Molecular diagnosis and multi drug resistance detetction of Mycobacterium tuberculosis in clinical specimens ans computational genome/phylogeny analysis of tuberculosis and non tuberculosis mycobacteria
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(2) 2012. [SCIENTIFIC PRODUCTION].
(3) Doctorat Es Science in Microbiology & Molecular Biology. Scientific production. 2012. SCIENTIFIC PRODUCTION. A- List of international Publications (06): 1. Fathiah ZAKHAM, Lamiae BELAYACHI, Dave USSERY, Mohammed AKRIM, Abdelaziz BENJOUAD, Rajae El AOUAD and Moulay Mustapha ENNAJI. 2011. Mycobacterial species as a case-study of comparative genome analysis. Cell. Mol. Biol. 57: 1462-1469.. 2. Fathiah ZAKHAM, Halima BAZOUI, Mohammed AKRIM, Sanae LAMRABET, Ouafae LAHLOU, Mohamed EL MZIBRI, Abdelaziz BENJOUAD, My Mustapha ENNAJI and Rajae ELAOUAD. 2012. Evaluation of conventional Molecular diagnosis of Mycobacterium tuberculosis in the clinical specimens from Morocco. J Infect Dev Ctries. 6(1):40-45. 3. Fathiah ZAKHAM, Mohammed AKRIM, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Rajae ELAOUAD and My Mustapha ENNAJI. 2012. Rapid Screening and Diagnosis of Tuberculosis: a real Challenge for the mycobacteriologist. Cell. Mol. Biol. 58: 1632-1640.. 4. Fathiah ZAKHAM, Oufae LAHLOU,. Mohammed AKRIM, Nada BOUKLATA,. Sanae JAOUHARI, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Mustapha ENNAJI and Rajae ELAOUAD. 2012. Comparison of a DNA based PCR approach with conventional methods for the detection of Mycobacterium tuberculosis in Morocco. Mediterr J Hematol Infect Dis. 4: (1) 5. Fathiah ZAKHAM, Othmane AOUANE, David USSERY, Abdelaziz BENJOUAD and Mouly Mustapha ENNAJI. 2012. Computational and comparative genomicsproteomics and Phylogeny analysis of twenty one mycobacterial genomes. BMC microbial informatics and experimentation. 7:2. 6. Fathiah. ZAKHAM,. Imane. CHAOUI,. Mohammed. ABID,. Moulay. Driss. MESSAOUDI, Moulay Mustapha ENNAJI, Mohamed ELMZIBRI. Automated sequencing for the direct detection of Multi drug Mycobacterium tuberculosis strains in the sputum specimens of Moroccan patients. (In preparation)..
(4) Doctorat Es Science in Microbiology & Molecular Biology. Scientific production. 2012. B- Publication of sequences in GenBank at National Center for Biotechnology Information NCBI (10). HSP65. VFS22. 04. 11.sqn. HSP65S4. JF921153. HSP65. VFS22. 04. 11.sqn. HSP65S2. JF921154. HSP65. VFS22. 04. 11.sqn. HSP65S3. JF921155. HSP65. VFS22. 04. 11.sqn. HSP65S5. JF921156. HSP65. VFS22. 04. 11.sqn. HSP65S7. JF921157. HSP65. VFS22. 04. 11.sqn. HSP65S8. JF921158. HSP65. VFS22. 04. 11.sqn. HSP65S9. JF921159. HSP65. VFS22. 04. 11.sqn. HSP65S6. JF921160. HSP65. VFS22. 04. 11.sqn. HSP65S10. JF921161. HSP65. VFS22. 04. 11.sqn. HSP65S1. JF921162. C- List of Communications (13): 1. Oral presentations (05) 1. Fathiah ZAKHAM. Diagnostic moléculaire de Mycobacterium tuberculosis dans les échantillons cliniques. The 8th edition of Doctorial in Morocco. Incubateur Universitaire de Marrakech « INMA ». 12-18th December 2010. 2. Fathiah ZAKHAM, Lamiae BELAYACHI, Dave USSERY, Mohammed AKRIM, Abdelaziz BENJOUAD, Rajae ElAOUAD and M M ENNAJI. International Bioinformatics Software School. Tangier- Morocco. Comparative Genomics and Proteomics for differentiation between fourteen Mycobacterial strains. IBSS 2011, 4-9th April 2011 3. Fathiah. ZAKHAM,. Halima. BAZOUI,. Mohammed. AKRIM,. Sanae. LAMRABET, Ouafae LAHLOU, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Rajae ELAOUAD and My Mustapha ENNAJI. Evaluation of conventional polymerase chain reaction for the diagnosis of Mycobacterium tuberculosis in the.
(5) Doctorat Es Science in Microbiology & Molecular Biology. Scientific production. 2012. clinical specimens from Morocco. International Congress: La recherché, la Biotechnologie et le consommateur au service de l’Environnement et de l’industrie Agroalimentaire » Faculty of Science Kenitra. 19th-20th May 2011. 4. Fathiah. ZAKHAM,. Othmane. AOUANE,. David. USSERY,. Abdelaziz. BENJOUAD and Mouly Mustapha ENNAJI. Computational genome and proteome analysis of Mycobacterium tuberculosis and non tuberculosis mycobacteria. International conference Humboldt Kolleg “New Prospects and Challenges for Science and Education in the MENA region. Marrakesh- Morocco. 9th- 11th March, 2012. 5. Fathiah ZAKHAM, Imane CHAOUI, Mohammed ABID, Moulay Driss MESSAOUDI, Moulay Mustapha ENNAJI, Mohamed ELMZIBRI. Rapid detection of Multi Drug Resistance Mycobacterium tuberculosis strains in the clinical specimens from Morocco. International conference on Antimicrobial Research. Lisbon, Portugal. 21st -23rd November, 2012. (accepted). 2. Poster presentations (08) 1. Fathiah ZAKHAM, Halima BAZOUI , M M Ennaji, Sanae LAMRABET, Ouafae LAHLOU, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Mohammed AKRIM, and Rajae ELAOUAD. 3rd International Congress of Biochemistry and Molecular Biology. Direct Molecular Diagnosis of Mycobacterium tuberculosis in the sputum specimens. Marrakech, April 20-25th, 2009. 2. Fathiah Zakham, Lamiae BELAYACHI, Dave USSERY, Mohammed AKRIM, Abdelaziz BENJOUAD, Rajae El AOUAD and Moulay Mustapha ENNAJI. Xth Spanish symposium on Bioinformatics. Comparative Genome Analysis of Mycobacterium sp (Mycobacterium Tuberculosis and other non tuberculosis Mycobacteria). Torremolinos- Malaga, Spain 27-29th. October, 2010. 3. Fathiah ZAKHAM, Lamiae BELAYACHI, Dave USSERY,. Mohammed. AKRIM, Abdelaziz BENJOUAD, Rajae El AOUAD and Moulay Mustapha ENNAJI.. International. Bioinformatics. Software. School. IBSS. 2011.. Computational Genomics and Proteomics are potential tools for the comparison.
