.

2.11. Comparative genomics and evolution of tubercle bacilli 79

bovis AF2122) may have wrongly suggested that M. bovis is an offspring clone of

M. tuberculosis. As Brosch et al. identified deletions in M. bovis by comparing it

with the only M. tuberculosis chromosome sequence available at that time, it was

inevitable to conclude that M. bovis was the terminal group in the lineage (Smith

2006a). The assumption that the RD9-deleted lineage (including M. bovis) de-

scended from an M. tuberculosis–like ancestor also implies, by parsimony, that the

most recent common ancestor of these strains was adapted to humans. The exact

host-association of M. africanum subtype I strains has not been examined so far.

There is some evidence that M. africanum, which is less virulent than other M.

tuberculosis complex genotypes, is currently extinct in settings where it was the

most prevalent strain only three decades ago. Instead, it is being replaced by im-

ported, more virulent genotypes (V. Vincent, unpublished results). The genetic

susceptibility of the indigenous African population to TB during World War I is a

well-known fact which supports the idea that TB caused by a more virulent geno-

type evokes a different, acute and even fatal disease, very different from that pro-

duced by M. africanum.

2.11. Comparative genomics and evolution of tubercle bacilli

The wealth of completed genome sequences, the development of microarray tech-

nology, and the decreasing cost of sequencing have enabled scientists to thoroughly

study the significance of strain to strain variation in bacteria such as Streptococcus

agalactiae and to define the “pan-genome” concept (Tettelin 2005). According to

this concept, any species is made up of a common and a strain-specific genetic

pool. Depending on the population structure of the studied organism and on the

levels of lateral gene transfer, the relative part of these two pools may vary signifi-

cantly. The core genome contains genes present in all strains, and the dispensable

genome contains genes present in two or more strains as well as genes unique to

single strains. Given that the number of unique genes is vast, the pan-genome of a

bacterial species might be orders of magnitude larger than any single genome

(Medini 2005).

LSP analysis is of particular interest in the M. tuberculosis complex, given the low

level of sSNPs (Sreevatsan 1997, Kato-Maeda 2001, Alland 2007). Figure 2-7

shows the non-randomness of deletions in the 16 clinical isolates that were tested

by microarray against the H37Rv genome. Some isolates contained unique dele-

tions whereas other deletions were shared by many isolates. This study was ex-

tended to 100 different and unique IS6110 RFLP types representing the global

genetic diversity of the M. tuberculosis complex observed in San Francisco over

80 Molecular Evolution of the Mycobacterium tuberculosis Complex

12 years (Tsolaki 2004). LSP size varied between 105 and 11,985 bp, with eight

deleted sequences larger than 5,000 bp. LSPs tend to occur in genomic regions that

are prone to repeated insertion-deletion events and may be responsible of a high

degree of genomic variation in the M. tuberculosis complex (Alland 2007). Chap-

ter 4 provides an exhaustive review on the comparative genomics of members of

the M. tuberculosis complex.

Figure 2-7: Circular map of genomic deletions among M. tuberculosis showing that the pattern

of deletions differs between clones and is not spatially random. The outer numbers show the

scale in mega base pairs (O=replication origin). In blue: genomic locations of deleted se-

quences. The outer circle summarizes the sum of all detected deletions. Color code (blue,

orange, green) is linked to number of deletions (respectively 1, 2 and 3 deletions). The thin red

line spans the genomic region of the genome where the number of deletions detected is

greater than expected by chance alone. CDC1551 appears as the third ring on this picture.

(Reproduced with permission from M. Kato-Maeda and P. Small)

2.12. Short-term evolutionary markers and database building

There are also ongoing debates about the true status of “M. prototuberculosis”

(Gutierrez 2005). Whereas some consider “M. prototuberculosis” to be the most

likely common ancestor to all M. tuberculosis complex members (Brisse 2006),

others do not believe in the fact that these smooth variants of the tubercle bacilli are

the true ancestors of today’s tubercle bacilli (Smith 2006b). According to Smith,

the computation providing a 3 million-year time frame is not reliable and there is

no reason to believe that “M. prototuberculosis” is a more likely ancestor to the M.

2.13. Conclusion and Perspectives 81

tuberculosis complex than any animal pathogen still to be characterized. There is

agreement, however, that the gene mosaicism found in “M. prototuberculosis” is

real. Also, it is widely acknowledged that further studies on the genetic diversity of

“M. prototuberculosis” will allow light to be shed on lateral genetic transfer and

homologous recombination events in the M. tuberculosis complex.

Research on the molecular evolution of the M. tuberculosis complex is today ad-

dressed by exploiting multiple markers such as the DR locus, insertion sequences,

deletion regions, mini-satellites, and SNPs, etc. However, in order to data-mine

these large polymorphism databases better, newer methods of data analysis are

needed in order to discover intelligible rules and to eliminate noisy data. Simplified

decision rules are also needed to distinguish emerging pathogenic clones from

those in extinction or from others reflecting ongoing TB transmission. A practical

consequence of such studies would be a simplification of typing methods, which in

turn, would result in a reduction of experimental constraints and an increase in the

number of samples processed. At the Institut Pasteur of Guadeloupe, a new version

of the spoligotyping database is currently incorporating MIRU-VNTR alleles and

will be released for web-based consultation in 2007. In the future, similar websites

will add new markers, allowing the performance of combined searches, including

country of isolation, country of origin and ethnicity of the patient, multiple geno-

typing data, as well as a fine analysis of their geographical distribution. Further

links of such databases to geographic information systems (GIS) for real-time map

construction and clinical expression of the disease might help to shed new light on

a stable association between populations of tubercle bacilli and their human hosts

over time and across environments, as well as providing brand new tools to tackle

the multifactorial nature of the variable clinical expression of the disease.

2.13. Conclusion and Perspectives

The description of the main branches of phylogeographically specific M. tubercu-

losis clonal complexes and the incipient unraveling of the molecular evolution of

the M. tuberculosis complex took very long and there are reasons to believe that the

task has just started. Some of the reasons are to be found within the complexity of

the problem itself. A likely ancient TB pathogen may have had the time to create a

large number of population-adapted genetic variants. Other challenges may lie in

the slow development of efficient methods to characterize the intra-species genetic

diversity of the M. tuberculosis complex. Also, we may invoke the recent introduc-

tion of new concepts, such as statistical phylogeography, whose application to TB

will require the construction of an adequate dataset and even more time for the

82 Molecular Evolution of the Mycobacterium tuberculosis Complex

requisite reconstruction (Knowles 2004). However, the increasing human mobility

worldwide is expected to blur the picture of the history of spread of the M. tuber-

culosis complex.

Lastly, a more precise understanding of the evolutionary genetic network of all M.

tuberculosis complex clonal complexes may also emerge thanks to new studies

using the recently standardized MIRU format (Supply 2006). Figure 2-8 illustrates

the minimum spanning tree approach, built on polymorphisms of 24 MIRUs, found

in a cosmopolitan sample including “M. prototuberculosis” isolates. The dotted

lines represent some doubtful links (for example, the ancestral position of Beijing,

relatively to CAS and EAI is totally speculative since this type of graph does not

represent phylogenetical links).

The longer a clone takes to evolve, the more extensive the observed genetic diver-

sity will be. In view of the assumedly ancient origin of TB, much work remains to

be done to unravel the true genealogy of the numerous clonal complexes of the M.

tuberculosis complex that have been described so far. Many others remain to be

discovered since the sampling is still very small compared to the extent of diversity

that is likely to exist.

Most of the scientific contributions reviewed in this chapter find an echo best

translated by Douglas Young’s concluding remarks in the lecture “Ten years of

research progress and what’s to come” (Young 2003): “Armed with powerful new

molecular tools and renewed momentum, laboratory-based researchers are begin-

ning to tackle the fundamental questions of persistence and pathogenesis of human

TB that have frustrated previous generations. Progress in fundamental under-

standing of disease process poses the exciting challenge of translating new ideas

into practical tools that will assist in the global control of TB”. It is quite satisfying

to see that the research conducted in the last 12 years is clearly advancing towards

a better understanding of the tubercle bacillus and its interaction with the host, the

mechanisms of pathogenicity involved, and the co-evolution of the bacterium and

its host through time and space.

References 83

Figure 2-8: Minimum spanning tree based on MIRU-VNTR relationships among tubercle bacilli.

Circles correspond to the different types identified by the set of 24 loci among the 494 M.

tuberculosis isolates from cosmopolitan origins, and 35 “M. prototuberculosis”. The corre-

sponding species names and spoligotype family designations (except T types) are indicated.

Linkage by a single, double, or triple locus variation is boldfaced. EAI = East-African Indian

(Indo-Oceanic in Gagneux’s 2006 terminology), CAS = Central Asian (East-African-Indian in

Gagneux’s 2006 terminology), Beijing/W (East-Asian in Gagneux’s 2006 terminology) LAM =

Latino-American and Mediterranean, X = European IS6110 low-banders, S = Sicily-Sardinia

clade (all these clades are designated as Euro-American lineages in Gagneux’s 2006 termi-

nology) (Reproduced from Supply 2006 with authorization)

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92 Molecular Evolution of the Mycobacterium tuberculosis Complex

Chapter 3: The Basics of Clinical Bacteriology

Lucía Barrera

3.1. The tubercle bacillus: a continuous taxon

Bacteria of the genus Mycobacterium are non-motile and non-sporulated rods.

They are grouped in the suprageneric rank of actinomycetes that, unusually, have a

high content (61-71 %) of guanine plus cytosine (G+C) in the genomic desoxyribo-

nucleic acid (DNA), and a high lipid content in the wall, probably the highest

among all bacteria. Mycobacterium and other closely related genera (i.e. Coryne-

bacterium, Gordona, Tsukamurella, Nocardia, Rhodococcus and Dietzia) have

similar cell wall compounds and structure, and hence show some phenotypic re-

semblance. Several mycolic acids in the envelope structure distinguish the myco-

bacteria. These quirky lipids may act as carbon and energy reserves. They are also

involved in the structure and function of membranes and membranous organelles

within the cell. Lipids constitute more than half of the dry weight of the mycobac-

teria. However, the lipid composition of the tubercle bacillus may vary during the

life cycle in culture, depending on the availability of nutrients. The waxy coat con-

fers the idiosyncratic characteristics of the genus: acid fastness, extreme hydropho-

bicity, resistance to injury, including that of many antibiotics, and distinctive im-

munological properties. It probably also contributes to the slow growth rate of

some species by restricting the uptake of nutrients.

Even exhibiting this common badge, the species within the genus Mycobacterium

show great diversity in many aspects. Most of them live and replicate freely in

natural ecosystems and seldom, if ever, cause disease. Only a few mycobacteria

became successful pathogens of higher vertebrates, preferentially inhabiting the

intracellular environment of mononuclear phagocytes. The host-dependent myco-

bacteria that cannot replicate in the environment are Mycobacterium leprae, Myco-

bacterium lepraemurium, Mycobacterium avium subsp. Paratuberculosis, and the

members of the Mycobacterium tuberculosis complex. Bacteria within the M. tu-

berculosis complex are able to reproduce in vitro, in contrast to M. leprae and M.

lepraemurium, which are uncultivable and require the intracellular milieu for sur-

vival and propagation.

Comprised within the M. tuberculosis complex and generically called the tubercle

bacillus, the various etiologic agents of tuberculosis (TB) have distinct hosts, zoo-

notic potential and reservoirs. M. tuberculosis, and the regional variants or subtypes

Mycobacterium africanum and “Mycobacterium canettii” are primarily pathogenic

94 The Basics of Clinical Bacteriology

in humans. Mycobacterium bovis and Mycobacterium microti are the causative

agents of TB

in animals, and can be transmitted to humans. Some particular strains

isolated from goats and seals have been named Mycobacterium caprae and Myco-

bacterium pinnipedi, although sometimes they are identified as M. bovis subspecies

or variants. It could be expected that the major evolutive shifts involved in adapta-

tion to different hosts would have entailed significant microbiological differentia-

tion. However, the above mentioned agents of TB together with the vaccine bacille

Calmette-Guérin (BCG) strains rank close to each other along a phenotypically

continuous taxon (David 1978, Wayne 1982, Vincent 1992, van Soolingen 1997,

van Soolingen 1998, Niemann 2000, Niemann 2002, Sola 2003, Mostowy 2005).

Phenotypic differentiation is consistently clear-cut between the extreme species

within the taxon, i.e. M. tuberculosis and M. bovis, but differences between species

comprised within these two extremes are much less defined. The close affiliation

among the members of the complex is endorsed by high genomic DNA similarity.

At the same time, some molecular markers allow species differentiation within the

complex (see chapter 2).

Table 3-1: Lineage of the agents of TB.

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=1760&lvl=3&lin=f

&keep=1&srchmode=1&unlock

Kingdom Bacteria

Phylum Actinobacteria

Class Actinobacteria

Subclass Actinobacteridae

Order Actinomycetales

Suborder Corynebacterineae

Family Mycobacteriaceae

Genus Mycobacterium

unique genus

Species M. tuberculosis

M. bovis

M. africanum

M. microti

"M. canettii”

M. caprae

M. pinnipedii

3.2. Microscopic morphology 95

In general, systematic and clinical mycobacteriologists accept new taxa at a slow

pace. This is why the taxonomic status of some new members of the complex is

still uncertain (see LPSN, http://www.bacterio.cict.fr and DSMZ,

http://www.dsmz.de/microorganisms/bacterial_nomenclature.php). At the same

time, the rank and species assignment have been questioned in other cases (Nie-

mann 2003, Niemann 2004). The value of phenotypic and genotypic traits in the

definition of a species in the complex should be reconsidered to meet new widely

accepted definitions.

