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SignificancePrevious attempts to identify the action of natural selection in the Mycobacterium tuberculosis complex (MTBC) were limited by sample size and averaging across time and lineages. We investigate changes in selective pressures across time for every single gene of the MTBC. We developed a methodology to analyze temporal signals of selection in a large dataset (∼5,000 complete genomes) and showed that 1) almost half of the genes seem to have been under positive selection at some point in time; 2) experimentally confirmed epitopes tend to accumulate more mutations in deeper branches than in external branches; and 3) temporal signals identify genes that were conserved in the past but under positive selection in the present, suggesting ongoing adaptation to the host.

Outbreak strains of Mycobacterium tuberculosis are promising candidates as targets in the search for intrinsic determinants of transmissibility, as they are responsible for many cases with sustained transmission; however, the use of low-resolution typing methods and restricted geographical investigations represent flaws in assessing the success of long-lived outbreak strains. We can now address the nature of outbreak strains by combining large genomic data sets and phylodynamic approaches. We retrospectively sequenced the whole genome of representative samples assigned to an outbreak circulating in the Canary Islands (the GC strain) since 1993, which accounts for ~20% of local tuberculosis cases. We selected a panel of specific single nucleotide polymorphism (SNP) markers for an in-silico search for additional outbreak-related sequences within publicly available tuberculosis genomic data. Using this information, we inferred the origin, spread, and epidemiological parameters of the GC strain. Our approach allowed us to accurately trace the historical and more recent dispersion of the GC strain. We provide evidence of a highly successful nature within the Canarian archipelago but limited expansion abroad. Estimation of epidemiological parameters from genomic data disagree with a distinctive biology of the GC strain. With the increasing availability of genomic data allowing for the accurate inference of strain spread and critical epidemiological parameters, we can now revisit the link between Mycobacterium tuberculosis genotypes and transmission, as is routinely carried out for SARS-CoV-2 variants of concern. We demonstrate that social determinants rather than intrinsically higher bacterial transmissibility better explain the success of the GC strain. Importantly, our approach can be used to trace and characterize strains of interest worldwide. IMPORTANCE Infectious disease outbreaks represent a significant problem for public health. Tracing outbreak expansion and unde

[ES] La resistencia a los antibióticos en la tuberculosis es un grave problema de salud mundial y un obstáculo creciente para el control de la enfermedad. En esta tesis utilizamos la genómica como herramienta para estudiar diferentes aspectos de la biología de la bacteria en relación con el desarrollo de resistencia a los antibióticos y aportar nuevos conocimientos para afrontar el reto de su erradicación. En primer lugar, caracterizamos los nuevos determinantes genómicos de la resistencia a isoniazida utilizando un enfoque basado en genómica funcional y asociación filogenética. El método consiste en la secuenciación de transposones de bacterias expuestas al antibiótico para determinar los genes implicados tanto en sensibilidad como en resistencia, y su filtrado con datos genómicos de una colección global de cepas clínicas. Hemos comprobado la importancia de las rutas metabólicas de síntesis de la pared bacteriana en el mecanismo de acción de los antibióticos, y también hemos descubierto nuevos genes implicados en el equilibrio redox celular que confieren resistencia de bajo nivel. Estos resultados pueden utilizarse para desarrollar nuevas técnicas de diagnóstico o dianas terapéuticas, y el método es aplicable a nuevos antibióticos para predecir futuros determinantes de resistencia. En segundo lugar, exploramos la diversidad bacteriana de la tuberculosis en su sitio natural de infección. Hemos analizado muestras de diferentes partes de la lesión pulmonar de pacientes en Georgia, un país con una alta incidencia de tuberculosis resistente, y hemos detectado un número significativo de infecciones policlonales (causadas por dos o más cepas diferentes). Estas diferentes cepas pueden a su vez ser resistentes a diferentes antibióticos, por lo que es fundamental detectarlas a tiempo. Las muestras quirúrgicas nos dieron una imagen más completa de la diversidad bacteriana que el esputo de rutina, y nos permitieron detectar infecciones policlonales con mayor precisión. Este ti, [EN] Antibiotic resistance in tuberculosis is a serious global health problem and a growing obstacle to the control of the disease. In this thesis we use genomics as a tool to study different aspects of the biology of the bacterium in relation to the development of antibiotic resistance and provide new knowledge to meet the challenge of its eradication. First, we characterize new genomic determinants of resistance to isoniazid by using an approach based on functional genomics and phylogenetic association. The method consists of transposon sequencing of bacterial populations exposed to the antibiotic to determine the genes involved in both sensitivity and resistance, and their filtering with genomic data from a global collection of clinical strains. We have verified the importance of the metabolic pathways of bacterial wall synthesis in the mechanism of antibiotic action, and also discovered new genes involved in cellular redox balance that confer low-level resistance to the bacteria. These results can be used to develop new diagnostic techniques or therapeutic targets, and the method is applicable to new antibiotics to predict future resistance determinants. Second, we explored the bacterial diversity of tuberculosis at its natural site of infection and the population dynamics of antibiotic resistance. We have analyzed samples from different parts of the lung lesion from patients in Georgia, a country with a high incidence of resistant tuberculosis, and detected a significant number of polyclonal infections (caused by two or more different strains). These different strains can in turn be resistant to different antibiotics, which makes it essential to detect them in time to offer the patient the most appropriate treatment possible. The surgical specimens gave us a more complete picture of bacterial diversity than sputum, the routine clinical specimen, and allowed us to detect polyclonal infections more accurately. The data show that we are underestimating these types