(6) Doctorat Es Science in Microbiology & Molecular Biology. Scientific production. 2012. between pathogenic and free living Mycobacteria. Tangier- Morocco 4-9th April, 2011. 4. Fathiah ZAKHAM, Oufae LAHLOU,. Mohammed AKRIM, Nada BOUKLATA,. Sanae JAOUHARI, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Mustapha ENNAJI and Rajae ELAOUAD. The insertion sequence IS6110 as a potential tool for the. detection of Mycobacterium tuberculosis in the critical cases of the Moroccan population. The 7th European congress of Tropical Medicine and International Health. Barcelona. Spain.3- 6th October. 2011. 5. Fathiah ZAKHAM, Oufae LAHLOU, Mohammed AKRIM, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Rajae ELAOUAD and Mustapha ENNAJI. Detection of. Mycobacterium tuberculosis in the clinical specimens by the PCR technique targeting the insertion sequence IS6110 in Morocco. International engagement: Responsible Bio Science and Secure Society, workshop III. Tunis, Tunisia, 31st October- 1st November, 2011. 6. Fathiah ZAKHAM, Mohammed AKRIM, Nada BOUKLATA, Mohamed ELMZIBRI, Abdelaziz BENJOUAD, Mustapha ENNAJI and Rajae ELAOUAD. Comparative. analysis of conventional and molecular detection of Mycobacterium tuberculosis in the critical cases of the Moroccan population. International conference Humboldt Kolleg “New Prospects and Challenges for Science and Education in the MENA region. Marrakesh- Morocco. 9th- 11th March, 2012. 7. Fathiah. ZAKHAM,. Othmane. AOUANE,. David. USSERY,. Abdelaziz. BENJOUAD, Mohamed ELMZIBRI and Mouly Mustapha ENNAJI. Evolutionary Relationships between pathogenic and free living mycobacterial species. Tuberculosis Conference 2012: Biology, Pathogenesis, Intervention strategies. Pasteur Institute Paris. 12th-15th September 2012. 8. Fathiah ZAKHAM, Imane CHAOUI, Mohammed ABID, Moulay Driss MESSAOUDI, Moulay Mustapha ENNAJI, Mohamed ELMZIBRI. Automated sequencing for the rapid detection of Multi Drug Resistant Mycobacterium tuberculosis strains in the sputum specimens of Moroccan patients. Tuberculosis Conference 2012: Biology, Pathogenesis, Intervention strategies. Pasteur Institute Paris. 12th-15th September 2012..
(7) Doctorat Es Science in Microbiology & Molecular Biology. Dedication. 2012. DEDICATION. I dedicate dedicate my modest work to every person who helped me in the process of fulfilling my thesis.. To my daughter daughter Romissa’a , my symbol of love To my parents, my symbol of confidence To my sisters: sisters: Najat, Mariam and Fatima, Fatima, my symbol of kindness To my friends, my symbol of fidelity and respect And to whom I’m grateful.. Fathiah ZAKHAM. Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(8) Doctorat Es Science in Microbiology & Molecular Biology. Foreword & Acknowledgement. 2012. FOREWORD & ACKNOWLEDGEMENT This present study was done within the framework between laboratory of Biochemistry and Immunology at Faculty of Science-Rabat (FSR)-University Mohammed V-AGDAL, directed by Pr. Abdelaziz BENJOUAD, the laboratory of Virology and Hygiene &Microbiology (LVHM) at Faculty of Sciences and Techniques (FSTM) - University Hassan II Mohammedia, Casablanca (UH2MC), directed by Pr. Moulay Mustapha Ennaji and Laboratory of Molecular Biology at the National Institute of Hygiene (NIH) of Rabat. This thesis was realized in collaboration with the Unit of Biology & Medical Research at the National Centre of Nuclear Energy, Sciences & Techniques (CNESTEN) of Rabat. The computational & bioinformatics analysis were performed in partnership with the Centre for Biological Sequence Analysis (CBS) - Technical University of Denmark, Lyngby, Denmark. This thesis was financially supported by the Academy Hassan II of Sciences & Techniques within the framework of IMMGEN project and The Ministry of Higher Education and Scientific Research (UH2MC) and the National Centre of Scientific and technical research (CNRST) for supporting a part of technical analysis. I want to express my sincere thanks to all, who had played a role in the drafting of this manuscript and helped bring this work into the light. My regards and my thanks are due to: Professor Abdelaziz BENJOUAD, the Director of the CNRST, the head of the UFR Biochemistry and immunology, who co supervised and directed me through the period of my study. In addition, I would like to thank Pr BENJOUAD for his commitment and his competence, despite the many concerns and responsibilities.. Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(9) Doctorat Es Science in Microbiology & Molecular Biology. Foreword & Acknowledgement. 2012. Professor Moulay Mustapha Ennaji, Full Professor and Director of LVHM- (FSTMUH2MC), who supervised and played the role of my Director of my thesis, Firstly, for accepting my candidacy within the PhD program, secondly for giving me a chance to continue my higher studies. Finally, for supervising and directing my work and for being one of my competent professors during the years of higher studies. Professor Rajae Elaouad, Professor at the Faculty of Medicine and Pharmacy of Rabat and director of the National Institute of Hygiene (NIH) for having pleasantly agreed to apply this research within her establishment. Dr. Mohammed Akrim, the head of the laboratory of Molecular Biology at NIH, for the honour that he agreed to supervise me at NIH, I am grateful for his invaluable councils, for being always there for me to correct and advise me during the period of my education, for his generosity, sympathy, and encouragements, for accepting to direct my research, guide me during the period of my presence at the laboratory and for devoting his time for reading and correction of this document.. Professor Said AMZAZI, the Dean of the faculty of Science in Rabat, for chairing the committee of the acceptation of this thesis, for the honour of being the president of the jury and hence I am extremely honoured. I warmly thank Professor Youssef BAKRI to accept judging and being a reporter of this thesis and for his invaluable judgment. I also want to express my special gratitude to Professor Houssin AZZEDOUG, the Vice Dean of the Faculty of Science Ain Achok- Casablanca, for accepting to be a reporter of my thesis.. Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(10) Doctorat Es Science in Microbiology & Molecular Biology. Foreword & Acknowledgement. 2012. I would like to thank Professor Khalid ZEROUALI at the Faculty of Medicine & Pharmacy of Casablanca, for accepting to judge this work and being a reporter in the jury. I express my sincere thanks to Dr. Mohammed ELMZIBRI, for his advice and help during my presence at CNESTEN, for devoting his time and efforts in reading and correcting the drafts of my scientific articles and for allowing me to finalize the last part of my thesis at his laboratory. I warmly thank Professor David USSERY at the CBS for his generous help & support, for enabling me to fulfil an important part of my thesis on the server of the CBS, for devoting his time in the correction of my articles and distinguished remarks. El FAHIME El Mostafa at the Technical Support Units for Scientific Research (UATRS) at the CNRST for his technical support. MELLOUL Merouane at the UATRS for his help, technical support and scientific councils. I do not forget the people who surrounded me every day, I express thanks to all the staff members of the National laboratory of Reference in Tuberculosis (LNRT) at NIH. I also thank all the personnel at the laboratory of Molecular Biology at NIH for their assistance and for their sympathy. I thank particularly ELOUDIYI H., LAMRABET S. and KASMI S. I express my special thanks to CHAOUI I. at CNESTEN for her help and scientific advice. I address my sincere acknowledgements to the Academy of Hassan II in Morocco for the funding of this project. I would like finally to thank every person who participated in the realization of this project.. Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(11) Doctorat Es Science Microbiology & Molecular Biology. Abstract. 2012. Abstract In spite of the availability of treatment and vaccine, tuberculosis (TB) remains a major public health concern and a great threat of humanity. Recently, an alarming situation has been emerged by the discovery of Multi and extensively drug resistant strains (MDR/XDR-TB). Worldwide, the health care providers face a vast problem for the rapid diagnosis of Mycobacterium tuberculosis (MTB), the causal agent of TB and MDR-TB strains. The conventional techniques for diagnosis of MTB are based on the microscopical examination, which lacks sensitivity and culture that requires several weeks of incubation. Furthermore, the conventional drug susceptibility testing (DST) takes long turnaround time and cumbersome procedure. This present study aim to evaluate the PCR based molecular techniques for the rapid and direct detection of MTB in clinical specimens by using the gene target hsp65 and the insertion sequence IS6110 for this purpose. Automated sequencing of hsp65 gene was used for the identification of MTB to species level. Moreover, the automated sequencing based PCR was also performed for the detection of mutations in specific genes (rpoB and katG) for rapid investigation of MDR-TB strains in sputum specimens. For better understanding the evolution of MTB strains, a comparative genome, proteome and phylogeny analysis was done by comparing the available whole mycobacterial genomes against each other and visualizing this comparisons in simple schemes presented by the definition of their pan and core genome, BLAST matrix and 16S r RNA phylogenetic tree. The results of molecular approach showed a good sensitivity and specificity of both targets hsp65 gene and the insertion sequence IS6110, with an excellent agreement between the culture (gold standard) and IS6110 PCR by applying Kappa index. Further, the sequencing of genes responsible of drug resistance rpoB and katG showed that MDR-TB cases were related to previously treated patients (relapse, failure, chronic and treatment abandon) and this will help the national TB program for establishing new strategies for the surveillance of those critical cases. Significantly, the computational and phylogeny analysis showed a high relatedness between the members of the complex MTB and a strong relationship between MTB and other pathogenic mycobacteria and this could provide a new insight of understanding the evolutionary events of those microorganisms and paving the way of new diagnosis targets.. Key words: Mycobacterium tuberculosis, polymerase chain reaction (PCR), diagnosis, evolution. Direct Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(12) Doctorat Es Science Microbiology & Molecular Biology. Abstract. 2012. Résumé Malgré la disponibilité des traitements et de vaccins, la tuberculose (TB) reste un problème majeur de santé publique et une grande menace de l'humanité. Récemment, une situation alarmante a été émergée par la découverte des souches multi-résistance aux médicaments (MDR-TB). Dans le monde, les personnels de santé sont confrontés à un vaste problème pour le diagnostic rapide de Mycobacterium tuberculosis (MTB), l'agent causal de tuberculose et de TB-MDR. Les techniques classiques de diagnostic de MTB sont basées sur l'examen microscopique, qui manque de sensibilité et de la culture qui nécessite plusieurs semaines d'incubation. En outre, les tests de sensibilité aux médicaments antituberculeux (DST) prennent du temps longue et procédure lourde. L’objectif de ce travail est d'évaluer les techniques basées sur la PCR pour la détection moléculaire rapide et directe de MTB dans les échantillons cliniques en utilisant le gène cible hsp65 et la séquence d'insertion IS6110. Le Séquençage automatisé du gène hsp65 a été utilisé pour l'identification de MTB à niveau de l'espèce. Par ailleurs, le séquençage automatisé basé sur la PCR a également été effectuée pour la détection de mutations dans des gènes spécifiques (rpoB et katG) pour la détection rapide des souches de MDR-TB dans les crachats Pour une meilleure compréhension de l'évolution des souches de MTB, une analyse comparative, génomique, protéomique et phylogénétique a été réalisée en comparant les génomes entiers disponibles des souches mycobactériennes uns contre les autres et la visualisation, présentées par la définition de pan core génome, la matrice BLAST et 16S r ARN arbre phylogénétique. Les résultats de l'approche moléculaire ont montré une bonne sensibilité et spécificité du gène hsp65 et la séquence d'insertion IS6110, avec une excellente accordance entre la culture et IS6110 PCR en appliquant l’indice de Kappa. En outre, le séquençage des gènes responsables de la résistance aux médicaments rpoB et katG a montré que les cas MDR-TB ont été liés à des patients préalablement traités (rechute, échec, chronique et l'abandon de traitement) et cela aidera le programme national de lutte antituberculeuse pour l'établissement de nouvelles stratégies pour la surveillance de ces cas critiques. De manière significative, l'analyse bioinformatique et la phylogénie ont montré une forte similarité entre les membres de la complexe MTB et une relation forte entre le MTB et d'autres mycobactéries pathogènes, ce qui pourrait donner un nouvel aperçu de la compréhension des événements évolutifs de ces microorganismes et ouvrant la voie de nouveaux cibles du diagnostic.. Les mots clés: Mycobacterium tuberculosis, PCR, diagnosis, evolution.. Direct Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
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(46) Doctorat Es Sciences in Microbiology & Molecular Biology. List of Abbreviation. 2012. List of abbreviations A: Adenine AIDS: Acquired Immunodeficiency Syndrome BACTEC MGIT 960: BACTEC Mycobacteria Growth Indicator Tube BACTEC 460 TB system: Fully Automated Instruments for Detection of M. tuberculosis BCG: Bacille Calmette–Guérin BLAST: Basic Local Alignment and Research Tool bp: Base Pair bPCR: Biotinylated PCR. C: Cytosine C°: Celsius degree CDST: Centre de Diagnostic de Santé de la Tuberculose CFU: Cell Forming Unit CMI: Cell-Mediated Immunity ddNTPs: Didesoxynucleotides DELM: Direction Epidémiologique de Lutte contre Maladies DNA: Deoxy Ribonucleic acid dNTP: Deoxy Nucleotides DOTS strategy: Directly Observed Therapy Strategy ED: Dense Electron layer EDTA: Ethylene Diamine Tetra-Acetic acid ELISA: Enzyme Linked Immunosorbent Assay ET: Electron Transparent layer g: Gram G: Guanine HIV: Human Immunodeficiency Virus HSP 65: Heat Shock Protein 65 INH: Institut National d’Hygiène L: Litre LAM: Lipoarabinomannan LiPA: Solid Phase Hybridization Assay Molecular Detection of MTB in the clinical specimens. F.ZAKHAM.