3.2. Microscopic morphology

The microscopic appearance does not allow the differentiation of the pathogenic

agents of TB, mainly M. tuberculosis, from other mycobacteria although some

characteristics may be indicative. In smears stained with carbol fuchsin or auramine

and examined under light microscope, the tubercle bacilli typically appear as

straight or slightly curved rods. According to growth conditions and age of the

culture, bacilli may vary in size and shape from short coccobacilli to long rods. A

typical curved shape has been described for M. microti (van Soolingen 1998). The

dimensions of the bacilli have been reported to be 1-10 µm in length (usually 3-

5 µm), and 0.2-0.6 µm in width. Therefore, the length of the microorganism is

comparable to the diameter of the nucleus of a lymphocyte. Unlike some fast

growing mycobacteria and other actinomycetales, M. tuberculosis is rarely pleo-

morphic, it does not elongate into filaments, and does not branch in chains when

observed in clinical specimens or culture. In the experimental macrophage infec-

tion, intracellular bacilli were described as being significantly elongated compared

to broth-grown bacilli and, remarkably, to display bud-like structures (Chauhan

2006).

When numerous and actively multiplying, the bacilli are strongly acid fast and

show an evident and distinctive tendency to form hydrophobic bundles (Figure 3-1

and 3-2). Free bacilli can also be seen, though, especially at the border of the

swarms. In unlysed host tissue, the bacilli are more numerous within the phagocytic

cells.

Once the disease has been controlled, dying bacilli become sparser, often faintly

and unevenly colored, due to partial loss of the internal contents. Of course, ir-

regular staining may also be the consequence of technical defectiveness of dyes or

staining procedures.

96 The Basics of Clinical Bacteriology

Figure 3-1: Ziehl-Neelsen staining of Mycobacterium tuberculosis growing in culture at 1000x

magnification.

Figure 3-2: Electron microscopy of Mycobacterium tuberculosis growing in culture (Courtesy

of M. Rohde -M. Singh).

The light microscope examination can not resolve the internal structures of the

tubercle bacillus with the exception of some intracellular lipid vacuoles appearing

as unstained spherules at regular intervals inside the bacilli (Draper 1982) and de-

posits of lipophilic material that might have a storage function (Garton 2002). De-

spite considerable efforts, a more subtle resolution of the ultrastructure of the ba-

cillus has not been achieved. This is probably due to technical problems arising

from biosafety, from the minute size of the bacilli, and from the large amounts of

3.3. Cell wall structure 97

complex lipids existing in their wall. With electron microscopy, some inner dense

granules can be identified. They are believed to consist of polyphosphate and might

be an energy store in the cell and also the site of oxidation-reduction reactions. In

sections of the cell, the plasma membrane is seen to proliferate into vesicular or

laminated internal bodies that might supply metabolic activities. Ribosomes, DNA

filaments and radial bands, the latter postulated to be remaining scars of cell divi-

sion, have also been described (Draper 1982, Brennan 1994).

Recently, the initiation of septum formation prior to division was clearly evidenced

by tagging the mid-cell rings with green fluorescent protein (Chauhan 2006). Also,

impressive images of the surface of M. bovis BGG were obtained by atomic force

microscopy (Verbelen 2006).

3.3. Cell wall structure

As the most distinctive anatomical feature of the bacillus, the cell envelope has

been the main object of research. Progressive chemical, molecular and ultrastruc-

tural research has produced robust knowledge on the synthetic pathways and

structure of the mycobacterial cell envelope (Draper 1982, Brennan 1994, Draper

2005, Kremer 2005). The envelope, which has been profusely represented by

schematic models, is composed of the plasma membrane, a cell wall, and an

outer capsule like layer.

The cytoplasmic membrane of mycobacteria does not seem to be peculiar except

for the presence of some lipopolysaccharides that are anyway shared by all actino-

mycetales (Mahapatra 2005). This vital interface provides osmotic protection,

regulates the traffic of specific solutes between the cytoplasm and the environment,

and subsumes the cell house-keeping tasks. The membrane contains proteins with

different functions, i.e. sensors measuring the concentration of molecules in the

environment, proteins translocating signals to genetic and metabolic machinery in

the cytoplasm, enzymes involved in metabolic processes and energy generation,

and carriers mediating selective passage of nutrients and ions. The enzymes inter-

vene in cell wall and membrane synthesis, septum formation during cell division,

assembly and secretion of extracytoplasmic proteins, and DNA replication. Still,

very little is known specifically about the membrane of M. tuberculosis.

The membrane is surrounded, as in almost all bacteria, by a cell wall that protects

the cell contents, provides mechanical support and is responsible for the character-

istic shape of the bacterium. The mycobacterial cell wall, however, is unique

among prokaryotes. The wall is constituted by an inner peptidoglycan layer, which

98 The Basics of Clinical Bacteriology

seems to be responsible for the shape-forming property and the structural integrity

of the bacterium. The structure of this stratum differs slightly from that of common

bacteria, as it presents some particular chemical residues and an unusual high num-

ber of cross-links. Indeed, the degree of peptidoglycan cross linking in the cell wall

of M. tuberculosis is 70-80 % whereas that in E. coli is 20-30 %.

Covalently bound to the peptidoglycan is a branched polysaccharide, the arabino-

galactan, whose outer ends are esterified with high molecular weight fatty acids

called mycolic acids. These components are peculiar as the arabinogalactan has

unusual components and linkages and the mycolic acids are typically long and

branched chains containing 60- to 90-carbon atoms. The genera Dietzia, Rhodococ-

cus, Nocardia, Gordona, and Mycobacterium have mycolic acids with increasing

average numbers of carbon atoms. The arrangements of these mycolic acids are

species-specific, a property that allows the identification of many species of myco-

bacteria by gas-liquid, high-performance liquid or thin-layer chromatography (see

chapter 14). The mycolic acids specific to M. tuberculosis are alpha, keto and

methoxymycolates containing 76 to 82, 84 to 89, and 83 to 90 carbons respectively.

The outer layer of the cell wall presents an array of free lipids such as phthiocerol

dimycoserosates (PDIM), phenolic glycolipids (PGL), trehalose-containing glyco-

lipids and sulfolipids (SL). The unusual “M. canettii”, with its smooth colony mor-

phology, has a unique phenolic glycolipid (van Soolingen 1997). M. bovis and M.

bovis BCG produce sizable amounts of a PGL designated as mycoside B, whereas

most M. tuberculosis strains are deficient in this component.

Traversing the whole envelope, some glycolipids such as the phosphatidyl-myo-

inositol mannosides, lipomannan (LM) and lipoarabinomanan (LAM), are anchored

to the plasma membrane and extend to the exterior of the cell wall. LAMs are spe-

cies-specific. The mycobacterial wall also contains interspersed proteins. Some are

in the process of being exported, some might be residents. Several of these proteins

are responsible for cell wall construction during the life of the bacillus. There are

also certain proteins called porins forming hydrophilic channels that permit the

passive passage of aqueous solutes through the mycolic acid layer. Mycobacterial

porins seem to be different from those of gram-negative bacteria.

While growing in a static liquid culture or within a human cell, M. tuberculosis

seems to accumulate an unbound pseudo-capsule. Apparently, when the medium is

disturbed, the capsule separates, leaving the lipophilic surface exposed. In fact, the

capsule components have largely been recognized in culture filtrates but its struc-

ture and location were resolved rather recently. The capsule contains proteins,

polysaccharides and minor amounts of inner lipids, which are apparently in con-

3.3. Cell wall structure 99

stant turnover. The constituents of the capsule might be shed in vivo within the

infected host cells. It has been proposed that the capsule might be protective and

bioactive. In addition, a number of envelope-associated substances have been de-

scribed, mostly lipids and glycolipids.

The tubercle bacillus shares most ultrastructural features with other members of the

genus, including non-pathogenic mycobacteria. Its distinctive ability to survive in

mammalian hosts, its pathogenicity and its immunogenic properties seem to derive,

at least in part, from the nature of some of the molecules of the bacterial wall (Ri-

ley 2006, Smith 2003).

The envelope of the tubercle bacillus seems to be a dynamic structure that can be

remodeled as the microorganism is either growing or persisting in different envi-

ronments (Kremer 2005). In fact, in growth conditions interfering with the synthe-

sis of the wall, M. tuberculosis may be induced to produce wall-deficient sphero-

plasts that apparently are not pathogenic unless they revert to being normal bacteria

(Ratnan 1976). Cell wall thickening was observed in oxygen-deficient conditions

(Cunningham 1998). Besides, the expression of genes that putatively code for

porins seems to be up regulated in certain environmental conditions, such as mildly

acidified culture medium, as well as inside the macrophage vacuoles (Draper

2005).

3.3.1. Acid fastness

Unlike Gram-negative bacteria, mycobacteria do not have an additional membrane

in the outer layers of the cell wall. They are structurally more closely related to

Gram-positive bacteria. However, mycobacteria do not fit into the Gram-positive

category as the molecules attached to the cell wall are distinctively lipids rather

than proteins or polysaccharides. Frequently, they do not retain the crystal violet

and appear as “ghosts” after Gram staining. The waxy cell wall of mycobacteria is

impermeable to aniline and other commonly used dyes unless these are combined

with phenol.

To discover the causative agent of TB, Robert Koch had to develop a specific

staining process using alkaline dyes. Soon after, Ehrlich discovered the acid fast-

ness of the tubercle bacillus, which has been the prominent characteristic of myco-

bacteria up until now. The expression “acid-fastness” describes the resistance of

certain microorganisms to decolorization with acid-alcohol solutions after staining

with arylmethane dyes such as carbol fuchsin. This feature is of utmost practical

100 The Basics of Clinical Bacteriology

importance in identifying the tubercle bacillus, particularly in pathological speci-

mens.

In spite of being a hallmark, the wall permeability to alkaline dyes and the mecha-

nisms preventing their removal by acids are still not totally understood in molecular

terms. Most of the current knowledge on this phenomenon was disclosed in pioneer

experiments. The beading observed inside the cells was interpreted as accumulation

of free dye rather than staining of particular structures, which led to the early hy-

pothesis that alkaline stains are retained in the cytoplasm (Yegian 1947). Later,

evidence was provided sustaining the role of lipids in trapping the dyes. Indeed,

there is a parallelism between the increasing degree of acid fastness displayed by

microorganisms in the genera Corynebacterium, Nocardia, and Mycobacterium,

and the increasing length of mycolic acid chains in their walls. This correspon-

dence suggests that the chemical binding of the dye to these molecules might be a

determinant for acid fastness.

Bacilli suspended in aqueous solution retain the acid fastness for a long time, even

after heating. However, the property is absolutely dependent on the integrity of the

bacillus. Unimpaired mycolic acids are required to hinder the penetration of water-

soluble dyes and bleaching acids (Goren 1978). The acid fastness of the bacillus is

obliterated by cell trauma or autolysis (Baisden 1942), infection by specific myco-

bacteriophages (Gangadharam 1976) or treatment with antibiotics targeting cell-

wall synthesis, such as isoniazid (INH) (Mohamad 2004). Acid fastness seems to

also be dependent on nutrients and oxygen tension, as suggested by fluctuations in

staining observed in different culture conditions (Nyka 1971). Dormant M. tuber-

culosis bacteria bearing cell wall alterations may remain undetected by the classic

Ziehl-Neelsen staining (Seiler 2003).

3.3.2. Cord formation

By microscopic observation, Robert Koch first described the arrangement of bacilli

in braided bunches and associated this phenomenon with virulent strains of M.

tuberculosis. He also detailed the aspect of cultures in blood serum as compact

scales which could be easily detached. In general, fresh virulent M. tuberculosis

bacilli produce rough textured colonies on solid media, expanded gummy veils on

the surface of liquid media and serpentines on microscopic smears. In contrast,

non-virulent mycobacteria and tubercle bacilli attenuated by prolonged cultures

usually develop smooth colonies on solid media, form discrete mats in liquid media

and distribute randomly in loose aggregates when smeared. The recognition of

these two peculiarities, cording and crumbly colony formation, provides a reliable

3.3. Cell wall structure 101

and timely clue to the experienced microbiologist for the presumptive distinction of

M. tuberculosis from other mycobacteria in cultured specimens and even in sputum

smears (see chapter 12).

These distinctive characteristics of the virulent bacilli have been attributed to the

trehalose 6, 6’-dimycolate. This compound, also known as cord factor, was de-

scribed as an extractable glycolipid consisting of two mycolic acid molecules

loosely bound in the outer layer of the cell wall (Noll 1956). A myriad of biological

activities related to pathogenicity, toxicity, and protection against the host response

have been attributed to this molecule. However, it does not seem to be essential for

bacterial multiplication in vitro (Indrigo 2002).

Several models were used to identify the role of the trehalose 6, 6’-dimycolate

(TDM) in the microscopic and macroscopic morphology of M. tuberculosis. In this

way it was demonstrated that beads coated with this substance generate an oriented

hydrophobic interaction and aggregate in elongated structures similar to cords

(Behling 1993). Later, the molecular packing of TDM was imitated (Almond

1996). Recently, immunohistochemistry was used to investigate the distribution of

TDM in M. tuberculosis culture pellicles. According to the results of this experi-

ment it was proposed that the TDM released by the microorganism molds a rigid

hydrophobic interphase that is responsible for the cultural and microscopic appear-

ance of virulent bacilli (Hunter 2006).

However, this phenomenon is not yet clearly understood. One matter of confusion

is the fact that TDM is also present in other non-cording avirulent mycobacteria.

Taking this into consideration, the activity of the cord factor in M. tuberculosis has

been ascribed to a particular surface conformation (Schabbing 1994) and to the

large amounts of this molecule released by the tubercle bacilli (Hunter 2006). The

localization of DNA sequences encoding cording has not yet been elucidated. Five

genes probably associated with cord formation were identified, but their real impli-

cation has not been demonstrated (Gao 2004).

So far, the characteristics of the TDM of “M. canettii”, a human pathogen that

produces unusually smooth colonies, have not been described.

