PALAEOMICROBIOLOGICAL EVIDENCES OF THE ORIGIN OF MYCOBACTERIUM TUBERCULOSIS. BY TITUS OLUKITBI - Ph.D. Trainee

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1 PALAEOMICROBIOLOGICAL EVIDENCES OF THE ORIGIN OF MYCOBACTERIUM TUBERCULOSIS BY TITUS OLUKITBI - Ph.D. Trainee

2 PART A GENERAL OVERVIEW OF MYCOBACTERIUM TUBERCULOSIS

3 INTRODUCTION First isolated in They cause tuberculosis They are complex organisms and are regarded as MTBC They are aerobic bacilli, non spore forming, nonmotile, rod shaped, cell wall rich in lipid They are acid fast bacilli (Zeihl Neelsen staining technique) and slow growing Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN

4 Non-selective media Selective media Sautomated system (Sussana et al., 2015) CULTURE MEDIA

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8 DIAGNOSIS Blood Test Imaging Test Sputum Test TREATMENENT isoniazid pyrazinamide ethambutol (Myambutol) Rifampin (Rifadin)

9 PALEOMICROBIOLOGY EVIDENCE OF MYCOBACTERIUM TUBERCULOSIS: PART B OSTEOLOGICAL APPROACH MOLECULAR PERSPECTIVES

10 HELEN D. D Paleomicrobiology of Human Tuberculosis doi: /microbiolspec.poh , Barbier M, Wirth T The evolutionary history, demography, and spread of the Mycobacterium tuberculosis complex. Microbiol Spectrum 4(4):TBTB doi: /microbiolspec.tbtb PALEOPATHOLOGY OF TUBERCULOSIS Archaeological Reports of Tuberculosis around the World CHINA 5700 BP Egypt (3500 to 2650 BC) INDIA 3500 BP ITALY 3000 BC ARGENTINA AND CHILE 500 AD-1000 AD PERU 700 AD JAPAN 2000 BP THAILAND BP PERU 750 AD-1350 AD

11 HELEN D. D Paleomicrobiology of Human Tuberculosis doi: /microbiolspec.poh PALEOPATHOLOGY OF TUBERCULOSIS Skeletal Changes Indicative of Tuberculosis Tuberculosis of the spine Pott s disease Cold abscess phthisis, scrofula, King s Evil, Lupus vulgaris Periostitis hypertrophic ostearthropathy serpens endocrania symmetrica

12 Multisystemic tuberculosis in Peruvian mummy 900 AD Period Agarwal Y, Chopra RK, Gupta DK, Sethi RS. The tuberculosis timeline: Of white plague, a birthday present, and vignettes of myriad hues. Astrocyte 2017;4:7-26

13 An ancient Egyptian painting depicting a man afflicted with Pott's disease of the vertebral spine. Agarwal Y, Chopra RK, Gupta DK, Sethi RS. The tuberculosis timeline: Of white plague, a birthday present, and vignettes of myriad hues. Astrocyte 2017;4:7-26

14 Collapse of vertebral bodies due to TB Nicklisch, N., Maixner, F., Ganslmeier, R., Friederich, S., Dresely, V., Meller, H., Zink, A., & Alt, K. (2012). Rib lesions in skeletons from early neolithic sites in Central Germany: On the trail of tuberculosis at the onset of agriculture American Journal of Physical Anthropology, 149 (3), DOI: /ajpa.22137

15 Pathological lesions in (A) the rib, Marvele and (B) vertebrae, Sukioniai (the 8th- 11th Century AD Marina F. and Rimantas J.Paleopathological and molecular evidence of human bone tuberculosis in Iron Age Lithuania

16 Molecular Detection of Archaeological and Historical Mycobacterium tuberculosis Ancient DNA (adna) adna develops hydrolytic and oxidative lesions Hydrolytic depurination loss of G and A Hydantoins formation (blocks extraction during PCR) by oxidative damage HELEN D. D Paleomicrobiology of Human Tuberculosis doi: /microbiolspec.poh , Formation of Maillards

17 FEMS Microbiology Letters Volume 213, Issue 2, pages , 9 JAN 2006 DOI: /j tb11298.x Disaggregation of samples with ethylenediamine-tetra acetic acid (EDTA) and proteinase K. Disruption of samples with lysis buffer based on guanidium thiocyanate or hydrochloride, silica capture or of adna, washing, drying.

18 Ancient DNA (adna) Analysis Temperature, the ph at the site, the availability of water oxygen, the fluctuations of all these factors over time the nature of the infection at the time of death of its host.

19 Pre Next Generation Sequence Restriction Fragment Length Polymorphism (RFLP) method- IS16110 Spoligotyping Variable Number Tandem Repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) SNPs Genome-wide comparisons Lipids biomarkers NGS MOLECULAR METHODS

20 Sequences in the TBD1 region received from strains of different geographic regions Gordon SV, Brosch R, Billault A, Garnier T, Eiglmeier K, Cole ST (1999) Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 32(3):

21 Development of a Hierarchical Variable-Number Tandem Repeat Typing Scheme for Mycobacterium tuberculosis in China Luo T, Yang C, Pang Y, Zhao Y, Mei J, Gao Q (2014) Development of a Hierarchical Variable-Number Tandem Repeat Typing Scheme for Mycobacterium tuberculosis in China. PLoS ONE 9(2): e

22 Global phylogeny of 1,601 MTBC isolates inferred from a total of 91,648 SNPs spanning the whole genome. Barbier M, Wirth T The evolutionary history, demography, and spread of the Mycobacterium tuberculosis

23 Methods used in M. Tuberculosis adna and lipid biomarker diagnoses Donoghue, Helen, et al. "Positive Diagnosis of Ancient Leprosy and Tuberculosis Using Ancient DNA and Lipid Biomarkers." Diversity 9.4 (2017): 46.

24 Molecular Approach Principle Application Advantages Disadvantages Restriction Fragment Length Polymorphism (RFLP) method- IS16110 Fingerprintbased The IS6100 RFLP analysis has been widely used for M. tuberculosis molecular typing (van Embden et al., 1993) The study of bacterial DNA, unraveled fine-scale genetic structures, and clearly segregated sister strains, Cumbersome and laborious. Besides, differentiation of strains is strongly dependent on the number of IS6100 copies. It requires mycobacterial culture, lasting from 20 to 40 days. Spoligotyping Variable Number Tandem Repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) Space Oligotyping The measure of the number of repetitions at each locus It differentiates MTBC strains from other environmental mycobacteria and clearly separates M. bovis from M. tuberculosis. Fast, Reproducible and efficient typing It has less discriminatory power than IS6100, when present in high copy numbers spoligo patterns are CRISPR structures that play a role in Eubacteria and Archaebacteria defense against phages Genotyping high-resolution typing Only minor indels or polymorphisms occur in these sequences (Barbier and Wirth, 2016)

25 PART C CASE STUDIES When did MTBC emerged? Are the previous findings correct?

26 This study addresses the following issues Does Mycobacterium tuberculosis emerged from M. bovis How long has M. tuberculosis been in existence? Where is the origin of MTBC? How does human evolution affect the spread of M. tuberculosis

27 (Comas et al., 2013) Genome-based phylogeny of MTBC

28 Out-of-Africa and Neolithic expansion of MTBC (Comas et al., 2013)

29 CONCLUSION M. tuberculosis emerged years ago and predates Neolithic era M. tuberculosis emerged from Africa and spread across globe The spread of tuberculosis during Neolithic era was not as a result of zoonotic infection but human high population density due to civilization

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