Infectious diseases have affected humanity for millennia and are among the strongest selective forces. Tuberculosis (TB) is an ancient disease, caused by the human-adapted members of the Mycobacterium tuberculosis complex (MTBC). The outcome of TB infection and disease is highly variable, and co-evolution between human populations and MTBC strains may account for some of this variability. Particular human genetic ancestries have been associated with higher susceptibility to TB, but sociodemographic aspects of the disease can confound such associations. Here, we studied 1000 TB patients from Dar es Salaam, Tanzania, together with their respective MTBC isolates, by combining human and bacterial genomics with clinical data. We found that the genetic background of the TB patient population was strongly influenced by migrations of Bantu-speaking populations from West Africa, which contrasts with the corresponding MTBC genotypes that were mainly introduced from outside Africa. These findings suggest a recent evolutionary history of co-existence between the human and MTBC populations in Dar es Salaam. We detected no evidence of an effect of human genetic ancestry, or MTBC phylogenetic diversity alone, nor their interaction, on TB disease severity. There was also no evidence of an association between human variation genome-wide and TB disease severity. Treatment-seeking, social, and environmental factors are likely to be the main determinants of disease severity at the point of care in this patient population.

Non-tuberculous mycobacteria (NTM) present a diagnostic challenge due to clinical overlap with tuberculosis (TB). The Xpert MTB/RIF Ultra assay occasionally detects rpoB probe amplification in specimens negative for the Mycobacterium tuberculosis complex (MTBC). We evaluated whether rpoB amplification could indicate NTM infection and support diagnostic decision-making. Specimens showing rpoB probe amplification but negative for MTBC by Xpert Ultra were selected from TB Screening and Treatment Centers (TBSTC) of icddr, b between January and August 2024. Samples underwent further testing using PCR targeting rpoB and IS6110, culture, and targeted next-generation sequencing (tNGS) to detect and identify NTM species. Among 50 specimens with rpoB amplification, rpoB PCR positivity was confirmed in all cases. Atypical mycobacterial growth was observed in 43 (86%) isolates. tNGS identified 34 isolates as NTM, with Mycobacterium gordonae (20.5%) and Mycobacterium abscessus subsp. abscessus (17.6%) being the most prevalent; 3 were MTBC, and the 6 were not detected. Receiver operating characteristic (ROC) analysis of rpoB2 threshold cycle (Ct) values demonstrated moderate diagnostic performance (AUC 0.807) with a Ct cutoff of 32.7 yielding 82.4% sensitivity, 77.8% specificity, and a positive predictive value of 93.3%. At a cutoff value of 32.7, the sensitivity and specificity for pulmonary samples were 90% and 100%, respectively, whereas for extrapulmonary samples they were 76% and 85%, respectively. These findings indicate that rpoB2 Ct values from the Xpert Ultra assay could provide additional diagnostic utility for identifying NTMs in MTBC-negative samples without additional costs.

Abstract Even though our meta-analysis ranks Mycobacterium tuberculosis genomes among the bacterial pathogens that are most straightforward to assemble, most available assemblies relied on short-read sequencing and contain genomic blind spots that miss functionally important genes. Complete genomes are essential for functional genomics, particularly for identifying small ORF-encoded proteins (SEPs; ≤100 amino acids), which can play critical biological roles yet are frequently missed by standard annotations. Here, we generated complete long-read assemblies for six clinical reference strains representing lineage 1 and the more pathogenic lineage 2, followed by comparative genomic and proteogenomic analyses. We additionally provide software to predict comprehensive sets of mycobacteria-specific proline-glutamic acid (PE) and PPE family genes, including lineage-specific variants. Using parallel accumulation–serial fragmentation mass spectrometry, we detected approximately two-thirds of each strain’s annotated proteome from unfractionated cell extracts. Extending our proteogenomic framework across related strains, and adding rigorous control of proteogenomic discovery rates using entrapment strategies, we revealed 12–24 previously unannotated proteins per strain, predominantly SEPs, 56–60 alternative translation start sites, and 9–17 expressed pseudogenes. Newly identified proteins included conserved and lineage-specific SEPs, an antitoxin, candidate antimicrobial peptides and novel proteins under purifying selection. Overall, applying this improved proteogenomics method to phylogenomically selected clinical reference strains provides a valuable approach for discovering candidate diagnostics or therapeutics, as illustrated here for a WHO-listed critical bacterial pathogen.

