Dissertationen zum Thema „Mycobacterium tuberculosis sequencing“

Tuberculosis has been an important disease throughout human history, shaping countless past populations. The archaeological study of the causative agents of tuberculosis, members of the Mycobacterium tuberculosis Complex (MTBC), is hindered by the non-diagnostic nature of tuberculosis-associated skeletal changes. As such, ancient DNA (aDNA) or palaeogenetic analyses have become an important tool for identifying tuberculosis in past populations. However, due to the age and variable preservation of aDNA, there are often issues with sporadic results and false negatives. The overall aim of the work presented here was to use different methods, including traditional target-specific PCR, to identify and detect tuberculosis aDNA in archaeological remains. The main objectives within this overarching aim were to first test a method called whole genome amplification (WGA), used to non-specifically amplify all the DNA within a sample, and its potential to improve the yield of aDNA from skeletal remains (Chapters 3 and 4). To determine the extent of its impact, WGA was used in a comparative context, where each archaeological sample analysed was separately subjected to two methods of MTBC detection - the traditional targeted PCR method and the same method assisted by the initial application of WGA. The results show that applying WGA before the traditional targeted PCR methodology to detect the presence of MTBC pathogens in skeletal remains is only useful and viable in some cases, likely depending on the age and preservation of the sample. The second objective was to use next generation sequencing to obtain more information on the aDNA composition of certain archaeological samples and answer questions beyond the scope of traditional target-specific PCR techniques (Chapter 5). Although most of the sequencing runs were variably unsuccessful, the composition of two samples, both known to probably contain tuberculosis aDNA, could be analysed. The samples both contained similar amounts of mycobacterial aDNA and varying amounts of both human and even potentially human intestinal flora DNA. Finally, the third objective was to determine if MTBC aDNA could be detected in a rib sample from Private William Braine of the lost Franklin Expedition using standard target-specific PCR (Chapter 6). In this case study, no evidence of tuberculosis ancient DNA was found. The work done through-out highlights the difficulties of ancient DNA research and, in Chapter 4, shows the importance of using more than a single sample to evaluate methods for application in palaeogenetic contexts.

Mycobacterium tuberculosis (Mtb) poses a global health catastrophe that has been compounded by the emergence of highly drug resistant Mtb strains. We used whole genome sequencing (WGS) to directly compare the accumulation of mutations in Mtb isolated from cynomolgus macaques with active, latent and early reactivation disease. Based on the distribution of single nucleotide polymorphisms (SNPs) observed, we calculated the mutation rates for these disease states. Our data suggest that during latency, Mtb acquires a similar number of chromosomal mutations as would be expected to emerge in a logarithmically growing culture over the same period of time despite reduced bacterial replication during latent infection. The pattern of polymorphisms suggests that the mutational burden in vivo is due to oxidative DNA damage. We next sought to determine why some strains of Mtb are preferentially associated with high-level drug resistance. We demonstrate that Mtb strains from the East Asian lineage acquire drug resistances in vitro more quickly than Mtb strains from the Euro-American lineage. Their higher drug resistance rate in vitro reflects a higher basal mutation. Moreover, the in vitro mutation rate correlates well with the bacterial mutation rate in humans as determined by whole genome sequencing of clinical isolates. Finally, using an agent-based model, we show that the observed differences in mutation rate predict a significantly higher probability of multi-drug resistance in patients infected with East Asian lineage strains of Mtb. Lastly, we sought to determine the mechanisms Mtb uses to proofread nascently polymerized DNA. Through fluctuation analysis of deletion mutants of two potential \(polIII\epsilon\) homologs, we demonstrate that neither is responsible for the maintenance of DNA replication fidelity. To explore the possibility that one of these homologs, Rv3711c, participates in an unknown redundant pathway, we used transposon capture and sequence (TraCS) to identify genes conditionally essential in an Rv3711c deletion mutant. Our analysis suggests that while Rv3711c does not participate in proofreading, it may act in an alternative novel DNA repair pathway. Taken together, our fluctuation analysis and TraCS data suggest that mycobacteria do not use canonical methods of proofreading to maintain genomic fidelity.

Mycobacterium tuberculosis (Mtb) remains a global challenge that has been worsened by the emergence of drug resistant strains of Mtb. We used publicly available Next Generation Sequencing (NGS) and drug susceptibility (DST) data to develop “Resistance sniffer”, an online software program for the rapid prediction of lineage and Mtb drug resistance. Based on the distribution of polymorphisms in the genomes of Mtb, we calculated the power of association between the polymorphisms in different clades of Mtb and resistance to 13 anti-TB drugs. Our data suggests that the development of drug resistance in Mtb is a stepwise process that involves the accumulation of polymorphisms in the Mtb genome. We carefully curated the polymorphisms based on their association powers to create a diagnostic key that captures patterns of these polymorphisms that can be used to predict lineage and drug resistance in Mtb. This diagnosis key was incorporated into the Resistance Sniffer tool, an online software program that we developed for the rapid diagnosis of drug resistance in Mtb. The tool was tested using sequence data from the South Africa Medical Research Council (SA-MRC). Our data suggests that the majority of the strains in SA may have been brought by the arrival of European settlers while the more resistant strains may have been introduced in the region by Asian travellers later on. We next sought to determine non-random associations between polymorphic sites in genomes of Mtb. Using the attributable risk (Ra) statistical methods, we distinguished between functional associations and associations that may have been due to genetic drift events for different Mtb clades. We then integrated the (Ra) data with drug susceptibility and annotation data to generate networks in Cytoscape 3.71. These networks were then used to infer evolutionary trajectories that drive the emergence and fixation of the drug resistant phenotype in different clades of Mtb. We demonstrate that strains from the Lineage 1.2 are associated with less complex functional associations compared to the strains from other clades such as the Asian and Euro-American clades. Our data also shows that the predisposition of strains from the Asian clades to develop multi-drug resistance may be attributed to a complex network of functional interactions of mutations in genes that are involved in several aspects of Mtb physiology such as cell wall modelling, lipid metabolism, stress response and DNA repair.
Dissertation (MSc)--University of Pretoria, 2019.
Biochemistry
MSc
Unrestricted