Efforts to eradicate tuberculosis are hampered by the rise and spread of antibiotic resistance. Several large-scale projects have aimed to specifically link clinical mutations to resistance phenotypes, but they were limited in both their explanatory and predictive powers. Here, we combine functional genomics and phylogenetic associations using clinical strain genomes to decipher the architecture of isoniazid resistance and search for new resistance determinants. This approach has allowed us to confirm the main target route of the antibiotic, determine the clinical relevance of redox metabolism as an isoniazid resistance mechanism and identify novel candidate genes harboring resistance mutations in strains with previously unexplained isoniazid resistance. This approach can be useful for characterizing how the tuberculosis bacilli acquire resistance to new antibiotics and how to forestall them.

Background. Outbreak strains are good candidates to look for intrinsic transmissibility as they are responsible for a large number of cases with sustained transmission. However, assessment of the success of long-lived outbreak strains has been flawed by the use of low-resolution typing methods and restricted geographical investigations. We now have the potential to address the nature of outbreak strains by combining large genomic datasets and phylodynamic approaches. Methods. We retrospectively sequenced the whole genome of representative samples assigned to an outbreak circulating in the Canary Islands (GC) since 1993; accounting for ~20% of local TB cases. We selected a panel of specific SNP markers to in-silico search for additional outbreak related sequences within publicly-available TB genomic data. Using this information we inferred the origin, spread and epidemiological parameters of the GC-outbreak. Findings. Our approach allowed us to accurately trace both the historical and recent dispersion of the strain. We evidenced its high success within the Canarian archipelago but found a limited expansion abroad. Estimation of epidemiological parameters from genomic data contradicts a distinct biology of the GC-strain. Interpretation. With the increasing availability of genomic data allowing for an accurate inference of strain spread and key epidemiological parameters, we can now revisit the link between Mycobacterium tuberculosis genotypes and transmission, as routinely done for SARS-CoV-2 variants of concern. We show that the success of the GC-strain is better explained by social determinants rather than intrinsically higher bacterial transmissibility. Our approach can be used to trace and characterize strains of interest worldwide.

Understanding Multidrug-resistant tuberculosis cases not detected by rapid phenotypic and genotypic routine clinical tests is essential to improve diagnostics and advance towards personalized TB treatments. Here, we combine WGS with single-colony phenotyping testing to diagnose a MDR strain infecting a patient over nine years. Our investigation revealed the failure of rapid testing assays and genome-based prediction tools to identify the MDR strain. The false negative outcome was caused by uncommon RIF and INH resistance mutations. Despite WGS data helped to personalize treatment, the patient developed XDR-TB, highlighting the importance of coupling new diagnostic methods with appropriate treatment regimens.

Polyclonal infections occur when at least two unrelated strains of the same pathogen are detected in an individual. This has been linked to worse clinical outcomes in tuberculosis, as undetected strains with different antibiotic resistance profiles can lead to treatment failure. Here, we examine the amount of polyclonal infections in sputum and surgical resections from patients with tuberculosis in the country of Georgia. For this purpose, we sequence and analyse the genomes of Mycobacterium tuberculosis isolated from the samples, acquired through an observational clinical study (NCT02715271). Access to the lung enhanced the detection of multiple strains (40% of surgery cases) as opposed to just using a sputum sample (0-5% in the general population). We show that polyclonal infections often involve genetically distant strains and can be associated with reversion of the patient's drug susceptibility profile over time. In addition, we find different patterns of genetic diversity within lesions and across patients, including mutational signatures known to be associated with oxidative damage; this suggests that reactive oxygen species may be acting as a selective pressure in the granuloma environment. Our results support the idea that the magnitude of polyclonal infections in high-burden tuberculosis settings is underestimated when only testing sputum samples.