Tuberculosis (TB) remains a major public health concern. Improving TB control programs and treatment success requires a deeper understanding of the factors that determine disease presentation and treatment outcomes. While the importance of patient factors is well established, our understanding of the bacterial determinants of disease presentation and treatment outcomes in TB remains limited. In this study, we analyzed the Mycobacterium tuberculosis complex (MTBC) genomes and the associated clinical data from 4529 TB patients in the country of Georgia covering a period of 13 years. We used multivariable modeling together with genome-wide association studies (GWAS) to identify patient and bacterial factors that determine TB disease manifestation and clinical outcomes. Multivariable modelling confirmed the role of demographic and clinical factors in determining treatment outcomes, as well as the efficacy of novel TB treatments containing bedaquiline. In addition, we found that several bacterial factors, including the MTBC lineage, the specific mutations conferring resistance to rifampicin and fluoroquinolones, as well as a high bacterial burden, were associated with unfavorable outcomes. GWAS analyses revealed no bacterial genetic mutations associated with treatment outcomes beyond the known drug resistance-conferring mutations. However, we found that mutations in the bacterial gene sufD were linked to a reduced risk of lung cavities and a lower bacterial burden within patients. By contrast, specific mutations conferring resistance to rifampicin and fitness compensatory mutations were associated with a higher bacterial burden. Our results show that both patient and bacterial factors determine disease presentation and clinical outcomes in TB. They also support the rationale of optimizing treatment regimens against drug-resistant TB with existing drugs based on the specific genetic features of the pathogen. Finally, our results highlight sufD as a possible therapeutic candidate.

Drug tolerance allows bacteria to survive extended exposure to bactericidal drugs and is thought to play a role in drug resistance evolution. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), multidrug-resistant TB outbreaks are frequently caused by strains belonging to two phylogenetic lineages of the human-adapted strains of the Mtb complex, namely, lineages (L) 2 and L4. We hypothesized that members of L2 and L4 are more intrinsically drug tolerant and, as such, more readily evolve drug resistance. To explore this, we devised a high-throughput in vitro assay to measure drug tolerance in Mtb. We selected a cohort of strains representative of the globally most frequent lineages, L1-L4. We measured tolerance to rifampicin and bedaquiline and found L3 and L4 strains to have higher tolerance compared to L1 and L2 strains. In addition, phylogenetically closely related strains exhibited similar levels of tolerance, suggesting that tolerance is heritable. Finally, we explored genes previously reported to be associated with tolerance in Mtb and found significant enrichment in mutations in genes involved in cell wall and cell processes, intermediary metabolism and respiration, as well as lipid metabolism in high-tolerance strains.

Repeats are the most diverse and dynamic but also the least well-understood component of microbial genomes. For all we know, repeat-associated mutations such as duplications, deletions, inversions and gene conversion might be as common as point mutations, but because of short-read myopia and methodological bias, they have received much less attention. Long-read DNA sequencing opens the perspective of resolving repeats and systematically investigating the mutations they induce. For this study, we assembled the genomes of 16 closely related strains of the bacterial pathogen Mycobacterium tuberculosis from Pacific Biosciences HiFi reads, with the aim of characterizing the full spectrum of DNA polymorphisms. We found that complete and accurate genomes can be assembled from HiFi reads, with read size being the main limitation in the presence of duplications. By combining a reference-free pangenome graph with extensive repeat annotation, we identified 110 variants, 58 of which could be assigned to repeat-associated mutational mechanisms such as strand slippage and homologous recombination. Whilst recombination events were less frequent than point mutations, they affected large regions and introduced multiple variants at once, as shown by three gene conversion events and a duplication of 7.3 kb that involved ppe18 and ppe57, two genes possibly involved in immune subversion. The vast majority of variants were present in single isolates, such that phylogenetic resolution was only marginally increased when estimating a tree from complete genomes. Our study shows that the contribution of repeat-associated mechanisms of mutation can be similar to that of point mutations at the microevolutionary scale of an outbreak. A large reservoir of unstudied genetic variation in this 'monomorphic' bacterial pathogen awaits investigation.