Mycobacterium tuberculosis, la bactérie causant la tuberculose, est un pathogène d'importance majeure à l'échelle mondiale. Depuis sa découverte en 1882 par Robert Koch, de nombreuses études se sont penchées sur les caractéristiques de cette bactérie et des souches proches, connues sous le nom de complexe Mycobacterium tuberculosis (MTBC). Dans le cadre de ce travail nous avons commencé par nous intéresser à l'espèce proche "Mycobacterium canettii", qui avait été identifiée au milieu du XXème siècle comme étant également capable de causer des cas de tuberculose chez l'Homme, tout en possédant des caractéristiques phénotypiques propres. Par le biais de l'étude de certains marqueurs phylogénétiques, nous avons pu établir que cette bactérie n'appartenait pas au MTBC au sens strict et pouvait donc être utilisée comme point d'ancrage dans le cadre de l'étude de la phylogénie et de l'émergence de ce dernier. C'est pourquoi nous avons choisi d'étudier la diversité de la collection de souches de "Mycobacterium canettii", qui proviennent toutes d'une même région du globe, la Corne de l'Afrique. L'étude de cette collection, construite au fil des ans par le Service de Santé des Armées (SSA), a permis de mettre en évidence l'émergence d'un groupe particulier de souches au sein de cette espèce, ainsi que d'obtenir des éléments permettant de préciser le positionnement du dernier ancêtre commun (MRCA) du MTBC. Du fait de l'origine géographique exclusive de ce taxon, nous avons ensuite décidé d'évaluer la diversité génétique des souches de Mycobacterium tuberculosis provenant de cette même région du globe. Cette seconde partie de l'étude, menée sur une collection à nouveau constituée par le SSA, a conduit à l'identification d'une nouvelle lignée au sein du MTBC, jusqu'alors inconnue. Cette découverte a un impact important sur la compréhension de l'émergence de Mycobacterium tuberculosis, car elle permet d'envisager un nouveau modèle d'apparition en interprétant cette lignée comme le descendant contemporain de l'écotype fondateur du MTBC. L'évolution de Mycobacterium tuberculosis peut ainsi être comprise suivant une progression liant "Mycobacterium canettii", pathogène occasionnel supposé environnemental, et cette nouvelle lignée. Une fois ce nouveau modèle proposé, nous avons tenté de le dater en extrapolant le taux de mutations observé lors d'évènements épidémiques contemporains, ce qui nous a permis de dater le MRCA du MTBC à environ 10 000 ans. Enfin nous avons mis en parallèle ces éléments concrets avec les connaissances paléo-ethnographiques actuelles concernant la Corne de l'Afrique pour proposer un modèle historiquement argumenté permettant d'expliquer la structuration phylogénétique actuelle du MTBC
Mycobacterium tuberculosis, the causative agent of tuberculosis, is a pathogen of world-wide impact. Since its discovery in 1882 by Robert Koch many studies have been focusing on the characteristics of this bacterium and of the most closely related strains known as the Mycobacterium tuberculosis complex (MTBC). In this work we started by studying the closest neighbor to the MTBC, the "Mycobacterium canettii" taxon, which is only found in one particular region of the world, the Horn of Africa. It t has been first identified in the middle of the XXth century as being able to cause tuberculosis in humans, but having at the same time peculiar phenotypic characteristics. Through the study of some phylogenetic markers we have been able to establish that this bacterium does not belong to the MTBC sensu stricto and can therefore be used as an outgroup in order to root the phylogeny to study the emergence of the MTBC. The next step was to study the genetic diversity of a collection of strains of "M. canettii",using the “next generation sequencing” (NGS) approach.. The analysis of this collection, built along the years by the French Army Health Service (SSA), has permitted to show the rapid emergence of a particular clone, as well as to get information enabling to precise the position of the most recent common ancestor (MRCA) of the MTBC. Because of the restricted geographic location of this species, it was also decided to assess the genetic diversity of strains of M. tuberculosis coming from the same part of the globe. This second part of the study, performed on a collection of strains also gathered by the SSA, has lead to the identification of a new, previously unknown, lineage of the MTBC. This discovery has a profound impact on the comprehension of the emergence of M. tuberculosis, as it permits to develop a new model of appearance by interpreting this lineage as the founder ecotype of the MTBC. The evolution of M. tuberculosis can therefore by understood along a path linking "M. canettii", opportunistic pathogen supposedly environmental, and this new lineage. After this proposal of a new model, we tried to date it by extrapolating

In 2013 an estimated 9 million patients were diagnosed with tuberculosis across the globe, leading to 1.5 million deaths. In the UK, just under 8,000 cases were notified. Where resources allow, tuberculosis control is based on the identification of outbreaks, and the timely diagnosis and appropriate treatment of infected patients. However, current methods for identifying tuberculosis outbreaks are limited in their specificity, whilst the definitive diagnostic tests remain culture-dependent and can hence take weeks before producing a result. Whole-genome sequencing (WGS) technology is now affordable, rapid and accurate, and in this thesis I explore its potential both for detecting transmission and for identifying the genetic variation underlying drug resistance. Understanding the degree of M. tuberculosis genetic diversity within and between epidemiologically related individuals is a prerequisite to using WGS to identify Mycobacterium tuberculosis transmission. In chapter 3 I outline how this diversity is rarely greater than 5 nucleotide variants and also describe how the pattern of genetic diversity within an outbreak relates to the epidemiologically recognised transmission patterns. In chapter 4 I apply the findings from chapter 3 to all tuberculosis cases in Oxfordshire over a 6-year period to show that although most patients with tuberculosis were born in a high-incidence country, the odds of transmission among UK-born patients are in fact greater. These findings have contributed to the decision by Public Health England to invest in the routine whole-genome sequencing of M. tuberculosis from 2015. In chapter 5 I explore whether the potential utility of future sequence data can be increased by also predicting phenotypic drug susceptibility. I therefore devise an algorithm to characterise relevant genetic variation associated with phenotypic drug resistance or susceptibility. I conclude that WGS has a significant contribution to make towards improving patient outcomes and decreasing onward transmission of disease.

Philosophiae Doctor - PhD
Mycobacterium tuberculosis, the causative agent of tuberculosis, is estimated to infect approximately one-third of the world’s population and is responsible for around 2 million deaths per year. The disease is endemic in South Africa which has one of the world’s highest tuberculosis incidence and death rates. The M. tuberculosis Beijing genotype are characterised by having an enhanced virulence capability over other M. tuberculosis strains and are the predominant strain observed in the Western Cape of South Africa. DNA methylation is a largely untapped area of research in M.tuberculosis and has been poorly described in the literature especially given its connection to virulence despite it being well characterised along with its role in virulence in other pathogenic bacteria such as E.coli. The overall aim was to characterise a global DNA methylation profile for two M. tuberculosis Beijing strains, hyper-virulent and hypo-virulent, using single molecule real time sequencing data technology. Moreover, to determine if adenine methylation in promoter regions has a possible functional role. This study identified and characterised the DNA methylation profile at the single nucleotide resolution in these strains using Pacific Biosciences single molecule real time sequencing data. A computational approach was used to discern DNA methylation patterns between the hyper and hypo-virulent strains with a view of understanding virulence in the hyper-virulent strain. Methylated motifs, which belong to known Restriction Modification (RM) systems of the H37Rv referencegenome were also identified. N6-methyladenine (m6A) and N4-methlycytosine (m4C) loci were identified in both strains. m6A were idenitified in both strains occuring within the following sequence motifs CACGCAG (Type II RM system), GATNNNNRTAC/GTAYNNNNATC (Type I RM system), while the CTGGAGGA motif was found to be uniquley methylated in the hyper-virulentstrain.Interestingly, the CACGCAG motif was significantly methylated (p = 9.9 x10 -63) at a higher proportion in intergenic regions (~70%) as opposed to genic regions in both the hyper-virulent and hypo-virulent strains suggesting a role in gene regulation. There appeared to be a higher proportion of m6A occuring in intergenic regions compared to within genes for hyper-virulent (61%) and hypo-virulent (62%) strains. The genic proportion revealed that 35% of total m6A occurred uniquely within genes for the hyper-virulent strain while 27.9% for uniquely methylated genes in hypo-virulent strain.