3.3.3. Permeability barriers

The tightly packed mycolic acids provide the bacillus with an efficient protection

and an exceptional impermeability. In addition to the capsule, an even thicker layer

of carbohydrate and protein outside the lipid layer impedes the diffusion of large

molecules, such as enzymes, and protects the lipid layer itself. The shell restricts

102 The Basics of Clinical Bacteriology

the permeability to most lipophilic molecules. Other substances can bypass this

barrier through the porins, although this mechanism is not very efficient: M. tuber-

culosis possesses a low number of porins compared to other bacteria and the porins

admit only small water soluble molecules (Niederweis 2003).

Several experiments have been performed that have provided the rationale for the

long believed concept that impermeability is at least one of the determinants for

two M. tuberculosis characteristics: its slow growth and its intrinsic drug resis-

tance. The penetration rate of β-lactam antibiotics into M. tuberculosis was found

to be comparable to that of Pseudomonas aeruginosa and approximately 100 times

lower than that of Escherichia coli (Chambers 1995). In recombination experi-

ments, the expression of the M. smegmatis porin MspA was followed by increased

susceptibility of the tubercle bacillus to β-lactam antibiotics and even to first-line

anti-tuberculous drugs. At the same time, the expression of the same porin in M.

bovis BCG stimulated the uptake of glucose and accelerated growth (Mailaender

2004).

Treatment with some drugs that are known to fray or somehow alter the surface

architecture of the cells was shown to increase the susceptibility of M. tuberculosis

(Verbelen 2006). In effect, at sub-inhibitory concentrations, ethambutol and di-

methyl sulfoxide enhanced the activity of anti-tuberculosis drugs against M. tuber-

culosis strains that were originally resistant to these drugs (Jagannath 1995). Simi-

larly, some antidepressants, such as chlorpromazine, have in vitro activity them-

selves against the tubercle bacillus (Ordway 2003).

3.4. Nutritional and environmental requirements for growth

The tubercle bacillus is prototrophic (i.e. it can build all its components from basic

carbon and nitrogen sources) and heterotrophic (i.e. it uses already synthesized

organic compounds as a source of carbon and energy). The microorganism macro-

molecular structure and physiological (metabolic) capabilities result in high adap-

tation to the specific environment. In turn, the nutritional quality of the environ-

ment determines the bacillus lifestyle and limitations, either in the natural habitat or

in culture media, as do various physical conditions such as oxygen availability,

temperature, pH and salinity.

As the environment changes, the bacillus is able to bring into play different

physiological pathways in order to survive even in harsh conditions. This is a

highly resourceful strategy, not only for pathogenicity but also for species persis-

tence. It has been shown that, during the course of infection in mice, M. tuberculo-

3.4. Nutritional and environmental requirements for growth 103

sis metabolism may shift from an aerobic, carbohydrate-metabolizing mode to one

that is more microaerophilic and utilizes lipids (Segal 1956). These demonstrations,

which were reported a long time ago, were supported in recent times by the com-

plete sequencing of the M. tuberculosis genome in which an unusually high number

of genes putatively involved in fatty acid metabolism were identified. This phe-

nomenon may be related to the ability of the pathogen to grow or persist in host

tissues where fatty acids may be the major carbon source (Neyrolles 2006) (see

chapter 4).

In vitro, the members of the M. tuberculosis complex are not fastidious unless

damaged by some noxious agents. In fact, the medium used by Koch to cultivate

M. tuberculosis was simply sterile coagulated blood serum. The tubercle bacilli can

also grow in salt solutions using glycerol as a carbon source, ammonium ions and

asparagine as nitrogen sources, and micronutrients. M. tuberculosis is able to me-

tabolize glycerol into pyruvate, whereas M. bovis is not. Indeed, the genome se-

quence analysis confirmed that all the genes required for the formation of pyruvate

are non-functional in M. bovis. Being defective in this metabolic process, M. bovis

grows much better in the presence of a pyruvate salt as a source of carbon. Albu-

min, which is normally provided by adding eggs or bovine serum albumin to the

culture media, promotes the growth of these microorganisms. Other subsidiary

media components may be used, such as Tween 80, a detergent that disperses the

bacilli in liquid media. It was postulated that bovine serum albumin may bind the

excess of oleate that can be released from the detergent up to toxic amounts. Biotin

and catalase have been incorporated to the Middlebrook series media to stimulate

the revival of damaged bacilli in clinical specimens (Wayne 1982).

Trace elements found by the microorganism in the water, inorganic ions, small

molecules, and macromolecules have either a structural or a functional role in the

cell. Magnesium and iron are essential for life. A deficiency in these elements fre-

quently reduces the virulence of bacterial pathogens, including the tubercle bacil-

lus. As iron is usually in the form of insoluble ferric salts in the environment, spe-

cial iron systems are required to incorporate this element into the cell. Exochelins

and mycobactins are the major siderophores used by mycobacteria to perform this

function. The former are hydrophilic peptides secreted into the environment for

iron gathering. The latter are hydrophobic compounds located within the cell wall

to introduce the iron into the cytoplasm. The mbt operon is putatively involved in

the synthase activities required to produce the mycobactin core (De Voss 2000).

The incorporation of mycobactin into culture media can promote the growth of

ailing M. tuberculosis isolates.

104 The Basics of Clinical Bacteriology

a b c d

Figure 3-3: Mycobacteria growing on Löwenstein-Jensen slants. a. Mycobacterium gordonae;

b. Mycobacterium fortuitum; c. Mycobacterium avium; d. Mycobacterium tuberculosis.

The tubercle bacillus requires oxygen as a final electron acceptor in aerobic respi-

ration. Molecular oxygen is reduced to water in the last step of the electron trans-

port system. In nature, the bacillus grows most successfully in tissues with high

oxygen partial tension, such as the lungs, particularly the well-aerated upper lobes.

Carbon dioxide is essential and may be taken from the atmosphere and also from

carbonates or bicarbonates. In the laboratory, an atmosphere of 5 to 10 % carbon

dioxide favors culture growth, at least during the early stage of incubation. On the

other hand, M. bovis is microaerophilic, i.e. it grows preferentially at a reduced

oxygen tension.

M. tuberculosis is mesophile and neutrophile as its multiplication is restricted to

conditions offered by warm-blooded animals: about 37ºC and a neutral pH. The

temperature and hydrogen ion concentration ranges, in which the bacillus is able to

multiply, are relatively narrow. High saline concentration such as that found in

media containing 5 % sodium chloride, inhibits the growth of the microorganism.

3.5. Generation time 105

3.5. Generation time

Under favorable laboratory conditions, M. tuberculosis divides every 12 to 24

hours. This pace is extremely slow compared to that of most cultivable bacteria,

which duplicate at regular intervals ranging from about 15 minutes to one hour.

Recently, the low multiplication rate of the tubercle bacillus was nicely exposed by

Chauhan et al. These authors demonstrated the small proportion of cells initiating

the septation process prior to division among tubercle bacilli growing either in

broth or inside macrophages (Chauhan 2006).

The slow growth rate might be partially determined by the cell wall impermeability

that limits nutrient uptake. However, only a minimal stimulus to bacterial multipli-

cation is achieved when the permeability is increased through treatment with some

compounds that interact with the cell envelope. Harshey and Ramakrishnan identi-

fied ribonucleic acid (RNA) synthesis to be a major factor associated with the long

generation time of the tubercle bacillus. They demonstrated that both the ratio of

RNA to DNA and the RNA chain elongation rate are ten-fold lower in M. tuber-

culosis compared to E. coli (Harshey 1977). Another unusual feature is the exis-

tence of a unique operon commanding RNA synthesis. Furthermore, when the

tubercle bacillus switches from the stationary to the active multiplying phase, its

total RNA content increases only twofold. Consequently, the protein synthesis must

be retarded (Verma 1999). The influence of nutrient availability on the ribosome

synthesis rate, which is a proxy of metabolic activity, remains controversial

(Hampshire 2004).

The low multiplication rate explains the typically sub-acute to chronic evolution of

the disease and the long time required to attain visible growth in vitro. Numerous

experiences using different nutrients and culture conditions have demonstrated that

some factors may abrogate a lag in adaptation of the bacilli in culture media but,

once growth is initiated, the replication cycle will still take no less than 12 hours.

This limitation in accelerating the tubercle bacillus growth could not be overcome.

Instead, the main achievements for diagnosis have been made through the use of

tools that enable the detection of a minimal quantity of bacilli in the media. First,

transparent agar medium allowing the detection of tiny colonies were introduced;

more recently, the addition of biosensors has been adopted to detect redox changes

produced by the bacilli metabolism (see chapters 12 and 14).

106 The Basics of Clinical Bacteriology

3.6. Metabolic and biochemical markers

In the laboratory, the classical phenotypic identification, speciation and subspecia-

tion of members of the M. tuberculosis complex include key diagnostic tests devel-

oped to detect certain metabolic intermediates and the activity of some enzymes

that are essential for life and pathogenicity. In addition to some susceptibility tests,

the investigation of niacin accumulation, nitrate reductase and urease activity al-

lows the distinction of M. tuberculosis complex and species differentiation within

the complex (see chapter 8). Most of the information on the structure and function

of these metabolites and enzymes has focused on M. tuberculosis and, to some

extent, on M. bovis. Much less is known about these features in other members of

the M. tuberculosis complex.

Niacin (nicotinic acid) plays a vital role in organic life, as it is involved in the oxi-

dation-reduction reactions of energy metabolism and in the DNA repair processes.

Although all mycobacteria produce niacin, most of them employ the majority of the

yielded metabolite in the synthesis of co-enzymes. In contrast, M. tuberculosis

produces and accumulates substantial amounts of niacin as a result of a very active

nicotinamide adenine dinucleotide degradation pathway and the inability to process

the resultant niacin (Kasarov 1972). In vitro, M. tuberculosis, “M. canettii”, and

some isolates of M. africanum excrete water-soluble niacin into the culture media,

the detection of which is extremely useful for definitive identification. This is an-

other hallmark that has not been investigated in molecular terms. Again, most of

the knowledge existing on this phenomenon and the tools for its detection were

produced a long time ago by bacteriological and chemical studies.

Like many aerobes, including other mycobacteria, the tubercle bacillus depends

upon certain enzymes to detoxify lethal oxygen radicals, such as peroxides and

which are

self-generated during respiration or produced by host phagocytes.

The main M. tuberculosis antioxidant enzyme that can hydrolyze H

is a heat-

labile catalase-peroxidase with both catalase and peroxidase activities. The thermal

lability of this enzyme is a marker of the M. tuberculosis complex. M. tuberculosis

also has an alternative alkyl-hydroperoxidase, which is postulated to compensate

for the lack of catalase activity. Paradoxically, the catalase is not only self-

protective but can also be self-destructive as it activates the anti-tuberculous pro-

drug INH. Mutations in the genes encoding both enzymes (katG and ahpC) are

involved in resistance to INH and thus, have been the subject of active investiga-

tion (see chapter 18). Understandably, resistance to INH may be associated with

irregular catalase activity. Among the biochemical markers commonly investigated

for mycobacteria identification in the clinical microbiological laboratory, this is the

only one that may be affected by drug resistance to some extent.

3.7. Resistance to physical and chemical challenges 107

Even though M. tuberculosis prefers ammonium and asparagine, it can deficiently

utilize nitrate and nitrite as sole sources of nitrogen in vitro. It has been speculated

that, in infected hosts, the microorganism might use nitrate as a nitrogen source

and/or as a terminal electron acceptor in the absence of oxygen. Whatever the

physiological function may be, M. tuberculosis has an enzyme bound to the cell

membrane that rapidly reduces nitrate and leads to the accumulation of nitrite.

Unlike those of other mycobacteria, M. tuberculosis nitrate reductase is perma-

nently very active in vitro regardless of the culture conditions. Under hypoxic con-

ditions or on exposure to nitric oxide, its activity may even be enhanced by induc-

tion of the protein NarK2. This protein is a nitrate transporter that might be able to

sense the redox state of the cell and adjust its own activity accordingly (Sohaskey

2005). The reductase activity may be hindered by very high concentrations of INH.

Furthermore, some isolates of the tubercle bacillus that are resistant to INH and

para-aminosalicylic acid (PAS) were found to be unable to reduce nitrate when

growing in minimal media (Hedgecock, 1962). The nitrate reductase activity seems

to be encoded by the constitutive narGHJI operon (Weber 2000), which is present

in both M. tuberculosis and M. bovis. However, M. bovis does not reduce nitrate. It

was demonstrated that a single nucleotide polymorphism at position 215 in the

promoter of this gene cluster determines different levels of enzyme activity in both

species (Sohaskey 2003). “M. canettii” and some isolates of M. africanum produce

detectable amounts of nitrite from nitrate in vitro.

M. tuberculosis is able to produce ammonia from urea by a urease-mediated reac-

tion. The ammonium can be then used by the microorganism for biosynthesis. The

urease is coded by the genes ureABC (Reyrat 1995) and it might also be important

for nitrogen acquisition as its activity increases when nitrogen sources are limited

(Clemens 1995). In addition, the consequent alkalinization of the microenviron-

ment by ammonium ions might inhibit the maturation of phagolysosomes and con-

tribute to the defective maturation of major histocompatibility complex class II

molecules of host monocytes (Sendide 2004).

3.7. Resistance to physical and chemical challenges

Although the tubercle bacillus is not a spore-forming bacterium, it has a remarkable

capacity to endure unfavorable conditions. The bacillus is able to circumvent de-

struction within the macrophages and to limit the access to the bacterial targets of

hydrophilic antiseptics and antibiotics (see Chapters 5, 11, and 18). For example,

chloride and bromide salts of cetylpyridium do not impair the viability of the tuber-

cle bacilli for at least 14 days (Tazir 1979, Pardini 2005). Therefore, these salts are

108 The Basics of Clinical Bacteriology

used as preservatives when the processing of specimens is delayed. Likewise, the

natural impermeability of the bacterium to common hydrophilic antimicrobial

agents is used in the clinical mycobacteriology laboratory. In effect, some broad

spectrum antibiotics are added to selective media to isolate the tubercle bacillus.