(47) Doctorat Es Sciences in Microbiology & Molecular Biology. List of Abbreviation. 2012. LJ: Löwenstein-Jensen medium MDR: Multi Drug Resistance mg: Milligram µg: Microgram MIC: Minimal Inhibitory Concentration min: Minute mL: Millilitre MTB: Mycobacterium tuberculosis MTBc: Mycobacterium tuberculosis complex NaOH: Sodium hydroxide NTM: Non Tuberculosis Mycobacteria PCR: Polymerase Chain Reaction PE: Glycine- rich Proteins, Pro-Glu pH: Hydrogen potential PM: Plasma Membrane PPE: Pro-Pro-Glu PRA : PCR- Restriction Enzyme Analysis qNRT- PCR: Quantitative Nested Real-Time PCR qPCR: Quantitative PCR. RD: Deleted Regions RFLP: Restriction enzyme Fragment Length Polymorphisme Sp: Species T: Thymine TB: Tuberculosis TBE buffer: Tris-Borate EDTA buffer TE buffer: Tris –EDTA WHO: World Health Organisation.. Molecular Detection of MTB in the clinical specimens. F.ZAKHAM.
(48) Doctorat Es Science in Microbiology & Molecular Biology. List of tables. 2012. List of tables Table 1: Master-Mix and the conditions of hsp65 gene PCR………………………………..42 Table 2: Master-Mix and the conditions of IS6110 PCR......………………………………...42 Table 3: Master-Mix and the conditions of rpoB gene PCR………………………………... 43 Table 4: Master-Mix and the conditions of KatG gene PCR………………………………...43 Table 5: Primers used for the realization of each PCR………………………………............44 Table 6: The conditions of the sequencing reaction…………………………………………45. Molecular Diagnosis of MTB in clinical specimens. F.ZAKHAM.
(49) Doctorat Es Science in Microbiology & Molecular Biology. List of Figures. 2012. List of figures Figure 1: Opened thorax of Egyptian mummy showed the presence of BK……………….6 Figure 2: double-barred cross, symbol of anti-tuberculosis crusade………………………..6 Figure 3: Model of mycobacterial cell envelope ………………………………………......12 Figure 4: Circular map of the chromosome of M. tuberculosis H37Rv ……………….......13 Figure 5: Scheme of the proposed evolutionary pathway of the tubercle bacilli..................16 Figure 6: Phylogenetic Position of MTB within the Genus Mycobacterium........................17 Figure 7: Principal genetic groups of the complex MTB......................................................17 Figure 8: Estimated incidence of tuberculosis in the world in 2011.....................................20 Figure 9: Estimated Incidence of TB in African Countries in 1990 and 2005…………….20 Figure 10: Pathogenesis of tuberculosis …………………………………………………..24 Figure 11: Mechanism of host defense against intracellular infection by mycobacteria ....24 Figure 12: Infected macrophages were acid-fast examined by light microscopy ………...30 Figure 13: Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium…....30 Figure 14: Steps of Spoligotyping method………………………………………………..34 Figure 15: Chromosome of MTB strains, genotyping by MIRUs…………………...........34. Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(50) Doctorat Es Science Microbiology & Molecular biology. List of appendix. 2012. List of appendix Appendix (1)……………………………………………………………………………..80 Appendix (2)……………………………………………………………………………..81 Appendix (3)……………………………………………………………………………..82. Molecular Diagnosis of MTB in clinical specimens. F.ZAKHAM.
(51) CONTENTS List of scientific production Dedication Foreword and Acknowledgment Abstract Resume List of abbreviation List of tables List of figures List of appendix Contents Problematic Hypothesis of thesis General introduction................ ………………………………………………….................1 Objectives of this thesis................……………………………………………………….....3 PART 1: Bibliographical review I. History of tuberculosis......................................................................................................4 I.1. History of disease......................................................................................................4 I.2. History of anti-tuberculosis therapy..........................................................................7 II. General bacterial characteristics…………………………………………………..........8 II.1. Taxonomy & classification of mycobacteria...........................................................8 II.2. General Characteristics of mycobacteria..................................................................9 II.3. Morphology………………………………………………………………………10 II.4. Mycobacterial cell envelop……………………………………………………….10 II.5. Metabolism of mycobacteria……………………………………………………..10 II.6. Genomics of mycobacteria..........................……………………………………...11 II.7. Molecular evolution of MTBC...............................................................................14 III. Epidemiology of tuberculosis………………………………………………………...18 III. 1. Tuberculosis in the world....................................................................................18 III. 2. Tuberculosis in Africa.........................................................................................19 III. 3. Tuberculosis in Morocco.....................................................................................19 IV. Immunology, Pathogenesis & Virulence …………………………….........................21 IV.1. Pathogenesis…………………………………………………………………….21 IV.2.Host Defenses……………………………………………………………………21.
(52) IV.3.Clinical Manifestations…………………………………………………………..22 IV.3.1. Pulmonary tuberculosis ……………………………………………………..22 IV.3.2.Tuberculous meningitis ……………………………………………………...23 IV.3.3. Miliary tuberculosis ………………………………………………………....23 IV.3.4. Renal and urogenital tuberculosis…………………………………………...23 IV.3.5 .Bone and joints tuberculosis ………………………………………………..23 V. Tuberculosis & Co-infection with HIV..........................................................................25 VI. Screening & Diagnosis of MTB.....................................................................................25 VI.1. Diagnosis of latent TB …………………………………………………………..25 VI.2. Diagnosis of active TB……………………………………………………...........26 VI.2.1. Radiological Examination..............................................................................26 VI.2.2. Bacteriological examination...........................................................................26 VI.2.2. A. Microscopic examination...............................................................27 Ziehl-Neelsen staining technique.................................................27 Auramine phenol technique..........................................................28 VI.2.2. B. Culture............................................................................................28 VI.2.2. C. Phenotypic and biochemical identification....................................28 VI.2.2. D. Drug susceptibility testing DST………………………………….29 VI.2.3. Molecular Examination.....................................................................................29 VI.2.3.A. DNA Amplification by PCR, including Real Time PCR……………….31 VI.2.3. B. Nucleic acid Probe……………………………………………………...32 VI.2.3.C. In situ Hybridization…………………………………………………….32 VI.2.3.D. Automated Sequencing…………………………………………………32 VI.2.3.E. Strain typing and DNA fingerprinting………………………………......32 VI.2.3.F. Microarray analysis………………………………………………………33 VII. Treatment and Control……………………………………………………………......35 IX. Drug resistance & extensively drug resistance...............................................................36. Study Plan...........................................................................................................................38 Review article (Publication I)……………………………………………………………...40. PART 2: Practical part A. Materials & Methods.