A tuberculose é uma doença infectocontagiosa causada por bactérias do complexo Mycobacterium tuberculosis (MTBC) que afeta humanos e/ou animais. Membros desse complexo evoluíram clonalmente e possuem grande similaridade genômica, diferenciando-se por polimorfismos de nucleotídeo único (SNPs) e regiões de diferença (RDs). Dentre os patógenos da tuberculose em animais, Mycobacterium bovis, causador da tuberculose bovina, é o membro do MTBC de maior importância global. Desta maneira, o presente estudo tem por objetivo o sequenciamento, a anotação e a análise da estirpe brasileira SP38 de M. bovis, seguido da genômica comparativa desse com outros genomas de M. bovis depositados no GenBank. Mycobacterium bovis SP38 apresenta um genoma tradicional de micobactéria tuberculosa, sendo esse único, circular com 4.347.646 pb, alto conteúdo de GC (65,6%) e 4.216 genes, incluindo 154 pseudogenes, 3 genes de rRNA (RNA ribossomal), 45 de tRNA (RNA transportador), 2 de ncRNA (RNA não codificante), 1 tmRNA (RNA transferência-mensageiro) e 4.011 sequências de DNA codificante (CDSs) (NZ_CP015773.1). Dentre as CDSs, a maioria (2.805 - 69,93%) foi anotado com função e 1.206 (30,07%) como hipotéticos. Para a genômica comparativa, os 31 genomas completos ou em drafts de M. bovis depositados no GenBank, 32 genomas de Mycobacterium bovis BCG e 23 genomas de Mycobacterium tuberculosis foram selecionados. Análises in silico dos padrões de RD resultaram na exclusão de três genomas anotados equivocadamente como M. bovis virulentos. A análise de genes ortólogos sugere que M. bovis está sob processo de decay genômico. A quantificação de sítios polimórficos indica uma maior variabilidade genética em números totais (8.335 em M. tuberculosis, 3.448 em M. bovis virulentos, e 1.088 em M. bovis BCGs) e comparações par-a-par (p ≤0,05) de M. tuberculosis em relação a M. bovis virulentos e BCGs, indicando uma maior pressão evolutiva sob M. tuberculosis, contrastando com o fato de que M. bovis é capaz de infectar um maior número de espécies hospedeiras que M. tuberculosis. A maioria desses sítios polimórficos estão localizados em CDSs hipotéticos (31,7% - 51,3%), sendo associados a família gênica PE/PPE, e apresentam uma proporção de mutações não sinônimas crescentes pela ordem M. bovis BCG, M. bovis virulentos e M. tuberculosis (48,90%, 51,92% e 59,52%, respectivamente). Essa menor proporção de mutações não sinônimas e a categorização funcional dissimilar entre CDSs contendo sítios polimórficos, indica que M. bovis BCG está sujeito a diferentes pressões seletivas quando comparado a M. bovis virulentos e M. tuberculosis. Por fim, a análise filogenética baseada em sítios polimórficos indica agrupamentos filogenéticos de M. bovis suportados pela classificação dos Complexos Clonais (CCs) e não por hospedeiros de origem dos isolados, confirmando que sítios polimórficos podem ser utilizados para classificação filogenética de linhagens genéticas desta espécie bacteriana. Além do mais, 2/28 (7,14%) genomas de M. bovis não puderam ser classificados nos CCs atualmente descritos, sugerindo a existência de complexos ainda não determinados. Este estudo representa o primeiro genoma de uma estirpe nacional de M. bovis a ser completamente sequenciado e a primeira análise de genômica comparativa de genomas desta espécie bacteriana.
Tuberculosis is an infectious disease caused by bacteria of the Mycobacterium tuberculosisComplex (MTBC) that affects human beings and/or animals. Members of this complex clonally evolved and have high genomic similarity, differentiated by single nucleotide polymorphisms (SNPs) and regions of difference (RDs). Among the animal tuberculosis pathogens, Mycobacterium bovis, the causative agent of bovine tuberculosis, is the MTBC member of greatest global importance. Therefore, the aim of the present study is to sequence, assemble and annotate the genome of the Brazilian strain SP38 of M. bovis, followed by the comparative genomics with other M. bovis genomes available in GenBank. Mycobacterium bovis SP38 has a traditional mycobacteria genome. It has a single and circular chromosome with 4,347,646 bp, high GC content (65.6%), and 4,216 genes, including 154 pseudogenes, 3 rRNA genes (ribosomal RNA), 45 tRNA (transfer RNA), 2 ncRNA (non-coding RNA), 1 tmRNA (transfer-messenger RNA), and 4,011 coding DNA sequences (CDSs) (NZ_CP015773.1). The majority of CDSs (2,805 - 69,93%) was annotated with function and 1,206 (30,07%) are hypothetical. For the comparative genomics analyses, the 31 genomes (complete and drafts) of M. bovis available in GenBank, 32 Mycobacterium bovis BCG and, 23 of Mycobacterium tuberculosis were chosen. In silico analysis of the RDs patterns resulted in the exclusion of three genomes, mistakenly annotated as virulent M. bovis. Orthologous gene analysis suggests that strains of M. bovis are under genomic decay. The quantification of polymorphic sites indicates the greater variability in absolute numbers (8,335 in M. tuberculosis, 3,448 in virulent M. bovis, and 1,088 in M. bovis BCG) and in pairwise comparisons (p≤0,05) of M. tuberculosis compared to virulent M. bovis and M. bovis BCG, suggesting that M. tuberculosis is under high evolutionary pressure. This is in contrast to the fact that M. bovis is capable of infecting a higher number of host species than M. tuberculosis. Most of these polymorphic sites are located in hypothetical CDSs (31.7% - 52.3%), being associated with PE/PPE family, and demonstrating a nonsynonymous mutations proportion of the following increasing order: M. bovis BCG, virulent M. bovis and M. tuberculosis (48.90%, 51.92% and 59.52%, respectively). This lower proportion of nonsynonymous mutations and the dissimilar functional categorization of CDSs with polymorphic sites indicates that M. bovis BCG is subjected to different selective pressure when compared to virulent M. bovis and M. tuberculosis. Finally, the phylogenetic analysis based on polymorphic sites indicates that the phylogenetic grouping of M. bovis is supported by Clonal Complexes (CCs), and not by the host of M. bovis isolates, confirming that polymorphic sites can be used for phylogenetic classification of genetic lineages of this bacterial species. Furthermore, 2/28 (7.14%) genomes of M. bovis could not be classified in the currently described CCs, suggesting the existence of complexes yet to be determined. This study represents the first genome of a Brazilian strain of M. bovis to be completely sequenced and the first comparative genomic analysis of the genomes of this bacterial species.