As already mentioned, M. tuberculosis complex organisms multiply within narrow

temperature and pH ranges, and at a high oxygen tension, which is indicative of the

effect produced by these physical conditions on the rates of enzymatic reactions.

However, the tubercle bacilli can withstand conditions far distant from those opti-

mal for propagation. The bacillus survives to some extent in the acid or alkaline

microenvironment as a result of its interaction with the defensive mechanism of the

host, as well as the acid contents of the stomach. Similarly, a significant proportion

of the bacilli population present in clinical specimens can endure a brief treatment

with diluted solutions of acids and alkalis such as sulfuric acid or sodium hydrox-

ide. This property is peculiar as most microflora present in the specimens are killed

by this treatment; thus, it is exploited to isolate mycobacteria (see chapter 12). The

stress generated by a low pH is more severe in a nutrient-limited environment.

High levels of magnesium are required for growth in mildly acidic media (Cotter

2003).

The microorganism also withstands very low temperatures. Its viability may be

increasingly preserved for a long term between 2-4°C to -70°C. When ultrafrozen,

the viability of the bacilli remains almost intact as well as the taxonomic, serologic,

immunologic, and pathogenic properties. After thawing, they may require re-

adaptation to recover full metabolic activity (Kim 1979). On the other hand, the

bacilli are very sensitive to heat, sunlight and ultraviolet (UV) irradiation. In spu-

tum or in aqueous suspension, they progressively lose viability between 30 and

37°C within one week. Exposed to direct UV irradiation, moderate loads of tuber-

cle bacilli die in a few minutes (Huber 1970, Collins 1971).

In addition, M. tuberculosis tolerates low oxygen tension as demonstrated in un-

disturbed liquid culture media where the self-generated microaerophilic sediment

contains non-dividing, yet viable, bacilli. The bacilli may survive for many years in

this condition but need a minimal concentration of oxygen to induce the switch into

a fermentative metabolism (Wayne 1982, Wayne 1984). Adaptation to microaero-

philic conditions was further substantiated when it was found that, unlike aerobi-

cally-cultured bacilli, those persisting at low oxygen tension were susceptible to

metronidazole, a drug that is known to be effective against anaerobic bacteria.

Using transmission electron microscopy, Cunningham and Spreadbury demon-

strated that the cell wall of the microorganism thickens notoriously in microaerobic

and anaerobic cultures, which might be a strategy to endure oxygen depletion

References 109

(Cunningham 1998). Under these conditions, a highly expressed and ubiquitous

16 kilo Dalton protein was identified. This heat-shock protein might play a role in

stabilizing the cell structures for long-term survival in the dormant state.

The tight structure of the cell wall of the tubercle bacillus is undoubtedly the shield

that preserves the posistion and function of the metabolic and replicating machin-

ery, even when inactive. At the same time, a succession of physiological mecha-

nisms, which are still poorly understood, are ready to shift this machinery towards

dormancy whenever necessary. This seems to be the main adaptive response of the

bacilli to almost all sub-optimal or even harsh conditions, in vitro, ex vivo, and in

vivo (see chapter 5).

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113

Chapter 4: Genomics and Proteomics

Patricia Del Portillo, Alejandro Reyes, Leiria Salazar, María del Carmen

Menéndez and María Jesús García

4.1. Impact of new technologies on Mycobacterium tuberculo-

sis genomics

A new wave in the analysis of the physiological secrets of microorganisms started

more than a decade ago with the reading of the first complete genome sequence,

corresponding to the bacterium Haemophilus influenzae (Fleishman 1995). Nowa-

days, the accessibility to hundreds of bacterial genome sequences has changed our

way of studying the bacterial world, including bacterial pathogens such as M. tu-

berculosis.

The overwhelming information displayed by genome sequences started the era of

“omics” technologies. These technologies are in accordance to the currently fast

times. A quick search in PubMed, limiting results to the last 10 years, showed more

than 27,000 papers devoted to “omics” issues: more than three thousand concerning

bacteria, and almost three hundred concerning Mycobacterium tuberculosis. Up to

five different “omics” methodologies have been described so far, all concerning the

global study of the target organism, analyzing all its genes, transcriptional products,

proteins, etc.

• Genomics involves the study of all genes that are present in the genomes

• Transcriptomics concerns the analysis of the cellular functions at the mes-

senger ribonucleic acid (mRNA) level

• Proteomics refers to the detection and identification of all proteins in a cell

• Metabolomics comprises the complete set of all metabolites formed by the

cell and its association with its metabolism

• Fluxomics compares the cellular networks (Fiehn 2003, Nielsen 2005)

In the tuberculosis (TB) field, only papers concerning genomics, transcriptomics,

and proteomics have been published. Integration of data derived from the several

“omics” by bioinformatics will probably allow a rational insight into M. tuberculo-

sis biology and its interactions with the host, leading to true control of the disease.

Undoubtedly, the biggest step in our knowledge on TB during the last decade was

the description of the complete genome sequence of the laboratory reference M.

114 Genomics and Proteomics

tuberculosis strain H37Rv (Cole 1998a). For example, the identification of genes

involved in the bacterial cell wall biosynthesis, the routes for lipid metabolism, the

location of insertion sequences and the variability in the PE_PPE genes allowed

scientists to merge the fragments of knowledge derived from the pre-genomic era

in a more comprehensive way. The sequence of the genome, and its comparison to

sequences of other microorganisms reported in several databases, allowed the as-

signation of precise functions to 40 % of the predicted proteins and the identifica-

tion of 44 % of orthologues (genes with very similar functions in a different spe-

cies), leaving 16 % as unique unknown proteins.

The elucidation of complete genome sequences and the development of microar-

ray-based comparative genomics have been powerful tools in the progress of new

areas by the application of robotics to basic molecular biology. Comparative ge-

nomics and genomic tools have also been used to identify factors associated with

the pathogenicity of M. tuberculosis, such as virulence factors and genes involved

in persistence of the pathogen in host cells. Moreover, these tools allowed a de-

scription of the evolutionary scenario of the genus (see Chapter 2).

Structural genomics was the starting point. As more accurate technologies became

available, the interest was focused into functional genomics. Thus, information on

specific mRNA actively synthesized by bacteria inside macrophages or during in

vitro starvation, opened ways to the analysis of gene expression. Microarray tech-

nology was applied to the detection of global gene activity in M. tuberculosis under

several environmental conditions. However, bacterial function cannot be under-

stood by looking at the mRNA level alone. A major barrier for genomic studies has

been the great number of genes with unknown function that have been identified.

Up to 60 % of the open reading frames (ORFs) had unknown functions after the

initial annotation of genes (identification of the protein unrevealed by the corre-

sponding ORF’s amino acid sequence) (Cole 1998a). The elucidation of protein

function was possible with the global analysis of bacterial proteins, giving insights

into the functional role of several so far unknown proteins. Thanks to the joint

contributions of biochemical techniques and mass spectrometry, up to 1,044 non-

redundant proteins were reported in different cellular fractions (Mawuenyega

2005). The upcoming task will be to assign them all a functional role. As more

results are obtained from the proteomic analysis, it is expected that the function of

more ORFs will be unveiled with the aid of new data on transcriptomics and pro-

teomics.

Genomics and other molecular tools allowed studies on gene expression and regu-

lation, which were unthinkable years ago. M. tuberculosis is a restricted human

pathogen; therefore it must have developed mechanisms enabling its quick and

4.2. M. tuberculosis genome 115

efficient adaptation to a variety of “intra-human” environments, which are, in fact,

its natural habitat. Understanding how the bacillus regulates its different genes

according to environmental changes will probably lead to the comprehension of

many interesting aspects of M. tuberculosis, including latency and host-adaptation.

This chapter will address the general basics, as well as the state-of-the-art ge-

nomics, transcriptomics and proteomics in relation to M. tuberculosis. Finally, a

general overview will be made on lipids, the most peculiar metabolites of this bac-

terium.

4.2. M. tuberculosis genome

4.2.1. Genomic organization and genes

TB research made huge progress with the availability of the genome sequence of

the type strain M. tuberculosis H37Rv (Cole 1998a). Expectations were generated

on the elucidation of some unique characteristics of the biology of the tubercle

bacillus, such as its characteristic slow growth, the nature of its complex cell wall,

certain genes related to its virulence and persistence, and the apparent stability of

its genome. This first available genome sequence of a pathogenic M. tuberculosis

strain helped to answer some of these questions and, what is even more stimulating,

to open many more. We describe herein the main characteristics of the M. tuber-

culosis genome sequences completed thus far and highlight some of the most inter-

esting questions answered and opened with this advance in TB research.

M. tuberculosis H37Rv (Cole 1998a) was revealed to possess a sequence of

4,411,529 bp, the second largest microbial genome sequenced at that time. The

characteristically high guanine plus cytosine (G+C content; 65.5 %) was found to

be uniform along most of the genome, confirming the hypothesis that horizontal

gene transfer events are virtually absent in modern M. tuberculosis (Sreevatsan

1997). Only a few regions showed a skew in this G+C content. A conspicuous

group of genes with a very high G+C content (> 80 %) appear to be unique in my-

cobacteria and belong to the family of PE or PPE proteins. In turn, the few genes

with particularly low (< 50 %) G+C content are those coding for transmembrane

proteins or polyketide synthases. This deviation to low G+C content is believed to

be a consequence of the required hydrophobic amino acids, essential in any trans-

membrane domain, that are coded by low G+C content codons.

Fifty genes were found to code for functional RNAs. As previously described

(Kempsell 1992), there was only one ribosomal RNA operon (rrn). This operon

was found to be located at 1.5 Mbp from the origin of replication (oriC locus).

116 Genomics and Proteomics

Most eubacteria have more than one rrn operon located much closer to the oriC

locus to exploit the gene-dosage effect during replication (Cole 1994). The posses-

sion of a single rrn operon in a position relatively distant from oriC has been pos-

tulated to be a factor contributing to the slow growth phenotype of the tubercle

bacillus (Brosch 2000a).

One of the most thoroughly studied characteristic of M. tuberculosis is the presence

and distribution of insertion sequences (IS). Of particular interest is IS6110, a se-

quence of the IS3 family that has been widely used for strain typing and molecular

epidemiology due to its variation in insertion site and copy number (van Embden

1993, see Chapter 9). Sixteen copies of IS6110 were identified in the genome of M.

tuberculosis H37Rv; some IS6110 insertion sites were clustered in sites named

insertional hot-spots. The same strain was found to harbor six copies of the more

stable IS1081, an insertion sequence that yields almost identical profiles in most

strains when analyzed by Restriction Fragment Length Polymorphism (RFLP)

(Sola 2001, Kanduma 2003). Another 32 different insertion sequences were found,

of which seven belonged to the 13E12 family of repetitive sequences; the other

insertion sequences had not been described in other organisms (Cole 1998b). Virtu-

ally all the ISs found in M. tuberculosis so far belong to previously described IS

families (Chandler 2002). The only exception is IS1556, which does not fit into any

known IS family (Cole 1999).

Two prophages were detected in the genome sequence; both are similar in length

and also similarly organized. One is the prophage PhiRv1, which in the M. tuber-

culosis H37Rv genome interrupts a repetitive sequence of the family 13E12. This

prophage is deleted or rearranged in other M. tuberculosis strains (Fleischmann

2002). The genome of M. tuberculosis possesses seven potential att sites for

PhiRv1 insertion, which explains the variability of its position between strains

(Cole 1999). The second prophage, PhiRv2 has proven to be much more stable,

with less variability among strains (Cole 1999).

Regarding protein coding genes, it was determined that M. tuberculosis H37Rv

codes for 3,924 ORFs accounting for 91 % of the coding capacity of the genome

(Cole 1998a). The alternative initiation codon GTG is used in 35 % of cases com-

pared to 14 % or 9 % in Bacillus subtilis or Escherichia coli respectively. This

contributes to the high G+C bias in the codon usage of mycobacteria.

A bias in the overall orientation of genes with respect to the direction of replication

was also found. On average, bacteria such as B. subtilis have 75 % of their genes in

the same orientation as that of the replication fork, while M. tuberculosis only has

59 %. This finding has led to the hypothesis that such a bias could also be part of

4.2. M. tuberculosis genome 117

the slow growing phenotype of the tubercle bacillus (Cole 1999). This conjecture,

however, does not take into account the fact that E. coli, a bacterium that grows

much faster than M. tuberculosis, has only 55 % of its genes in the same direction

as the replication origin (Li 2005).

From the predicted ORFs, all proteins have been classified in 11 broad functional

groups (Table 4-1), more precisely classified into COG functional categories

(http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=135) according to the National

Center for Biotechnology Information (NCBI) of the United States (US). The

analysis of the codon usage showed a preference for G+C-rich codons. It was also

found that the number of genes that arose by duplication is similar to the number

seen in E. coli or B. subtilis, but the degree of conservation of duplicated genes is

higher in M. tuberculosis. The lack of divergence of duplicated genes is consistent

with the hypothesis of a recent evolutionary descent or a recent bottleneck in my-

cobacterial evolution (Brosch 2002, Sreevatsan 1997, see chapter 2).

From the genome sequence it is clear that M. tuberculosis has the potential to

switch from one metabolic route to another including aerobic (e.g. oxidative phos-

phorylation) and anaerobic respiration (e.g. nitrate reduction). This flexibility is

useful for survival in the changing environments within the human host that range

from high oxygen tension in the lung alveolus to microaerophilic/anaerobic condi-

tions within the tuberculous granuloma. Another characteristic of the M. tuberculo-

sis genome is the presence of genes for synthesis and degradation of almost all

kinds of lipids from simple fatty acids to complex molecules such as mycolic acids.

In total, there are genes encoding for 250 distinct enzymes involved in fatty acid

metabolism, compared to only 50 in the genome of E. coli (Cole 1999).