(53) I. Specimens Collection.......................................................................................................41 II. Decontamination of specimens…………………………………………………….......41 II.1. Decontamination of specimens for hsp65 amplification...........................................41 II.2.Decontamination of specimens for IS6110, rpoB and katG amplification................41 III.1. TE boiling extraction………………………………………………………….......41 IV. Reaction of PCR……………………………………………………………………...42 The amplification of hsp65 gene.............................................................................42 The amplification of the insertion sequence IS6110...............................................42 The amplification of the gene rpoB.........................................................................43 The amplification of the katG gene.........................................................................43 V. Visualisation of the PCR products………………………………………………........44 VI. Purification of the PCR products ………………………………………………........44 VII. Sequencing Reaction………………………………………………………………..44 VIII. Purification of the products of the reactions of sequences…………………….......45 IX. Automated Detection of the sequences……………………………………………...46 X. Analysis of the sequences………………………………………………………….....46. B- Results Chapter 1: (Publications II& III)…………………………………………………………47 Chapter 2: (Publications IV)……………………………………………………………...51 Chapter 3: (Publications V & VI)………………………………………………………...54. PART 3: General Discussion ………………………………………………………….....58. PART 4: Conclusion & Perspectives Conclusion …………………………………………………………………………...........66 Perspectives ……………………………………………………………………….............67. PART 5: Bibliographical References References ………………………………………………………………………………..68 Appendix………………………………………………………………………………....80. RESUME.
(54) Doctorat Es Sciences in Microbiology & Molecular Biology. Problematic. 2012. Problematic. Tuberculosis (TB) is a major public health problem over the world, with a high incidence rate in Morocco and has an impact on the socioeconomic status of the country, since it infects the youngest productive age group (15- 45 years old) as reported by the Ministry of health. In many cases, the achievement of TB management is difficult due to the lack or late diagnosis of the disease. The diagnosis is mainly based on the bacilloscopy and sometimes on culture if requested and even so, the long incubation time of these fastidious microorganisms pose another problem. Therefore, the early diagnosis and appropriate chemotherapy are the keys of TB management and the solutions for preventing the dissemination of the disease in the community.. Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(55) Doctorat Es Sciences in Microbiology and Molecular Biology. Hypothesis of study. 2012. Hypothesis of study Are molecular techniques appropriate for the rapid diagnosis and identification of MTB in clinical specimens in Morocco? Are molecular techniques appropriate for the detection of mutations in the genes associated with MDR-TB and management of drug resistant TB cases? Could the Study of the whole entire available genome sequences of mycobacterial strains allow building a new vision of the evolution of Mycobacterium?.
(56) Doctorat Es Science in Microbiology & Molecular Biology. General Introduction. 2012. General Introduction Tuberculosis (TB) is as ancient as humanity, killing million of people through the ages and remains a major public health problem. According to the latest reports of the World Health Organization (WHO), one-third of the global population is infected with Mycobacterium tuberculosis (MTB) the causative agent of TB and approximately 9 million new cases of TB arise annually and over 1.1 million deaths among HIV-negative people and 0.35 million deaths among HIV-positive people (WHO, 2011). Due to demographic factors, socio-economic status, neglected TB control and HIV epidemic, there are many undiagnosed TB cases (WHO, 2011). Unfortunately, most of patients in the developing countries do not have an access to TB diagnosis and treatment leading to the acquisition of MTB drug resistance or multi drug resistance MDR-TB. With the emergence of MTB/MDR strains, there is a crucial need for the rapid diagnosis of the microorganism, detection of MDR-TB strains and development of new anti-tuberculosis drugs(Brooks et al., 1998). The conventional laboratory diagnosis of tuberculosis is based on the method of ZiehlNeelsen acid –fast bacilli stain and culture of MTB (Cheesbrough, 2000; Gupte, 1999). ZiehlNeelsen stain is a rapid and cheap method but it lacks sensitivity. The culture is time consuming and requires viable microorganism, and this is a problem especially in the treated patients (Nagesh et al., 2001). Indeed, several rapid methods to diagnose MTB have been developed, such as DNA probes, and BACTEC system but they require sophisticated equipment. The polymerase chain reaction (PCR) can amplify a small fragment of DNA with high specificity for the diagnosis of infectious diseases (Abraham et al., 2012; Ani et al., 2009; De Wit et al., 1990; Saltini, 1998; Takahashi et al., 2007; Zakham et al., 2012). In the first part of our study, we evaluated the PCR as a rapid and direct molecular method for diagnosis of MTB in clinical specimens by targeting the gene hsp65 and the insertion sequence IS6110. The hsp65 gene is present in all mycobacteria (Ringuet et al., 1999) and the insertion sequence IS6110 is specific to the complex MTB (Thorne et al., 2011) and both of those targets are useful for the diagnosis. 1 Molecular Diagnosis of MTB in Clinical Specimens. F. ZAKHAM.
(57) Doctorat Es Science in Microbiology & Molecular Biology. General Introduction. 2012. Then, we compared the results of molecular approach with the results of conventional methods to evaluate the direct PCR diagnosis of TB in clinical samples. Moreover, the identification of MTB strains to species level was also performed by sequencing the hsp65 gene for some specimens and the resulted sequences were registered at NCBI (GenBank) and the accession numbers are available online. Further, for the direct detection of MDR-TB, both of genes rpoB and katG were used as targets for the investigation of the resistance against the main first line TB drugs Rifampicin and Isoniazid (Brooks et al., 1998). Automated sequencing of these genes allowed the identification of new mutations associated with drug resistance. This approach provides a same-day drug resistance diagnosis from culture and even clinical samples with high sensitivity and specificity (Neonakis et al., 2008). Studying the genomics of MTB and other non tuberculosis mycobacterial strains is crucial for better understanding the evolutionary events and consequently, the conception of their environmental niches, mechanisms of adaptation into human and animal being, pathogencity, virulence determinants that paved the way for appropriate conditions of survival within their hosts and the development of new tools of diagnosis and drug targets for better controlling those threatening diseases (Cole, 2002). Thus, a comparison between mycobacteral (tuberculosis and non tuberculosis) strains was described and phylogeny analysis performed by applying computational tools (pan and core genome, BLAST matrix, 16S ribosomal RNA phylogenetic tree) on a set of mycobacterial genomes collection, which could provide a key insight and a strong foundation for future investigations on the genetics, evolution, natural physiology, and virulence of these important pathogens.. 2 Molecular Diagnosis of MTB in Clinical Specimens. F. ZAKHAM.