La capacidad de Mycobacterium tuberculosis para sobrevivir dentro del macrófago contribuye grandemente a su patogenicidad, latencia y persistencia durante la infección. Este bacilo induce alteraciones en el ambiente intrafagosomal e inhibe la maduración del fagosoma, favoreciendo su supervivencia intracelular. M. tuberculosis PknG secuestra al macrófago precisamente al evitar la fusión fagosoma-lisosoma. En este sentido, PknG representa una familia de dianas novedosas para enfrentar la necesidad de nuevos antimicrobianos para la tuberculosis latente. Aquí, apuntamos a: (i) elucidar la base estructural-molecular del ATP y Mg2+ como cofactores de PknG; (ii) caracterizar los parámetros cinéticos que gobiernan la formación del complejo PknG:ATP; e, (iii) identificar péptidos capaces de unirse a PknG para investigar experimentalmente su interactoma usando enfoques combinatorios como “Phage Display”. Nuestros resultados confirman que PknG se une exclusivamente al ATP con una constante de disociación (KD) de 108.8  22.9 µM. El Mg2+ estabiliza térmicamente a PknG de forma ATP-dependiente. Análisis de estado pre-estacionario muestran que la unión y disociación del ATP es rápida en el complejo PknG:ATP. Usando PknGN-Ext, TPR resolvimos la estructura cristalina en el estado unido al ADP mientras que demostramos que el ATP imposibilita la cristalización. Los análisis bioinformáticos de las librerías enriquecidas por Phage Display identificaron 57 potenciales peptidos que interactuarían con PknG. Una comparación cercana con el proteoma de M. tuberculosis proporcionó un subconjunto de 20 proteínas que podrían interactuar con PknG. Nuestros resultados confirmaron cinco proteínas asociadas a PknG previamente reportadas: PknG, DnaK chaperona, transportador ABC Rv1747, Proteína Ribosomal L23 y Factor de Elongación Tu, resaltando la validez de nuestra plataforma para descubrir el interactoma de PknG. Así, nuestros resultados revelan interacciones proteína-proteína putativas que podrían participar en la supervivencia micobacteriana, mientras que también proporcionan bases sólidas para desarrollar drogas antituberculosas al interrumpir estas interacciones o explotar estos peptidos tipo compuesto líder.
The ability of Mycobacterium tuberculosis to survive inside the macrophage greatly contributes to its pathogenicity, latency and persistence during infection. This bacillus induces alterations in the intraphagosomal environment and inhibits phagosome maturation, thus promoting mycobacterial survival. M. tuberculosis PknG hijacks the macrophage precisely by avoiding phagosome-lysosome fusion. In this sense, PknG represents a family of novel targets to cope with the need for new antimicrobials for latent tuberculosis. Here, we aimed to: (i) elucidate the structural-molecular basis of ATP and Mg2+ as PknG cofactors; (ii) characterize the kinetic parameters governing PknG:ATP complex formation; and, (iii) identify PknG-binding peptides to experimentally query PknG’s interactome using combinatorial approach such as Phage Display. Our results confirm that PknG exclusively binds to ATP with a dissociation constant (KD) of 108.8  22.9 µM. Mg2+ thermally stabilizes PknG in an ATP-dependent manner. Pre-steady-state analyses show that ATP binding and dissociation are rapid in the PknG:ATP complex. Using PknGN-Ext, TPR we solved the ADP-state crystal structure while showing that ATP precludes crystallization. Phage Display and bioinformatic analyses identified 57 potential PknG binders. A close comparison to the M. tuberculosis proteome provided a subset of 20 proteins that may interact with PknG. Our results confirmed five previously reported PknG-associated proteins: PknG, DnaK chaperone, ABC transporter Rv1747, Ribosomal Protein L23 and Elongation Factor Tu, highlighting our platform’s validity to uncover the PknG interactome. Altogether, our results reveal putative protein-protein interactions that may play a role in mycobacterial survival, while also providing solid bases for the development of anti-tuberculosis drugs by disrupting these interactions or exploiting these lead-like peptide molecules.
Tesis

>Magister Scientiae - MSc
Next generation sequencing (NGS) technology platforms have accelerated ability to produce completed genome assemblies. Recently, collaborators at Tygerberg Medical School outsourced the sequencing of Oryx bacillus, a member of the Mycobacterium tuberculosis complex (MTC). A total of 31,271,059 short reads were generated and required filtering, assembly and annotation using bioinformatics algorithms. In this project, an NGS assembly pipeline was implemented, tailored specifically for SOLiD sequence data. The raw reads were aligned to seven fully sequenced and annotated MTC members, namely, Mycobacterium tuberculosis H37Rv, H37Ra, CDC1551, F11, KZN 1435, Mycobacterium bovis AF2122/97 and Mycobacterium bovis BCG str. Pasteur 1173P2 using NovoalignCS. Depth and breadth of sequence coverage across each base of the reference genome was calculated using BEDTools, and structural variation. Structural variation at the nucleotide level including deletions, insertions and single nucleotidepolymorphisms (SNPs) were called using three tools, GATK, SAMtools and Nesoni. These variations were further filtered using in-house PERL scripts. Putative functional roles for the alterations at the DNA level were extrapolated from the overlap with essential genes present in annotated MTC members. Approximately 20,730,631 short reads (59.78%) out of a total of 31,271,059 reads aligned to the seven reference genomes. The per base sequence coverage calculations revealed an average of 1,243 unaligned regions. These unaligned regions overlapped with mycobacterial regions of difference (RD) and genetic phage elements acquired by the MTC through horizontal gene transfer and are genes prevalent in the clinical isolates of M. tuberculosis. A total of 2,680 genetic variations were identified and categorised into 845 synonymous and 1,724 non-synonymous SNPs together with 44 insertions and 67 deletions. Some of the variant alleles overlapped known genes to be involved in TB drug resistance. While the biological significance of our findings remain to be elucidated, it nonetheless deserves further attention, because SNPs have the potential to impact on strain phenotype by gene disruption. Therefore, any hypotheses generated from these large-scale analyses will be tested by our collaborators at Tygerberg medical school.

Le phénomène émergent de la tuberculose multirésistante et ultrarésistante est un problème de santé publique à l’échelle mondiale. Dans les pays en développement, ce problème est accru du fait que les laboratoires de diagnostic de la tuberculose manquent d’équipement et d’outils de diagnostics pour identifier ces cas pour prescrire une chimiothérapie adaptée. La première partie de ce travail de doctorat a permis à travers le séquençage du locus pncA, de mettre en évidence que la résistance au Pyrazinamide survient généralement et de manière significative lorsque la souche est multirésistante, c’est-à-dire après l’acquisition de la résistance à la Rifampicine et à l’Isoniazide. Le pourcentage des souches résistantes au PZA est même plus élevé chez les souches MDR résistantes aux FQs. Dans la seconde partie de l’étude, nous proposons une méthode alternative à la culture de bacilles dans un environnement confiné de type P3. À partir d’échantillons cliniques non cultivés (expectoration) et grâce au GeneXpert MTB/RIF, au séquençage de gènes et au spoligotypage, nous avons pu identifier 19 souches multirésistantes, une transmission active de souches sensibles appartenant aux clades LAM10, T1, MANU, H3 et enfin une épidémie sous-jacente de 5 souches Beijing multirésistantes
The emerging phenomenon of the MDR and XDR-TB is a worldwide public health issue. In developing countries, this problem is amplified due to the fact that TB diagnostic laboratories lack equipment and diagnostic tools to identify these cases and therefore prescribe appropriate chemotherapy. In the first part of this doctoral work, the sequencing of the pncA gene allowed us to show that the resistance to Pyrazinamide occurs significantly when the strain is MDR, corresponding to the acquisition of resistance to Rifampicin and Isoniazid; and that after the acquisition of Fluoroquinolones and to injectable antibiotics of second line (Amykacine, Kanamycine, Capreomycine) resistance by MDR strains, this rate increases even more. In the second part of the study, we propose an alternative method to the culture of bacilli in a BSL3 confined environment. From uncultivated clinical samples (sputum) and through GeneXpert MTB/RIF, sequencing of genes and spoligotyping, we identified 19 MDR strains, active transmission of sensitive strains belonging to clades LAM10, T1, MANU, H3 and finally as well as an underlying epidemic of 5 Beijing MDR strains.In the first study, 272 retrospective samples of Mycobacterium tuberculosis isolates were selected from two large cosmopolitan cities: Northern Paris (Bichat-Claude Bernard Hospital, 101 strains) and Southwest of Shanghai (Songjiang district, 171 Strains). These strains were selected according to their known phenotypic sensitivity to Rifampicin (RIF) and Isoniazid (INH). These phenotypic resistances were confirmed by the HAIN genotype analysis tools MTBDRplus and by the sequencing of the rpoB and katG/inhA genes. To determine the extensively drug resistance strains (XDR), we sequenced the gyrA/gyrB and rrs genes to identify genetic mutations associated with resistance to Fluoroquinolones (FQs) and second-line injectable antibiotics: Amikacin (AMK)-Kanamycin ( KAN)-Capreomycin (CAP), respectively. Finally, we sequenced the pncA gene of all isolates to identify the genetic mutations associated with resistance to Pyrazinamide (PZA). The strains were genotyped by spoligotyping and MIRU-VNTR.In the second study, from October 2014 to February 2015, 159 morning sputum samples with smear-positive smear after Ziehl-Neelsen staining were collected at the three main diagnostic laboratories for tuberculosis in Libreville, Gabon. These clinical samples were transported to the National Laboratory of Public Health in Libreville for analysis with the GeneXpert MTB/RIF automaton to confirm the microscopic diagnosis and to determine the resistance of bacilli to Rifampicin. Of the 159 samples, 29 samples had a sputum volume less than 1 ml, the minimum required according to the manufacturer's recommendations. For the 130 sputum samples analyzed by the GeneXpert automaton, 375 μl of the remaining GeneXpert solution not introduced into the cartridge was introduced into a 50 ml conical tube containing 25 ml of phosphate buffer (autoclaved solution) to neutralize the pH of the GeneXpert solution. The conical tube is centrifuged for 15 minutes at 4,500 rpm, the pellet is taken up in 100 μl of TE and then transferred to a 100 μl microtube which is subsequently heated for 30 minutes at 90°C. After a cycle of freezing (-40 ° C. for 1 h)-defrosting, the microtube is briefly centrifuged and the supernatant is transferred to a new microtube. From this new microtube we amplified by PCR and then sequenced the rpoB, katG/inhA, pncA, gyrA, rrs and rpsL genes to identify mutations associated with resistance to Rifampicin, Isoniazid, Pyrazinamide, Fluoroquinolones, Antibiotics in second lines: Amikacin-Kanamycin-Capreomycin and Streptomycin (SM), respectively. All the samples were genotyped by the multiplexed spoligotyping applied to the Luminex MagPix