Concerning transcriptional regulation, M. tuberculosis codifies for 13 putative

sigma factors and more than 100 regulatory proteins (see section 4.3 of this chap-

ter).

Among the most interesting protein gene families found in M. tuberculosis are the

PE and PPE multigene families, which account for almost 10 % of the genome

capacity. The names PE and PPE derive from the motifs of Pro-Glu (PE) and Pro-

Pro-Glu (PPE) found near the protein N-terminus in most cases. These proteins are

believed to play an important role in survival and multiplication of mycobacteria in

different environments (Marri 2006). There are about 100 members of the PE fam-

ily, which is further divided into three sub-families, the most important of which is

the polymorphic GC-rich sequences (PGRS) class that contains 61 members. Pro-

teins in this class contain multiple tandem repetitions of the motif Gly-Gly-Ala,

hence, their glycine concentration is superior to 50 %. The PE_PGRS proteins

118 Genomics and Proteomics

have been found to be exclusive to the M. tuberculosis complex (Marri 2006) and

resemble the Epstein-Barr virus nuclear antigens (EBNA), which are known to

inhibit antigen presentation through the histocompatibility complex (MHC) class I

(Cole 1999).

Interestingly, the analysis of the desoxyribonucleic acid (DNA) metabolic system

of M. tuberculosis indicates a very efficient DNA repair system, in other words,

replication machinery of exceptionally high fidelity. The genome of M. tuberculo-

sis lacks the MutS-based mismatch repair system. However, this absence is over-

come by the presence of nearly 45 genes related to DNA repair mechanisms

(Mizrahi 1998), including three copies of the mutT gene. This gene encodes the

enzyme in charge of removing oxidized guanines whose incorporation during repli-

cation causes base-pair mismatching (Mizrahi 1998, Cole 1999).

With the aim of making the information publicly available and the search and

analysis of information easier, the Pasteur Institute

(http://www.pasteur.fr/recherche/unites/Lgmb/) has created a database system in-

corporating not only all genes and annotation but other search tools such as Blast or

FastA, that allow the user to search for homologue sequences of a query sequence

inside the M. tuberculosis genome. This database is freely available for use on the

Internet and is known as the Tuberculist Web Server

http://genolist.pasteur.fr/TubercuList/).

As more information was generated, databases grew bigger, more experimental

information became available, and better and more accurate algorithms for gene

identification and prediction were released. The initial genome annotation in M.

tuberculosis H37Rv strain soon became out of date. For this reason, a re-annotation

of that genome sequence was published in 2002. This re-annotation incorporated 82

additional genes. The gene nomenclature was not altered; the new genes have the

name of the preceding gene followed by A, B or D, for example, two new ORFs

were described between Rv3724 and Rv3725, hence, they were named Rv3724A

and Rv3724B. The letter C was not included since it usually stands for “comple-

mentary”, which means that the gene is located in the complementary strand. As

expected, the classes that exhibited the greatest numbers of changes were the un-

known category and the conserved hypothetical category (Table 4-1). The re-

annotation of the genome sequence allowed the identification of four sequencing

errors making the current sequence size change from 4,411,529 to 4,411,532 bp

(Camus 2002).

4.2. M. tuberculosis genome 119

As shown in Table 4-1, the information obtained from a single sequenced genome

is enormous. The advances made on the analysis of such information have acceler-

ated TB research.

Table 4-1: Functional classification of M. tuberculosis H37Rv and re-annotation*

Class Function

Number of

genes

(1998)

Number of

genes

(2002)

0 Virulence, detoxification, adaptation 91 99

1 Lipid metabolism 225 233

2 Information pathways 207 229

3 Cell-wall and cell processes 516 708

4 Stable RNAs 50 50

5 Insertion sequences and phages 137 149

6 PE and PPE proteins 167 170

7 Intermediary metabolism and respiration 877 894

8 Proteins of unknown function 606 272

9 Regulatory proteins 188 189

10 Conserved hypothetical proteins 910 1,051

* Data taken from Fleischman 2002

4.2.2. Comparative genomics

In recent times, new technologies have been developed at an overwhelming pace,

in particular those related to sequencing and tools for genome sequence data man-

agement, storage and analysis. As of April 2007, 484 microbial genomes have been

finished and projects are underway aimed at the sequencing of other 1,155 micro-

organisms (http://www.genomesonline.org/gold.cgi). Mycobacteria are not an

exception in this titanic genome-sequencing race; since 1998, when the first myco-

bacterial genome sequence was published (Cole 1998a); many genome projects

have been initiated. Until April 2007, 34 projects on the genome sequencing of

different mycobacterial species are finished or in-process. Of these, 15 are directed

towards M. tuberculosis strains, and 5 towards other members of the M. tuberculo-

sis complex. This information will be invaluable to improve the knowledge about

M. tuberculosis in the next few years. Currently, there are only two M. tuberculosis

(H37Rv and CDC1551) and two M. bovis (AF2122/97 and BCG Pasteur) genome

sequences annotated and published. For this reason, these are the strains that have

been used as reference strains for comparative genomics both in vitro and in silico.

120 Genomics and Proteomics

The pioneer of in vitro assays of comparative mycobacterial genomics involved

comparison of restriction profiles using low frequency restriction enzymes and

pulsed-field gel electrophoresis (PFGE). These studies allowed a rough analysis of

differences among M. bovis bacille Calmette-Guérin (BCG) isolates (Zhang 1995)

and most importantly, contributed to the construction of the first physical maps,

which were essential for the generation of the first genome sequence (Philipp

1996).

The next step in comparative genomics was the use of genomic subtractive hybridi-

zation or bacteria artificial chromosome hybridization for the identification of re-

gions of difference among the strains under analysis (Mahairas 1996, Gordon

1999). Mahairas et al. (Mahairas 1996) used subtractive hybridization to identify

regions of difference that account for the avirulent phenotype of the vaccine strain

M. bovis BCG. As a result of their studies, they identified three regions of differ-

ence (RD1-RD3) in the genome of M. tuberculosis H37Rv that appeared to be

absent from M. bovis BCG. Further studies of these regions showed that RD3 cor-

responded to the prophage PhiRv1, a sequence that has been shown to vary among

M. tuberculosis clinical isolates and laboratory strains (see section 4.2.1). RD2 was

only deleted in isolates of M. bovis BCG that were re-cultured after 1925. Finally,

RD1 turned out to be the only sequence deleted from all M. bovis BCG strains and

present in pathogenic strains. However, complementation assays did not reconsti-

tute the full virulent phenotype in M. bovis BCG (Mahairas 1996). The RD1 region

contains eight ORFs, including members of the Early Secretory Antigenic Target 6

(ESAT-6) gene cluster (Brosch 2000a). The ESAT-6 proteins have been shown to

act as potent stimulators of the immune system (Brodin 2002).The genome of

H37Rv contains 23 copies of ESAT-6 family proteins distributed in 11 different

regions. Except for esxQ, all are clustered in pairs belonging to the ESAT-6 and

CFP-10 protein families (Stanley 2003, Gey Van Pittius 2001).

Gordon et al. (Gordon 1999) used ordered bacteria artificial chromosome arrays to

determine genomic differences between M. tuberculosis H37Rv and M. bovis BCG.

As a result, they identified 10 regions of difference, including the three previously

described (Mahairas 1996). Interestingly, two of the newly described regions (RD5

and RD8) also contained members of the ESAT-6 family of proteins. In addition,

RD5 contained three genes coding for phospholipase C, a gene with a putative role

in mycobacterial pathogenesis (Johansen 1996). Several members of the PE and

PPE family proteins were also found in the regions of difference. One copy of

IS1532 was identified in RD6 and one copy of IS6110 in RD5. Furthermore, the

study searched for regions present in M. bovis BCG but absent from M. tuberculo-

sis H37Rv. Two regions with this characteristic were found and were named RvD1

4.2. M. tuberculosis genome 121

and RvD2 standing for H37Rv Deleted. Almost all ORFs from these regions code

for unknown proteins, so the role of these deletions has not been elucidated.

Until 2002, most studies concerning comparative genomics were based on differ-

ences among the strain type M. tuberculosis H37Rv and other tuberculous bacilli

(Behr 1999, Brosch 1999, Brosch 2002). Different approaches using DNA hybridi-

zation techniques, such as microarrays, allowed identification of regions of differ-

ence with more accuracy and sensitivity than previous methodologies. In total, 16

regions of difference have been found in M. tuberculosis H37Rv that were deleted

from M. bovis BCG. The basic idea behind the identification of regions of differ-

ence between the avirulent strain M. bovis BCG and the virulent laboratory strain

M. tuberculosis H37Rv was the identification of specific deletions in all BCG

strains that could be responsible for their lack of virulence. However, nine of the

regions of difference were also absent in pathogenic isolates of M. bovis.

Other studies have been done comparing M. tuberculosis H37Rv to its avirulent

counterpart M. tuberculosis H37Ra (Brosch 1999), in which other Rv-deleted re-

gions were identified. These regions, named RvD3 to RvD5, were found to be

products of homologous recombination of adjacent IS6110, as with RvD2. Finally,

only RD1 was found to be absent in all M. bovis BCG strains and present in other

members of the complex.

The regions of difference were used as markers of the molecular evolution of M.

tuberculosis (Brosch 2002) and are represented in Figure 4-1. The use of deletions

as molecular markers has been described in Chapter 2.

Besides the above mentioned deletions, two duplications were identified in the M.

bovis BCG genome (Brosch 2000b). These duplications, named DU1 and DU2,

apparently arose from independent events. DU1 seems to be restricted to the BCG

Pasteur strain and comprises the OriC locus, indicating that BCG Pasteur is diploid

for OriC and some other neighboring genes. The DU2 region has been found in all

BCG substrains tested and includes the sigma factor sigH, which has been related

to the heat-shock response (Brosch 2001). Some excellent reviews are available on

comparative genomics, made before the publication of the second M. tuberculosis

genome (Cole 1998a, Brosch 2000a, Brosch 2000c, Brosch 2001, Domenech 2001,

Cole 2002a, Cole 2002b).

In 2002, the second M. tuberculosis genome sequence was completed, namely the

clinical strain CDC1551, which had been previously involved in a TB outbreak.

This strain was considered to be highly transmissible and virulent for human beings

(Fleischmann 2002). With the sequence of this second strain, a first approach to the

bioinformatic analysis of intraspecies variability became possible. In the initial

122 Genomics and Proteomics

comparison by sequence alignment, H37Rv presented a total of 37 insertions

(greater than 10bp) relative to strain CDC1551; from these, 26 affected ORFs while

the remaining 11 were intergenic. On the other hand, CDC1551 presented 49 inser-

tions relative to M. tuberculosis H37Rv; 35 affecting ORFs and 14 intergenic. A

total of 80 ORFs were inserted in either genome, 25 (31.2 %) of them were hypo-

thetical or conserved hypothetical ORFs, while 36 (45 %) corresponded to the

family of PE/PPE proteins, showing the potential role of this family of proteins in

antigenic variability and thus in pathogenicity.

Deletion M. tuberculosis

H37Rv

M. africanum M. microti M.

bovis

M. bovis

BCG

RD2

RD14

RD1

RD4

RD12

RD13

RD7

RD8

RD10

RD9

RvD1

TbD1

Figure 4-1: Distribution of deleted regions in M. tuberculosis complex members. Dark gray

filled cells indicate the presence in all strains tested, light gray indicate the presence in some

strains, white is absence from all strains tested. Data taken from (Gordon 1999, Brosch 2002,

Brosch 2000b, Marmiesse 2004)

Only one major rearrangement was found, consisting of the PhiRv1 (RD3),which

was found in the genome of M. tuberculosis H37Rv on coordinates 1,779,312 asso-

ciated with a protein of the REP13E12 family. On the genome of CDC1551, it was

found to be located on the complementary strand at coordinates 3,870,803, also

associated with a REP13E12 protein. M. tuberculosis CDC1551 was found to have

four copies of IS6110 while M. tuberculosis H37Rv had 16. Interestingly, four of

the 16 IS6110 copies found in M. tuberculosis H37Rv lacked the characteristic 3 to

4 base pair direct repeat and were adjacent to regions deleted in M. tuberculosis

H37Rv relative to M. tuberculosis CDC1551, which suggests homologous recom-

bination.

4.2. M. tuberculosis genome 123

Since 2002, a large number of studies has been based on Large Sequence Polymor-

phisms (LSPs) and Single Nucleotide Polymorphisms (SNPs), identified by the

comparison of the first two M. tuberculosis genome sequences (Hughes 2002, Gu-

tacker 2002). These studies have been complemented with data obtained from the

genome sequence of a third organism of the M. tuberculosis complex. The com-

plete genome of Mycobacterium bovis AF2122/97, a fully virulent strain isolated

from a diseased cow in 1997 in Great Britain, was published in 2003 (Garnier

2003). This genome was composed of 4,345,492 bp with a G+C content of

65.63 %, 3,952 putative coding genes, one prophage (PhiRv2), and four IS ele-

ments. As expected, similarity of more than 99.95 % was found with a complete

colinearity, without evidence of extensive rearrangements. With regard to LSP,

most of them have been described above as regions of difference. Sequencing con-

firmed the absence of 11 regions of difference, and the presence of only one inser-

tion in comparison to the sequenced M. tuberculosis genomes: the region named M.

tuberculosis specific deletion 1 (TbD1), is a reflection that deletion events relative

to M. tuberculosis have shaped the M. bovis genome. The comparison of the three

genomes reflects the high degree of conservation among the members of the M.

tuberculosis complex, as well as the divergence of M. bovis related to M. tubercu-

losis strains.

For specific proteins or genes that vary between M. bovis and M. tuberculosis, a

detailed list can be found in Garnier et al. (Garnier 2003). However, it is important

to mention that the greatest degree of variation among these bacilli is found in

genes encoding cell wall components and secreted proteins. Extensive variations

have been found in genes of the PE/PPE family of proteins as well as in genes from

the ESAT-6 family, where six of the more than 20 members are absent or altered in

M. bovis. Some other changes are registered in genes coding for lipid synthesis and

secretion as the mmpL and mmpS family of genes. Deletions responsible for the M.

bovis requirement of pyruvate as a carbon source were also identified (Garnier

2003).