The occurrence of multiple strains of a bacterial pathogen such as M. tuberculosis or C. difficile within a single human host, referred to as a mixed infection, has important implications for both healthcare and public health. However, methods for detecting it, and especially determining the proportion and identities of the underlying strains, from WGS (whole-genome sequencing) data, have been limited. In this paper we introduce SplitStrains, a novel method for addressing these challenges. Grounded in a rigorous statistical model, SplitStrains not only demonstrates superior performance in proportion estimation to other existing methods on both simulated as well as real M. tuberculosis data, but also successfully determines the identity of the underlying strains. We conclude that SplitStrains is a powerful addition to the existing toolkit of analytical methods for data coming from bacterial pathogens and holds the promise of enabling previously inaccessible conclusions to be drawn in the realm of public health microbiology

Tuberculosis (TB) surveillance is scarce in most African countries, even though it is the continent with the greatest disease incidence according to the World Health Organization. Liberia is within the 30 countries with the highest TB burden, probably as a consequence of the long civil war and the recent Ebola outbreak, both crippling the health system and depreciating the TB prevention and control programmes. Due to difficulties working in the country, there is a lack of resistance surveys and bacillus characterization. Here, we use genome sequencing of Mycobacterium tuberculosis clinical isolates to fill this gap. Our results highlight that the bacillus population structure is dominated by lineage 4 strains that harbour an outstanding genetic diversity, higher than in the rest of Africa as a whole. Coalescent analyses demonstrate that strains currently circulating in Liberia were introduced several times beginning in the early year 600 CE until very recently coinciding with migratory movements associated with the civil war and Ebola epidemics. A higher multidrug-resistant (MDR)-TB frequency (23.5 %) than current estimates was obtained together with non-catalogued drug-resistance mutations. Additionally, 39 % of strains were in genomic clusters revealing that ongoing transmission is a major contribution to the TB burden in the country. Our report emphasizes the importance of TB surveillance and control in African countries where bacillus diversity, MDR-TB prevalence and transmission are coalescing to jeopardize TB control programmes.

Tuberculosis resistance diagnostics have vastly improved in recent years thanks to the development of standardised phenotypic and molecular testing methods. However, these methods are either slow or limited in the number of resistant genotypes they can detect. With the advent of next-generation sequencing (NGS) we can sidestep all those problems, as we can sequence whole tuberculosis genomes at increasingly smaller costs and requiring less and less DNA. In this review, we explain how accumulated knowledge in the field has allowed us to go from phenotypic testing to molecular methods to Whole Genome Sequencing (WGS) for resistance diagnostics. We compare current diagnostic methods with WGS as to their efficacy in detecting resistant cases, and show how forthcoming advances in NGS technologies will be crucial in widespread implementation of WGS as a diagnostic tool.

[ES] La bacteria de la tuberculosis mata a más de un millón y medio de personas al año y representa un problema de salud global. Las técnicas genómicas están permitiendo entenderla mejor de tal manera que la cantidad de datos genómicos, transcriptómicos, proteómicos y metabolómicos ha ido aumentando. Puesto que es una enfermedad global, la bacteria que la causa es también heterogénea. Recientemente, en el laboratorio se ha caracterizado a nivel genómico una colección global de cepas de tuberculosis. Esto nos ha dado un arsenal de polimorfismos presentes en las cepas circulantes de todo el mundo que pueden tener un impacto en el fenotipo (diferencias en expresión génica y proteica, epidemiología, presentación clínica). Particularmente muchos de esos polimorfismos afectan zonas reguladoras de la transcipción. Para empezar a entender ese impacto se propone este trabajo de investigación. El hecho de que dichas zonas estén o no conservadas a nivel global puede indicar su importancia para la virulencia y el desarrollo de la enfermedad. En este trabajo se plantea estudiar dichas regiones. Para ello, se han identificado diferentes elementos reguladores (sitios de unión de factores de transcripción, regiones de inicio de la transcripción y la traducción así como ARNs pequeños). Usando la información de mutaciones acumuladas en nuestra muestra genómica global clasificaremos dichas regiones como esenciales (conservadas evolutivamente) y no esenciales., [EN] Mycobacterium tuberculosis is a bacterium that kills more than one and a half million people each year, constituting a global health issue. Modern genomic techniques are allowing to understand it better, so the amount of genomic, transcriptomic, proteomic and metabolomic data is constantly rising. As it is a global disease, bacteria which cause it are heterogeneous. Recently, a global collection of M. tuberculosis strains has been characterized in the lab, providing us with a vast set of polymorphisms present in the different strains which could have a great impact on their phenotype and clinical presentation or epidemiology. Particularly lots of these polymorphisms affect transcriptional regulatory regions in the genome. This work’s goal is to begin understanding that impact studying those regions. For that purpose, different regulatory regions have been identified and using the information stacked in the global sample of strains, we can classify them as essential (evolutionarily conserved) or nonessential.

The 5′ untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg2+-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg2+. This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg2+ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.