Tuberculosis remains a major public health issue globally, with an estimated 9.2 million new cases in 2006. A new threat to TB control is the emergence of drug resistant strains. These strains are harder to cure as standard anti-tuberculosis first line treatments are ineffective. Multi Drug Resistant Tuberculosis (MDR-TB) is defined as Mycobacterium tuberculosis that has developed resistance to at least rifampicin and isoniazid, and these strains now account for greater than 5% of worldwide cases. Mutations within the Rifampicin Resistance Determining Region (RRDR) of the rpoB gene are present in greater than 95% of strains that show rifampicin resistance by conventional drug susceptibility testing. As rifampicin mono resistance is extremely rare, and rifampicin resistance is usually associated with isoniaizd resistance, the RRDR region of the rpoB gene is a very useful surrogate marker for MDR-TB. Many molecular assays have been attempted based on this theory and have had varied levels of success. The three methods evaluated in this study are DNA sequencing of the rpoB, katG and inhA genes, the Genotype MTBDRplus line probe assay (Hain Lifesciences) and a novel method incorporating Real-Time PCR with High Resolution Melt analysis targeted at the RRDR using the Rotorgene 6000 (Corbett Lifesciences). The sensitivity for the detection of rifampicin resistance was far better using DNA sequencing or the commercially available line probe assay than detection by the Real-Time PCR method developed in this study.

La tuberculose (TB), provoquée par Mycobacterium tuberculosis, est une des trois maladies prioritaires dans le monde. Les TB multi-résistantes (MDR) et ultra-résistantes (XDR-TB) représentent des obstacles majeurs pour la lutte antituberculeuse. Dans les pays à MDR-TB élevée, comme le Vietnam, la détection insuffisante de la résistance aux antibiotiques est un des facteurs principaux qui favorisent la transmission des souches résistantes. De plus, dans ces pays, encore très peu de choses sont connues sur la résistance à la pyrazinamide et aux antibiotiques de seconde ligne et sur les déterminants génétiques liés à ces résistances. Dans ce contexte, ce travail vise donc à acquérir des connaissances sur la résistance aux antibiotiques au Vietnam et à étudier comment M. tuberculosis évolue de l’état sensible à l’état ultra-résistant.260 isolats cliniques collectés au Vietnam entre 2005 et 2009 ont été inclus. Diverses techniques et analyses ont été utilisées: tests de sensibilité aux médicaments (développement d'un test à temps réduit), spoligotypage et MIRU-VNTR (24 loci) et séquençage de gènes. Les données ont été analysées par des analyses statistiques et phylogénétiques. Ce travail s’est d’abord focalisé sur la caractérisation d’isolats hautement résistants et sur la résistance à la pyrazinamide. Une forte proportion d'isolats quadruple résistants aux antibiotiques de première ligne a été identifiée comme pré-XDR et XDR et en majorité appartenant à la famille Beijing. L'analyse moléculaire a également révélé une forte proportion d'isolats, en particulier MDR, quadruple résistants et de la famille Beijing, portant des mutations associées à la résistance à la pyrazinamide.L'analyse génétique et phylogénétique globale a ensuite montré une grande diversité de profils de mutations dans chaque famille et chaque cluster MIRU-VNTR. Ces données suggèrent que M. tuberculosis peut suivre des chemins évolutifs variés pour devenir ultra-résistant. La prédominance de mutations et de combinaisons de mutations associées à un haut niveau de résistance et à un faible coût en termes de fitness suggère un effet cumulatif des mutations et un rôle de l’épistasie dans l'acquisition de la résistance multiple. De plus, une fréquence élevée de mutations compensatoires associées à la résistance à la rifampicine a été détectée chez les isolats très résistants. Ces processus semblent donc influencer fortement l'évolution de la résistance dans notre échantillon. Il est à noter que les mutations liées à des niveaux de résistance élevée et à de faibles coûts en termes de fitness, ainsi que les mutations compensatoires étaient plus particulièrement associées à la famille Beijing.En conclusion, ce travail fournit des connaissances uniques sur la résistance aux antibiotiques chez M. tuberculosis au Vietnam. En particulier, ces données prédisent une évolution de la résistance vers une situation de plus en plus préoccupante. Premièrement, la famille Beijing, en cours d’invasion au Vietnam, apparaît associée à de hauts niveaux de résistance, de faible coût en termes de fitness et aux mutations compensatoires. Deuxièmement, le risque élevé de résistance à la pyrazinamide remet en question son efficacité et son utilisation dans les traitements contre la MDR et la XDR-TB. Troisièmement, les données suggèrent une évolution de M. tuberculosis vers un potentiel de résistance plus élevé par effet cumulatif des mutations associés à la résistance et l’existence de phénomènes d’épistasie. Comme les échantillons étudiés dans ce travail ont été collectés, l’étape suivante est de valider nos hypothèses sur des données actualisées.Enfin, ce travail avec les données déjà publiées a permis d’établir, pour la première fois, un inventaire des mutations associées à la résistance aux antibiotiques chez M. tuberculosis au Vietnam. Cette base de données sera utilisée pour le développement d'une puce à ADN pour la détection rapide de la résistance aux antibiotiques au Vietnam
Tuberculosis (TB) is one of the deadliest infectious diseases worldwide, mainly caused by Mycobacterium tuberculosis. Multidrug resistant (MDR) and extensively drug resistant (XDR) TB are currently main challenges for TB control. In high MDR-TB burden countries like Vietnam, one of the main factors of drug resistant strain spread is the insufficient capacity of drug resistance detection. Besides, still little is known in these countries about the resistance to second line and pyrazinamide drugs (key drugs in the MDR-TB treatment) and the genetic determinants linked to these resistances. In this context, this work aimed to acquire knowledge on drug resistance in Vietnam and to understand how M. tuberculosis evolved from sensitive to highly drug resistance form by molecular analysis.260 clinical isolates collected in Vietnam between 2005 and 2009 were included. Various techniques and analyses were used: drug susceptibility testing (development of a test with a reduced turn-around time), spoligotyping and 24-MIRU-VNTR typing and gene sequencing. The data were analyzed by statistical and phylogenetic analyses.First, this work was focused on highly drug resistant M. tuberculosis clinical isolates and pyrazinamide resistance. A high proportion of quadruple first-line drug resistant isolates (resistant to isoniazid, rifampicin, streptomycin and ethambutol) have been characterized as pre-XDR and XDR isolates, belonging especially to Beijing family. The molecular analysis revealed also high proportion of drug resistant isolates carrying highly confident pyrazinamide resistance-associated mutations, particularly in MDR and quadruple resistant isolates and in Beijing family.Second, the genetic and phylogenetic analyses showed high diversity of mutation patterns within each family and each MIRU-VNTR cluster suggesting various evolutionary trajectories towards first and second-line drug resistance. The predominance of specific mutations and combinations of mutations associated with high level of resistance and low fitness cost suggests a cumulative effect of mutations and a role for epistasis in multiple-drug resistance acquisition. In addition, high frequency of fitness-compensatory mutations associated with rifampicin resistant mutations was detected in highly drug resistant isolates. These processes may drive the evolution of drug resistance in this sample and lead to a successful spread of highly drug resistant strains. It is worth noting that Beijing family was specifically linked to high-level drug resistance and low fitness cost mutations and to compensatory mutations.In conclusion, this work provides knowledge on the resistance to the first and second-line anti-TB drugs in clinical M. tuberculosis samples collected in Vietnam between 2005 and 2009. These data predict an evolution towards a more problematic situation in terms of drug resistance. First, because the Beijing family, which is currently invading Vietnam, is associated with highly drug resistance, mutations linked to high-level drug resistance and low fitness cost and compensatory mutations. Second, the high risk of pyrazinamide resistance in our sample challenges the efficacy and the use of this drug in MDR-TB treatment. Third, our data suggest an evolution of M. tuberculosis towards a higher potential of drug resistance because of a probable cumulative effect of drug resistant mutations and epistatic interactions. Since the samples under study were collected between 2005-2009, the next step is to test our hypotheses on a recent sampling. Finally, this study together with published data allowed making, for the first time, an inventory of the drug resistance associated mutations in M. tuberculosis isolates from Vietnam

Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species. While Mycobacterium marinum, M. fortuitum and M. chelonae are the most frequently reported species to be involved in the disease, several new mycobacteria species have also recently been implicated. Conventional detection / identification of fish mycobacteria is based on histopathology, culture and biochemical characteristics. In this study complementary molecular approaches were developed to assist in Mycobacterium identification. First, a highly specific and sensitive multiplex PCR-based assay, targeting two genes (hsp65 and 16S RNA), was established to simultaneously detect the genus Mycobacterium and identify M. marinum, M. fortuitum or M. chelonae from culture or infected fish tissue, based on presence / absence of specific amplicons. In addition, PCR-restriction enzyme analysis (PRA) and DNA sequence analysis of the 16S-23S internal transcribed spacer (ITS) region and a 441 bp fragment of the hsp65 gene demonstrated the limitations of multiplex PCR (and commercial line probe assays) to differentiate among the species of the M. fortuitum complex. However DNA sequence analysis of the hsp65 gene fragment was found to reliably identify M. fortuitum from closely related species, M. conceptionense and M. senegalense. Reliable identification of novel species (or very similar species) of aquatic mycobacteria requires more extensive DNA sequence comparisons. Thus, multigene (polygenetic) analyses, as used here, provide rapid, accurate and reliable species identification of aquatic mycobacteria. Furthermore, a number of novel species of aquatic mycobacteria, M. stomatepiae, ‘M. angelicum’, ‘M. aemonae’ and M. salmoniphilum were discovered using the polygenetic analysis approach. Correct identification of Mycobacterium species by DNA sequence comparisons relies on accurate database information. Difficulties in this study in assigning M. marine and M. gordonae to their correct taxa suggest errors in the current public sequence repositories. The above methods were successfully applied to detect and identify mycobacteria in field samples including formalin-fixed, paraffin-embedded (FFPE) fish tissue, water and frozen fish tissue.

Mycobacterium bovis, agent étiologique de la tuberculose bovine (bTB), affecte principalement les bovins mais évolue également dans des systèmes multi-hôtes. La France possède le statut UE indemne de bTB depuis 2000, mais la prévalence de la maladie a augmenté régulièrement ces cinq dernières années. Son contrôle passe par la connaissance des nouveaux facteurs de risque. Pour les déceler, nous avons étudié l’évolution spatio-temporelle de la bTB via la caractérisation des souches isolées de foyers français depuis 1978. A partir de plus de 2.000 souches, environ 600 profils génétiques ont été définis par spoligotypage et typage MLVA. Les groupes majoritairement identifiés, SB0120, SB0134, SB0121 et la «famille F4», sont différenciés plus finement par le MLVA qui augmente la puissance des études d’épidémiologie moléculaire. La diminution de la variabilité génétique et des changements de la distribution géographique des souches s’explique par des modifications dans les pratiques d’élevages et par la prolifération de certains génotypes évoluant dans des systèmes multi-hôtes. L’identification des groupes clonaux existants en France est confirmée par l’étude de mutations SNP effectuée suite au séquençage total de 82 génomes sélectionnés. De nouveaux outils de typage visant à affiner l’identification des souches, définir des profils de transmission méconnus et établir des liens épidémiologiques entre animaux sauvages et de rente sont envisagés
Myobacterium bovis is the causative agent of bovine tuberculosis (bTB), principally affecting cattle but also evolving in multi-host livestock-wildlife systems. Prevalence is regularly increasing in France, an EU bTB-free state since 2000’s, after a 50 year collective fighting-campaign. To control the disease, it is necessary to acknowledge new risk factors. For identifying them, we have studied the spatial-temporary evolution of the disease by characterizing M. bovis strains causative of French outbreaks since 1978. Within more than 2,000 strains, around 600 profiles could be defined by spoligotyping and MLVA. SB0120, SB0134, SB0121 and the « F4 family » are the major spoligotypes isolated. In these groups, the refinement of differentiation can be increased by MLVA typing for powerful molecular epidemiological studies. Decreases in genetically and geographical variability could be explained by changes in husbandry practices and by the proliferation of unique genotypes in multi-host systems. Identification of clonal groups coexisting in France is confirmed by the study of SNPs mutations deduced from the whole genome sequencing of 82 representative strains. New typing tools for refining strains identification and disclosing unknown transmission patterns between livestock and wildlife are foreseeable