The analysis of the genome sequence of members of the M. tuberculosis complex

has led to great advances in the knowledge of the biology and pathogenesis of these

bacteria. The sequencing of whole genomes of Mycobacterium leprae (Cole 2001),

Mycobacterium avium subspecies paratuberculosis (Li 2005) and of other mem-

bers of the genus, such as Mycobacterium smegmatis and M. bovis, has also made

huge contributions to the understanding of the lifestyle of mycobacteria. Recently,

a report compared the metabolic pathways shared among five of the mycobacterial

genomes that have been sequenced (the genome sequence of M. smegmatis was not

included on this report) (Marri 2006). The characteristics of the sequenced ge-

124 Genomics and Proteomics

nomes of organisms in the genus Mycobacterium are presented in Table 4-2. The

main differences were found in ISs, the PE/PPE gene family, genes involved in

lipid metabolism and those encoding hypothetical proteins. The members of the M.

tuberculosis complex had the highest number of IS elements, which might suggest

higher intra-species variability in M. tuberculosis compared to other species of

mycobacteria.

Table 4-2: Features of sequenced genomes of species belonging to the Mycobacterium ge-

nus*

Feature

M. tuberculo-

sis H37Rv

M. tuberculo-

sis

CDC1551

M. bovis

AF2122/97C

M. leprae

M. avium

subsp.

paratuber-

culosis

M. smegma-

tis

Genome

size (bp)

4,411,529 4,403,836 4,345,492 3,268,203 4,829,781 6,988,209

Protein

coding

genes

3,927 4,186 3,920 1,604 4,350 6,897

G+C (%) 65.6 65.6 65.6 57.79 69.3 67.40

Protein

coding

(%)

91.3 ~ 91 90.8 49.5 91.5 92.42

Gene

density

(bp/gene)

1,114 1,052 1,099 2,037 1,112 1,013

Average

gene

length

1,012 952 995 1,011 1,015 936

tRNAs454545454547

rRNA

operon

111112

*Data taken from Li 2005, Marri 2006

The comparison of the proteins encoded within the five sequenced genomes re-

vealed a core, or a number of shared proteins, of 1,326 proteins, compared to the

219 core genes described by macroarray and bioinformatic analyses (Marmiesse

2004). Unique genes ranged between 966 (M. avium subsp paratuberculosis) and

26 (M. tuberculosis H37Rv) depending on the genome, and most of these proteins

are hypothetical. Regarding the PE/PPE family proteins, it is worth mentioning that

M. tuberculosis and M. bovis contained the highest number of these proteins, while

neither M. leprae nor M. avium subsp paratuberculosis have PE_PGRS proteins.

Also, a wide variation has been noted in the mmpL gene family, known to partici-

4.2. M. tuberculosis genome 125

pate in lipid transport and secretion. It has been proposed that these variations

could be involved in host specificity (Marsh 2005).

4.2.3. Comparing genomes of clinical strains of M. tuberculosis

Genome comparison has shown that gene content can vary between strains of M.

tuberculosis. The analysis of complete genome sequences identified SNPs, LSPs,

and regions of difference (RDs) when clinical isolates of M. tuberculosis were

compared (Fleischmann 2002, Gutacker 2002, Tsolaki 2004, Filliol 2006).

The microarray approach allows the comparison of a large number of genomes,

providing information on the diversity, frequency, and phenotypic effects of poly-

morphisms in the population (Tsolaki 2004). This kind of genomic analysis is also

useful for the investigation of outbreaks. Particularly when applied to genomics,

DNA microarrays allow the identification of sequences present in the M. tuberculo-

sis reference strain, but absent from different clinical isolates. Unfortunately, the

microarray technique cannot detect genes present in a clinical isolate that are absent

in the reference strain. These changes can originate from small deletions, deletions

in homologous repetitive elements, point mutations, genome rearrangements,

frame-shift mutations, and multi-copy genes (Ochman 2001, Schoolnik 2002).

Fleischeman et al. suggested that genetic variation among M. tuberculosis strains

might denote selective pressure, and therefore might play an important role in bac-

terial pathogenesis and immunity (Fleischmann 2002). Although associations be-

tween host and pathogen populations seems to be highly stable, the evolutionary,

epidemiological, and clinical relevance of genomic deletions and genetic variation

regions remain ill-defined, as do the molecular bases of virulence and transmissi-

bility (Hirsh 2004).

Up to six M. tuberculosis lineages adapted to specific human populations have

been described by Gagneux et al. using comparative genomics and molecular

genotyping tools: the Indo-Oceanic lineage, East-Asian lineage, East-African-

Indian lineage, Euro-American lineage, and two West-African lineages (Gagneux

2006, see chapter 2). Specific deletions associated with the hypervirulent Bei-

jing/W strains of M. tuberculosis were identified (Tsolaki 2005). Evidently, these

differences cannot include sequences present in clinical isolates that are absent

from M. tuberculosis H37Rv, so they necessarily represent a small part of the total

potential genetic variability. Up to 13 complete genome sequences of representa-

tive M. tuberculosis clinical isolates are currently under progress

(http://www.genomesonline.org/gold.cgi). That number accounts for near half of all

126 Genomics and Proteomics

the mycobacterial strains that are currently undergoing complete genome sequenc-

ing.

All major functional categories are represented among deleted genes in clinical

isolates of M. tuberculosis. Mobile genetic elements (insertion sequences or pro-

phages) are frequently deleted. DNA loss frequently results from the activity of the

insertion sequence IS6110 (Brosch 1999, Gordon 1999). The rate of deletion in

genes involved in intermediary metabolism and respiration, and in cell wall synthe-

sis is surprisingly high. Some of the genes encoding for potential antigens (plcA,

plcD, lpqH, lppA, esx, or PE/PPE genes) might be deleted under the influence of

host selective pressures, which would confer an adaptational advantage during

infection or help transmission (Tsolaki 2005). Some of these missing genes (e.g.

esx genes) encode proteins from the ESAT-6 family (Marmiesse 2004).

The use of microarray-based comparative genomics for the study of the genetic

variability of pathogens provides interesting information. Not only the identifica-

tion of the deleted or absent genes is important, but also the differential hybridiza-

tion signal between samples is of interest. These differential signals can indicate

sequence divergence or a difference in the copy number, which may provide an

insight into strain evolution and pathogenesis (Taboada 2005).

4.2.4. Functional genomics

Functional genomics is the analysis of the biological function of the genes and their

products within a cell or organism. Unlike genomics and proteomics, functional

genomics focus on gene transcription, translation, and protein-protein interactions.

Genes operate as long as they are expressed and their expression is regulated at the

transcriptional or post-transcriptional level.

Functional genomics uses mRNA expression profiling to provide a picture of the

transcriptome in a specific condition or time, in order to identify co-regulated genes

that perform common metabolic and biosynthetic functions. A set of co-regulated

genes is known as a regulon.

Microarrays have additional applications in functional genomics apart from gene

expression studies, and other uses have also been reported. Using this new power-

ful technique, Sassetti et al. developed a method to map transposon insertion sites

in order to identify essential genes in mycobacteria. The probes were synthesized

from a transposon library and then used for hybridization in the array. A number of

mutants carrying an insert in each gene were obtained, which were later isolated

and identified (Sassetti 2001).

4.3. Gene expression in M. tuberculosis 127

The results of studies on comparative mycobacterial genomics have been validated

by functional analysis, involving transcriptomics and proteomics. In fact, gene

knock-out followed by transcript analysis and proteome definition seems to be the

way to identify essential genes. For example, M. tuberculosis genes that encode

functions essential for growth are prime choices for further investigation as targets

for the development of new drugs or diagnostic methods (Cole 2002b).

Subsequently, research derived from comparative genomic studies was directed

towards the study of particular genes. That is the case of the deletion designated as

RD750 (corresponding to the Rv1519 gene) in the genome of the M. tuberculosis

strain named CH, from a large outbreak that occurred in a community of Indian

immigrants in the United Kingdom (Rajakumar 2004) and belonging to the East

African-Indian lineage. Complementation and combination of in vitro and in vivo

assay systems indicated the participation of the gene Rv1519 in the persistence and

outbreak potential of this M. tuberculosis lineage in human populations (Gagneux

2006, Newton 2006).

Construction and transcription analysis of the appropriate mutant have revealed the

functional role of the Rv3676 gene, a member of the cyclic adenosine monophos-

phate (cAMP) receptor protein family of transcription factors. This factor is re-

quired for virulence of M. tuberculosis in the mouse model. The functional map

obtained from the transcriptome revealed information about regulatory pathways.

The global transcription profiling experiments, comparing the wild type M. tuber-

culosis H37Rv strain and Rv3676 mutant grown in vitro, identified some of the

genes that are co-regulated, directly or indirectly, by Rv3676 in M. tuberculosis

(Rickman 2005).

4.3. Gene expression in M. tuberculosis

4.3.1. Control of gene expression

The ability of M. tuberculosis to survive within host cells requires a complex and

tightly controlled gene regulation. The genes that are used under different condi-

tions could be readily inferred from the corresponding mRNAs. Thanks to the de-

velopment of highly specific and sensitive technologies, such as microarrays and

quantitative real-time Polymerase Chain Reaction (qRT-PCR), it is now possible to

analyze the global expression from both the bacillus and the infected host. Taken

together, all this could help us to understand the adaptative machinery of M. tuber-

culosis.

128 Genomics and Proteomics

The deciphering of the complete M. tuberculosis genome sequence has unveiled its

well-equipped machinery, which accounts for its high degree of adaptability. Thir-

teen putative sigma (σ) factors and 192 regulatory proteins seem to be involved in

the control of M. tuberculosis gene expression (Cole 1998a). Interchangeable σ

factors regulate the function of RNA polymerase, initiating transcription and con-

ferring promoter specificity to the holoenzyme (Kazmierczak 2005, Mooney 2005).

To date, consensus promoter sequences have been proposed for six σ factors, be-

sides the housekeeping σ factor, σ

(for a review, see Rodriguez 2006). Gene ex-

pression levels could be further modified by the action of transcriptional activators

and repressors: regulatory proteins (Barnard 2004). These regulatory proteins in-

clude 11 two-component systems, five unpaired response regulators, seven wbl

genes, and more than 130 other putative transcriptional regulators (Cole 1998a).

The differential expression of these regulatory gene products throughout different

stages of the lifespan of M. tuberculosis must be determinant for the pathogen’s

successful infection and/or persistence within the human host. In recent years, a

number of reports have correlated the response of several of these transcriptional

regulators to a variety of environmental stresses (for a summary, see Table 4-3 at

http://www.tuberculosistextbook.com/pdf/Table 4-3.pdf), such as cold shock, heat

shock, hypoxia, iron or zinc starvation, nitric oxide, surface stress and oxidative

stress (Manganelli 1999, Raman 2001, Sherman 2001, Shires 2001, Manganelli

2002, Stewart 2002, Park 2003, Rodriguez 2003, Voskuil 2003, Canneva 2005,

Geiman 2006). However, the biological signals that stimulate the expression of the

majority of them are still poorly recognized. Likewise, the connections between the

different regulatory circuits of the complex network that controls gene expression

in M. tuberculosis are incompletely established. An example of the intricacy of this

network is the genetic regulation of sigB, which is induced by σ

in response to

surface stress (Manganelli 2001) or by σ

under heat shock and oxidative stress

(Manganelli 2002). The regulation of sigB expression seems to be more complex

than the above cited, given that σ

and σ

-dependent promoters were identified in

the regulatory promoter region of sigB; and σ

-dependent transcription was origi-

nated upstream to sigB (Dainese 2006). It has been shown that σ

is also responsi-

ble of the induction of sigE after heat shock and exposure to diamine (Raman

2001). DNA microarray experiments with M. tuberculosis mutants revealed that

some σ factors control the expression of their own structural gene (Manganelli

2002, Geiman 2004, Sun 2004, Raman 2004, Dainese 2006). Autoregulation has

also been demonstrated for the six two-component systems studied so far in M.

tuberculosis, which are senX3-regX3 (Himpens 2000), trcRS (Haydel 2002),

prrAB (Ewann 2004), dosRS (Bagchi 2005), mprAB (He 2005), and phoPR (Gupta

4.3. Gene expression in M. tuberculosis 129

2006). Two-component signal transduction systems are composed of a histidine

kinase sensor and a cytoplasmic response regulator that is activated by the cognate

histidine kinase (West 2001). One of these systems, dosRS, is induced by hypoxia,

exposure to ethanol or the nitric oxide donor S-nitrosoglutathione (Sherman 2001,

Voskuil 2003, Kendall 2004). This regulon is responsible for the transcriptional

changes during oxygen limitation, which is considered an important stimulus for

the entry of M. tuberculosis into a dormant state (Wayne 2001). For this reason, the

genes included under control of dosRS are considered members of the dormancy

regulon. Recently, the induction of sigB and sigE has been shown to depend on the

two-component system MprA/MprB when the bacilli are subjected to surface stress

(He 2006).

The transcriptional regulator WhiB3 seems to positively regulate the expression of

the housekeeping σ factor named sigA, by interacting with the subregion 4.2 of σ

factor (Steyn 2002). WhiB3 is encoded by one of the seven whiB-like genes de-

scribed in the M. tuberculosis genome (Cole 1998a) and belongs to the wbl family

of genes, which encodes putative transcription factors, which are unique to actino-

mycetes (Molle 2000, Soliveri 2000). A recent analysis has demonstrated that the

expression of M. tuberculosis whiB-like genes is modified in response to anti-

mycobacterial agents and environmental stress conditions (Geiman 2006). Addi-

tionally, whiB1 transcription is regulated by cAMP levels via direct binding of the

activated form of the product of Rv3676 (CRP protein-cAMP) to a consensus site

adjacent to the whiB1 promoter (Agarwal 2006).

