HIV co-infection is associated with reduced Mycobacterium tuberculosis transmissibility in sub-Saharan Africa.

Persons living with HIV are known to be at increased risk of developing tuberculosis (TB) disease upon infection with Mycobacterium tuberculosis (Mtb). However, it has remained unclear how HIV co-infection affects subsequent Mtb transmission from these patients. Here, we customized a Bayesian phylodynamic framework to estimate the effects of HIV co-infection on the Mtb transmission dynamics from sequence data. We applied our model to four Mtb genomic datasets collected in sub-Saharan African countries with a generalized HIV epidemic. Our results confirm that HIV co-infection is a strong risk factor for developing active TB. Additionally, we demonstrate that HIV co-infection is associated with a reduced effective reproductive number for TB. Stratifying the population by CD4+ T-cell count yielded similar results, suggesting that, in this context, CD4+ T-cell count is not a better predictor of Mtb transmissibility than HIV infection status alone. Together, our genome-based analyses complement observational household contact studies, and more firmly establish the negative association between HIV co-infection and Mtb transmissibility.

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania.

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1ß, and IL-1ß compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host-pathogen interaction in human tuberculosis.

Phylogenetic Analysis of Pyruvate-Ferredoxin Oxidoreductase, a Redox Enzyme Involved in the Pharmacological Activation of Nitro-Based Prodrugs in Bacteria and Protozoa.

The present frontrunners in the chemotherapy of infections caused by protozoa are nitro-based prodrugs that are selectively activated by PFOR-mediated redox reactions. This study seeks to analyze the distribution of PFOR in selected protozoa and bacteria by applying comparative genomics to test the hypothesis that PFOR in eukaryotes was acquired through horizontal gene transfer (HGT) from bacteria. Furthermore, to identify other putatively acquired genes, proteome-wide and gene enrichment analyses were used. A plausible explanation for the patchy occurrence of PFOR in protozoa is based on the hypothesis that bacteria are potential sources of genes that enhance the adaptation of protozoa in hostile environments. Comparative genomics of Entamoeba histolytica and the putative gene donor, Desulfovibrio vulgaris, identified eleven candidate genes for HGT involved in intermediary metabolism. If these results can be reproduced in other PFOR-possessing protozoa, it would provide more validated evidence to support the horizontal transfer of pfor from bacteria.

The relative transmission fitness of multidrug-resistant Mycobacterium tuberculosis in a drug resistance hotspot.

Multidrug-resistant tuberculosis (MDR-TB) is among the most frequent causes of death due to antimicrobial resistance. Although only 3% of global TB cases are MDR, geographical hotspots with up to 40% of MDR-TB have been observed in countries of the former Soviet Union. While the quality of TB control and patient-related factors are known contributors to such hotspots, the role of the pathogen remains unclear. Here we show that in the country of Georgia, a known hotspot of MDR-TB, MDR Mycobacterium tuberculosis strains of lineage 4 (L4) transmit less than their drug-susceptible counterparts, whereas most MDR strains of L2 suffer no such defect. Our findings further indicate that the high transmission fitness of these L2 strains results from epistatic interactions between the rifampicin resistance-conferring mutation RpoB S450L, compensatory mutations in the RNA polymerase, and other pre-existing genetic features of L2/Beijing clones that circulate in Georgia. We conclude that the transmission fitness of MDR M. tuberculosis strains is heterogeneous, but can be as high as drug-susceptible forms, and that such highly drug-resistant and transmissible strains contribute to the emergence and maintenance of hotspots of MDR-TB. As these strains successfully overcome the metabolic burden of drug resistance, and given the ongoing rollout of new treatment regimens against MDR-TB, proper surveillance should be implemented to prevent these strains from acquiring resistance to the additional drugs.

Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.

Using population-specific add-on polymorphisms to improve genotype imputation in underrepresented populations.

Genome-wide association studies rely on the statistical inference of untyped variants, called imputation, to increase the coverage of genotyping arrays. However, the results are often suboptimal in populations underrepresented in existing reference panels and array designs, since the selected single nucleotide polymorphisms (SNPs) may fail to capture population-specific haplotype structures, hence the full extent of common genetic variation. Here, we propose to sequence the full genomes of a small subset of an underrepresented study cohort to inform the selection of population-specific add-on tag SNPs and to generate an internal population-specific imputation reference panel, such that the remaining array-genotyped cohort could be more accurately imputed. Using a Tanzania-based cohort as a proof-of-concept, we demonstrate the validity of our approach by showing improvements in imputation accuracy after the addition of our designed add-on tags to the base H3Africa array.