The Mycobacterium tuberculosis complex (MTBC) phylogenetic lineages 1-4 (L1-L4) are the main causes of human tuberculosis (TB). Until now, most of the focus in the TB field has been on MTBC L2 and L4, as these two lineages are geographically widespread and have been repeatedly associated with multidrug resistance. By comparison, MTBC L1 has received little attention, partially because of its restricted geographical range that mainly includes low- to middle-income countries in South and Southeast Asia, and East Africa. However, recent estimates indicate that MTBC L1 is in fact the most common cause of human TB in terms of absolute numbers of TB patients, particularly among several high TB burden countries. As more L1 strains are being sampled in L1-endemic countries, the high genetic diversity of this geographically restricted MTBC lineage is slowly uncovered. This discovery has also impacted L1 nomenclature, which has been modified as new distinct L1 clades were identified. In parallel to the genomic discoveries ushered by progress in whole genome sequencing, clinical researchers have also studied several phenotypes that better describe L1 TB disease. L1 strains have been shown to have increased vulnerability to oxidative stress, which was associated with decreased virulence in animal and in vitro models. L1 infection also shows possible association with extrapulmonary TB and asymptomatic TB. However, despite belonging to the same lineage, L1 strains display phenotypic diversity that can be attributed to high within-lineage genetic diversity and possibly the interaction of different L1 genotypes with different human host genotypes. Among the clinical phenotypes that show heterogeneity are bacterial factors, immune profiles, and clinical virulence. The traditional view regarding the reduced transmissibility in L1 is now being challenged by new data indicating that L1 may be as transmissible as L2 or L4. Lastly, although historically referred to as being negatively associated with drug resistance, there is indication that the contribution of L1 to TB drug resistance is significant and that it may evolve drug resistance in ways distinct from those of other MTBC lineages.

The risk and prognosis of tuberculosis (TB) are influenced by a complex interplay between human and bacterial genetic factors. While previous genomic studies have largely examined human and bacterial genomes separately, we adopted an integrated approach to uncover host–pathogen interactions. We leveraged paired human and Mycobacterium tuberculosis (M.tb) genomic data from 1000 adult TB patients from Tanzania and used a “genome-to-genome” approach to search for associations between human and M.tb genetic variants and to identify interacting genetic loci. Our analyses revealed two significant host–pathogen genetic associations. The first significant association (p = 4.7e-11) links a human intronic variant in PRDM15 (rs12151990), a gene involved in apoptosis regulation, with an M.tb variant in Rv2348c (I101M), which encodes a T cell-stimulating antigen. The second significant association (p = 6.3e-11) connects a human intergenic variant near TIMM21 and FBXO15 (rs75769176) – also associated with TB severity (p = 0.04) – with an M.tb variant in FixA (T67M). While FBXO15 is involved in the regulation of antigen processing and TIMM21 affects mitochondrial function, FixA's role remains undefined due to limited functional characterization. Additionally, we observed that a group of M.tb T cell epitope variants were significantly associated with HLA-DRB1 variation, suggesting that, despite their rarity, certain epitopes may still be subjected to immune selective pressure. Together, these findings identify previously unknown sites of genomic conflicts between humans and M.tb, advancing our understanding of how this pathogen evades selection pressure and persist in human populations.

With the COVID-19 pandemic receding, tuberculosis (TB) is again the number one cause of human death to a single infectious agent. TB is caused by bacteria that belong to the Mycobacterium tuberculosis complex (MTBC). Recent advances in genome sequencing have provided new insights into the ecology and evolution of the MTBC. This includes the discovery of new phylogenetic lineages within the MTBC, a deeper understanding of the host tropism among the various animal-adapted lineages, enhanced knowledge on the evolutionary dynamics of antimicrobial resistance and transmission, as well as a better grasp of the within-host MTBC diversity. Moreover, advances in long-read sequencing are increasingly highlighting the relevance of structural genomic variation in the MTBC. These findings not only shed new light on the biology and epidemiology of TB, but also give rise to new questions and research avenues. The purpose of this Review is to summarize these new insights and discuss their implications for global TB control. In this Review, Gagneux and colleagues discuss ecological and evolutionary concepts related to the biology and epidemiology of the Mycobacterium tuberculosis complex, including new phylogenetic lineages, host tropism among the various animal-adapted lineages, the evolutionary dynamics of antimicrobial resistance and transmission, as well as within-host diversity and structural genomic variation.

Mycobacterium tuberculosis complex (MTBC) lineages show substantial variability in virulence, but the epidemiological consequences of this variability have not been studied in detail. Here, we aimed for a lineage-specific epidemiological characterization by applying phylodynamic models to genomic data from different countries, representing the most abundant MTBC lineages. Our results suggest that all lineages are associated with similar durations and levels of infectiousness, resulting in similar reproductive numbers. However, L1 and L6 are associated with a delayed onset of infectiousness, leading to longer periods between subsequent transmission events. Together, our findings highlight the role of MTBC genetic diversity in tuberculosis disease progression and transmission.