A post-translational regulation has also been reported for several σ factors. An-

tagonist proteins, known as anti-σ factors, can negatively regulate some σ factors

by sequestering them and preventing their association with RNA polymerase.

Many of these anti-σ factors are located downstream of their cognate σ factor-

encoding gene and both genes are usually co-transcribed (Bashyam 2004). The

functions of five specific anti-σ factors of M. tuberculosis have so far been exam-

ined: RseA (Rodrigue 2006); RshA (Song 2003); RslA (Hahn 2005, Dainese 2006);

RsbW or UsfX (Beaucher 2002); and RskA (Saïd-Salim 2006). Interestingly,

RsbW, the σF-specific antagonist, is post-translationally regulated by two anti-anti-

σ factors: RsfA and RskB (Beaucher 2002, Parida 2005).

Although the function of many of these mycobacterial transcriptional regulators

and signal transduction systems remains poorly defined, recent studies have begun

to provide evidence of the biological role of these regulatory circuits throughout

each stage of the lifecycle of M. tuberculosis inside the human host. The expression

of sigA, sigE and sigG (Manganelli 2001, Capelli 2006, Volpe 2006), that of some

130 Genomics and Proteomics

two-component systems (Ewann 2002, Haydel 2004, Walters 2006), as well as that

of the transcriptional regulator whiB3 are induced during macrophage infection.

The role of these transcriptional regulators in pathogenesis and virulence became

even more evident in animal model experiments, where disruption or deletion of

these genes was shown to affect M. tuberculosis virulence in mice (Parish 2003a,

Parish 2003b, Sun 2004, Manganelli 2004, Raman 2004, Hahn 2005, Walters

2006).

Studies on mutagenesis and the expression profile of several regulators during the

growth of M. tuberculosis in the macrophages and in organs of experimental ani-

mal models are currently underway. These regulators can modify bacterial physiol-

ogy and are able to modulate host-pathogen interactions in response to environ-

mental signals.

4.3.2. In vitro gene expression

M. tuberculosis is an obligate mammalian pathogen that is able to infect many

different cells, including macrophages, dendritic cells, alveolar-epithelial cells, and

neutrophils. It is also able to reside extracellularly in the lung, inside granulomas.

As mentioned previously, the tubercle bacillus adapts its transcriptome to the envi-

ronment in which it replicates. The adaptation of a bacterium to harsh environ-

ments involves the transcriptional activation of genes whose final products help the

bacterium to reprogram its physiology, thus ensuring survival. Among the genetic

determinants that the bacterium must modulate are those involved in intermediary

and secondary metabolism, cell wall processes, stress responses and signal trans-

duction pathways.

By utilizing the microarray technology, quantitative RT-PCR and laboratory gener-

ated mutants, studies on M. tuberculosis global gene expression have been under-

taken using broth cultures, cell cultures, and animal models. None of these models

reproduce several key features of TB in the human infection; and, unfortunately, no

data is available from human tissues.

Table 4-4 summarizes the most important genes whose expression is modulated by

the transcriptional regulators mentioned previously (see section 4.3.1).

4.3. Gene expression in M. tuberculosis 131

Table 4-4: Regulation of cell process genes

CHP = conserved hypothetical protein

For example, analysis of a mutant of M. tuberculosis sigC showed that this σ factor

induces the expression of some virulence-associated genes. On the contrary, the

genes hspX (encoding the α-crystalline homologue), senX3 (sensor kinase), mtrA

(response regulator), and fbpC (mycolyl transferase and fibronectine binding pro-

tein or antigen 85C) were down-regulated in that mutant strain at different times of

the growth curve (Sun 2004). Genes induced by σ

include the resuscitation pro-

moting factor rfpC, several chaperone genes and genes involved in lipid metabo-

lism and cell wall processes (Raman 2004). Thirty-nine genes were shown to be

under the control of σ

. These include genes coding for some heat shock proteins

Cell process genes

Transcrip-

tional

regulation

Condition

regulated

Down

regulated

σC

Growth

curve

Two-component systems: senX3, mtrA,

hspX (α-crystallin), fbpC (antigen 85C)

σD

Exponen-

tial growth

rpfC (resuscitation factor), Rv1815,

Rv3413c

PE_PGRS

family genes

σH

Diamine

exposure,

heat

stress

Heat shock proteins: hsp, clp, trxB2C

operon, transcriptional regulators: sigE,

sigB

σM

Log-phase

growth

Esx family genes, PPE1, PPE19

PPE60, kasA-

kasB, fas,

pks2, pks3

σE

Exponen-

tial growth,

SDS

exposure

Icl1 (isocitrate lyase), heat-shock proteins,

transcriptional regulators: sigB, mprAB

σL

sigL-rslA, pks10-pks7, mpt53-Rv2877c,

Rv1139c-Rv1138c

σF

Stationary-

phase

growth

HP and CHP family of proteins, transcrip-

tional regulators: sigC, sigF, and MarA,

GntR TetR family, cell envelope: murB

DosS-DosR

Standing

cultures

hspX (α-crystallin), Rv3130c (CHP),

Rv1738 (CHP), Rv0572c (CHP)

PhoPR

Exponen-

tial growth

Cell envelope components

MprA

Mid-

exponen-

tial phase

∼95 genes of M. tuberculosis. PE/PPE

gene family, HP, CHP

132 Genomics and Proteomics

(hsp and clp), the trxB2C operon and some transcriptional regulators (Manganelli

2002). Quantification of mRNA by primer extension under different stresses dem-

onstrated that the transcription of trxB2, dnaK, clp and sigE could be induced from

-dependent promoters located upstream of these genes (Raman 2001). σ

seems

to regulate the expression of proteins involved in fatty acid degradation, such as the

isocitrate lyase (coded by icl1), two proteins related to fatty acid degradation

(fadE23 and fadE24), heat shock proteins, and the transcriptional regulators sigB

and mprAB (Manganelli 2001). Recently, it was reported that σ

induces the ex-

pression of two pairs of secreted proteins of the Esx family and two PPE genes,

while it negatively regulates PPE60 expression as well as the expression of several

genes involved in surface lipid biosynthesis and transport (Raman 2006).

At least four small operons appear to be directly regulated by σ

: sigL-rslA, pks10-

pks7, mpt53-Rv2877c, and Rv1139c-Rv1138c, which clearly have a σ

-consensus

promoter sequence in their regulatory region (Hahn 2005, Dainese 2006). The pks

genes are involved in the biosynthesis of phthiocerol dimycocerosate, a component

of the cell envelope associated with virulence (Sirakova 2003); and the mpt operon

contains genes involved in fatty acid transport (Sonden 2005). DNA microarrays of

a mutant of M. tuberculosis lacking a functional sigF, have revealed that this σ

factor is able to induce gene expression almost exclusively during the stationary

phase of growth, supporting the hypothesis of a major role of σ

in the adaptation

to the stationary phase. Among the σ

-targeted genes, 50 % coded for hypothetical

proteins or proteins of unknown function, some were transcriptional re-

pressor/activators (MarR, GntR and TetR family of DNA binding regulators) and

others were found to be involved in the biosynthesis and structure of the cell enve-

lope (Geiman 2004).

A complete genomic microarray analysis has also been performed on M. tuberculo-

sis strains mutated in two-component regulatory systems. The dormancy-related

two-component system dosRS was inactivated in M. tuberculosis using different

methodologies (Parish 2003b, Park 2003). It was shown that the expression of this

two-component system is highly induced under hypoxia (Sherman 2001b, Park

2003). A consensus dosR-specific binding motif was reported to be located up-

stream of hypoxic response genes (Park 2003, Kendall 2004). The microarray ex-

pression profiles of mutants in each of the components (dosR and dosS) showed

that DosR is required for the expression of genes usually induced under oxygen

limitation, such as hspX gene. Several putative operons with unknown function

were also strongly regulated by DosRS.

4.3. Gene expression in M. tuberculosis 133

Up to 30 genes were found to be up-regulated, and another 68 genes down-

regulated in a mutant of M. tuberculosis senX3-regX3. However, it has not been

clearly determined if the changes found in gene expression were directly or indi-

rectly related to the lack of this two-component regulatory system (Parish 2003a).

Recently, the global transcriptional profile of the two-component systems PhoP and

MprA has been reported. One of these studies provided evidence that the

PhoP/PhoR system is a positive transcriptional regulator of genes involved in the

synthesis of the cell envelope of M. tuberculosis (Walters 2006). On the other hand,

MprA regulates sigB and sigE and many other genes previously reported to be

associated to various stress conditions (He 2006).

In order to analyze the mechanisms involved in bacilli intracellular survival, myco-

bacterial gene expression was determined in M. tuberculosis infected macrophages

from different sources. Macrophages play a crucial role in TB infection because

they represent both the effector cells for bacterial killing and the primary habitat in

which the persisting bacilli reside. Macrophages have been investigated at different

time points post-infection for the differential expression of various two-component

system regulators (regX3, phoP, prrA, mprA kdpE, tcr, devR and tcrX) (Haydel

2004). More recently, the gene expression profile of M. tuberculosis grown in hu-

man macrophages compared to that of bacteria growing in synthetic culture me-

dium was published (Capelli 2006). In this work, the authors reported that ap-

proximately one-third (32 %) of the genes upregulated by M. tuberculosis in

macrophages correspond to conserved hypothetical proteins, with unknown func-

tion; this finding highlights the considerable gap that still remains in the knowledge

of how this bacterium survives intracellularly. Genes involved in cell wall proc-

esses (19.5 %), regulation and information pathways (16 %), and PE family pro-

teins (3.6 %) were also upregulated. Interestingly, the authors observed high induc-

tion of the sigma factor sigG and 13 other putative transcriptional regulators.

Upregulation of sigA, sigE, and sigG was also reported in a similar study (Volpe

2006). The whiB3 gene was also induced in M. tuberculosis during infection of

naïve bone marrow-derived macrophages in comparison to bacteria in broth culture

mid-log growth (Banaiee 2006).

4.3.3. In vivo gene expression

The use of microarrays for profiling transcriptomes of bacteria inside the host cell

has been limited by the paucity of bacterial mRNA in samples containing a pre-

ponderance of mammalian RNA. Therefore, while significant work has been per-

134 Genomics and Proteomics

formed on the gene expression profile of the host, information on M. tuberculosis

expression inside infected hosts is still limited.

So far, there is only one publication concerning global mycobacterial transcription

expression in the animal model, using microarray as the analytical method (Talaat

2004). Differential expression levels of M. tuberculosis during infection in Balb/c

or Severe Combined Immunodeficiency (SCID) mice were evaluated and com-

pared to the levels found in mycobacteria grown in broth culture. These authors

identified up to 40 genes whose expression significantly changed during Balb/c and

SCID mouse infection. These genes include rubB, dinF, and fdxA. The same genes

were also found to be induced 24 hours post-infection in murine bone marrow

macrophages (Schnappinger 2003). Additionally, several genes were regulated up

or down only in Balb/c mice, such as proZ (transport system permease protein),

aceAa (probable isocitrate lyase involved in lipid metabolism), and genes encoding

for regulatory proteins, such as sigK, sigE and kdpE. The authors concluded that

the expression profile of M. tuberculosis in SCID mice resembles the profile found

in bacilli grown in vitro, while the expression profile in Balb/c mice resembles that

reported in multiplication within the macrophages (Schnappinger 2003). Excep-

tionally, some genes were found to be expressed only in Balb/c mice.

A small number of studies applied quantitative RT-PCR to investigate the expres-

sion of mycobacterial genes in the animal model. These studies focused on the

analysis of a few particular genes. Examination of lungs of infected C57BL/6 mice

showed that the transcriptional regulator genes whiB3, fdxA (electron transfer),

hspX (α-crystalline), acg (unknown function), Rv1738 (unknown function), and

Rv2626c (unknown function) were markedly induced during the course of infection

(Banaiee 2006). A gene required for extrapulmonary dissemination (hbhA) was

also upregulated in the lung but not in the spleen during the early stages of infec-

tion (Delogu 2006). While the expression of PE_PGRS16 was up-regulated in the

spleens and lungs of infected mice, the expression of PE_PGRS26 was down-

regulated (Dheenadhayalan 2006). A study on human lung biopsies revealed a high

variability in expression profiles of specific M. tuberculosis genes among the

specimens analyzed (Timm 2003). The biopsies were obtained from four HIV-

negative patients with chronic active TB that was unresponsive to therapy. Al-

though some differences were observed when comparing human and murine lung,

the authors admitted that it was difficult to ascertain whether the infection stage in

the analyzed human lung specimens could be correlated with the persistent infec-

tion in mice.

4.4. M. tuberculosis proteome 135

4.4. M. tuberculosis proteome

With the availability of the genomes of M. tuberculosis H37Rv, M. tuberculosis

CDC1551, M. bovis, and the ongoing sequencing projects, attention in the coming

years must be focused on the interpretation of the sequences determining the

structure and function of the proteins. Proteomics, the global study of proteins that

are translated in a given physiological state is one of the most important and ambi-

tious goals in M. tuberculosis research. The proteome of an organism implies not

only an inventory of its gene products but also the transduction rate and the post-

transcriptional events that occur in the organism (Betts 2002). Classical studies of

proteomics involve two dimensional electrophoresis (2-DE), in which proteins are

first separated by the isoelectric point and then by the molecular weight (O’Farrel

1975). Every spot of protein is then isolated, hydrolyzed and subjected to tech-

niques of mass spectrometry (MS), tandem MS (MS/MS); matrix-assisted laser

desorption/ionization mass spectrometry (MALDI/MS) and, matrix-assisted laser

desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS). For a

good review on the different techniques used in protein mapping, readers are re-

ferred to Patterson et al (2000). Techniques different from two dimensional elec-

trophoresis have also been implemented. For instance, the use of one dimension

electrophoresis has been shown to be very useful for the separation of hydrophobic

proteins (Simpson 2000). Other approaches that do not involve the use of gels, such

as two-dimensional liquid chromatography (LC) and the subsequent analysis by

MS (2 DLC/MS), have been shown to be very efficient in the identification of

hydrophobic and membrane proteins (Isobe 1991). In 1999, the isotope-coded

affinity tag (ICAT) technology was reported (Gygi 1999). In this, mixtures of pro-

teins from bacteria in two different conditions are covalently labeled with isotopi-

cally labeled heavy or light forms of the reagents. The samples are combined and

subjected to proteolysis. After purification of the labeled peptides through affinity

tag, which is part of the reagents, they are analyzed by LC-MS/MS. This new tech-

nology has proven to be very useful in the quantitation of complex mixtures of

proteins.