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.

Genomic epidemiological analysis identifies high relapse among individuals with recurring tuberculosis and provides evidence of recent household-related transmission of tuberculosis in Ghana.

OBJECTIVE: To retrospectively investigate the cause of recurring tuberculosis (rcTB) among participants with pulmonary TB recruited from a prospective population-based study conducted between July 2012 and December 2015. METHODS: Mycobacterium tuberculosis complex isolates obtained from rcTB cases were characterized by standard mycobacterial genotyping tools, whole-genome sequencing, and phylogenetic analysis carried out to assess strain relatedness. RESULTS: The majority (58.3%, 21/36) of study participants with rcTB episodes had TB recurrence within 12 months post treatment. TB strains with isoniazid (INH) resistance were found in 19.4% (7/36) of participants at the primary episode, of which 29% (2/7) were also rifampicin-resistant. On TB recurrence, an INH-resistant strain was found in a larger proportion of participants, 27.8% (10/36), of which 40% (4/10) were MDR-TB strains. rcTB was attributed to relapse (same strain) in 75.0% (27/36) of participants and 25.0% (9/36) to re-infection. CONCLUSION: Our findings indicate that previous unresolved infectiondue to inadequate treatment, may be the major cause of rcTB.

Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum. Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum, and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

A new nomenclature for the livestock-associated Mycobacterium tuberculosis complex based on phylogenomics.

Background:  The bacteria that compose the Mycobacterium tuberculosis complex (MTBC) cause tuberculosis (TB) in humans and in different animals, including livestock. Much progress has been made in understanding the population structure of the human-adapted members of the MTBC by combining phylogenetics with genomics. Accompanying the discovery of new genetic diversity, a body of operational nomenclature has evolved to assist comparative and molecular epidemiological studies of human TB. By contrast, for the livestock-associated MTBC members, Mycobacterium bovis, M. caprae and M. orygis, there has been a lack of comprehensive nomenclature to accommodate new genetic diversity uncovered by emerging phylogenomic studies. We propose to fill this gap by putting forward a new nomenclature covering the main phylogenetic groups within M. bovis, M. caprae and M. orygis. Methods:  We gathered a total of 8,736 whole-genome sequences (WGS) from public sources and 39 newly sequenced strains, and selected a subset of 829 WGS, representative of the worldwide diversity of M. bovis, M. caprae and M. orygis. We used phylogenetics and genetic diversity patterns inferred from WGS to define groups. Results:  We propose to divide M. bovis, M. caprae and M. orygis in three main phylogenetic lineages, which we named La1, La2 and La3, respectively. Within La1, we identified several monophyletic groups, which we propose to classify into eight sublineages (La1.1-La1.8). These sublineages differed in geographic distribution, with some being geographically restricted and others globally widespread, suggesting different expansion abilities. To ease molecular characterization of these MTBC groups by the community, we provide phylogenetically informed, single nucleotide polymorphisms that can be used as barcodes for genotyping. These markers were implemented in KvarQ and TB-Profiler, which are platform-independent, open-source tools. Conclusions:  Our results contribute to an improved classification of the genetic diversity within the livestock-associated MTBC, which will benefit future molecular epidemiological and evolutionary studies.

HIV Coinfection Is Associated with Low-Fitness rpoB Variants in Rifampicin-Resistant Mycobacterium tuberculosis.

We analyzed 312 drug-resistant genomes of Mycobacterium tuberculosis isolates collected from HIV-coinfected and HIV-negative TB patients from nine countries with a high tuberculosis burden. We found that rifampicin-resistant M. tuberculosis strains isolated from HIV-coinfected patients carried disproportionally more resistance-conferring mutations in rpoB that are associated with a low fitness in the absence of the drug, suggesting these low-fitness rpoB variants can thrive in the context of reduced host immunity.

Whole Genome Sequencing and Spatial Analysis Identifies Recent Tuberculosis Transmission Hotspots in Ghana.