Les microARN sont des petits ARN non-codant impliqués dans la régulation de multiples fonctions biologiques dont la réponse immunitaire. L'infection par un pathogène induit un changement transcriptomique fort chez l'hôte. Cependant, la variabilité de ces dérégulations reste encore mal décrite. Cette thèse avait pour principaux objectifs de mieux comprendre la spécificité et la variabilité de la réponse des microARN chez l'homme ainsi que mettre en évidence les bases génétiques de cette diversité en utilisant comme modèle l'infection des cellules dendritiques par Mycobacterium tuberculosis (MTB). Nous avons utilisé une approche ex vivo et des techniques à haut débit dans le but de décrire la réponse des microARN suite à l'infection par MTB dans la population générale, et de la comparer à celle induite par d'autres mycobactéries et bactéries intracellulaires. Nous montons que l'infection modifie profondément l'expression des microARN ainsi que la diversité de leurs isoformes, dont un certain nombre de microARN sont impliqués dans une réponse très conservée. Nos résultats soulignent aussi l'effet de l'infection sur les réseaux de régulation de l'expression des gènes impliquant les microARN et montrent que l'expression de 3% de ces transcrits peut-être corrélée à un marqueur génétique. Grâce à l'intégration de ces différentes analyses, nous proposons certains microARN candidats qui pourraient jouer un rôle dans la variabilité de la réponse immunitaire. L'ensemble de nos résultats constitue la première tentative de compréhension de l'architecture génétique de la réponse des microARN et apporte un nouvel éclairage sur le rôle de ces transcrits dans la réponse antibactérienne
MicroRNAs (miRNAs) are important epigenetic regulators of gene expression that play a key role in many biological processes, including the immune response. Although infection is accompanied by marked changes in the transcriptional profiles of host cells, little is known about the variability of host miRNA responses to infection. In this thesis, we aimed to define the extent and specificity of pathogen-induced miRNA transcriptional responses of host cells, and to characterise the genetic basis of miRNA variability upon infection, using the model of Mycobacterium tuberculosis (MTB) infection of human dendritic cells. To this end, we have combined ex vivo approaches with a range of high-throughput genomic techniques to profile miRNA responses to MTB at the population-level and to compare this response with other mycobacterial and non-mycobacterial infections. We show that miRNAs display marked changes in expression and in isomiR distribution upon infection that are highly consistent across diverse bacteria, demonstrating the presence of a strong core miRNA response to bacterial infection. Our results highlight the impact of infection on miRNA-mediated gene regulatory networks and show that the expression of 3% of miRNAs are controlled by proximate expression quantitative trait loci (eQTLs) and identify a number of candidate miRNAs that may play a role in variability in the immune response to infection. Together, these results provide the first assessment of the impact of genotype-environment interactions on the regulation of miRNA expression, as well as offering novel insights into the specificity of these miRNAs in the response to mycobacterial infections

Orientador: Marcelo de Carvalho Ramos
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O M. Avium é um microrganismo oportunista e sua infecção é feeqüentemente encontrada em pacientes com aids no Brasil, apesar do largo uso da quimioterapia antiretroviral altamente efetiva. Este estudo documenta a relevância desse problema. Dentro de uni número significante de pacientes (n=39) infectados com o M. avium, os isolados puderam ser recuperados de uma variedade de espécimes clínicos. Todos os isolados (n=45) foram tipados pela técnica de RFLP usando a seqüência 1S1245. A maioria dos pacientes (n=35) eram infectados pelo HIV. Somente duas cepas não puderam ser tipadas por causa da ausência da seqüência detectável pela 1S1245. Nas 43 cepas restantes os "blots" apresentaram de 6 a 23 bandas. Uma média de 17 seqüências foram observadas para cada cepa. Para alguns pacientes, mais de um isolado pode ser recuperado. Em dois pacientes deste grupo com doença disseminada, o M. avium pode ser recuperado mais de uma vez. De cada paciente, pelo menos duas amostras com diferentes genótipos foram recuperadas de locais estéreis, indicando que eles tinham infecções policlonais. Esses achados têm sido relatados por outros autores. Em um estudo recente, SAAD et aI., 2000, demonstrou que isolados de infecções policlonais e diferentes "fmgerprints" podem apresentar diferentes suscetibilidade antimicrobiano. Quatro "clusters" de pacientes puderam ser identificados. O maior "cluster" foi composto de oito pacientes. Estes resultados indicam que um mecanismo de transmissão recente ocorreu. A fonte de contaminação desses microrganismos não pode ser determinada. Assim, a transmissão pessoa a pessoa não apresentou uma importância significativa na transmissão desse microrganismo. Nós supomos que esses pacientes adquiriram o microrganismo de fontes hospitalares como água, alimento ou mesmo do ambiente
Abstract: not informed.
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

Orientador: Marcelo de Carvalho Ramos
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A técnica do estudo do polimorfismo de fragmentos de restrição, com a pesquisa da seqüência de inserção IS6110 (IS6110-RFLP), é o método de genotipagem mais empregado mundialmente para a caracterização de isolados de M. tuberculosis. Ela pode ser empregada para o estudo de surtos, epidemias ou para estudos de genética populacional. Em Moçambique, onde a tuberculose tem uma elevada prevalência, não há informação suficiente sobre os padrões genotípicos obtidos com a IS6110-RFLP de cepas locais de M. tuberculosis. A descrição dos padrões obtidos com essa metodologia pode ser útil localmente para propósitos epidemiológicos ou, internacionalmente, para descrever o relacionamento de cepas isoladas em Moçambique com outras áreas do mundo. Neste estudo, uma coleção de 158 isolados de M. tuberculosis, identificados com o emprego da análise de fragmentos de restrição após a amplificação de trecho do gene hsp65 (hsp65-PRA), recuperados de pacientes infectados pelo HIV com tuberculose pulmonar e que residiam em Maputo, Moçambique, foram genotipados. O número de seqüências IS6110 obtido variou de 1 to 18, com 21.5% dos isolados exibindo menos de seis cópias. Um total de 10 ¿clusters¿ foram caracterizados, um com três isolados e os demais com dois cada. Os isolados que exibiram menos de seis seqüências não foram incluídos na análise, dado o baixo poder discriminatório do método. Baseado no coeficiente de similaridade, 85% dos isolados tinham mais do que 65% de homologia. Esses dados mostram que, isolados de M. tuberculosis obtidos em Moçambique, África, podem ser analisados, para fins epidemiológicos com o auxílio dessa técnica de genotipagem. Entretanto, um considerável número de isolados exibiu um número pequeno de cópias da seqüência IS6110 e um segundo marcador genético, como a espoligotipagem, deve ser utilizado
Abstract: IS6110 RFLP has been the most widely used genetic subtyping method for M. tuberculosis strains, to characterize disease outbreaks or for evolutionary genetics studies. In Mozambique, where tuberculosis exhibits a high prevalence, there is not enough information about IS6110-RFLP patterns of local M. tuberculosis strains. The description of the fingerprinting patterns obtained with this methodology can be useful locally for epidemiological purposes, and internationally to investigate the relatedness of strains isolated in Mozambique to other areas of the world. In this study, a collection of 158 isolates of M. tuberculosis strains, as identified by using hsp65-PRA, recovered from HIV-infected patients with pulmonary tuberculosis residing in Maputo, Mozambique, was genotyped. The number of IS6110 copies ranged from 1 to 18, with 21.5% of strains exhibiting less than six copies. A total of 10 clusters were found, one consisting of three strains and all the others of two strains. Isolates showing less than six bands were not included in the cluster analyses due to low discriminatory power of the analysis. Based on similarity coefficients 85% of strains had more than 65% homology. This data show that M. tuberculosis strains obtained in Mozambique, Africa can be analyzed for epidemiological purposes with the use of this genotyping technique. However, a considerable number of strains exhibited a low number of IS6110 copies, and a second genetic marker as spoligotyping has to be used.
Mestrado
Clinica Medica
Mestre em Clinica Medica