Before the disclosure of the M. tuberculosis genome, antigens and proteins were

identified by one- and two-dimension polyacrilamyde gel electrophoresis, and the

use of cumbersome immunological methods (Nagai 1991, Garbe 1996). With the

advance in high-resolution 2-DE and analytical chemistry, M. tuberculosis pro-

teome is at present a reality. Pioneering studies in the proteomic field included the

mapping of 32 N-terminal sequences by MS and the identification of culture filtrate

proteins by 2-DE and immunodetection (Sonnenberg 1997).

136 Genomics and Proteomics

Very soon after the publication of its genome, bioinformatic tools were applied to

predict the proteomic profile of M. tuberculosis (Tekaia 1999). The in silico analy-

sis showed characteristics of the tubercle bacillus as the duplication of numerous

genes, especially those involved in gene regulation and in lipid metabolism, and

those coding for the PE/PPE protein family. The study also showed a reduced rep-

ertoire of proteins devoted to transport, which might reflect the intracellular life-

style. The bioinformatics-predicted proteome was compared to 2-DE protein maps

obtained from M. tuberculosis whole cell lysates, which were separated in a broad

pH range between 2.3 and 11.0. This work demonstrated that proteins with a mo-

lecular mass below 10 kDa were not predicted from the genome sequence and also

that experimentally basic and high molecular mass proteins could not be resolved

by 2-DE (Urquhart 1998). Since 1999, the huge amount of data in proteomics has

led to the creation of 2-DE databases, where images generated in different laborato-

ries can be stored and analyzed. These databases are accessible on the internet at:

http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/overview.cgi?gel=16

(Mollenkopf 1999) and http://www.ssi.dk/sw14644.asp (Rosenkrands 2000b).

4.4.1. Structural proteomics of M. tuberculosis

Thanks to recent technological advances, the subcellular protein profile of M. tu-

berculosis can now be drawn. The global analysis of compartmentalized proteins

will shed light on host-pathogen interactions, metabolic pathways and cell commu-

nication, just to mention some of the mechanisms related to pathogenesis. In addi-

tion, many pathogenic bacteria secrete proteins that are involved in virulence (Fin-

lay 1997) and thus culture filtrates of M. tuberculosis could be a source for identifi-

cation of virulence factors. Cell wall proteins play a fundamental role in cell archi-

tecture, resistance of the pathogen to chemical injury and dehydratation, and many

other key functions of this microorganism. Thus, the identification of proteins lo-

calized in this subcellular fraction may lead, in the near future, to the development

of new diagnostic tests and drugs. Membrane proteins demand special attention,

because they are involved in host-pathogen interactions, nutrient transport, quorum

sensing mechanisms, etc. Knowledge of these proteins could be the clue to the

development of novel vaccines. Finally, the identification of cytosol proteins and

the intricate network of their interaction will reveal metabolic pathways that can be

targets for the design of rational drugs against TB. Even though we are still far

from identifying the almost 4,000 genes predicted by genomics, the number of

identified proteins increases each year and shows how genomic and proteomic

technologies complement each other.

4.4. M. tuberculosis proteome 137

The biochemical methods developed for the separation of the cell wall, membrane

and cytosol fractions have facilitated proteomic studies in M. tuberculosis (Hirsch-

field 1990, Lee 1992). Jungblut et al. identified 53 proteins from cell lysates and 54

from culture filtrates using 2-DE and MALDI/MS (Jungblut 1999). These authors

performed comparative proteomics in M. tuberculosis, which will be discussed

later in this chapter.

Reference maps of cellular fractions and culture filtrate proteins were constructed

using 2-DE, N-terminal sequencing and antibodies against previously identified

antigens (Rosenkrands 2000b). As many as 1,184 spot proteins were visualized

after silver staining. Only 10 % of them were identified. In order to map less abun-

dant proteins, different methods were applied for their separation, which allowed

the identification of 12 novel proteins, five of them with a known function (Ro-

senkrands 2000b). The study also showed the identification of a protein that was

not predicted by genomics and revealed the presence of alternative start codons.

The implementation of immobilized pH gradient for 2-DE and MALDI/MS al-

lowed the identification of 288 proteins (Rosenkrands 2000a). Six proteins were

identified, all of them with molecular masses between 13,200 and 7,200 kDa and

with isoelectric point (pI) ranges between 4.5 and 5.9. Five of these proteins were

correctly identified in the genome of the clinical strain CDC1551 (Jungblut 2001).

In spite of the enormous usefulness of 2-DE in proteomic studies, there are certain

disadvantages inherent to its technique, such as the low resolution of proteins with

very high or very low molecular masses, or proteins that are very acidic, very basic

or hydrophobic. But in particular, the technique is biased towards the preferential

identification of the most abundant proteins. Therefore, less abundant proteins,

such as transcriptional regulators, are rarely detected when whole cell lysates are

analyzed (Gygi 1999). To overcome these inconveniences, alternative techniques

have been applied in proteomic studies. For example ICAT reagent method and

LC-MS/MS were used as a complement of 2-DE-MS/MS. Using these approaches,

388 M. tuberculosis proteins were quantified and identified. Each one of these

techniques has been shown to be adequate for the identification of certain classes of

proteins. For example, the ICAT method performed better for the identification of

cell membrane and high molecular mass proteins, while 2-DE showed better results

in the identification of low molecular mass and cysteine-free proteins (Schmidt

2004). Interestingly, none of these techniques allowed the identification of proteins

in the following subclasses: cell division, IS elements, repeated sequences, phages,

PE/PPE families, cytochrome P450 enzymes, cyclases, and chelatases.

138 Genomics and Proteomics

By 2004, only about 400 proteins had been identified in the proteome of M. tuber-

culosis, probably due to the limitations of 2-DE-based separation methods. The use

of automated two-dimensional, capillary high-performance liquid chromatography

(HPLC) coupled with MS gave a wider and more accurate proteomic profile of M.

tuberculosis (Mawuenyega 2005). Proteins of the cell wall, membrane and cytosol

subcellular fractions could be identified. The number of identified proteins in-

creased to 1,044 non-redundant proteins, 67 % more than those obtained by con-

ventional 2-DE. This study identified proteins in extreme pI ranges, among them

the most acidic proteins ( PE_PGRS, Rv3512) with a pI of 3.89 and the most basic

proteins (rps2, a 30S ribosomal protein) with a pI of 12.18. Proteins of high mo-

lecular mass, such as the 230,621 Da polyketide synthase ppsC, were also identi-

fied. A total of 705 proteins were identified in the membrane, 306 were localized in

the cell wall, and 356 in the cytosol fraction. Forty-seven were present in all ana-

lyzed fractions. The study also included a computational analysis of protein net-

works, one of the most exciting fields in the coming years. Readers are invited to

consult the supplementary table of this work (Mawuenyega 2005).

M. tuberculosis is an intracellular pathogen, the bacillus is engulfed by alveolar

macrophages where it can survive and grow by altering the intracellular compart-

ments to preclude the normal maturation to phagolysosomes or to prevent fusion of

phagosomes to lysosomes (Clark-Curtiss 2003). The interaction between host and

pathogen is thought to be mediated by membrane proteins. Therefore, the charac-

terization of membrane proteins is a topic of intensive research. As mentioned

before, most of the studies regarding M. tuberculosis proteomics have been carried

out by 2-DE. However, the number of membrane and membrane-associated pro-

teins has been underestimated by the 2-DE technology due to the hydrophobic

nature of this class of proteins and their low solubility. In order to overcome these

problems, fractions of cellular membranes were prepared by differential centrifu-

gation and separated by one-dimensional electrophoresis. The separated bands were

then excised and hydrolyzed prior to LC and MS/MS (Gu 2003). This approach

allowed the identification of up to 739 membrane and membrane-associated pro-

teins. Very hydrophobic proteins, including those with 15 transmembrane helices,

were detected in this study. The use of alternative solubilizing agents, such as Tri-

ton X-114, has proven to be a good choice for membrane fractionation. The deter-

gent was shown to be useful in the identification of nine novel proteins that have

been already incorporated in the M. tuberculosis proteome (Sinha 2005). Interest-

ingly, when analyzing the interferon-gamma (IFN-γ) response of BCG-vaccinated

healthy individuals from an endemic area to these newly identified proteins, the

strongest response was found to be that against ribosomal proteins. Other mem-

4.4. M. tuberculosis proteome 139

brane associated proteins, such as ESAT-6, did not contribute significantly to the

T-cell response in these individuals.

4.4.2. Comparative proteomics

The comparative proteomic analysis using 2-DE and MALDI/MS was applied to

compare proteins present in two virulent laboratory M. tuberculosis strains (H37Rv

and Erdman strains) with those present in two M. bovis BCG strains (Chicago and

Copenhagen BCG strains) (Jungblut 1999). The results showed that, as expected,

the two M. tuberculosis strains differed from each other in only a few proteins. Of

the 18 variant proteins, 16 were identified. L-alanine dehydrogenase (Rv2780) was

not detected in the Erdman strain, and the protease IV was absent in this strain. On

the other hand, the Soj protein and the hypothetical protein Rv2641 were absent

from the M. tuberculosis H37Rv proteome. Some of the 18 proteins were over-

expressed in one or the other strain, and some shifted their mobility probably due to

the presence of amino acid substitutions. The comparison of M. tuberculosis

H37Rv with M. bovis BCG revealed the presence of 13 protein spots exclusive to

the tubercle bacilli, six of which were identified. The differential proteins com-

prised L-alanine dehydrogenase (40 kDa protein), isopropyl malate synthase nico-

tinate-nucleotide pyrophosphatase (Rv1596), MPT64 (Rv1980c), and two hypo-

thetical conserved proteins (Rv2449c and Rv0036c). On the other hand, M. tuber-

culosis H37Rv lacked eight spots compared to M. bovis BCG.

In another study using 2-DE and MS, a comparison of the proteins present in M.

tuberculosis and M. bovis BCG revealed the presence of 56 unique protein spots in

M. tuberculosis and 40 in the attenuated strain BCG (Mattow, 2001). Of these, 32

were identified as exclusive proteins of M. tuberculosis, of which 12 had been

previously reported to be deleted in M. bovis BCG. The remaining 20 spots were

newly identified as absent from M. bovis BCG.

A third comparative proteomic study of M. tuberculosis and M. bovis BCG was

performed using 2-DE and ICAT technology (Schmidt 2004). This work demon-

strated the presence of only three exclusive proteins in M. tuberculosis H37Rv. One

is Rv0223c, a protein belonging to the aldehyde dehydrogenase family. The second

is Rv0570, a ribonucleotide reductase class II. The third is a hypothetical protein

named Rv1513.

The studies on comparative proteomics allowed the identification of isopropyl

malate synthase exclusively in the M. tuberculosis proteome. Recently, this enzyme

was included in a new class of virulence factors known as ‘anchorless adhesins’

140 Genomics and Proteomics

(Kinhicard 2006) that were absent from the avirulent BCG, thus proving the use-

fulness of this methodology.

Of special interest in the coming years will be the proteomic comparison between

circulating M. tuberculosis strains differing in virulence, transmissibility, tissue

tropism, and/or ability to acquire drug resistance. As a matter of fact, the proteomic

profile of M. tuberculosis H37Rv has already been compared with that of the clini-

cal strain CDC1551 at different time points during in vitro growth (Betts 2000).

Subscribing the low structural DNA polymorphism observed in M. tuberculosis

(Sreevatsan 1997), the resulting patterns of the protein-spot were found to be both

highly reproducible and highly similar between the two strains during growth. One

unique protein was identified in M. tuberculosis CDC1551, namely Rv0927c, a

probable alcohol dehydrogenase. Similarly, a spot corresponding to the HisA pro-

tein, which is involved in the histidine biosynthetic pathway, was detected in the M.

tuberculosis H37Rv proteome but was absent from the M. tuberculosis CDC1551

protein profile. Oddly enough, both genes were found to be present in both M.

tuberculosis strains. Thus, the described proteomic differences between H37Rv and

CD1551 might be ascribed to post-translational events or to degradation during the

manipulation of the specimens. Another interesting feature in the same study was

the mobility variations of the transcriptional regulator MoxR, which the authors

attributed either to amino acid changes or to post-translational modifications. A

BlastP analysis of both genomes showed a single amino acid substitution of histid-

ine in M. tuberculosis H37Rv to asparagine in M. tuberculosis CDC1551 that might

explain the variation in mobility.

Transcriptional regulation differences between strains might be the key to under-

standing how virulence factors are involved in a variety of roles, including host-cell

invasion, survival within the host cell, and long-term persistence. Therefore, com-

parative proteomic studies are of special interest in the post genomic era, helping to

understand the manifestation of disease produced by different strains involved in

the current TB epidemic.

4.4.3. Environmental proteomics

The information obtained by genomic studies is static, because DNA is not essen-

tially affected by the environment. In contrast, the proteomic profile of an organism

in a particular physiological situation complements and helps to decipher its inter-

action with the environment. The study of the M. tuberculosis proteome in different

physiological states is one of the most fascinating fields of research. Being an in-

tracellular pathogen, the bacillus is challenged by a variety of environmental