Whole genome sequencing (WGS) is progressively being used to investigate the transmission dynamics of Mycobacterium tuberculosis complex (MTBC). We used WGS analysis to resolve traditional genotype clusters and explored the spatial distribution of confirmed recent transmission clusters. Bacterial genomes from a total of 452 MTBC isolates belonging to large traditional clusters from a population-based study spanning July 2012 and December 2015 were obtained through short read next-generation sequencing using the illumina HiSeq2500 platform. We performed clustering and spatial analysis using specified R packages and ArcGIS. Of the 452 traditional genotype clustered genomes, 314 (69.5%) were confirmed clusters with a median cluster size of 7.5 genomes and an interquartile range of 4-12. Recent tuberculosis (TB) transmission was estimated as 24.7%. We confirmed the wide spread of a Cameroon sub-lineage clone with a cluster size of 78 genomes predominantly from the Ablekuma sub-district of Accra metropolis. More importantly, we identified a recent transmission cluster associated with isoniazid resistance belonging to the Ghana sub-lineage of lineage 4. WGS was useful in detecting unsuspected outbreaks; hence, we recommend its use not only as a research tool but as a surveillance tool to aid in providing the necessary guided steps to track, monitor, and control TB.

A sister lineage of the Mycobacterium tuberculosis complex discovered in the African Great Lakes region.

The human- and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region - involved in the cobalamin/vitamin B12 synthesis - and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

An African origin for Mycobacterium bovis.

BACKGROUND AND OBJECTIVES: Mycobacterium bovis and Mycobacterium caprae are two of the most important agents of tuberculosis in livestock and the most important causes of zoonotic tuberculosis in humans. However, little is known about the global population structure, phylogeography and evolutionary history of these pathogens. METHODOLOGY: We compiled a global collection of 3364 whole-genome sequences from M.bovis and M.caprae originating from 35 countries and inferred their phylogenetic relationships, geographic origins and age. RESULTS: Our results resolved the phylogenetic relationship among the four previously defined clonal complexes of M.bovis, and another eight newly described here. Our phylogeographic analysis showed that M.bovis likely originated in East Africa. While some groups remained restricted to East and West Africa, others have subsequently dispersed to different parts of the world. CONCLUSIONS AND IMPLICATIONS: Our results allow a better understanding of the global population structure of M.bovis and its evolutionary history. This knowledge can be used to define better molecular markers for epidemiological investigations of M.bovis in settings where whole-genome sequencing cannot easily be implemented. LAY SUMMARY: During the last few years, analyses of large globally representative collections of whole-genome sequences (WGS) from the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages have enhanced our understanding of the global population structure, phylogeography and evolutionary history of these pathogens. In contrast, little corresponding data exists for M. bovis, the most important agent of tuberculosis in livestock. Using whole-genome sequences of globally distributed M. bovis isolates, we inferred the genetic relationships among different M. bovis genotypes distributed around the world. The most likely origin of M. bovis is East Africa according to our inferences. While some M. bovis groups remained restricted to East and West Africa, others have subsequently dispersed to different parts of the world driven by cattle movements.

The molecular clock of Mycobacterium tuberculosis.

The molecular clock and its phylogenetic applications to genomic data have changed how we study and understand one of the major human pathogens, Mycobacterium tuberculosis (MTB), the etiologic agent of tuberculosis. Genome sequences of MTB strains sampled at different times are increasingly used to infer when a particular outbreak begun, when a drug-resistant clone appeared and expanded, or when a strain was introduced into a specific region. Despite the growing importance of the molecular clock in tuberculosis research, there is a lack of consensus as to whether MTB displays a clocklike behavior and about its rate of evolution. Here we performed a systematic study of the molecular clock of MTB on a large genomic data set (6,285 strains), covering different epidemiological settings and most of the known global diversity. We found that sampling times below 15-20 years were often insufficient to calibrate the clock of MTB. For data sets where such calibration was possible, we obtained a clock rate between 1x10-8 and 5x10-7 nucleotide changes per-site-per-year (0.04-2.2 SNPs per-genome-per-year), with substantial differences between clades. These estimates were not strongly dependent on the time of the calibration points as they changed only marginally when we used epidemiological isolates (sampled in the last 40 years) or three ancient DNA samples (about 1,000 years old) to calibrate the tree. Additionally, the uncertainty and the discrepancies in the results of different methods were sometimes large, highlighting the importance of using different methods, and of considering carefully their assumptions and limitations.

Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues.

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.

Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium canettii and Members of the Mycobacterium tuberculosis Complex.