(58) Doctorat Es Science in Microbiology & Molecular Biology. Objectives. 2012. Objectives of Thesis The main objective: Rapid and direct molecular diagnosis of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis and molecular detection of Multi Drug Resistance (MDR-TB) by PCR based techniques in clinical specimens.. Specific objectives:. 1. Evaluation of polymerase chain reaction (PCR) to amplify the MTB specific insertion sequence IS6110 and the gene hsp65 for identification of MTB. 2. Alignment of hsp65 gene sequences for the identification of MTB complex to the species level. 3. Determination of the specificity and the sensitivity of the PCR diagnostic techniques. 4. Comparison of the routine microbiological laboratory testing and molecular diagnosis for detection of MTB (validation of results). 5. Rapid identification of MDR-TB by DNA sequencing of rpoB and KatG genes for the characterization of the mutations responsible of drug resistance. 6. Comparison. of. entire. genomes. and. phylogenic. analysis. between. Mycobacterium tuberculosis and other mycobacterial (Pathogenic and environmental) species, which permits the study of more complex evolutionary events for better improving the techniques of diagnosis by uncovering new gene targets.. 3 Molecular Diagnosis of MTB in clinical specimens. F. ZAKHAM.
(59) Doctorat Es Science in Microbiology & Molecular Biology. I.. Bibliographical part. 2012. HISTORY OF TUBERCULOSIS (TB):. I.1. History of the disease: Tuberculosis (TB) is an ancient disease infected mankind since millennia and has a long history. Remarkably, it was fired by different terms; consumption, phthisis, scrofula, Pott's disease and the White Plague…etc. Moreover, the presence of Mycobacterium tuberculosis (MTB), the causal agent of TB in the tissues and skeletal remains show that prehistoric humans (5000 BC) had tuberculosis (Daniel, 2006). Additionally, tubercular decay and skeletal abnormalities, including characteristic of Pott’s deformities, had been detected in the spines of Egyptian mummies (Figure 1) and documented in several studies (Daniel, 2006), either by direct conventional microscopic examination (Zimmerman, 1977) or by the detection of mycobacterial DNA using molecular techniques (Donoghue et al., 2004; Nerlich et al., 1997; Ziskind and Halioua, 2007). The existence of pre- Colombian TB was also registered in America, particularly in the Peruvian mummies (Daniel, 2006). Furthermore, according to recent archeological studies carried out on the Siberian skeletal remains from the iron age and based on the single-nucleotide polymorphic loci PCR and the analysis of the regions of difference (RDs) of the MTB complex (MTBC), the presence of M. bovis in those remains was also confirmed (Taylor et al., 2007). Consequently the scientists attributed this persistence and survival to the high amount of lipids, mycolic acid in the mycobacterial cell wall and the high GC content of the DNA (Leao and Portaels, 2007). The term phthisis showed first in the Greek literature; In 460 BC, Hippocrates talked about TB under the term "phthisis," the Greek "decay" and provided the clinical information specific enough to pulmonary TB and described the disease as widespread and almost fatal (Leao and Portaels, 2007). Moreover, he mentioned that phthisis attacks young adults aged between 18- 35 years (Daniel, 2006). Significantly, the accurate pathological and anatomical description of TB began to appear during the 17th century by Franciscus Sylvius in his Opera Medica, published in 1679, in which was the first description of the progression of the lesions from tubercles to ulcers and cavities. The earliest references to the infectious nature of TB also appeared in 17th century Italian medical literature (Leao and Portaels, 2007). 4 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(60) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. In 1720, the English physician Benjamin Marten was the first to speculate, in his publication, A New Theory of Consumption that TB could be caused by "wonderfully minute living creatures," which could engender the lesions and symptoms of the disease (Leao and Portaels, 2007). The discovery of the stethoscope by Doctor Laennec in 1821 developed the first step toward the thoracic auscultation of TB patients (Daniel, 2006). Obviously, owing to the variety of TB symptoms and clinical manifestations, TB was not identified as a unified disease until the 1820s, and was not named tuberculosis until 1839 by Johann Lukas Schönlein (Leao and Portaels, 2007). In 1854, Hermann Brehmer thought that tuberculosis was a curable disease and the introduction of the sanatorium cure provided the first step toward treatment for tuberculosis. In 1865, the French military doctor Jean-Antoine Villemin demonstrated the contagiousness nature of TB by inoculating a small amount of purulent liquid from a tuberculous cavity in rabbits and he postulated that a specific microorganism caused the disease (Daniel, 2006). In 1882, Robert Koch, a German physician, announced that the causal agent of TB had been identified and cultivated; initially, Koch's discovery was rejected by the scientific world, before being accepted worldwide. In 1905 he received the Nobel Prize for Medicine and Physiology for his scientific research (Sakula, 1982). In the late 18th and 19th centuries, Europe was booming and had an industrial progress, while the TB endemic was peaked, which can be explained by the increase of the population in developing cities and malnutrition described by that time (Leao and Portaels, 2007). A further significant advance came in 1895 when Wilhelm Konrad von Rontgen discovered the radiation that now bears his name and actually the radiological examination is mandatory as the first step of diagnosis. As a result of the international efforts for combating against TB, the Central Bureau for the Prevention of Tuberculosis was founded in Berlin in 1902 and Dr. Gilbert Sersiron suggested that the fight against TB was comparable to crusade and it would be appropriate to adopt the emblem of a crusader, then the Duke of Lorraine, bearing the double-barred cross that signified courage and success to crusaders (Leao and Portaels, 2007) became the worldwide symbol of the fight against TB (Figure 2).. 5 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(61) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Figure 1: Opened thorax of Egyptian mummy Large arrow=pleural adhesions of right lung; small arrow= destruction of boney elements of lumbar vertebral bodies L4 and L5(Nerlich et al., 1997). Figure 2: double-barred cross, symbol of anti-tuberculosis crusade (Leao and Portaels, 2007).. 6 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(62) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Another important development was developed by the French bacteriologist Calmette and Guerin in 1921, who used specific culture media to lower the virulence of the bovine TB bacterium, creating the basis for the BCG (Bacille Calmette Guérin) vaccine, even though relatively ineffective still in widespread use today (Daniel, 2006). I.2. History of Anti tuberculosis Therapy: Before the introduction of antibiotics, many remedies were used for the treatment of TB, sometimes by offering good diets and herbal extracts. From 1943, the era of drug therapy was started, which created an important milestone in the history of TB and precisely in 1943 the discovery of paraamino salicylic acid (PAS) by Jorgen Lehmann and of thiosemicarbazone by Gerhard Domagk, yielded the first therapeutic agents with efficacy in the treatment of TB (Daniel, 2006). In 1944 Albert Schatz, Elizabeth Bugie, and Selman Waksman reported the utilization of streptomycin (SM), the first antibiotic and first bactericidal agent effective against MTB (Daniel, 2006). In 1952, Domag discovered isoniazid (INH), the first oral mycobactericidal drug, a key compound for the treatment of TB. The PAS + SM combination therapy then evolved into a triple therapy INH + PAS + SM, which provided curative effect on 90 to 90% of patients(Leao and Portaels, 2007). The problematic of the long duration (24 to 30 months) of treatment was a serious challenge and in 1960, the duration of treatment was reduced to 18 months after the replacement of PAS by ethambutol (EMB). The next step in the evolution of anti-tuberculous therapy was the discovery of rifampicin (RIF) in 1957 (Daniel, 2006).. In 1980, pyrazinamide (PZA) was introduced into the scheme of treatment and its introduction in the presence of INH and RIF allowed curing more than 95% of patients within 6 months.. 