No
Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.
European Research Council grant (INTERRUPTB; no. 311725), European Research Council grant (TB-ACCELERATE; no. 638553), Foundation for Innovative New Diagnostics, German Center for Infection Research (DZIF), Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy (EXC 22167–390884018), FWO Odysseus G0F8316N, US National Institutes of Health BD2K K01 (MRF ES026835), Agence Nationale de la Recherche (ANR-16-CD35-0009)

yes
The next-generation short-read sequencing technologies that generate comprehensive, whole-genome data with single-nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long read ONT sequencing data with potential applications for M. tuberculosis. Only eight studies presented results of ONT sequencing of M. tuberculosis, of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. Summary: The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data is available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

Dissertação de mestrado em Bioinformática
Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis is an infectious disease that remains a global public health problem where approximately one-third of the world population have been at least in contact and is latently infected with. Whole genome sequencing has revolutionized the investigation of mycobacterial genomes. The application of this technology has provided innovative understandings into the evolution of the Mycobacterium tuberculosis due to recent studies reporting conflicting findings on its genomic stability, particularly during the evolution of drug resistance in modern lineages. To address this question we focused on understanding the genotypic and epidemiological factors that influence the spread and fitness of this bacterium by analyzing deep –sequencing data of 85 patient samples from Central Asia. Samples were part of a larger study of 399 clinical isolates of newly diagnosed patients with pulmonary TB collected between 2012 and 2013 at the NCTLD in Tbilisi, Georgia. All the samples were mapped against H37Rv strain. We focused on single-nucleotide polymorphisms to reconstruct models for molecular evolution, using Maximum Likelihood and Bayesian Inference methods. 84% of our population belongs to the Beijing lineage, associated with the massive spread of multidrug-resistant strains. Relationship between mutations on rpoB and rpoC were associated with drug resistance to rifampicin and mutations on pncA region also demonstrated to be related with drug resistance to pyrazinamide. Furthermore we found that the amount of variation accumulated within a patient can be as high as that observed between patients along, what we assume to be, a chain of transmission. Intrapatient diversity was found in all of the follow up patients. Our study adds new data to the understandings of the variability among Mycobacterium tuberculosis strains in an intra and interpatient microevolution scenario.
A Tuberculose provocada pelo agente patogénico intracelular Mycobacterium tuberculosis é uma doença infeciosa que continua a ser um dos maiores problemas de saúde global, estimando-se que aproximadamente um terço da população tenha estado em contacto e esteja infetada de forma latente. Whole genome sequencing surgiu como um método revolucionário da investigação de genomas de micobactérias. A sua aplicação têm proporcionado conhecimentos inovadores relativamente à evolução da Mycobacterium tuberculosis devido a estudos recentes que reportam resultados contraditórios sobre a sua estabilidade genómica, particularmente durante a evolução da sua resistência a antibióticos em linhagens consideradas modernas. Para abordar esta questão, focámo-nos na análise e compreensão dos fatores genotípicos e epidemiológicos que influenciam a capacidade de disseminação e o fitness desta bactéria através da análise de dados deep-sequencing provenientes de amostras de 85 pacientes provenientes da Ásia Central. As amostras pertencem a um estudo maior composto por 399 isolados clínicos de pacientes recentemente diagnosticados com tuberculose pulmonar recolhidas entre 2012 e 2013 no National Center of Tuberculosis and Lung Diseases (NCTLD) em Tbilisi, Geórgia. Todas as amostras foram mapeadas contra a estirpe H37Rv. Para a reconstrução de modelos de evolução molecular, focámo-nos apenas em single-nucleotide polymorphisms e utilizámos dois métodos distintos, Maximum Likelihood e Bayesian Inference. Cerca de 84% da nossa população pertence à linhagem Beijing, associada com a propagação em massa de estirpes resistentes a múltiplos antibióticos. Além disso, as mutações no rpoB e rpoC foram associadas à resistência a rifampicina e mutações na região pncA também demonstraram estar relacionadas com a resistência à pirazinamida. Verificou-se ainda que a quantidade de variabilidade genética acumulada dentro de um paciente pode ser tão alta quanto a observada entre pacientes ao longo, do que supomos ser, uma cadeia de transmissão. Todos os pacientes que foram acompanhados durante tratamento apresentaram variabilidade genética. O nosso estudo acrescenta novos dados relativamente à variabilidade entre diferentes estirpes de Mycobacterium tuberculosis tendo em conta um panorama de microevolução intra e inter paciente.

Yes
Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important challenges to integrate WGS into surveillance and research. We assessed the global status of Mtb WGS and developed a 3-week training course coupled with long-term mentoring and WGS infrastructure building. Training focused on genome sequencing, bioinformatics and development of a locally relevant WGS research project. The aim of the long-term mentoring was to support trainees in project implementation and funding acquisition. The focus of WGS infrastructure building was on the DNA extraction process and bioinformatics. Compared to their TB burden, Asia and Africa are grossly underrepresented in Mtb WGS research. Challenges faced resulted in adaptations to the training, mentoring and infrastructure building. Out-of-date laptop hardware and operating systems were overcome by using online tools and a Galaxy WGS analysis pipeline. A case studies approach created a safe atmosphere for students to formulate and defend opinions. Because quality DNA extraction is paramount for WGS, a biosafety level 3 and general laboratory skill training session were added, use of commercial DNA extraction kits was introduced and a 2-week training in a highly equipped laboratory was combined with a 1-week training in the local setting. By developing and sharing the components of and experiences with a sequencing and bioinformatics training program, we hope to stimulate capacity building programs for Mtb WGS and empower high-burden countries to play an important role in WGS-based TB surveillance and research.
Vlaamse Interuniversitaire Raad-secretariaat voor universitaire ontwikkelingssamenwerking (ET2018JOI008A10); the Research Foundation Flanders under FWO Odysseus (grant G0F8316N); the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (64751); the South African Medical Research Council.

Yes
Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting.
ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Yes
Background In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments. Methods From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis. Results Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16–101)/mm3 and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical. Conclusions Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly.

no
The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Yes
Background: Mycobacterium tuberculosis rapid diagnostic tests (RDTs) are widely employed in routine laboratories and national surveys for detection of rifampicinresistant (RR)-TB. However, as next-generation sequencing technologies have become more commonplace in research and surveillance programs, RDTs are being increasingly complemented by whole genome sequencing (WGS). While comparison between RDTs is difficult, all RDT results can be derived from WGS data. This can facilitate continuous analysis of RR-TB burden regardless of the data generation technology employed. By converting WGS to RDT results, we enable comparison of data with different formats and sources particularly for low- and middle-income high TB-burden countries that employ different diagnostic algorithms for drug resistance surveys. This allows national TB control programs (NTPs) and epidemiologists to utilize all available data in the setting for improved RR-TB surveillance. Methods: We developed the Python-based MycTB Genome to Test (MTBGT) tool that transforms WGS-derived data into laboratory-validated results of the primary RDTs—Xpert MTB/RIF, XpertMTB/RIF Ultra, GenoType MDRTBplus v2.0, and GenoscholarNTM+MDRTB II. The tool was validated through RDT results of RR-TB strains with diverse resistance patterns and geographic origins and applied on routine-derived WGS data. Results: The MTBGT tool correctly transformed the single nucleotide polymorphism (SNP) data into the RDT results and generated tabulated frequencies of the RDT probes as well as rifampicin-susceptible cases. The tool supplemented the RDT probe reactions output with the RR-conferring mutation based on identified SNPs. The MTBGT tool facilitated continuous analysis of RR-TB and Xpert probe reactions from different platforms and collection periods in Rwanda. Conclusion: Overall, the MTBGT tool allows low- and middle-income countries to make sense of the increasingly generated WGS in light of the readily available RDT.
Erasmus Mundus Joint Doctorate Fellowship grant 2016- 1346.