Repeats are the most diverse and dynamic, but also the least well understood component of microbial genomes. For all we know, repeat-associated mutations such as duplications, deletions, inversions, and gene conversion might be as common as point mutations, but because of short-read myopia and methodological bias they have received much less attention. Long-read sequencing opens the perspective of resolving repeats and systematically investigating the mutations they induce. For this study, we assembled the genomes of 16 closely related strains of the bacterial pathogen Mycobacterium tuberculosis from PacBio HiFi reads, with the aim of characterizing the full spectrum of DNA polymorphisms. We find that complete and accurate genomes can be assembled from HiFi reads, with read size being the main limitation in the presence of duplications. By combining a reference-free pangenome graph with extensive repeat annotation, we identified 110 variants, 58 of which can be assigned to repeat-associated mutational mechanisms such as strand slippage and homologous recombination. While recombination events are less frequent than point mutations, they can affect large regions and introduce multiple variants at once, as shown by three gene conversion events and a duplication of 7.3 kb that involve ppe18 and ppe57, two genes possibly involved in immune subversion. Our study shows that the contribution of repeat-associated mechanisms of mutation can be similar to that of point mutations at the microevolutionary scale of an outbreak. A large reservoir of unstudied genetic variation in this “monomorphic” bacterial pathogen awaits investigation. ### Competing Interest Statement The authors have declared no competing interest.

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug-resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensive resistance (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides the analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definitions. Sub-lineage 4.3 (52%) was the most frequent genotype, and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected twenty novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. A cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug resistance prediction, presenting the best accuracy values for five of the nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) or genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil and the genetic and clinical data correlations and demonstrated its utility for surveillance efforts regarding the spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant (XDR) TB are threatening global TB control. The World Health Organization has recently endorsed new regimens for the treatment of MDR-TB that rely on the new and repurposed drugs bedaquiline, pretomanid and linezolid with or without moxifloxacin (BPaL(M)). As BPaL(M) is being rolled-out, resistance to these new drugs is already emerging, leading to acquired XDR-TB. Importantly, instances of transmitted XDR-TB have been reported. The spread of highly drug-resistant M. tuberculosis (MTB) strains pose at risk novel TB treatments that took decades to develop. In this study, we analyzed 6,926 MTB genomes from a 13-year nationwide study in Georgia, a known geographical hotspot of MDR-TB, together with more than 80,000 MTB genomes from public sources to estimate the relative contribution of transmission to the burden of XDR-TB. We show that XDR-TB is already geographically widespread, occurring in at least 27 countries across four continents. Moreover, we estimated that a quarter of the XDR-TB cases identified are likely the consequence of transmission. Our findings call for urgent improvements in the global diagnostic capacity, infection control, and surveillance of XDR-TB. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the Swiss National Science Foundation (grants 320030-227432, and CRSII5_213514) and the European Research Council (883582-ECOEVODRTB). Calculations were performed at sciCORE (http://scicore.unibas.ch/) scientific computing core facility at the University of Basel and on the Euler cluster at ETH Zuerich. Sequencing was carried out at the Genomics Facility Basel of the University of Basel and the Department of Biosystems Science and Engineering at ETHZ in Basel, Switzerland. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The institutional Review Board of the NCTLD in Tbilisi, Georgia and the Ethics Commission of North- and Central Switzerland granted ethical approval for this study. The ethics committees waived the need for individual patient consent since only limited and anonymized clinical data were used. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Supplementary File 1 contains the accession numbers with additional information for all XDR strains analyzed.

AbstractMycobacterium tuberculosiscomplex (MTBC) lineages show substantial variability in virulence, but the epidemiological consequences of this variability have not been studied in detail. Here, we aimed for a lineage-specific epidemiological characterization by applying phylodynamic models to genomic data from different countries, representing the most abundant MTBC lineages. Our results show that all lineages are associated with similar durations and levels of infectiousness, resulting in similar reproductive numbers. However, L1 and L6 are associated with a delayed onset of infectiousness, leading to longer periods between subsequent transmission events. Together, our findings highlight the role of MTBC phylogenetic diversity in tuberculosis disease progression and transmission.

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 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-1β, and IL-1β 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.

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.

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.