7 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(63) Doctorat Es Science in Microbiology & Molecular Biology. II.. Bibliographical part. 2012. GENERAL BACTERIAL CHARACTERISTICS. II.1. Taxonomy & Classification of Mycobacteria The causal agent of TB is a bacterium called Mycobacterium tuberculosis (MTB) or Koch Bacillus, which belongs to mycobacteria. Mycobacteria have been classified into the family Mycobacteriaceae within the order Actinomycetales based upon similarities in staining and motility, lack of spore formation and catalase production (Baron, 1996). However, in almost cases; the MTB is the causal agent of human TB, this illness can be caused by other Mycobacteria in some cases with special conditions (Brooks et al., 1998), these Mycobacteria belong to the members of MTB complex (MTBC), which are characterized in exception of Mycobacterium canetti, by the RD-9 deleted ancestry (Cole, 2002; Smith and Upton, 2011; Soo et al., 2006): •. Mycobacterium africanum: That can provoke human tuberculosis, principally in the. west of Africa (Demers et al., 2010). •. Mycobacterium bovis: which infects Badgers, cattle, deer, elephants, goats, lions, seals,. etc (Cole, 2002) and can spread to humans through inhalation of infectious droplet and by ingestion of raw milk. Furthermore, M. bovis has been associated with extrapulmonary tuberculosis in infants and children, generally occurring due to the consumption of milk, which had not been pasteurized or boiled, from infected cattle (Thoen et al., 2006), especially in the HIV infected infants (Houde and Dery, 1988). Therefore the zoonotic risk for human represents a serious problem, mostly for those living at the animal-human interface (Michel et al., 2011). •. Mycobacterium bovis, Bacille Calmette–Guérin (BCG): this has been largely used as a. vaccine against human TB (Bastos et al., 2009; Dietrich et al., 2003) and can be obtained by the attenuation of M.bovis and deletion of the RD1 region (Cole, 2002) •. Mycobacterium microti: which infect voles, Cats, dogs (Cole, 2002; Rufenacht et al.,. 2011) and human as reported recently in immunocompetent patients in France by Panteix et al.(Panteix et al., 2011). •. Mycobacterium canetti: which infects human in limited geographical location, in the horn. of Africa (Fabre et al., 2011). 8 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(64) Doctorat Es Science in Microbiology & Molecular Biology. •. Bibliographical part. 2012. Mycobacterium pinnipedii: that infects seals, sea lions and marine mammals (Bigi et al.,. 2005; Kiers et al., 2008). •. Mycobacterium caprae: which infects domestic animals such as goats, sheep, ewes,. camels, horses, pigs, dogs, cats, wild animal species and human being(Cvetnic et al., 2006; Mendoza et al., 2011; Rodríguez et al., 2011). •. Mycobacterium mungi sp. nov: that infects mongoose in Botswana and live in close. association with humans (Alexander et al., 2010). Furthermore, it has been demonstrated that these members are sharing a common ancestor in their evolutionary events (Hewinson et al., 2006; Zakham et al., 2011). It is worth mentioning that there are other mycobacteria called atypical mycobacteria or non tuberculosis mycobacteria (NTM). These mycobacteria are ubiquitous in the environment and in extreme circumstances (immunosuppression, HIV infection, underlying diseases……etc), some of them become pathogenic for humans and can induce opportunistic infections or mycobacterioses(Gupta et al., 2010). These mycobacteria often are described based on their growth rate and pigmentation with and without exposure to light (Baron, 1996). The last group of mycobacteria is represented by M leprae, the causal agent of leprosy in humans and characterized by the inability to be cultured in vitro (Kaplan and Cohn, 1986).. II.2. General Characteristics of Mycobacteria The mycobacteria are rod-shaped, aerobic bacteria that do not form spores. Although they do not stain easily, once it stained they resist decolorization by acid or alcohol and are therefore called “acid fast “bacilli (Brooks et al., 1998). Furthermore, Mycobacteria are characterized by the presence of long-chain fatty acids, called mycolic acids in their cell walls (Baron, 1996). Consequently, this unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence(Hotter et al., 2005).. 9 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(65) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. II.3. Morphology In tissue, tubercle bacilli are thin straight, curved rods measuring about 0.4×3µm and coccoid and filamentous forms are seen on artificial media, with variable morphology from one species to another. Mycobacteria can not be classified as either gram positive or gram negative. Once stained by basic dyes they can not be decolorized by alcohol, regardless of treatment with iodine. True tubercle bacilli are characterized by “acid fastness”, 95% ethyl alcohol containing 3% hydrochloric acid (acid –alcohol) quickly decolorizes all bacteria except mycobacteria (Brooks et al., 1998).. II.4. Mycobacterial cell envelop Essentially, there are four major layers in the cell envelope of mycobacteria. The first layer contains the cytoplasmic, or plasma membrane (PM, Figure 3). The second layer is called the electron dense (ED) layer because of its staining properties when observed by transmission electron microscopy. In this area is found the peptidoglycan/ arabinogalactan moieties that make up the basic structural component of the cell wall. Adjacent to this is a layer that appears to be electron transparent (ET layer) upon observation by transmission electron microscopy. One of the primary components in the ET layer is mycolic acid. In the outer layers of the cell envelope a variety of components can be found, depending upon which mycobacterial species is being considered. These areas are sometimes referred to as the L1 and L2 layers. The outer layers are the most important with regard to initial host interaction (Barrow, 1997). Other important wall components are trehalose dimycolate (so-called cord factor, as it is thought to induce growth in serpentine cords on artificial medium) and mycobacterial sulfolipids, which may play a role in virulence. Another constituent which may contribute to pathogenesis is lipoarabinomannan (LAM).. II.5. Metabolism of mycobacteria Mycobacteria, like most actinomycetes, can grow in vitro on an extremely wide range of carbohydrates. These oxidized carbon sources include alkanes, alcohols, ketones, and many mono-, di-, and tri-carboxylic acids.With the exception of several glycolytic enzymes, the hexose monophosphate shunt, the Entner–Doudoroff pathway, and phosphoglucose isomerase, carbon metabolism in mycobacteria has not been well characterized. Glycerol is 10 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(66) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. considered to be the preferred carbon source when mycobacteria are grown in vitro, although its utilization is blocked until all other carbon sources, including amino acids, have been consumed (Av-Gay and Sobouti, 2000; Masood et al., 1985). II.6. Genomics of Mycobacteria The first sequenced mycobacterial genome was that of M. tuberculosis H37Rv (Cole et al., 1998) and significantly, the genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high GC content that is reflected in the biased amino-acid content of the proteins. Two years latter this genome was re-annotated by Camus et al. (Camus et al., 2002). Annotation of the M. tuberculosis genome shows that this bacterium has some unique features and interestingly, a large part of its coding sequences is devoted to the production of enzymes involved in the metabolism of fatty acids. Over 200 genes are annotated as encoding enzymes for the metabolism of fatty acids, comprising 6% of the total. Among these are approximately 100 that are predicted to function in the oxidation of fatty acids and this could be related to the ability of this pathogen to grow in the tissues of the infected host, where fatty acids may be the major carbon source ( Figure 4). This genome is characterized also by the presence of two new families of glycine-rich proteins (PE and PPE families) with a repetitive structure that may represent a source of antigenic variation (Cole et al., 1998). Recently, different databases were established and provided the complete genome annotation of the reference strain and other tuberculosis strains, such: TubercuList and TB database (TBDB) (Galagan et al., 2010; Lew et al., 2011).. 