Using 894 phylogenetically diverse genomes of the Mycobacterium tuberculosis complex (MTBC), we simulated in silico the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g., it can distinguish some strains of Mycobacterium canettii and variants of Mycobacterium bovis that are not intrinsically resistant to pyrazinamide) and limitations (e.g., Mycobacterium orygis cannot be differentiated from Mycobacterium africanum).

Insights into the genetic diversity of Mycobacterium tuberculosis in Tanzania.

BACKGROUND: Human tuberculosis (TB) is caused by seven phylogenetic lineages of the Mycobacterium tuberculosis complex (MTBC), Lineage 1-7. Recent advances in rapid genotyping of MTBC based on single nucleotide polymorphisms (SNP), allow for phylogenetically robust strain classification, paving the way for defining genotype-phenotype relationships in clinical settings. Such studies have revealed that, in addition to host and environmental factors, strain variation in the MTBC influences the outcome of TB infection and disease. In Tanzania, such molecular epidemiological studies of TB however are scarce in spite of a high TB burden. METHODS AND FINDINGS: Here we used SNP-typing to characterize a nationwide collection of 2,039 MTBC clinical isolates representative of 1.6% of all new and retreatment TB cases notified in Tanzania during 2012 and 2013. Four lineages, namely Lineage 1-4 were identified within the study population. The distribution and frequency of these lineages varied across regions but overall, Lineage 4 was the most frequent (n = 866, 42.5%), followed by Lineage 3 (n = 681, 33.4%) and 1 (n = 336, 16.5%), with Lineage 2 being the least frequent (n = 92, 4.5%). We found Lineage 2 to be independently associated with female sex (adjusted odds ratio [aOR] 2.14; 95% confidence interval [95% CI] 1.31 - 3.50, p = 0.002) and retreatment cases (aOR 1.67; 95% CI 0.95 - 2.84, p = 0. 065) in the study population. We found no associations between MTBC lineage and patient age or HIV status. Our sublineage typing based on spacer oligotyping on a subset of Lineage 1, 3 and 4 strains revealed the presence of mainly EAI, CAS and LAM families. Finally, we detected low levels of multidrug resistant isolates among a subset of 144 retreatment cases. CONCLUSIONS: This study provides novel insights into the MTBC lineages and the possible influence of pathogen-related factors on the TB epidemic in Tanzania.

Reference set of Mycobacterium tuberculosis clinical strains: A tool for research and product development.

The Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB) in humans and various other mammals. The human-adapted members of the MTBC comprise seven phylogenetic lineages that differ in their geographical distribution. There is growing evidence that this phylogeographic diversity modulates the outcome of TB infection and disease. For decades, TB research and development has focused on the two canonical MTBC laboratory strains H37Rv and Erdman, both of which belong to Lineage 4. Relying on only a few laboratory-adapted strains can be misleading as study results might not be directly transferrable to clinical settings where patients are infected with a diverse array of strains, including drug-resistant variants. Here, we argue for the need to expand TB research and development by incorporating the phylogenetic diversity of the MTBC. To facilitate such work, we have assembled a group of 20 genetically well-characterized clinical strains representing the seven known human-adapted MTBC lineages. With the "MTBC clinical strains reference set" we aim to provide a standardized resource for the TB community. We hope it will enable more direct comparisons between studies that explore the physiology of MTBC beyond the laboratory strains used thus far. We anticipate that detailed phenotypic analyses of this reference strain set will increase our understanding of TB biology and assist in the development of new control tools that are broadly effective.

Dispersal of Mycobacterium tuberculosis Driven by Historical European Trade in the South Pacific.

Mycobacterium tuberculosis (Mtb) is a globally distributed bacterial pathogen whose population structure has largely been shaped by the activities of its obligate human host. Oceania was the last major global region to be reached by Europeans and is the last region for which the dispersal and evolution of Mtb remains largely unexplored. Here, we investigated the evolutionary history of the Euro-American L4.4 sublineage and its dispersal to the South Pacific. Using a phylodynamics approach and a dataset of 236 global Mtb L4.4 genomes we have traced the origins and dispersal of L4.4 strains to New Zealand. These strains are predominantly found in indigenous Maori and Pacific people and we identify a clade of European, likely French, origin that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests the expansion of European trade networks in the early 19th century drove the dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of Maori in the 20th century. Our results offer new insight into the expansion and dispersal of Mtb in the South Pacific and provide a striking example of the role of historical European migrations in the global dispersal of Mtb.