11 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(67) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Figure 3: Model of mycobacterial cell envelope (Barrow, 1997). PM: The plasma membrane, ED: the electron dense, ET: electron transparent, L1 and L2: fibrillar layers, PM: membrane proteins, PG: peptidoglycan, AG: arabinogalactan, LAM: lipoarabinomannan, GPL: glycopeptidolipid, aGPL: apolar glycopeptidolipid.. 12 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(68) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Figure 4: Circular map of the chromosome of M. tuberculosis H37Rv (Cole et al., 1998).. The outer circle shows the scale in Mb, with 0 representing the origin of replication. The first ring from the exterior denotes the positions of stable RNA genes (tRNAs are blue, others are pink) and the direct repeat region (pink cube); the second ring inwards shows the coding sequence by strand (clockwise, dark green; anticlockwise, light green); the third ring depicts repetitive DNA (insertion sequences, orange; 13E12 REP family, dark pink; prophage, blue); the fourth ring shows the positions of the PPE family members (green); the fifth ring shows the PE family members (purple, excluding PGRS); and the sixth ring shows the positions of the PGRSsequences (dark red). The histogram (centre) represents G + C content, with <65% G + C in yellow, and >65% G + C in red.. 13 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(69) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. II.7. Molecular evolution of MTBC The MTBC is one of the most successful pathogens and the sufficient evidence of the common ancestor, evolution, demographical spread of these microorganisms was surrounded by a mystery (Wirth et al., 2008). A decade ago the scientists thought that the members of MTBc were evolved from M.bovis (Donoghue et al., 2004), but this hypothesis has been rejected. Recently, the ambiguity regarding this question mark was uncovered and a new scenario was demonstrated by studying 20 variable deleted regions within the members of the MTBC (Brosch et al., 2002) and based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and ‘‘modern’’ strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters (Brosch et al., 2002). Moreover, consecutive loss of DNA, reflected by region of difference RD9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the ancestor of the present MTB strains before TbD1 occurred (Figure 5). Significantly, M.canettii or the smooth MTB stated to be the common ancestor, which did not lack those regions, unlike M. bovis that lost several genes that are present in MTB and other smooth MTB (Brosch et al., 2002; Ernst et al., 2007). Therefore, M.canettii seems to be direct descendant of tubercle bacilli that existed before the M. africanum, M. bovis lineage separated from the MTB lineage and more recently the microarray results demonstrated that those deletions occurred for the ancestral genes whose function are not necessary for surviving (KatoMaeda et al., 2001) and the minimal set of essential genes are still conserved. Furthermore, it has become clear that the members of MTBC were originated from a single ancestor resulted from an evolutionary bottleneck and a clonal expansion (Brosch et al., 2002; Ernst et al., 2007; Gutierrez et al., 2005) occurred 20,000 to 35,000 years ago. In addition the progenitor of MTBC offspring was restricted in a limited geographical region (East Africa) and called “M. prototuberculosis”(Gutierrez et al., 2005).. 14 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(70) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Notably, most of the pathogenic or slow growing mycobacteria are sharing a high similarity and a strong phylogeny relationship (Figure 6) (Devulder et al., 2005; van Ingen et al., 2011) and interestingly, several studies confirmed that the pathogenic mycobacteria were originated from a free living progeny (Rastogi and Sola, 2007) and due to the genome reduction and the acquisition of new genes by horizontal gene transfer (HGT) (Jang et al., 2008; Rosas-Magallanes et al., 2006; Veyrier et al., 2009) and gene rearrangement, their capacity of parasitism and infectiousness was developed for enabling them to cause severe and dangerous illnesses. Remarkably, the pathogenic slow growing mycobacteria had undergone to genome reduction (Wassenaar et al., 2009) and consequently they have a single rRNA operon and a low number of tRNA, comparing with the RGM and this is due to their long division time (Ussery et al., 2009). In this context, the loss of genes played an important role in the evolution of slow growing mycobacterial pathogens (Brosch et al., 2001) and some of those genomes were suffered of an extensive genome downsizing like the complex of MLP (Cole et al., 2001). On otherwise the members of the complex MTB were subjected to moderate genome reduction and concomitantly acquired new genes towards the speciation to the parasitic life style in mammalian macrophages. In 1997, Sreevatsan used two non-synonymous SNP but functionally neutral to establish three major genetic groups called PGG1, PGG2 and PGG3 (PGG "Principal Genetic Group"). The two SNPs used are: the codon 463 (Leu463 Arg) of the katG gene encoding catalase-peroxidase KatG and codon 95 (Thr95Ser) gene gyrA encoding GyrA, the A subunit of gyrase (Figure 7)(Sreevatsan et al., 1997). These two SNPs are not involved in antibiotic resistance. All isolates of MTB can be classified in these three genetic groups. According to studies, it appears that PGG1 MTB groups are ancestral to those of PGG 2 and those of PGG 2 have the same ancestors of PGG3 (Gutacker et al., 2002). It is also worth mentioning that the PGG1 MTB strains are related to M. bovis, the causative agent of bovine tuberculosis, M. microti and M. africanum.. 15 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(71) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Figure 5: Scheme of the proposed evolutionary pathway of the tubercle bacilli illustrating successive loss of DNA in certain lineages (gray boxes). The scheme is based on the presence or absence of conserved deleted regions and on sequence polymorphisms in five selected genes (Brosch et al., 2002). Blue arrows indicate that strains are characterized by katG463. CTG (Leu), gyrA95 ACC (Thr), typical for group 1 organisms. Green arrows indicate that strains belong to group 2 characterized by katG463 CGG (Arg), gyrA95 ACC (Thr).The red arrow indicates that strains belong to group 3, characterized by katG463 CGG (Arg), gyrA95 AGC (Ser).. 16 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
(72) Doctorat Es Science in Microbiology & Molecular Biology. Bibliographical part. 2012. Figure 6: Phylogenetic Position of the Tubercle Bacilli within the Genus Mycobacterium(Gutierrez et al., 2005). Figure 7: Principal genetic groups of the complex MTB (Sreevatsan et al., 1997). 17 Molecular Diagnosis of MTB in Clinical Specimens. F.ZAKHAM.
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